Heterodimeric antibodies and antigen-binding fragments thereof

Abstract

Novel polypeptide complexes useful for enhancing precise light chain pairing in bispecific or multispecific molecules are provided. Further provided are nucleic acids comprising nucleotide sequences encoding the polypeptide complexes, vectors comprising the nucleic acids, host cells comprising the nucleic acids or vectors, pharmaceutical compositions comprising the polypeptide complexes, and uses of the polypeptide complexes for treating or preventing a disease, condition or symptom.

Description

[Technical field]

[0001]

[0001] This disclosure relates generally to the field of protein engineering, specifically to the area of ​​engineered antibodies, and more specifically to bispecific antibodies engineered to have high selectivity in the cognate pairing of immunoglobulin light and heavy chains. [Background technology]

[0002]

[0002] Antibodies, also known as immunoglobulins (Ig), are large proteins present in the plasma or other body fluids of vertebrates, produced by plasma cells (i.e., differentiated B cells) and utilized by the immune system to bind and neutralize foreign agents (e.g., pathogenic bacteria, viruses, and parasites, etc.) that invade the host. Primarily due to their highly specific ability to recognize and bind to various target molecules (i.e., antigens), antibodies have traditionally been used as important research tools for scientific research as well as important disease screening and diagnostic tools, and have recently emerged as promising therapeutic agents to manage or treat a variety of human diseases, including certain autoimmune / inflammatory disorders, such as psoriasis, rheumatoid arthritis, and multiple sclerosis, and most notably, cancer (e.g., breast cancer, colorectal cancer, non-Hodgkin's lymphoma, etc.).

[0003]

[0003] Natural antibodies generally contain two identical heavy (H) chains and two identical light (L) chains in an immunoglobulin unit, so that despite the presence of two antigen-binding sites, such naturally occurring antibodies specifically target only one antigen. Although such single-antigen-targeted (i.e., monospecific) antibody drugs have achieved success in treating a significant number of human diseases, they have not achieved significant efficacy in treating many complex diseases that are usually caused by multiple factors, such as cancer.

[0004]

[0004] Therefore, there is a great need to design multispecific antibody drugs that can simultaneously bind to two or more different antigens in one therapeutic molecule, thereby generating additive, complementary or synergistic effects that exceed the effects of individual monospecific antibodies. Such multispecific antibodies can be useful, for example, to bring more than two or more different antigens into close proximity, thereby promoting their interaction. In one example, the immune system can promote the recognition or elimination of tumor or pathogen cells by bringing immune cells into close proximity to tumor-associated or pathogen antigens. In another example, multispecific antibodies can bind to different (and preferably non-overlapping) epitopes of one antigen, which can be useful in enhancing the recognition or binding to target antigens, especially antigens that are prone to mutation (e.g., viral antigens).

[0005]

[0005] Towards this goal, many attempts have been made to design new immunoglobulin formats that exhibit multispecificity, most notably bispecificity, binding to two or more epitopes simultaneously. Regarding bispecific antibodies, which are the predominant type of multispecific antibody, various bispecific formats have been designed, which can be classified into IgG-like specific antibodies and non-IgG-like bispecific antibodies (e.g., DVD-Ig, CrossMab, BiTE, etc.) (Spiess et al., Molecular Immunology, 67(2), pp.95-106(2015)). However, such formats usually have limitations in specificity, stability, solubility, yield, short half-life and immunogenicity.

[0006] Among such bispecific antibody formats, IgG-like bispecific antibodies are essentially monoclonal antibodies with one Fc region and two Fab arms that specifically target and bind two different antigens or two different epitopes of a single antigen, respectively. To facilitate downstream development, it is desirable to be able to produce such bispecific molecules as conveniently as conventional IgG, i.e., from a single host cell (e.g., quadroma), with high expression levels and purity. However, because the pairing of the cognate light-heavy chains and the assembly of the two different half antibodies are typically not automatically controllable, this traditional method can be quite inefficient and of low quality, with a high probability of mispairing resulting in significant heterogeneity of the product.

[0007]

[0007] Several strategies have been designed to reduce light chain mispairing. The CrossMab platform developed by Roche essentially swaps domains in the CH1 and CL regions (Schaefer et al., Proceedings of the National Academy of Sciences of the United States of America, 108(27), pp. 11187-11192 (2011)). Another strategy developed by MedImmune introduces alternative disulfide bonds into the CH1 and CL regions (Mazor et al., mAbs, 7(2), pp. 377-389 (2015); U.S. Patent No. 9527927; and U.S. Patent Publication No. 20180022807). Another strategy developed by Amgen introduces new electrostatics in the CH1-CL region (Liu et al., Journal of Biological Chemistry, 290(12), pp. 7535-7562 (2015); and US Patent Publication No. 20140154254). Another strategy developed by Lilly (Lewis et al., Nature Biotechnology, 32(2), pp. 191-198 (2014)) and Genentech (Dillon et al., mAbs, 9(2), pp. 213-230 (2017)) introduces mutations in both the variable and constant domains. However, each of these existing solutions has its own disadvantages, and thus there is still a great need for better solutions to reduce light chain mispairing. Summary of the Invention

[0008]

[0008] The present disclosure provides novel polypeptide complexes useful for enhancing correct light chain pairing in bispecific or multispecific molecules.

[0009]

[0009] In one aspect, the present disclosure provides a polypeptide complex comprising a first target binding domain comprising a first target binding portion operably linked to a first constant portion, the first constant portion comprising a first heavy chain constant region 1 (CH1) associated with a first light chain constant region (CL), the first CH1 region comprising a first amino acid residue at EU position n1 and the first CL region comprising a second amino acid residue at EU position n2, the n1:n2 position pair being selected from the group consisting of 128:118 and 173:160, and the first amino acid residue and the second amino acid residue form a covalent bond. In some such embodiments, the first CH1 region further comprises a third amino acid residue at EU position n3 and the first CL region further comprises a fourth amino acid residue at EU position n4, the n3:n4 position pair being selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, and the third and fourth amino acid residues form a non-covalent bond. In some embodiments, the first CH1 region further comprises a third amino acid residue at EU position n3 and the first CL region further comprises a fourth amino acid residue at EU position n4, the n3:n4 position pair being selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, and the third and fourth amino acid residues are oppositely charged.

[0010] In some embodiments, the first target binding domain comprises or is an antigen binding domain. In some embodiments, the first target binding moiety comprises or is an antigen binding moiety.

[0011]

[0011] In some embodiments, the polypeptide complex provided herein further comprises a second target binding domain comprising a second target binding portion operably linked to a second constant portion, the second constant portion comprising a second CH1 region associated with a second CL region, the first CH1 region does not substantially bind to the second CL region, and the second CH1 region does not substantially bind to the first CL region.

[0012] In some embodiments, the second target binding domain comprises or is an antigen binding domain. In some embodiments, the second target binding moiety comprises or is an antigen binding moiety.

[0013]

[0013] In some embodiments, the second CH1 region comprises a first corresponding amino acid residue at EU position n1' and the second CL region comprises a second corresponding amino acid residue at EU position n2', the n1':n2' position pair being identical to the n1:n2 position pair, and the first corresponding amino acid residue at EU position n1' does not form a covalent bond with the second amino acid residue at EU position n2, and / or the second corresponding amino acid residue at EU position n2' does not form a covalent bond with the first amino acid residue at EU position n1.

[0014] In some embodiments, the first corresponding amino acid residue at EU position n1' and the second corresponding amino acid residue at EU position n2' do not form a covalent bond.

[0015] In some embodiments, the second CH1 region further comprises a third corresponding amino acid residue at EU position n3', and the second CL region further comprises a fourth corresponding amino acid residue at EU position n4', wherein the n3':n4' position pair is identical to the n3:n4 position pair; (a) the fourth corresponding amino acid residue at EU position n4' and the third amino acid residue at EU position n3 are oppositely uncharged or similarly charged, and / or (b) the third corresponding amino acid residue at EU position n3' and the fourth amino acid residue at EU position n4 are oppositely uncharged or similarly charged.

[0016]

[0016] In some embodiments, the third corresponding amino acid residue at EU position n3' and / or the fourth corresponding amino acid residue at EU position n4' is uncharged.

[0017]

[0017] In some embodiments, the second CH1 region further comprises a fifth corresponding amino acid residue at EU position n5', and the second CL region further comprises a sixth corresponding amino acid residue at EU position n6', the fifth corresponding amino acid residue and the sixth corresponding amino acid residue form a covalent bond, and the n5':n6' position pair is different from the n1:n2 position pair.

[0018] In some embodiments, the n5':n6' position pair is selected from the group consisting of 220:214 (IgG1), 131:214 (IgG2 and IgG4), 128:118 and 173:160. In some embodiments, the n5':n6' position pair is 220:214 (IgG1). In some embodiments, the n5':n6' position pair is 131:214 (IgG2 and IgG4). In some embodiments, the n5':n6' position pair is 128:118 and the n1:n2 position pair is 173:160, or the n5':n6' position pair is 173:160 and the n1:n2 position pair is 128:118.

[0019] In some such embodiments, the first CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and the first CL region has an amino acid residue other than cysteine ​​at EU position 214. In some such embodiments, the second CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and the second CL region has an amino acid residue other than cysteine ​​at EU position 214. In some such embodiments, neither the first nor the second CH1 region has a cysteine ​​residue at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and / or neither the first nor the second CL region has a cysteine ​​residue at EU position 214.

[0020]

[0020] In some embodiments, the first CH1 region further comprises a fifth amino acid residue at EU position n5 and the first CL region further comprises a sixth amino acid residue at EU position n6, wherein the n5:n6 position pair is identical to the n5':n6' position pair, and the fifth corresponding amino acid residue at EU position n5' does not form a covalent bond with the sixth amino acid residue at EU position n6, and / or the sixth corresponding amino acid residue at EU position n6' does not form a covalent bond with the fifth amino acid residue at EU position n5.

[0021]

[0021] In some embodiments, the fifth amino acid residue at EU position n5 and the sixth amino acid residue at EU position n6 do not form a covalent bond.

[0022]

[0022] In some embodiments, the second CH1 region further comprises a seventh corresponding amino acid residue at EU position n7', and the second CL region further comprises an eighth corresponding amino acid residue at EU position n8', the n7':n8' position pair being selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, the seventh corresponding amino acid residue and the eighth corresponding amino acid residue are oppositely charged, and the n7':n8' position pair is different from the n3:n4 position pair.

[0023] In some embodiments, the n7':n8' position pair is selected from the group consisting of 183:176, 141:116, and 126:121.

[0024] In some embodiments, the n7':n8' position pair is 183:176 and the n3:n4 position pair is selected from the group consisting of 141:116, 126:121 and 218:122. In some embodiments, the n7':n8' position pair is 141:116 and the n3:n4 position pair is selected from the group consisting of 183:176, 126:121 and 218:122. In some embodiments, the n7':n8' position pair is 126:121 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 218:122. In some embodiments, the n7':n8' position pair is 218:122 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 126:121.

[0025]

[0025] In some embodiments, the first CH1 region further comprises a seventh amino acid residue at EU position n7 and the second CL region further comprises an eighth amino acid residue at EU position n8, wherein the n7:n8 position pair is identical to the n7':n8' position pair, and wherein the seventh corresponding amino acid residue at EU position n7' and the eighth amino acid residue at EU position n8 are not oppositely charged or are similarly charged, and / or the eighth corresponding amino acid residue at EU position n8' and the seventh amino acid residue at EU position n7 are not oppositely charged or are similarly charged.

[0026]

[0026] In some embodiments, the seventh amino acid residue at EU position n7 and / or the eighth amino acid residue at EU position n8 is uncharged.

[0027]

[0027] In this specification, a covalent bond is essentially a chemical bond formed between a first amino acid residue and a second amino acid residue, covalently linking the first CH1 region and the first CL region in the polypeptide complex. Such a chemical bond can be a disulfide bond formed between two cysteine ​​residues, but such a chemical bond can also be of different types.

[0028]

[0028] In some embodiments, the covalent bond is a disulfide bond.

[0029]

[0029] In some embodiments, the disulfide bond is formed between two cysteine ​​residues.

[0030]

[0030] In some embodiments, the first amino acid residue at EU position n1 and the second amino acid residue at EU position n2 are both cysteine ​​residues, and / or the fifth corresponding amino acid residue at EU position n5' and the sixth corresponding amino acid residue at EU position n6' are both cysteine ​​residues.

[0031] In some embodiments, the first CH1 region comprises a substitution of L128C (EU position n1) and the first CL region comprises a substitution of F118C (EU position n2). In some such embodiments, the second CH1 region comprises a substitution of V173C (EU position n5') and the second CL region comprises a substitution of Q160C (EU position n6') of the kappa light chain or E160C (EU position n6') of the lambda light chain.

[0032] In some embodiments, the first CH1 region comprises a substitution of V173C (EU position n1) and the first CL region comprises a substitution of Q160C (EU position n2) of the kappa light chain or E160C (EU position n2) of the lambda light chain. In some such embodiments, the second CH1 region comprises a substitution of L128C (EU position n5') and the second CL region comprises a substitution of F118C (EU position n6').

[0033] In some embodiments, the third amino acid residue at EU position n3 is a positively charged amino acid residue and the fourth amino acid residue at EU position n4 is a negatively charged amino acid residue. In some embodiments, the third amino acid residue at EU position n3 is a negatively charged amino acid residue and the fourth amino acid residue at EU position n4 is a positively charged amino acid residue.

[0034] In some embodiments, the seventh corresponding amino acid residue at EU position n7' is a positively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a negatively charged amino acid residue. In some embodiments, the seventh corresponding amino acid residue at EU position n7' is a negatively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a positively charged amino acid residue.

[0035]

[0035] In some embodiments, the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E).

[0036]

[0036] In some embodiments, at least one, two, three or four of the first amino acid residue at EU position n1, the second amino acid residue at EU position n2, the third amino acid residue at EU position n3 and the fourth amino acid residue at EU position n4 are introduced by substitution.

[0037] In some embodiments, the third and fourth amino acid residues of the n3:n4 position pair are S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K, S183D:S176R , S183D:S176H, S183E:S176K, S183E:S176R, S183E:S176H, A141K:F116D, A141K:F116 E, A141R:F116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141D:F116 R, A141D:F116H, A141E:F116K, A141E:F116R, A141E:F116H, F126K:S121D, F126K:S12 1E, F126R:S121D, F126R:S121E, F126H:S121D, F126H:S121E, F126D:S121K, F126D:S12 1R, F126D:S121H, F126E:S121K, F126E:S121R, F126E:S121H, K218D:D122K, K218D:D122H, K218D:D122R, K218E:D122K, K218E:D122H, and K218E:D122R.

[0038]

[0038] In some embodiments, at least one, two, three or four of the fifth corresponding amino acid residue at EU position n5', the sixth corresponding amino acid residue at EU position n6', the seventh corresponding amino acid residue at EU position n7' and the eighth corresponding amino acid residue at EU position n8' are introduced by substitution.

[0039] In some embodiments, the seventh and eighth corresponding amino acid residues of the n7':n8' position pair are S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K, S183D:S176 R, S183D:S176H, S183E:S176K, S183E:S176R, S183E:S176H, A141K:F116D, A141K:F116E, A1 41R:F116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141D:F116R, A141D: F116H, A141E:F116K, A141E:F116R, A141E:F116H, F126K:S121D, F126K:S121E, F126R:S121 D, F126R:S121E, F126H:S121D, F126H:S121E, F126D:S121K, F126D:S121R, F126D:S121H, F1 and the n7':n8' position pair is different from the n3:n4 position pair.

[0040] In some embodiments, the first target binding domain comprises a first combination of substitutions at the (n1+n2):(n3+n4) positions and / or the second target binding domain comprises a second combination of substitutions at the (n5'+n6'):(n7'+n8') positions, wherein the first combination of substitutions and / or the second combination of substitutions are (L128C+S183K):(F118C+S176D), (L128C+S183K):(F118C+S176E), (L128C+S183R):(F118C+S176D), (L128C+S183R ... ):(F118C+S176E), (L128C+S183H):(F118C+S176D), (L128C+S183H):(F1 18C+S176E), (L128C+S183D):(F118C+S176K), (L128C+S183D):(F118C+S 176R), (L128C+S183D):(F118C+S176H), (L128C+S183E):(F118C+S176K) , (L128C+S183E):(F118C+S176R), (L128C+S183E):(F118C+S176H), (V173 C+A141K):(Q160C(or E160C)+F116D),(V173C+A141K):(Q160C(or E160C)+F116E),(V173C+A141R):(Q160C(or E160C)+F116D),(V173C+A141R):(Q160C(or E160C)+F116E),(V173C+A141H):(Q160C(or E160C)+F116D),(V173C+A141H):(Q160C(or E160C)+F116E),(V173C+A141D):(Q160C( or E160C)+F116K), (V173C+A141D):(Q160C(or E160C)+F116R), (V173C+A141D):(Q160C(or E160C)+F116H), (V173C+A141E):(Q160C(or E160C)+F116K), (V173C+A141E):(Q160C(or E160C)+F116R), (V173C+A141E):(Q160C(or E160C)+F116H), (V173C+S183K):(Q160C(or E160C)+S176D),(V173C+S183K):(Q160C(or E160C)+S176E), (V173C+S183R):(Q160C(or E160C)+S176D), (V173C+S183R):(Q160C(or E160C)+S176E), (V173C+S183H):(Q160C(or E160C)+S176D), (V173C+S183H):(Q160C(or E160C)+S176E), (V173C+S183D):(Q160C(or E160C)+S176K), (V173C+S183D):(Q1 60C(or E160C)+S176R), (V173C+S183D):(Q160C(or E160C)+S176H), (V173C+S183E):(Q160C(or E160C)+S176K), (V173C+S183E):(Q160C(or E160C)+S176R), (V173C+S183E):(Q160C(or E160C)+S176H), (L128C+F126K):(F118C+S121D), (L128C+F126K):(F118C+S121E), (L128C+F126R):(F1 18C+S121D), (L128C+F126R):(F118C+S121E), (L128C+F126H):(F118C+S 121D), (L128C+F126H):(F118C+S121E), (L128C+F126D):(F118C+S121K) , (L128C+F126D):(F118C+S121R), (L128C+F126D):(F118C+S121H), (L12 8C+F126E):(F118C+S121K), (L128C+F126E):(F118C+S121R), (L128C+F12 6E):(F118C+S121H),(V173C+F126K):(Q160C(or E160C)+S121D),(V173C+F126K):(Q160C(or E160C)+S121E),(V173C+F126R):(Q160C(or E160C)+S121D),(V173C+F126R):(Q160C(or E160C)+S121E),(V173C+F126H):(Q160C(or E160C)+S121D),(V173C+F126H):(Q160C(or E160C)+S121E),(V173C+F126D):(Q160C(or E160C)+S121K), (V173C+F126D):(Q160C(or E160C)+S121R), (V173C+F126D):(Q160C(or E160C)+S121H), (V173C+F126E):(Q160C(or E160C)+S121K), (V173C+F126E):(Q160C(or E160C) +S121R), (V173C+F126E):(Q160C(or E160C)+S121H), (L128C+K218D):(F118C+D122K), (L128C+K218D):(F118C+D122H), (L128C+K218D):(F118C+D122R), (L128C+K218E):(F118C+D122K), (L128C+K218E):(F118C+D122 2H), (L128C+K218E):(F118C+D122R), (V173C+K218D):(Q160C(or E160C)+D122K), (V173C+K218D):(Q160C(or E160C)+D122H), (V173C+K218D):(Q160C(or E160C)+D122R), (V173C+K218E):(Q160C(or E160C)+D122K ), (V173C+K218E):(Q160C(or E160C)+D122H) and (V173C+K218E):(Q160C(or E160C)+D122R), with the proviso that when both the first substitution combination and the second substitution combination are selected, the n5':n6' position pair is different from the n1:n2 position pair and the n7':n8' position pair is different from the n3:n4 position pair.

[0041] In some embodiments, the first target binding moiety comprises a first polypeptide fragment operably linked to a first CL region, and the second target binding moiety comprises a second polypeptide fragment operably linked to a second CL region, the first polypeptide fragment having a different amino acid sequence than the second polypeptide fragment. In some embodiments, either the first polypeptide fragment or the second polypeptide fragment is not present in the polypeptide complex.

[0042] In some embodiments, the polypeptide complex may be a fusion protein comprising two or more polypeptide fragments operably linked to a CH1 region and a CL region, respectively. In some embodiments, the first target binding moiety further comprises a third polypeptide fragment operably linked to the first CH1 region, and the second target binding moiety comprises a fourth polypeptide fragment operably linked to the second CH1 region. In some embodiments, the third polypeptide fragment has a different amino acid sequence than the fourth polypeptide fragment. In some embodiments, either the third polypeptide fragment or the fourth polypeptide fragment is absent from the polypeptide complex.

[0043] In some embodiments, the first polypeptide fragment and the third polypeptide fragment each contain a target binding site and can bind to its target molecule. For example, the first polypeptide fragment and the third polypeptide fragment can bind to the same target molecule or to different target molecules. In another example, the first polypeptide fragment and the third polypeptide fragment can have either the same or different amino acid sequences. In some embodiments, either the first polypeptide fragment or the third polypeptide fragment is not present in the polypeptide complex.

[0044] Similarly, the second and fourth polypeptide fragments each contain a target binding site and can bind to their target molecules. For example, the second and fourth polypeptide fragments can bind to the same target molecule or to different target molecules. In another example, the second and fourth polypeptide fragments can have either the same or different amino acid sequences. In some embodiments, either the second or fourth polypeptide fragment is not present in the polypeptide complex. In some embodiments, one, two or three of the four polypeptide fragments are not present in the polypeptide complex.

[0045] In some embodiments, the first and third polypeptide fragments may associate to form a first target binding site. Similarly, the second and fourth polypeptide fragments may associate to form a second target binding site. In some embodiments, the first and second target binding sites may bind to the same target molecule, or to different portions on the same target molecule, or to different target molecules.

[0046]

[0046] In some embodiments, the first polypeptide fragment and the third polypeptide fragment each comprise a first target binding site or associate with each other to form a first target binding site, and / or the second polypeptide fragment and the fourth polypeptide fragment each comprise a second target binding site or associate with each other to form a second target binding site.

[0047] In some embodiments, the first target-binding moiety may be a first antigen-binding moiety and / or the second target-binding moiety may be a second antigen-binding moiety, hi some embodiments, the antigen-binding moiety is derived from one or more antibody fragments.

[0048] In some embodiments, the first antigen-binding portion may comprise a first VL region and a first VH region, which associate to form a first antigen-binding site. In some embodiments, the second antigen-binding portion may comprise a second VL region and a second VH region, which associate to form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site may bind to the same antigen, or different epitopes on the same antigen, or different antigens.

[0049]

[0049] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain are comprised within an antibody, optionally a bispecific antibody or a multispecific antibody.

[0050]

[0050] In some embodiments, the second antigen-binding domain and the first antigen-binding domain bind to different antigens or bind to different epitopes on the same antigen.

[0051] In some embodiments, (a) one of the first antigen-binding domain and the second antigen-binding domain binds to a tumor-associated antigen and the other binds to an immune-related target; or (b) one of the first antigen-binding domain and the second antigen-binding domain binds to a first tumor-associated antigen, and the other binds to a second tumor-associated antigen.

[0052]

[0052] In some embodiments, the first and / or second antigen binding domain is a chimeric, humanized or fully human antigen binding domain.

[0053]

[0053] In some embodiments, the first and / or second antigen-binding portion is selected from the group consisting of a nanobody, an Fv fragment, an scFv, a disulfide-stabilized Fv fragment, a (dsFv)2, a bispecific dsFv and a diabody.

[0054]

[0054] In some embodiments, the first and / or second antigen-binding domain is selected from the group consisting of a Fab domain, Fab' and F(ab')2.

[0055]

[0055] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain comprises one or more CDRs operably linked to a CH1 region and a CL region.

[0056] In some embodiments, the first antigen-binding domain is a first Fab domain and / or the second antigen-binding domain is a second Fab domain.

[0057]

[0057] In the present specification, the polypeptide complex may be an antibody or a fragment thereof having a Fab domain. Examples of the polypeptide complex may include, but are not limited to, the scope of the present disclosure, monospecific antibodies, bispecific antibodies, trifunctional antibodies, Fab, FAb', F(ab')2, etc. The polypeptide complex may be extended to any other molecule that contains a Fab domain / region.

[0058] In some embodiments, the second Fab domain comprises: (a) one or more light chain CDRs and / or light chain framework regions distinct from the first Fab domain, and optionally (b) comprises one or more heavy chain CDRs and / or heavy chain framework regions that differ from the first Fab domain.

[0059]

[0059] In some embodiments, the polypeptide complex further comprises an Fc region operably linked to the first target binding domain and the second target binding domain.

[0060] In some embodiments, the Fc region is derived from IgG, IgA, IgM, IgE, or IgD. In some embodiments, the Fc region is derived from IgG. In some embodiments, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc region is derived from IgG1.

[0061]

[0061] In some embodiments, the Fc region is a heterodimer.

[0062]

[0062] In some embodiments, the heterodimeric Fc region contains one or more mutations that promote heterodimerization.

[0063] In some embodiments, the heterodimeric Fc region comprises a first Fc polypeptide comprising a first Fc mutation and / or a second Fc polypeptide comprising a second Fc mutation; a) the first Fc mutation comprises T366W or S354C and the second Fc mutation comprises Y349C, T366S, L368A or Y407V; b) the first Fc mutation comprises D399K or E356K and the second Fc mutation comprises K392D or K409D; c) the first Fc mutation comprises E356K, E357K or D399K and the second Fc mutation comprises K370E, K409D or K439E; d) the first Fc mutation comprises S364H or F405A and the second Fc mutation comprises Y349T or T394F; e) the first Fc mutation comprises S364H or T394F and the second Fc mutation comprises Y394T or F405A; f) the first Fc mutation comprises K370D or K409D and the second Fc mutation comprises E357K or D399K; or g) the first Fc mutation comprises L351D or L368E and the second Fc mutation comprises L351K or T366K; Numbering follows the EU index.

[0064] In another aspect, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide complex provided herein, or a portion thereof.

[0065]

[0065] In another aspect, the disclosure provides a vector comprising a nucleic acid provided herein.

[0066]

[0066] In another aspect, the disclosure provides a host cell comprising a nucleic acid provided herein or a vector provided herein.

[0067]

[0067] In another aspect, the disclosure provides a pharmaceutical composition comprising a polypeptide conjugate as provided herein and a pharma ceutically acceptable carrier.

[0068]

[0068] In another aspect, the present disclosure provides a conjugate comprising a polypeptide complex as provided herein and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracer molecule, an anticancer drug and a cytotoxic molecule.

[0069]

[0069] In another aspect, the disclosure provides a composition comprising a polypeptide complex as provided herein or a conjugate as provided herein and a pharma- ceutically acceptable carrier.

[0070] In another aspect, the disclosure provides a method for treating or preventing a disease, condition or symptom, the method comprising administering a therapeutically effective amount of a polypeptide complex provided herein, a pharmaceutical composition provided herein, a conjugate provided herein, or a composition provided herein to a subject in need thereof.

[0071]

[0071] In some embodiments, the disease is selected from the group consisting of cancer, an inflammatory disease, an infectious or parasitic disease, a cardiovascular disease, a neurological disorder, a neuropsychiatric condition, an injury, an autoimmune disease, a metabolic disease, a neurodegenerative disease or a blood clotting disorder.

[0072]

[0072] In another aspect, the present disclosure provides a method for detecting the presence or level of an antigen, comprising the steps of contacting a sample suspected of containing the antigen with a polypeptide complex provided herein and determining complex formation between the antigen and the polypeptide complex.

[0073]

[0073] The foregoing and other features of the present invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0074] [Figure 1]

[0074] Figure 1 shows the SDS-PAGE results of DIC002 (Figure 1A), DIC007 (Figure 1B) and DIC0014 (Figure 1C). [Figure 2-1]

[0075] FIG. 2 shows the SEC-HPLC results of DIC002 (FIG. 2A), DIC007 (FIG. 2B) and DIC014 (FIG. 2C). [Figure 2-2] FIG. 2 shows the SEC-HPLC results of DIC002 (FIG. 2A), DIC007 (FIG. 2B) and DIC014 (FIG. 2C). [Figure 2-3] FIG. 2 shows the SEC-HPLC results of DIC002 (FIG. 2A), DIC007 (FIG. 2B) and DIC014 (FIG. 2C). [Diagram 3]

[0076] FIG. 3 shows the LC-MS results of intact DIC014 (FIG. 3A) and deglycosylated DIC014 (FIG. 3B). [Figure 4-1]

[0077] FIG. 4 shows the SDS-PAGE results of DIC003 (FIG. 4A), DIC004 (FIG. 4B), DIC005 (FIG. 4C), DIC006 (FIG. 4D), DIC009 (FIG. 4E) and DIC010 (FIG. 4F). [Figure 4-2] FIG. 4 shows the SDS-PAGE results of DIC003 (FIG. 4A), DIC004 (FIG. 4B), DIC005 (FIG. 4C), DIC006 (FIG. 4D), DIC009 (FIG. 4E) and DIC010 (FIG. 4F). [Figure 5-1]

[0078] FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 5-2] FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 5-3]FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 5-4] FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 5-5] FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 5-6] FIG. 5 shows the SEC-HPLC results of DIC003 (FIG. 5A), DIC004 (FIG. 5B), DIC005 (FIG. 5C), DIC006 (FIG. 5D), DIC009 (FIG. 5E) and DIC010 (FIG. 5F). [Figure 6]

[0079] FIG. 6 shows the SDS-PAGE results of DIC015 (FIG. 6A) and DIC016 (FIG. 6B). [Figure 7-1]

[0080] FIG. 7 shows the SEC-HPLC results of DIC015 (FIG. 7A) and DIC016 (FIG. 7B). [Figure 7-2] FIG. 7 shows the SEC-HPLC results of DIC015 (FIG. 7A) and DIC016 (FIG. 7B). [Figure 8]

[0081] FIG. 8 shows the LC-MS results of intact DIC015 (FIG. 8A) and deglycosylated DIC015 (FIG. 8B). [Figure 9]

[0082] FIG. 9 shows the LC-MS results of intact DIC016 (FIG. 9A) and deglycosylated DIC016 (FIG. 9B). [Figure 10-1]

[0083] Figure 10 shows the LC-MS results of intact DIC009 (Figure 10A) and deglycosylated DIC009 (Figure 10B). Figure 10C shows a partial enlargement of Figure 10B. [Figure 10-2] Figure 10 shows the LC-MS results of intact DIC009 (Figure 10A) and deglycosylated DIC009 (Figure 10B). Figure 10C shows a partial enlargement of Figure 10B. [Figure 11-1]

[0084] Figure 11 shows the LC-MS results of intact DIC010 (Figure 11A) and deglycosylated DIC010 (Figure 11B). Figure 11C shows a partial enlargement of Figure 11B. [Figure 11-2] Figure 11 shows the LC-MS results of intact DIC010 (Figure 11A) and deglycosylated DIC010 (Figure 11B). Figure 11C shows a partial enlargement of Figure 11B. [Figure 12-1]

[0085] FIG. 12 shows the binding affinity of DIC010 to human PD1 (FIG. 12A), human TGFβR2 (FIG. 12B) and human TGFβR3 (FIG. 12C) as measured by SPR assay. [Figure 12-2] FIG. 12 shows the binding affinity of DIC010 to human PD1 (FIG. 12A), human TGFβR2 (FIG. 12B) and human TGFβR3 (FIG. 12C) as measured by SPR assay. [Figure 13]

[0086] FIG. 13 shows the efficacy of DIC010 at various doses (0 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg) on ​​the human PD1-MC38 syngeneic mouse model. [Figure 14]

[0087] FIG. 14 shows the binding area analysis of the CH—CL interface. [Figure 15]

[0088] FIG. 15 shows binding area analysis of the CH1-VH and CL-VL interfaces. [Figure 16]

[0089] FIG. 16 shows the amino acid residues selected for mutation testing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075]

[0090] Provided herein are polypeptide complexes having engineered target binding domains, and bispecific antibodies engineered to have high selectivity for immunoglobulin light chain-heavy chain cognate pairings.

[0076]

[0091] Before providing a detailed description of the present invention, the following are noted and defined.

[0077]

[0092] All the descriptions provided herein are intended to merely illustrate various embodiments of the present invention provided in this disclosure.Therefore, the specific modifications discussed should not be interpreted as limitations on the scope of the present disclosure.It will be apparent to those skilled in the art that various equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification.

[0078]

[0093] All references cited in this disclosure, including patent applications, issued patents, published articles or other publications, are incorporated herein by reference in their entirety for the purpose of providing methods that may be used in connection with the descriptions provided herein. With respect to any terms provided in one or more publications that are similar or identical to terms explicitly defined in this disclosure, the definition of the term explicitly provided in this disclosure shall prevail in all respects.

[0079]

[0094] All technical and scientific terms used in this disclosure are generally assumed to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless expressly defined otherwise.

[0080]

[0095] As used herein, and throughout this disclosure, the articles "a," "an," and "the" should be construed to mean "one or more" or "at least one," unless otherwise specified. By way of example, "a polypeptide complex" means one polypeptide complex or two or more polypeptide complexes.

[0081]

[0096] As used herein, terms such as "about," "approximately," "around," and the like refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies to the extent of 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the referenced quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the terms "about" or "approximately," when used in front of a numerical value, indicate a value in the range of plus or minus 15%, 10%, 5%, or 1%.

[0082]

[0097] As used in this specification, the terms "comprise," "comprises," "comprising," "include," "includes," "including," "contain," "contains," "containing," "have," "has," "having," and the like are used synonymously, inclusively, and do not exclude additional elements, features, steps, acts, operations, etc.

[0083]

[0098] As used herein, the term "or," when used, is used in an inclusive sense (rather than an exclusive sense), e.g., linking a list of elements, so the term "or" refers to one, some, or all of the elements in the list.

[0084]

[0099] As used herein, the phrase "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. A phrase referring to "at least one of" a list of items is to be interpreted as referring to any combination of such items, including single members. As an example, "at least one of A, B, or C" is intended to encompass A, B, C, A and B, A and C, B and C, and A, B and C. Conjunction language, such as the phrase "at least one of X, Y, and Z," is otherwise understood to convey that an item, term, etc., may be at least one of X, Y, or Z, depending on the context of general usage, unless specifically stated otherwise. Thus, such conjunctive language is generally not intended to imply that a particular embodiment requires at least one of X, at least one of Y, and at least one of Z for each present.

[0085]

[0100] As used herein, reference to "one embodiment," "an embodiment," "a particular embodiment," "related embodiment," "a certain embodiment," "additional embodiments," "some embodiments," "particular embodiments," or "further embodiments," or combinations thereof, should be understood to mean that a particular feature, structure, or characteristic described in connection with the particular embodiment is included in at least one embodiment of the disclosure. Thus, the presence or appearance of the foregoing phrases in various places throughout the disclosure are not necessarily all referring to the same embodiment. Moreover, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0086]

[0101] Conditional language as used herein, such as, among others, "can," "could," "might," "may," "for example," and the like, is generally intended to convey that a particular embodiment includes certain features, elements and / or steps, but not other embodiments, unless expressly stated otherwise or understood otherwise in the context of use. Thus, such conditional language is generally not intended to imply that features, elements and / or steps are optionally required in one or more embodiments.

[0087] I. Terminology and Definitions

[0102] This section provides definitions of some general terms. Definitions of other terms can be found in other sections of this disclosure below.

[0088]

[0103] As used herein and throughout the rest of this disclosure, the term "polypeptide" is used interchangeably with "peptide", "protein", etc. to refer to a polymer of amino acid residues or an assembly of multiple polymers of amino acid residues. The term applies to both naturally occurring and non-naturally occurring amino acid polymers, and is also intended to encompass amino acid polymers in which one or more amino acid residues are artificial or synthetic chemical mimetics of the corresponding naturally occurring amino acid. The term "protein" typically refers to a large polypeptide. The term "peptide" typically refers to a short polypeptide. A polypeptide sequence is usually written with the left-hand end of the polypeptide sequence being the amino terminus (N-terminus) and the right-hand end of the polypeptide sequence being the carboxyl terminus (C-terminus).

[0089]

[0104] As used herein, the terms "polypeptide complex," "protein complex," and the like refer to a complex comprising one or more polypeptide / protein subunits associated with each other, which is capable of performing a particular function, e.g., specifically recognizing and binding to a particular target antigen.

[0090]

[0105] As used herein, the term "amino acid" refers to the basic unit of protein, peptide, polypeptide or amino acid polymer, and further refers to naturally occurring or synthetic amino acids, as well as any amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by genetic code, as well as later modified amino acids, such as hydroxyproline, gamma-carboxyglutamic acid and O-phosphoserine. As used in this application, naturally occurring amino acids include the group of naturally occurring carboxy alpha-amino acids including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V). As used herein, "amino acid analog" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The alpha carbon refers to the first carbon atom that is bonded to a functional group, e.g., a carbonyl. The beta carbon refers to the second carbon atom connected to the alpha carbon, and in this system, the atoms are designated alphabetically by the Greek letters. As used herein, "amino acid mimetics" refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0091]

[0106] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleotide", "polynucleotide", and the like are intended to refer to nucleotide polymers of any length, may include both DNA and RNA, and may be single-stranded or double-stranded. As used herein, the nucleotides in a nucleic acid may include deoxyribonucleotides, ribonucleotides, modified nucleotides (e.g., methylated nucleotides) or bases, or their analogs. A nucleic acid or polynucleotide may typically be polymerized using a DNA or RNA polymerase or a synthetic reaction. As used herein, the term also refers to synthetic and / or non-naturally occurring nucleic acid molecules (e.g., containing nucleotide analogs or modified backbone residues or linkages). The term encompasses nucleic acids that contain analogs of natural nucleotides. The term also encompasses nucleic acid-like structures with synthetic backbones. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed group and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0092]

[0107] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, multispecific, bispecific, bivalent or multivalent antibody that binds to a specific antigen. A more detailed description of antibodies as well as related terms is provided below.

[0093]

[0108] In mammals, e.g., humans, there are five different antibody classes / isotypes (i.e., IgA, IgD, IgE, IgG, and IgM, corresponding to the five Ig heavy chain types α, δ, ε, γ, and μ, respectively) depending on the different heavy chain types present in the immunoglobulin, which typically have different molecular and biological properties, functional group locations, physiological functionality, and pathological significance in diseases. A particular antibody class may further include subclasses. For example, in humans, IgA may include IgA1 and IgA2 subclasses, and IgG may include four subclasses designated as IgG1, IgG2, IgG3, and IgG4, respectively. With the immunoglobulin monomer as the basic functional unit, mammalian antibodies may exist as monomers (e.g., IgD, IgE, and IgG), dimers (IgA), tetramers (IgM), or pentamers (IgM). In mammals, two types of light chains exist, including kappa (κ) chains and lambda (λ) chains.

[0094]

[0109] A natural or naturally occurring antibody, such as IgG, generally comprises two identical heavy (H) chains and two identical light (L) chains in an immunoglobulin basic unit. Each light chain is linked to a heavy chain by one disulfide covalent bond or a linkage formed between a pair of cysteine ​​residues present in each light chain and heavy chain, respectively, and the two heavy chains are further linked to each other by several disulfide bonds formed between cysteine ​​residues of each heavy chain. The tetramer thus formed is essentially Y-shaped in the antibody, and the end of each fork arm contains an identical antigen binding site (i.e., paratope) that specifically interacts with the corresponding epitope of the antigen.

[0095]

[0110] As used herein, the term "domain" refers to a globular structure formed by one or more regions of one or more polypeptide chains that contain peptide loops (e.g., 3-4 peptide loops), which are stabilized, for example, by a beta-pleated sheet and / or intrachain disulfide bond(s). Examples may include Fab domains (see below for more details). It should be noted that in this disclosure, the two terms "domain" and "region" may be used interchangeably.

[0111] More specifically, in natural antibodies, from the N-terminus to the C-terminus, each heavy chain comprises a variable region (VH or HCVR) followed by three or four constant regions ("CH", in IgA, IgG, IgG contains three CH regions, CH1, CH2 and CH3, and in IgE and IgM contains four CH regions, CH1, CH2, CH3 and CH4), and each light chain comprises a variable region (VL or LCVR) and a constant region (CL). In Y-shaped antibodies, the variable region of each light chain (i.e., VL region) is juxtaposed with or associated with the variable region of the paired heavy chain (i.e., VH region) to together form the antigen-binding site of the antibody.

[0096]

[0112] The term "variable region" or "VR" as used herein refers to the region in the heavy or light chain of an antibody that is responsible for antigen binding. In a naturally occurring antibody, the heavy chain variable region (VH or HCVR) contains the heavy (H) chain CDRs, including three highly variable loops called "complementarity determining regions" (CDRs), i.e., HCDR1, HCDR2, HCDR3, and the light chain variable region (VL or LCVR) contains the light (L) chain CDRs, including LCDR1, LCDR2, and LCDR3. The CDR boundaries of an antibody may be defined or identified by the conventional methods of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol. Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are inserted into adjacent sections known as "framework regions" (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. In natural antibodies, each VH and VL comprises four FRs, and the CDRs and FRs are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. However, it should be understood that the term "variable region" as used herein does not necessarily have to include all three CDRs or all four FRs, and should be interpreted to include any variant or derivative of the natural variable region of a natural antibody, so long as any variant or derivative of the natural variable region of a natural antibody retains antigen-binding activity.

[0097]

[0113] The term "variant" with respect to a polypeptide or polynucleotide encompasses any and all different forms of the polypeptide or polynucleotide, including, but not limited to, a polypeptide or polynucleotide fragment, mutant, fusion, derivative, mimetic, or any combination thereof.

[0098]

[0114] The term "derivative" with respect to a polypeptide or polynucleotide refers to a chemically modified polypeptide or polynucleotide in which one or more clearly defined number of substituents are covalently attached to one or more specific amino acid residues of a polypeptide or one or more specific nucleotides of a polynucleotide. Exemplary chemical modifications to a polypeptide can be, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation, or conjugation with one or more moieties of one or more amino acids. Exemplary chemical modifications to a polynucleotide can be (a) terminal modifications, such as 5'- or 3'-terminal modifications, (b) nucleobase (or "base") modifications, including base replacement or removal, (c) sugar modifications, including modifications at the 2', 3', and / or 4' positions, and (d) backbone modifications, including modification or replacement of phosphodiester linkages.

[0099]

[0115] The term "constant region" or "constant portion" as used herein refers to the region in the heavy or light chain of an antibody that is not directly involved in antigen binding.It should be understood that the term "constant region" or "constant portion" as used herein does not necessarily include the full-length natural constant region of natural antibody, and should be interpreted as including any variant or derivative of such a natural constant region or constant portion, so long as such variant or derivative retains the ability to, for example, support the stability of the antigen binding domain or retain the intended biological function, such as effector function, secretion, transplacental mobility, Fc receptor binding, complement binding, etc.

[0100]

[0116] The term "CL region" refers to the constant region of an immunoglobulin light chain adjacent to the VL region. The CL region can range from about EU position 108 to about EU position 216 in an immunoglobulin light chain. In a native antibody, the constant region of the light chain (i.e., the CL region) is associated with the first constant region (i.e., the CH1 region) of the paired heavy chain.

[0101]

[0117] The term "CH1 region" as used herein includes the first (most amino terminal) constant region of an immunoglobulin heavy chain, which extends from about EU position 118 to at least about EU position 220 (e.g., can extend to EU position 221, etc.). The CH1 region is adjacent to the VH region and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0102]

[0118] The term "hinge region" in reference to an antibody includes the portion of a heavy chain molecule that connects the CH1 region to the CH2 region. The length of the hinge region may vary depending on the boundaries of the definition of the CH1 region and the CH2 region. The hinge region is usually flexible, and therefore allows the two N-terminal antigen-binding regions to move independently.

[0103]

[0119] The term "CH2 region" as used herein refers to that portion of a heavy chain immunoglobulin molecule that extends from about EU position 231 to EU position 340, for example.

[0104]

[0120] The term "CH3 region" as used herein refers to a portion of a heavy chain immunoglobulin molecule that extends from the N-terminus of the CH2 domain to approximately 110 residues, for example, from approximately EU position 341 to EU position 445, 446 or 447. The CH3 domain typically forms the C-terminal portion of antibodies such as IgG, IgA and IgD. However, in some immunoglobulins such as IgE and IgM, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ chain of IgM and the ε chain of IgE).

[0105]

[0121] Throughout this disclosure, numbers indicating the positions of amino acid residues in the constant region of an antibody, e.g., the heavy chain constant region 1 (CH1) and the light chain constant region (CL) in the constant portion, are based on the EU numbering system or EU index as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); and Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). EU numbering is also available from the IMGT scientific charts accessible from the international ImMunoGeneTics information system website.

[0106]

[0122] "Fab" as used herein refers to a single antigen-binding domain derived from an antibody, where the domain has a single heavy chain fragment associated with a single light chain fragment through one or more covalent bonds that are not peptide bonds. In some embodiments, the single heavy chain fragment in the Fab domain comprises an HCVR and a CH1 region. In some embodiments, the single light chain fragment in the Fab domain comprises an LCVR and a CL domain. In some embodiments, the CH1 region is associated with the HCVR by a covalent bond, e.g., a disulfide bond. In a natural antibody, the Fab domain, which substantially corresponds to one arm of an antibody, typically retains the ability to recognize and bind to the corresponding antigen.

[0107]

[0123] "Fc", as used herein, refers to the portion derived from an antibody consisting of the second (CH2) and third (CH3) constant regions of a first heavy chain bound to the second and third constant regions of a second heavy chain via one or more covalent bonds that are not peptide bonds, e.g., disulfide bonds, in the example of IgG. The Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0108]

[0124] "Antigen" or "Ag" as used herein refers to a compound, composition, peptide, polypeptide, protein, or substance (e.g., polypeptide, carbohydrate, nucleic acid, lipid, or other compound, naturally occurring or synthetic) capable of stimulating antibody production or T-cell response in cell culture or animal, including compositions added to cell culture (e.g., hybridoma) or injected or absorbed into animals (e.g., compositions containing cancer-specific proteins). Antigens react with products of specific humoral or cellular immunity (e.g., antibodies), including those induced by heterologous antigens. Antigens may include exogenous antigens, endogenous antigens, autoantigens, neoantigens, and antigens associated with specific pathogens or diseases. Exogenous antigens enter the body by inhalation, ingestion, or injection, and may be presented by antigen-presenting cells (APCs) by endocytosis or phagocytosis to form MHCII complexes. Endogenous antigens are produced within normal cells as a result of cellular metabolism, intracellular viral or bacterial infection, which may form MHCII complexes. Autoantigens (e.g., peptides, DNA, or RNA, etc.) are recognized by the immune system of patients suffering from autoimmune diseases, but under normal conditions, these antigens should not be targeted by the immune system. Neoantigens are completely absent from the normal body and are produced by certain diseases, e.g., tumors or cancer. In certain embodiments, the antigens are associated with certain diseases (e.g., tumors or cancer, autoimmune diseases, infectious and parasitic diseases, cardiovascular diseases, neurological disorders, neuropsychiatric conditions, injuries, inflammation, blood clotting disorders). In certain embodiments, the antigens are associated with the immune system (e.g., immune cells, e.g., B cells, T cells, NK cells, macrophages, etc.).

[0125] "Epitope" refers to a region of an antigen to which a binding agent (e.g., an antibody) binds. Epitopes can be formed from both contiguous amino acids (also called linear or contiguous epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (also called configurational or conformational epitopes). Epitopes formed from contiguous amino acids are typically arranged linearly along the major amino acid residues on the protein, and small segments of contiguous amino acids can be retained upon degradation from antigen binding to major histocompatibility complex (MHC) molecules or exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three or more, usually at least 5, approximately 7, or approximately 8-10 amino acids in a unique spatial conformation.

[0109]

[0126] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally an antigen-binding fragment or its variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments, most, but not all, of the above definitions of such antibody fragments are further provided below.

[0110]

[0127] As used herein, terms such as "antigen-binding fragment" refer to an antibody fragment formed from a portion of an antibody that contains one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments may include, but are not limited to, variable domains, variable regions, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds. Antigen-binding fragments may contain one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. Further detailed formatting of antigen-binding moieties is described in Spiess et al., 2015 (see above) and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0111]

[0128] As used herein, the term "target binding moiety" refers to a protein or polypeptide fragment or domain capable of binding to a target molecule, which may be any type of chemical or biological entity. For example, the target molecule may be a small molecule compound or a macromolecule, such as a peptide, polypeptide, protein, or nucleic acid. The target molecule may be a disease-related molecule, such as a tumor surface antigen, an immune checkpoint molecule, a cell surface receptor, an infectious agent, a cytokine, a growth factor, etc. A person skilled in the art can easily select from a wide variety of targets of interest. In some embodiments, the target binding moiety of the present disclosure comprises or is an antigen binding moiety.

[0112]

[0129] As used herein, the term "antigen-binding portion" refers to the portion of an antigen-binding domain that is responsible for antigen binding. For example, an antigen-binding portion may include one or more CDRs, but does not necessarily include a constant region / portion. Examples of antigen-binding portions may include, but are not limited to, variable domains, variable regions, nanobodies, Fv fragments, scFv, disulfide-stabilized Fv fragments, (dsFv)2, bispecific dsFv, or diabodies.

[0113]

[0130] As used herein, the term "target binding domain" refers to a domain that comprises a target binding portion and a constant portion that comprises CH1 region and CL region.For example, target binding domain can be a fusion protein that comprises a target binding portion and a constant portion.In some embodiments, the target binding domain of the present disclosure comprises or is an antigen binding domain.

[0114]

[0131] As used herein, the term "antigen-binding domain" refers to an antigen-binding fragment that includes an antigen-binding portion and a constant portion. Examples of antigen-binding domains can include, but are not limited to, Fab, Fab', or F(ab')2.

[0115]

[0132] "Fcab" refers to a modified Fc fragment that also contains an antigen binding site. Fcabs can exist as fragments or can be inserted into a complete immunoglobulin by swapping the Fc region, thereby obtaining antibodies with bispecific or even trispecific activity.

[0116]

[0133] "Fragment D (Fd)" with respect to an antibody refers to the amino terminal half of a heavy chain fragment that is capable of combining with a light chain to form a Fab.

[0117]

[0134] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that has a complete antigen-binding site. The Fv fragment consists of a single light chain variable domain bound to a single heavy chain variable domain. Many Fv designs are available, including dsFv, where the association between the two domains is enhanced by the introduction of a disulfide bond, and scFv, which can be formed using a peptide linker that connects the two domains as a single polypeptide. Fv constructs have also been generated that contain the variable domains of a heavy or light immunoglobulin chain bound to the variable and constant domains of the corresponding immunoglobulin heavy or light chain. Fvs can also multimerize to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0118]

[0135] "CrossMab" refers to a technology in which unmodified light chains are paired with corresponding unmodified heavy chains and modified light chains are paired with corresponding modified heavy chains, thereby generating antibodies with reduced mispairing in the light chains.

[0119]

[0136] A "BiTE®" is a bispecific T cell binding molecule comprising a first scFv with a first antigen specificity in an LCVR-HCVR orientation linked to a second scFv with a second specificity in an HCVR-LCVR orientation.

[0120]

[0137] As used herein, the term "multispecific antibody" refers to an artificial or engineered antibody that can simultaneously bind to at least two different epitopes. A bispecific antibody is essentially a type of multispecific antibody. In addition, other multispecific antibodies may include trispecific antibodies that have three different antigen binding specificities, tetraspecific antibodies that have four different antigen binding specificities, etc.

[0121]

[0138] As used herein, the term "bispecific antibody" refers to an antibody that contains two physically separable antigen-binding portions / sites that differ from each other in antigen specificity. Typically, bispecific antibodies are artificial antibodies that have fragments derived from two different monoclonal antibodies and can bind to two different epitopes. The two epitopes can be on the same antigen, or they can be on two different antigens. Being structurally identical and therefore having the same antigen specificity is in contrast to naturally occurring antibodies that have two physically separable antigen-binding portions. In the heterodimeric antibodies disclosed herein, each of the two different Fab domains (i.e., the first Fab domain and the second Fab domain) contains a different antigen-binding portion that specifically binds to a different epitope, typically comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) whose sequences differ from each other. In an attempt to increase the probability of proper cognate pairing to obtain highly selective and efficient bispecific antibodies, several strategies exist to promote light chain-heavy chain cognate pairing and heavy chain-heavy chain cognate pairing.

[0122]

[0139] The term "affinity," as used herein, refers to the strength of non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0123] II. Polypeptide Complexes

[0140] The present disclosure provides novel polypeptide complexes useful for promoting selective pairing of light and heavy chains (i.e., cognate pairing) in constructs comprising at least two different light chains associated with their respective heavy chain(s) through one or more non-peptide bonds.

[0124]

[0141] In one aspect, the disclosure provides a polypeptide complex comprising a first target binding domain comprising a first target binding moiety operably linked to a first constant portion, the first constant portion comprising a first heavy chain constant region 1 (CH1) associated with a first light chain constant region (CL), the first CH1 region comprising a first amino acid residue at EU position n1 and the first CL region comprising a second amino acid residue at EU position n2, the n1:n2 position pair being selected from the group consisting of 128:118 and 173:160, and wherein the first amino acid residue and the second amino acid residue form a covalent bond.

[0125]

[0142] As used herein, the term "operably linked" or "operably linked" refers to the juxtaposition, with or without a spacer or linker, of two or more biological sequences of interest in a relationship that allows them to function as intended. When used in relation to a polypeptide, it is intended to mean that the polypeptide sequences are linked in such a way that the linked product can have the intended biological function. For example, a target binding moiety may be operably linked to a constant moiety to produce a stable product with antigen binding activity. This term may also be used in relation to a polynucleotide. In one example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequence is linked in such a way that it allows for the expression regulation of the polypeptide derived from this polynucleotide.

[0126]

[0143] In certain embodiments, the linker used herein is selected from the group consisting of cleavable linker, non-cleavable linker, peptide linker, flexible linker, rigid linker, helical linker and non-helical linker. Any suitable linker known in the art can be used. In certain embodiments, the linker comprises a peptide linker. For example, the linker useful in the present disclosure can be rich in glycine and serine residues. Examples include linkers with single or repeating sequences containing threonine / serine and glycine, such as TGGGG (SEQ ID NO: 24), GGGGS (SEQ ID NO: 25) or SGGGG (SEQ ID NO: 26) or tandem repeats thereof (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeats). In certain embodiments, the linker used in the present disclosure comprises GGGGSGGGSGGGGGS (SEQ ID NO: 27). Alternatively, the linker can be a peptide long chain comprising one or more consecutive or tandem repeats of the amino acid sequence GAPGGGGGAAAAAGGGGG (SEQ ID NO:28). In certain embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive or tandem repeats of SEQ ID NO:28. In certain embodiments, the peptide linker comprises a GS linker. In certain embodiments, the GS linker comprises one or more repeats of GGGS (SEQ ID NO:29) or SEQ ID NO:25. In certain embodiments, the linker comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs:24-29.

[0127]

[0144] As used herein, the letter "n" followed by a number indicates the position of a given amino acid residue in the first target binding domain provided herein. For example, the position "n1" indicates the first specific position in the first target binding domain, and the position "n2" indicates the second specific position in the first target binding domain. All positions in the antibody sequence are based on the EU numbering system or EU index, and therefore, the positions are sometimes referred to as EU positions herein. The positions can be in the heavy chain portion of the first target binding domain or in the light chain portion of the first target binding domain. For example, according to this context, the position of n1 can be in the first CH1 region, and the position of n2 can be in the first CL region.

[0128]

[0145] As used herein, the term "position pair" refers to a pair of EU positions in an antibody fragment, e.g., a target binding domain. Position pairs are expressed herein using two numbers separated by a colon (:). For example, the n1:n2 position pair of 128:118 means that n1 is EU position 128 (e.g., in the first CH1 region) and n2 is EU position 118 (e.g., in the first CL region).

[0129]

[0146] Herein, the specificity of the cognate pairing of heavy and light chains can be improved by forming a covalent bond (e.g., disulfide bond) by configuring the first and second amino acid residues at one of EU position pairs 128:118 and 173:160.The amino acid residues at such EU position pairs in natural antibodies are not involved in the formation of natural covalent bonds, such as disulfide bonds.Therefore, the polypeptide complex provided herein is useful for reducing the mispairing of the first target binding domain and the second target binding domain, and has the possibility of not interfering with the covalent bond formation of such EU position pairs.

[0130]

[0147] As used herein, "disulfide bond" refers to a covalent bond with the structure RSS-R'. The amino acid cysteine, for example, contains a thiol group that can form a disulfide bond with a second thiol group from another cysteine ​​residue. Disulfide bonds can form between the thiol groups of two cysteine ​​residues present on two polypeptide chains, respectively, under physiological conditions, thereby forming interchain bridges or interchain bonds to form interchain (if both originate from different chains) or intrachain (if both originate from the same chain) disulfide bonds. With respect to antibodies, the term "interchain disulfide bond" refers to disulfide bonds formed between sulfhydryl groups of cysteine ​​residues in disulfide bond-forming cysteine ​​pairs in the immunoglobulin hinge region, or between the light and heavy chains of the respective constant regions. In this disclosure, unless otherwise indicated, "disulfide bond" refers to the disulfide bond formed between the immunoglobulin heavy and light chains in the CH1 and CL regions, respectively, which can be either the native / naturally occurring disulfide bond (e.g., EU position pair 220:214 (IgG1) or 131:214 (IgG2 and IgG4)) or the disulfide bond at EU position pair 128:118 and / or 173:160.

[0131]

[0148] According to some embodiments, the covalent bond formed between the first amino acid residue and the second amino acid residue is a disulfide bond, and further optionally, the disulfide bond is formed between two cysteine ​​residues. In other words, in this embodiment of the polypeptide complex, the covalent bond formed between the first CH1 and the first CL region in the first target binding domain is a disulfide bond formed between two cysteine ​​residues at EU position pair 128:118 or 173:160. In one embodiment, one or both of the first and second amino acid residues are introduced by substitution of the corresponding natural or wild-type amino acid residue at EU position in one of the two position pairs indicated above in the first CH1 and the first CL region with a cysteine ​​residue. For example, the first amino acid residue at EU position n1 and the second amino acid residue at EU position n2 are both cysteine ​​residues. More specifically, such substitutions can be either (i) L128C:F118C or (ii) V173C:Q160C (or E160C). In some embodiments, the first CH1 region comprises a substitution of L128C (EU position n1) and the first CL region comprises a substitution of F118C (EU position n2). However, it should be noted that in addition to the introduction of a disulfide bond, the covalent bond can also be of different types of covalent bonds. Thus, examples can include an isopeptide bond formed between lysine and asparagine or aspartic acid, or a covalent bond formed between a non-natural amino acid (Uaa) and a cysteine ​​residue (Nat Methods. 2013; 10 (9): doi: 10.1038 / nmeth.2595), etc.

[0132]

[0149] As used herein, words such as "introduced," "introduce," and the like refer to the condition of an element or structure not present in an original version of a larger entity or structure, and are to be interpreted as being brought into the new version as a change, modification, or newly added element or structure.

[0133]

[0150] The terms "associated with," "in association with," and the like, when referring to two regions of a polypeptide complex (e.g., an antibody or antigen-binding fragment thereof), refer to the situation in which the two regions are operatively linked to one another, with or without a spacer or linker, such that they are in a relationship that allows them to function as intended. For example, a variable region of an antibody may be associated with a constant region to provide a stable product that has antigen-binding activity. "Associated with," "in association with," "linked to," "coupled to," "fused to," "connected to," and "bound to" may be used interchangeably in this disclosure.

[0134]

[0151] According to some preferred embodiments of the polypeptide complex, the first target binding domain is further modified (e.g., mutated) such that the native disulfide bond in the first target binding domain is disrupted. Thus, in one embodiment, the first target binding domain comprises a substitution or deletion of a cysteine ​​residue at EU position 220 (IgG1) or EU position 131 (IgG2 and IgG4) of the heavy chain, and / or another substitution or deletion of a cysteine ​​residue at EU position 214 of the light chain.

[0135]

[0152] As used in the present disclosure, a polypeptide complex can be an antibody or antibody fragment that comprises a target binding domain.Examples of polypeptide complexes can include, but are not limited to the scope of the present disclosure, full-length antibodies (e.g., monospecific antibodies, bispecific antibodies, trispecific antibodies, bivalent antibodies, multivalent antibodies, etc.), antigen binding domains that comprise fragments thereof (e.g., Fab, Fab', F(ab')2, etc.), or protein complexes that comprise antibodies or fragments thereof.Also, a polypeptide complex can be any other type of molecule, as long as such molecules comprise target binding domains / regions with the above characteristics.

[0136]

[0153] In some such embodiments, the first CH1 region further comprises a third amino acid residue at EU position n3, and the first CL region further comprises a fourth amino acid residue at EU position n4, wherein the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 126:121, and wherein the third amino acid residue and the fourth amino acid residue form a non-covalent bond.

[0137]

[0154] By combining the covalent bond between the residues of n1:n2 position pair and the non-covalent bond (e.g., electrostatic interaction) of n3:n4 position pair, the present inventors unexpectedly find that the polypeptide complex provided herein is unexpectedly advantageous in reducing light chain mismatch and significantly improving the purity of expression product.Such unique constructs are not only easy to manufacture, but also produce much higher purity and yield than comparable constructs that lack such combination.

[0138]

[0155] As used herein, the term "non-covalent bond" refers to the non-covalent interaction between two peptide chains in a protein complex. Examples of non-covalent bonds include hydrogen bonds, electrostatic interactions, salt bridges or hydrophobic-hydrophilic interactions, knob-into-hole, or combinations thereof. Both non-covalent and covalent bonds (e.g., disulfide bonds) form a type of interchain bond, which is beneficial to the folding, conformation, stability, mobility and function of any protein, polypeptide or their complex.

[0139]

[0156] In some embodiments, non-covalent bond is electrostatic interaction.In some embodiments, the third amino acid residue and the fourth amino acid residue are oppositely charged.Herein, by configuring one or more of the third amino acid residues and the fourth amino acid residues of four EU position pairs 183:176, 141:116, 126:121 and 218:122 to be oppositely charged, one or more interactions between each pair of oppositely charged amino acid residues are substantially generated to form one or more non-covalent bonds between each pair, and then promote the specific cognate pairing of heavy chain and light chain in the polypeptide complex disclosed herein.

[0140]

[0157] As used herein, "electrostatic interaction" refers to non-covalent bond, and generally includes ionic interaction, hydrogen bond and halogen bond.Electrostatic interaction can be formed in polypeptide or between various subunits / chains of protein complex, for example, between Lys(K) and Asp(D), between Lys(K) and Glu(E), between Glu(E) and Arg(R), or between Glu(E), Trp(W) on the first chain and Arg(R), Val(V) or Thr(T) on the second chain.

[0141]

[0158] "Salt bridges" are short-range electrostatic interactions primarily due to the anionic carboxylate of either Asp (D) or Glu (E) and the cationic ammonium from Lys (K) or guanidinium from Arg (R), which are spatially proximal pairs of oppositely charged residues in a protein or polypeptide structure. Charged and polar residues in a mostly hydrophobic interface can act as hot spots for binding. Residues with ionizable side chains, such as His, Tyr and Ser, among others, can also participate in the formation of salt bridges.

[0142]

[0159] "Knobs and holes", as used herein, refers to the interaction between two polypeptides, where one polypeptide has a protuberance (i.e., "knob") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan), and the other polypeptide has a cavity (i.e., "hole") with an amino acid residue with a small side chain (e.g., alanine or threonine), and the protuberance can be positioned within the cavity to promote the interaction of the two polypeptides to form a heterodimer or complex. In general, the "knobs and holes" technique involves substantially introducing mutations into each of the two polypeptides in the Fc region to restrict heavy chain-heavy chain combinations (Ridgway et al., Protein Engineering, 9(7), pp.617-21(1996); Merchant et al., Nature Biotechnology, 16(7), pp.677-681(1998)). Methods for generating polypeptides with knobs and holes are known in the art and are described, for example, in U.S. Patent No. 5,731, 168. It should be noted that although the oppositely charged third and fourth amino acid residues at one or more of the three EU position pairs 183:176, 141:116 and 126:121 do not form knob-and-hole-type non-covalent interactions, the first CH1 and first CL regions have the potential to include other introduced residues (substitutions, insertions, modifications, etc.) that do.

[0143]

[0160] As used herein, the term "oppositely charged" with respect to two amino acid residues means that both of the two amino acid residues are charged, and at physiological pH (e.g., a pH value of approximately 7 to 7.5, preferably 7.4), one residue is positively charged while the other is negatively charged. For example, the first amino acid residue may be positively charged while the second amino acid residue is negatively charged, or the first amino acid residue may be negatively charged while the second amino acid residue is positively charged. Specifically, two amino acid residues are not "oppositely charged" if (a) one amino acid residue is charged and the other amino acid residue is uncharged, (b) both amino acid residues are uncharged, or (c) both amino acid residues are "similarly charged", i.e., one amino acid residue is positively charged and the other amino acid residue is also positively charged, or one amino acid residue is negatively charged and the other amino acid residue is also negatively charged.

[0144]

[0161] As used herein, the term "positively charged amino acid residue" or "positively charged amino acid residue" refers to an amino acid residue having a side chain that is positively charged under physiological conditions (e.g., a pH of about 7 to 7.5, preferably 7.4). Such positively charged amino acid residues are typically natural amino acid residues, such as lysine residues (K), arginine residues (R) and histidine residues (H), but may also include analogs, mimetics and modifications of other amino acids that exhibit a positive charge under physiological conditions.

[0145]

[0162] As used herein, the term "negatively charged amino acid residue" or "negatively charged amino acid residue" refers to an amino acid residue having a side chain that is negatively charged under physiological conditions (e.g., a pH of about 7 to 7.5, preferably 7.4). Such negatively charged amino acid residues are typically natural amino acid residues, such as aspartic acid residues (D) and glutamic acid residues (E), but may also include analogs, mimetics, and modifications of other amino acids that exhibit a negative charge under physiological conditions.

[0146]

[0163] In some embodiments, the polypeptide complexes provided herein further comprise a second target binding domain comprising a second target binding moiety operably linked to a second constant portion, the second constant portion comprising a second CH1 region associated with a second CL region, wherein the first CH1 region does not substantially bind to the second CL region, and the second CH1 region does not substantially bind to the first CL region.

[0147]

[0164] The term "does not substantially bind to" as used herein means that a given CH1 region and a mismatched CL region are significantly less likely to bind to each other and form a binding complex compared to a given CH1 region and its paired CL region.For example, a first CH1 region is paired with a first CL region and is not substantially bound to a second CL region, and in such a case, the first CH1 region and the second CL region are significantly less likely to bind to each other and form a binding complex, for example, the amount of binding complex between the first CH1 region and the second CL region is much less than the amount of binding complex between the first CH1 region and the first CL region (for example, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less).In some embodiments, element A is not substantially bound to element B means that element A is not covalently bound to element B. For example, in some embodiments, the first CH1 region is not covalently linked to the second CL region and the second CH1 region is not covalently linked to the first CL region.

[0148]

[0165] In some embodiments, the second CH1 region comprises a first corresponding amino acid residue at EU position n1' and the second CL region comprises a second corresponding amino acid residue at EU position n2', wherein the n1':n2' position pair is identical to the n1:n2 position pair, and wherein the first corresponding amino acid residue at EU position n1' does not form a covalent bond with the second amino acid residue at EU position n2, and / or the second corresponding amino acid residue at EU position n2' does not form a covalent bond with the first amino acid residue at EU position n1.

[0149]

[0166] Without being bound by any theory, it is believed that such a situation avoids cross-linking of the respective heavy and light chain parts forming the first and second target binding domains, thereby avoiding or reducing their mispairing. Nevertheless, it should be noted that in the second target binding domain, the first corresponding amino acid residue at EU position n1' can form a covalent bond with the second corresponding amino acid residue at EU position n2'. For example, if the first amino acid residue at EU position n1 and the second amino acid residue at EU position n2 form a disulfide bond, the first corresponding amino acid residue at EU position n1' and the second corresponding amino acid residue at EU position n2' can form a covalent bond that is not a disulfide bond, and the first and second corresponding amino acid residues that form a non-disulfide bond at EU positions n1' and n2' do not bind to the first and second amino acid residues that form a disulfide bond at EU positions n1 and n2, respectively.

[0150]

[0167] As used herein, the letter "n" followed by a number with an apostrophe (') indicates a given amino acid residue position in a second target binding domain provided herein, which position corresponds to a given position in a first target binding domain. For example, position "n1'" indicates a position in the second target binding domain that corresponds to position n1 in the first target binding domain, and position "n2'" indicates a position in the second target binding domain that corresponds to position n2 in the first target binding domain. In other words, when position n1 is determined, position n1' is also determined, and vice versa. For example, if EU position n1 is EU position 128 in the first CH1 region of the first target binding domain, then EU position n1' is EU position 128 in the second CH1 region of the second target binding domain.

[0151]

[0168] An amino acid residue in a second target binding domain that corresponds to its counterpart in the first target binding domain is referred to herein as a "corresponding amino acid residue." Once position n1 is determined, position n1' is determined, and both the residue at position n1 as well as the corresponding amino acid residue at position n1' are determined.

[0152]

[0169] In some embodiments, the first corresponding amino acid residue at EU position n1' and the second corresponding amino acid residue at EU position n2' do not form a covalent bond. In such embodiments, the CH1 region and the CL region of the second target binding domain are not associated with each other by a covalent bond at EU positions n1' and n2'. For example, the first and second corresponding amino acid residues at EU positions n1' and n2' are natural / wild type amino acid residues, for example, selected from the amino acid pair L128:F118, V173:Q160 (or E160). In another example, the first and second corresponding amino acid residues at EU positions n1' and n2' are mutated amino acid residues that do not form a covalent bond.

[0153]

[0170] The term "mutation" or "mutated" with respect to an amino acid residue, as used herein, refers to the substitution, replacement, insertion, addition or modification of the amino acid residue. The term "substitution" or "substituted" with respect to an amino acid residue, as used herein, refers to the replacement of an amino acid residue X (i.e., the amino acid residue before replacement, "X") with an amino acid residue Z (i.e., the amino acid residue after replacement, "Z") at position p in a peptide, polypeptide or protein, represented by XpZ. In one example, S183K represents the replacement of the original natural serine residue (S) at EU position 183 of the CH1 region of an immunoglobulin heavy chain with a lysine residue (K).

[0154]

[0171] In some embodiments, the second CH1 region further comprises a third corresponding amino acid residue at EU position n3' and the second CL region further comprises a fourth corresponding amino acid residue at EU position n4', wherein the n3':n4' position pair is identical to the n3:n4 position pair, and the fourth corresponding amino acid residue at EU position n4' and the third amino acid residue at EU position n3 are not oppositely charged or are similarly charged.

[0155]

[0172] In some embodiments, the second CH1 region further comprises a third corresponding amino acid residue at EU position n3' and the second CL region further comprises a fourth corresponding amino acid residue at EU position n4', wherein the n3':n4' position pair is identical to the n3:n4 position pair, and the third corresponding amino acid residue at EU position n3' and the fourth amino acid residue at EU position n4 are not oppositely charged or are similarly charged.

[0156]

[0173] As used herein, the third and fourth corresponding amino acid residues at EU positions n3' and n4' of the second target binding domain do not interfere with the electrostatic interaction of the third and fourth amino acid residues at EU positions n3 and n4 of the first target binding domain.

[0157]

[0174] In some embodiments, the third and fourth corresponding amino acid residues at EU positions n3' and n4' are natural / wild type amino acid residues, e.g., selected from the following amino acid pairs: S183:S176, A141:F116, F126:S121, K218:D122. In some embodiments, one or both of the third and fourth corresponding amino acid residues at EU positions n3' and n4' are mutated amino acid residues in the CH1 or CL region of the heavy or light chain of the second target binding domain. For example, one or both of the third and fourth corresponding amino acid residues at EU positions n3' and n4' are mutated amino acid residues that are similarly charged to the fourth and third amino acid residues at EU positions n4 and n3, respectively, of the first target binding domain.

[0158]

[0175] In some embodiments, the third corresponding amino acid residue at EU position n3' and / or the fourth corresponding amino acid residue at EU position n4' are uncharged. For example, the second target binding domain does not have an electrostatic interaction between the third corresponding amino acid residue at EU position n3' and the fourth corresponding amino acid residue at EU position n4'.

[0159]

[0176] In some embodiments, the second CH1 region further comprises a fifth corresponding amino acid residue at EU position n5', and the second CL region further comprises a sixth corresponding amino acid residue at EU position n6', wherein the fifth corresponding amino acid residue and the sixth corresponding amino acid residue form a covalent bond, and the n5':n6' position pair is different from the n1:n2 position pair.

[0160]

[0177] Herein, the second CH1 region and the second CL region are also associated with each other by covalent bond, but the position of the amino acid residue that forms the covalent bond is different from the position of the amino acid residue that forms the covalent bond in the first target binding domain.For example, both n5':n6' position pair and n1:n2 position pair are independently selected from the group consisting of 220:214 (IgG1), 131:214 (IgG2 and IgG4), 128:118 and 173:160, with the proviso that n5':n6' position pair is different from n1:n2 position pair.For example, if n5':n6' position pair is 220:214 (IgG1) or 131:214 (IgG2 and IgG4), then n1:n2 position pair is 128:118 or 173:160. In another example, the n5':n6' position pair is 128:118 and the n1:n2 position pair is 173:160. In yet another example, the n5':n6' position pair is 173:160 and the n1:n2 position pair is 128:118.

[0161]

[0178] In some embodiments, the fifth and sixth corresponding amino acid residues at EU positions n5' and n6' are natural / wild type amino acid residues, e.g., selected from the amino acid residues C220:C214 (IgG1), C131:C214 (IgG2 and IgG4), L128:F118, V173:Q160 (or E160), with the proviso that the n5':n6' position pair is different from the n1:n2 position pair. In some embodiments, the fifth corresponding amino acid residue at EU position n5' and the sixth corresponding amino acid residue at EU position n6' are both cysteine ​​residues. For example, the second CH1 region comprises a substitution of L128C (EU position n5') and the second CL region comprises a substitution of F118C (EU position n6'). In another example, the second CH1 region comprises a substitution of V173C (EU position n5') and the second CL region comprises a substitution of Q160C (EU position n6') of a kappa light chain or E160C (EU position n6') of a lambda light chain.

[0162]

[0179] In some embodiments, one or both of the fifth and sixth corresponding amino acid residues at EU positions n5' and n6' are mutated amino acid residues in the CH1 or CL region of the heavy or light chain of the second target binding domain. For example, one or both of the fifth and sixth corresponding amino acid residues at EU positions n5' and n6' are mutated amino acid residues that form a covalent bond, with the proviso that the n5':n6' position pair is different from the n1:n2 position pair.

[0163]

[0180] As known in the art, the native / wild-type disulfide bond is formed between a cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4) of the heavy chain and a cysteine ​​at EU position 214 of the light chain. In some embodiments, at least one of the first CH1 region and the second CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4), and / or at least one of the first CL region and the second CL region has an amino acid residue other than cysteine ​​at EU position 214. By doing so, the native / wild-type disulfide bond in at least one of the first target binding domain and the second target binding domain is disrupted. For example, the first CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4), and the first CL region has an amino acid residue other than cysteine ​​at EU position 214. In another example, the second CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and the second CL region has an amino acid residue other than cysteine ​​at EU position 214.

[0164]

[0181] As used herein, by constructing a covalent bond (e.g., a disulfide bond) and one or more non-covalent bonds between the immunoglobulin light and heavy chains of the respective CL and CH1 regions, and more preferably simultaneously disrupting the native disulfide bond, the accuracy and specificity of pairing to the light and heavy chains in the polypeptide complex can be maximized, which in turn can further increase the production of heterodimeric antibodies or antigen-binding fragments thereof with bispecific activity, as described in more detail below.

[0165]

[0182] It is further noted that in the above preferred embodiments, by simultaneously disrupting the native disulfide bond at EU position pair 220:214 (IgG1) or 131:214 (IgG2 and IgG4) of the same domain (i.e. the first target binding domain), potential interference with any of the introduced cysteine ​​residues by any native cysteine ​​residues at positions 220 (IgG1) or 131 (IgG2 and IgG4) or 214 can be reduced or eliminated, which can further improve the accuracy and specificity of the cognate pairing.

[0166]

[0183] In some embodiments, neither the first nor the second CH1 region has a cysteine ​​residue at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and / or neither the first nor the second CL region has a cysteine ​​residue at EU position 214. By doing so, the native / wild-type disulfide bond in both the first and second target binding domains is disrupted.

[0167]

[0184] In some embodiments, the first CH1 region further comprises a fifth amino acid residue at EU position n5, and the first CL region further comprises a sixth amino acid residue at EU position n6, and the n5:n6 position pair is identical to the n5':n6' position pair, and the fifth corresponding amino acid residue at EU position n5' does not form a covalent bond with the sixth amino acid residue at EU position n6, and / or the sixth corresponding amino acid residue at EU position n6' does not form a covalent bond with the fifth amino acid residue at EU position n5. Without being bound by any theory, it is believed that such a situation avoids cross-linking of the respective heavy and light chain portions forming the first and second target binding domains, thereby avoiding or reducing their mispairing. Nevertheless, it should be noted that in the first target binding domain, the fifth amino acid residue at EU position n5 can form a covalent bond with the sixth amino acid residue at EU position n6. For example, if the fifth corresponding amino acid residue at EU position n5' and the sixth corresponding amino acid residue at EU position n6' form a disulfide bond, then the fifth amino acid residue at EU position n5 and the sixth amino acid residue at EU position n6 may form a covalent bond that is not a disulfide bond, and the fifth and sixth amino acid residues that form a non-disulfide bond at EU positions n5 and n6 are not bound to the fifth and sixth corresponding amino acid residues that form a disulfide bond at EU positions n5' and n6', respectively.

[0168]

[0185] In some embodiments, the fifth amino acid residue at EU position n5 and the sixth amino acid residue at EU position n6 do not form a covalent bond. In such embodiments, the CH1 region and the CL region of the first target binding domain are not associated with each other by a covalent bond at EU positions n5 and n6. For example, the fifth and sixth amino acid residues at EU positions n5 and n6 are natural / wild type amino acid residues, for example, selected from the amino acid pairs L128:F118, V173:Q160 (or E160). In another example, the fifth and sixth amino acid residues at EU positions n5 and n6 are mutated amino acid residues that do not form a covalent bond.

[0169]

[0186] In some embodiments, the second CH1 region further comprises a seventh corresponding amino acid residue at EU position n7', and the second CL region further comprises an eighth corresponding amino acid residue at EU position n8', wherein the n7':n8' position pair is selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, wherein the seventh corresponding amino acid residue and the eighth corresponding amino acid residue are oppositely charged, and the n7':n8' position pair is different from the n3:n4 position pair.

[0170]

[0187] Herein, the second CH1 region and the second CL region also associate with each other through electrostatic interaction, but the position of the amino acid residue that forms electrostatic interaction is different from the position of the amino acid residue that forms electrostatic interaction in the first target binding domain.For example, both n7':n8' position pair and n3:n4 position pair are independently selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, with the proviso that n7':n8' position pair is different from n3:n4 position pair.In some embodiments, both n7':n8' position pair and n3:n4 position pair are independently selected from the group consisting of 183:176, 141:116 and 126:121, with the proviso that n7':n8' position pair is different from n3:n4 position pair. In some embodiments, the n7':n8' position pair is selected from the group consisting of 183:176, 141:116 and 126:121. For example, the n7':n8' position pair is 183:176 and the n3:n4 position pair is selected from the group consisting of 141:116, 126:121 and 218:122. In another example, the n7':n8' position pair is 141:116 and the n3:n4 position pair is selected from the group consisting of 183:176, 126:121 and 218:122. In yet another example, the n7':n8' position pair is 126:121 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 218:122. In yet another example, the n7':n8' position pair is 218:122 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 126:121.

[0171]

[0188] In some embodiments, the seventh and eighth corresponding amino acid residues at EU positions n7' and n8' are natural / wild type amino acid residues, e.g., selected from the amino acid pairs S183:S176, A141:F116, F126:S121 and K218:D122, with the proviso that the n7':n8' position pair is different from the n3:n4 position pair. In some embodiments, one or both of the seventh and eighth corresponding amino acid residues at EU positions n7' and n8' are mutated amino acid residues in the CH1 or CL region of the heavy or light chain of the second target binding domain. For example, one or both of the seventh and eighth corresponding amino acid residues at EU positions n7' and n8' are mutated amino acid residues that are oppositely charged, with the proviso that the n7':n8' position pair is different from the n3:n4 position pair.

[0172]

[0189] In some embodiments, the first CH1 region further comprises a seventh amino acid residue at EU position n7 and the second CL region further comprises an eighth amino acid residue at EU position n8, wherein the n7:n8 position pair is identical to the n7':n8' position pair, and wherein the seventh corresponding amino acid residue at EU position n7' and the eighth amino acid residue at EU position n8 are not oppositely charged or are similarly charged, and / or the eighth corresponding amino acid residue at EU position n8' and the seventh amino acid residue at EU position n7 are not oppositely charged or are similarly charged.

[0173]

[0190] As used herein, the 7th and 8th amino acid residues at EU positions n7 and n8 of a first target binding domain do not interfere with the electrostatic interaction of the 7th and 8th corresponding amino acid residues at EU positions n7' and n8' of a second target binding domain.

[0174]

[0191] In some embodiments, the 7th and 8th amino acid residues at EU positions n7 and n8 are natural / wild type amino acid residues, e.g., selected from the amino acid pairs S183:S176, A141:F116, F126:S121 and K218:D122. In some embodiments, one or both of the 7th and 8th amino acid residues at EU positions n7 and n8 are mutated amino acid residues in the CH1 or CL region of the heavy or light chain of the second target binding domain. For example, one or both of the 7th and 8th amino acid residues at EU positions n7 and n8 are mutated amino acid residues that are similarly charged to the corresponding 8th and 7th amino acid residues at EU positions n8' and n7' of the second target binding domain.

[0175]

[0192] In some embodiments, the seventh amino acid residue at EU position n7 and / or the eighth amino acid residue at EU position n8 are uncharged. For example, the first target binding domain has no electrostatic interaction between the seventh amino acid residue at EU position n7 and the eighth amino acid residue at EU position n8.

[0176]

[0193] In some embodiments at least one, two, three or four of the first amino acid residue at EU position n1, the second amino acid residue at EU position n2, the third amino acid residue at EU position n3 and the fourth amino acid residue at EU position n4 are introduced by substitution.

[0194] As used herein, each of the first, second, third and fourth introduced amino acid residues may be an amino acid residue that is not present in the natural / wild-type version of the amino acid and is therefore "introduced" into one of the EU positions shown above in the first CH1 or first CL region of the immunoglobulin (i.e., 128:118 and 173:160 for the first and second introduced amino acid residues and 183:176, 141:116, 126:121 and 218:122 for the third and fourth introduced amino acid residues). Any such introduced amino acid residue may be a substituted amino acid replacing a wild-type residue, or may be a newly added / inserted residue not present in the wild-type polypeptide, or may be a modified or artificial residue.

[0177]

[0195] In some other embodiments, at least one, two, three or four of the first amino acid residue at EU position n1, the second amino acid residue at EU position n2, the third amino acid residue at EU position n3 and the fourth amino acid residue at EU position n4 may be newly inserted / added amino acid residue(s) at EU position n1, n2, n3 and / or n4 rather than a substitution(s).

[0178]

[0196] In some and other embodiments, one or both of the third and fourth amino acid residues at EU positions n3 and n4 may be a non-natural amino acid analog(s) or mimetic(s) or a positively or negatively charged chemically modified amino acid residue(s).

[0179]

[0197] In certain embodiments, there may be two or more pairs of oppositely charged third and fourth amino acid residues at EU position pairs 183:176, 141:116, 126:121 and 218:122. In one example, the polypeptide complex may be configured such that each of the two amino acid residue pairs at both EU position pairs 183:176 and 141:116 are mutated (e.g., substituted, inserted or modified) to be oppositely charged. In another example, all four amino acid residue pairs at EU position pairs 183:176, 141:116, 126:121 and 218:122 are mutated (e.g., substituted, inserted or modified) to be oppositely charged.

[0180]

[0198] Thus, for each n3:n4 pair selected from EU position pairs 183:176, 141:116, 126:121 and 218:122, two alternative schemes can be applied. In the first scheme, the third amino acid residue at EU position n3 is a positively charged amino acid residue, such as lysine (K), arginine (R) or histidine (H), and the fourth amino acid residue at EU position n4 is a negatively charged amino acid residue, such as aspartic acid (D) or glutamic acid (E). In the second scheme, the third amino acid residue at EU position n3 is a negatively charged amino acid residue, such as aspartic acid (D) or glutamic acid (E), and the fourth amino acid residue at EU position n4 is a positively charged amino acid residue, such as lysine (K), arginine (R) or histidine (H).

[0181]

[0199] To summarize all the combinations in the above two schemes, the third and fourth amino acid residues of the n3:n4 position pair are S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K ... 83D:S176R, S183D:S176H, S183E:S176K, S183E:S176R, S183E:S176H, A141K:F116D, A14 1K:F116E, A141R:F116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141 D:F116R, A141D:F116H, A141E:F116K, A141E:F116R, A141E:F116H, F126K:S121D, F126K :S121E, F126R:S121D, F126R:S121E, F126H:S121D, F126H:S121E, F126D:S121K, F126D: The substitution is selected from the group consisting of S121R, F126D:S121H, F126E:S121K, F126E:S121R, F126E:S121H, K218D:D122K, K218D:D122H, K218D:D122R, K218E:D122K, K218E:D122H, and K218E:D122R.

[0182]

[0200] Similarly, in some other embodiments, the seventh corresponding amino acid residue at EU position n7' is a positively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a negatively charged amino acid residue. In some other embodiments, the seventh corresponding amino acid residue at EU position n7' is a negatively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a positively charged amino acid residue.

[0183]

[0201] In some embodiments, at least one, two, three or four of the fifth corresponding amino acid residue at EU position n5', the sixth corresponding amino acid residue at EU position n6', the seventh corresponding amino acid residue at EU position n7' and the eighth corresponding amino acid residue at EU position n8' are introduced by substitution.

[0184]

[0202] In some other embodiments, at least one, two, three or four of the fifth corresponding amino acid residue at EU position n5', the sixth corresponding amino acid residue at EU position n6', the seventh corresponding amino acid residue at EU position n7' and the eighth corresponding amino acid residue at EU position n8' may be newly inserted / added amino acid residue(s) at EU position n5', n6', n7' and / or n8' rather than a substitution(s).

[0185]

[0203] In some and other embodiments, one or both of the seventh and eighth corresponding amino acid residues at EU positions n7' and n8' may be a non-natural amino acid analog(s) or mimetic(s) or a positively or negatively charged chemically modified amino acid residue(s).

[0186]

[0204] In certain embodiments, there may be two or more pairs of oppositely charged seventh and eighth corresponding amino acid residues at EU position pairs 183:176, 141:116, 126:121 and 218:122. In one example, the polypeptide complex may be configured such that each of the two amino acid residue pairs at both EU position pairs 183:176 and 141:116 are mutated (e.g., substituted, inserted or modified) to be oppositely charged. In another example, all four amino acid residue pairs at EU position pairs 183:176, 141:116, 126:121 and 218:122 are mutated (e.g., substituted, inserted or modified) to be oppositely charged.

[0187]

[0205] Thus, for each n7':n8' pair selected from EU position pairs 183:176, 141:116 and 126:121, two alternative schemes can be applied. In the first scheme, the seventh corresponding amino acid residue at EU position n7' is a positively charged amino acid residue, e.g., lysine (K), arginine (R) or histidine (H), and the eighth corresponding amino acid residue at EU position n8' is a negatively charged amino acid residue, e.g., aspartic acid (D) or glutamic acid (E). In the second scheme, the seventh corresponding amino acid residue at EU position n7' is a negatively charged amino acid residue, e.g., aspartic acid (D) or glutamic acid (E), and the eighth amino acid residue at EU position n8' is a positively charged amino acid residue, e.g., lysine (K), arginine (R) or histidine (H).

[0188]

[0206] To summarize all the combinations in the above two schemes, the seventh and eighth corresponding amino acid residues of the n7':n8' position pair are S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K, S 183D:S176R, S183D:S176H, S183E:S176K, S183E:S176R, S183E:S176H, A141K:F116D, A141K: F116E, A141R:F116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141D:F116R, A141D:F116H, A141E:F116K, A141E:F116R, A141E:F116H, F126K:S121D, F126K:S121E, F126R :S121D, F126R:S121E, F126H:S121D, F126H:S121E, F126D:S121K, F126D:S121R, F126D:S121H , F126E:S121K, F126E:S121R, F126E:S121H, K218D:D122K, K218D:D122H, K218D:D122R, K218E:D122K, K218E:D122H, and K218E:D122R, and the n7':n8' position pair is different from the n3:n4 position pair.

[0189]

[0207] In some embodiments, the first target binding domain comprises a first combination of substitutions at the (n1+n2):(n3+n4) positions and / or the second target binding domain comprises a second combination of substitutions at the (n5'+n6'):(n7'+n8') positions, wherein the first combination of substitutions and / or the second combination of substitutions are (L128C+S183K):(F118C+S176D), (L128C+S183K):(F118C+S176E), (L128C+S183R):(F118C+S176D), (L128C+S183R):(F1 18C+S176E), (L128C+S183H):(F118C+S176D), (L128C+S183H):(F118C+ S176E), (L128C+S183D):(F118C+S176K), (L128C+S183D):(F118C+S176R ), (L128C+S183D):(F118C+S176H), (L128C+S183E):(F118C+S176K), (L1 28C+S183E):(F118C+S176R), (L128C+S183E):(F118C+S176H), (V173C+A 141K):(Q160C(or E160C)+F116D),(V173C+A141K):(Q160C(or E160C)+F116E),(V173C+A141R):(Q160C(or E160C)+F116D),(V173C+A141R):(Q160C(or E160C)+F116E),(V173C+A141H):(Q160C(or E160C)+F116D),(V173C+A141H):(Q160C(or E160C)+F116E),(V173C+A141D):(Q160C(or : E160C)+F116K), (V173C+A141D):(Q160C(or E160C)+F116R), (V173C+A141D):(Q160C(or E160C)+F116H), (V173C+A141E):(Q160C(or E160C)+F116K), (V173C+A141E):(Q160C(or E160C)+F116R), (V173C+A141E):(Q160C(or E160C)+F116H), (V173C+S183K):(Q160C(or E160C)+S176D),(V173C+S183K):(Q160C(or E160C)+S176E), (V173C+S183R):(Q160C(or E160C)+S176D), (V173C+S183R):(Q160C(or E160C)+S176E), (V173C+S183H):(Q160C(or E160C)+S176D), (V173C+S183H):(Q160C(or E160C)+S176E), (V173C+S183D):(Q160C(or E160C)+S176K), (V173C+S183D):(Q1 60C(or E160C)+S176R), (V173C+S183D):(Q160C(or E160C)+S176H), (V173C+S183E):(Q160C(or E160C)+S176K), (V173C+S183E):(Q160C(or E160C)+S176R), (V173C+S183E):(Q160C(or E160C)+S176H), (L128C+F126K):(F118C+S121D), (L128C+F126K):(F118C+S121E), (L128C+F126R):(F1 18C+S121D), (L128C+F126R):(F118C+S121E), (L128C+F126H):(F118C+S 121D), (L128C+F126H):(F118C+S121E), (L128C+F126D):(F118C+S121K) , (L128C+F126D):(F118C+S121R), (L128C+F126D):(F118C+S121H), (L12 8C+F126E):(F118C+S121K), (L128C+F126E):(F118C+S121R), (L128C+F12 6E):(F118C+S121H),(V173C+F126K):(Q160C(or E160C)+S121D),(V173C+F126K):(Q160C(or E160C)+S121E),(V173C+F126R):(Q160C(or E160C)+S121D),(V173C+F126R):(Q160C(or E160C)+S121E),(V173C+F126H):(Q160C(or E160C)+S121D),(V173C+F126H):(Q160C(or E160C)+S121E),(V173C+F126D):(Q160C(or E160C)+S121K), (V173C+F126D):(Q160C(or E160C)+S121R), (V173C+F126D):(Q160C(or E160C)+S121H), (V173C+F126E):(Q160C(or E160C)+S121K), (V173C+F126E):(Q160C(or E160C)+ S121R) and (V173C+F126E):(Q160C(or E160C)+S121H), (L128C+K218D):(F118C+D122K), (L128C+K218D):(F118C+D122H), (L128C+K218D):(F118C+D122R), (L128C+K218E):(F118C+D122K), (L128C+K218E):(F118C+D122 2H), (L128C+K218E):(F118C+D122R), (V173C+K218D):(Q160C(or E160C)+D122K), (V173C+K218D):(Q160C(or E160C)+D122H), (V173C+K218D):(Q160C(or E160C)+D122R), (V173C+K218E):(Q160C(or E160C)+D122K) , (V173C+K218E):(Q160C(or E160C)+D122H), and (V173C+K218E):(Q160C(or E160C)+D122R), with the proviso that when both the first substitution combination and the second substitution combination are selected, the n5':n6' position pair is different from the n1:n2 position pair, and the n7':n8' position pair is different from the n3:n4 position pair.

[0190]

[0208] In some embodiments, the first or second target binding domain comprises an L128C substitution in the first or second CH1 region and an F118C substitution in the first or second CL region.

[0191]

[0209] In some embodiments, the first or second target binding domain comprises a V173C substitution in the first or second CH1 region and a Q160C (or E160C) substitution in the first or second CL region.

[0192]

[0210] In some embodiments, the first or second target binding domain comprises an L128C and S183D substitution in the first or second CH1 region and an F118C and S176K substitution in the first or second CL region.

[0193]

[0211] In some embodiments, the first or second target binding domain comprises an L128C and F126D substitution in the first or second CH1 region and an F118C and S121K substitution in the first or second CL region.

[0194]

[0212] In some embodiments, the first or second target binding domain comprises a L128C and A141D substitution in the first or second CH1 region and a F118C and F116K substitution in the first or second CL region.

[0195]

[0213] In some embodiments, the first or second target binding domain comprises an L128C and a K218D substitution in the first or second CH1 region and an F118C and a D122K substitution in the first or second CL region.

[0196]

[0214] In some embodiments, the first or second target binding domain comprises a V173C and S183D substitution in the first or second CH1 region and a Q160C and S176K substitution in the first or second CL region.

[0197]

[0215] In some embodiments, the first or second target binding domain comprises a V173C and A141D substitution in the first or second CH1 region and a Q160C and F116K substitution in the first or second CL region.

[0198]

[0216] In some embodiments, the first or second target binding domain comprises a V173C and S183D substitution in the first or second CH1 region.

[0199]

[0217] In some embodiments, the first or second target binding domain comprises a V173C and S183D substitution in the first or second CH1 region and a Q160C (or E160C) and S176K substitution in the first or second CL region.

[0200]

[0218] In some embodiments, the first or second target binding domain comprises an L128C and a C220S (IgG1) or a C131S (IgG2 and IgG4) substitution in the first or second CH1 region and an F118C and a C214S substitution in the first or second CL region.

[0201]

[0219] In some embodiments, the first or second target binding domain comprises a V173C and a C220S (IgG1) or a C131S (IgG2 and IgG4) substitution in the first or second CH1 region, and a Q160C (or E160C) and a C214S substitution in the first or second CL region.

[0202]

[0220] In some embodiments, the first or second target binding domain comprises L128C, C220S for IgG1 (or C131S for IgG2 and IgG4) and S183D substitutions in the first or second CH1 region, and F118C, C214S and S176K substitutions in the first or second CL region.

[0203]

[0221] In some embodiments, the first or second target binding domain comprises L128C, C220S for IgG1 (or C131S for IgG2 and IgG4) and F126D substitutions in the first or second CH1 region, and F118C, C214S and S121K substitutions in the first or second CL region.

[0204]

[0222] In some embodiments, the first or second target binding domain comprises L128C, C220S for IgG1 (or C131S for IgG2 and IgG4) and A141D substitutions in the first or second CH1 region, and F118C, C214S and F116K substitutions in the first or second CL region.

[0205]

[0223] In some embodiments, the first or second target binding domain comprises L128C, C220S for IgG1 (or C131S for IgG2 and IgG4) and K218D substitutions in the first or second CH1 region, and F118C, C214S and D122K substitutions in the first or second CL region.

[0206]

[0224] In some embodiments, the first or second target binding domain comprises V173C, C220S for IgG1 (or C131S for IgG2 and IgG4) and S183D substitutions in the first or second CH1 region, and Q160C, C214S and S176K substitutions in the first or second CL region.

[0207]

[0225] In some embodiments, the first or second target binding domain comprises V173C, C220S for IgG1 (or C131S for IgG2 and IgG4) and A141D substitutions in the first or second CH1 region, and Q160C, C214S and F116K substitutions in the first or second CL region.

[0208]

[0226] In some embodiments, the first or second target binding domain comprises the following substitutions in the first or second CH1 region: V173C, C220S for IgG1 (or C131S for IgG2 and IgG4), and S183D.

[0209]

[0227] In some embodiments, the first or second target binding domain comprises V173C, C220S for IgG1 (or C131S for IgG2 and IgG4) and S183D substitutions in the first or second CH1 region, and Q160C (or E160C), C214S and S176K in the first or second CL region.

[0210]

[0228] In some embodiments, the first target binding moiety provided herein comprises a first polypeptide fragment operably linked to a first CL region, and the second target binding moiety provided herein comprises a second polypeptide fragment operably linked to a second CL region, and the first polypeptide fragment has a different amino acid sequence than the second polypeptide fragment. In some embodiments, either the first polypeptide fragment or the second polypeptide fragment is not present in the polypeptide complex.

[0211]

[0229] As used herein, a "different amino acid sequence" refers to one amino acid sequence that differs from another amino acid sequence, for example, in length, type of amino acid, or function.

[0212]

[0230] In some embodiments, the polypeptide complex may be a fusion protein comprising two or more polypeptide fragments each operably linked to a CH1 region and a CL region. In some embodiments, the first target binding moiety further comprises a third polypeptide fragment operably linked to the first CH1 region, and the second target binding moiety comprises a fourth polypeptide fragment operably linked to the second CH1 region. In some embodiments, the third polypeptide fragment has a different amino acid sequence from the fourth polypeptide fragment. In some embodiments, either the third polypeptide fragment or the fourth polypeptide fragment is not present in the polypeptide complex.

[0213]

[0231] In some embodiments, the first polypeptide fragment and the third polypeptide fragment each contain a target binding site and can bind to its target molecule.For example, the first polypeptide fragment and the third polypeptide fragment can bind to the same target molecule or bind to different target molecules.In another example, the first polypeptide fragment and the third polypeptide fragment can have either the same or different amino acid sequence.

[0214]

[0232] Similarly, the second polypeptide fragment and the fourth polypeptide fragment each contain a target binding site and can bind to its target molecule.For example, the second polypeptide fragment and the fourth polypeptide fragment can bind to the same target molecule or bind to different target molecules.In another example, the second polypeptide fragment and the fourth polypeptide fragment can have either the same or different amino acid sequence.

[0215]

[0233] In some embodiments, the first polypeptide fragment and the third polypeptide fragment can be associated to form a first target binding site.Similarly, the second polypeptide fragment and the fourth polypeptide fragment can be associated to form a second target binding site.In some embodiments, the first target binding site and the second target binding site can bind to the same target molecule, or to different parts on the same target molecule, or to different target molecules.

[0216]

[0234] In some embodiments, the first target-binding moiety may be a first antigen-binding moiety and / or the second target-binding moiety may be a second antigen-binding moiety. In some embodiments, the antigen-binding moiety is derived from one or more antibody fragments.

[0217]

[0235] In some embodiments, the first antigen-binding portion may comprise a first VL region and a first VH region, which associate to form a first antigen-binding site. In some embodiments, the second antigen-binding portion may comprise a second VL region and a second VH region, which associate to form a second antigen-binding site. The first antigen-binding site and the second antigen-binding site may bind to the same antigen, or different epitopes on the same antigen, or different antigens.

[0218]

[0236] In some embodiments, the first and / or second antigen-binding domain provided herein is selected from the group consisting of a Fab domain, a Fab' and a F(ab')2. The first and / or second antigen-binding domain comprises one or more CDRs operably linked to a CH1 region and a CL region. In some embodiments, the first antigen-binding domain comprises a first Fab domain. In some embodiments, the second antigen-binding domain comprises a second Fab domain. In some embodiments, the second Fab domain comprises one or more light chain CDRs and / or light chain framework regions that are different from the first Fab domain. In some embodiments, the second Fab domain further comprises one or more heavy chain CDRs and / or heavy chain framework regions that are different from the first Fab domain.

[0219]

[0237] In some embodiments, the first Fab domain and / or the second Fab domain provided herein also encompass various variants of the Fab domain. For example, the variant of the Fab domain may include one or more modifications or substitutions in one or more of the amino acid residues in the CH1 region and / or the CL region. Such variants may have one or more desirable properties conferred by the modification(s) or substitution(s), such as improved antigen binding affinity, improved glycosylation pattern, reduced glycosylation risk, reduced deamination, enhanced effector function(s), improved FcRn receptor binding, extended pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., introduction of one or more cysteine ​​residues). In some embodiments, the Fab domain provided herein does not include HCVR or LCVR. In some embodiments, the Fab domain provided herein includes HCVR, LCVR, CH1 region and CL region.

[0220]

[0238] In some embodiments, the first and second Fab domains disclosed herein are two different Fab domains, meaning that the two Fab domains each have one or more differences in terms of primary sequence, side chain modifications, or conformation of both the light chain and heavy chain portions that make up the Fab domain.

[0221] III. Heterodimeric Antibodies or Antigen-Binding Fragments Thereof

[0239] In some embodiments, the first antigen-binding domain and / or the second antigen-binding domain are comprised within an antibody, optionally a bispecific or multispecific antibody.

[0222]

[0240] In some embodiments, the present disclosure provides a heterodimeric protein complex comprising a first antigen-binding domain and a second antigen-binding domain as provided herein. In some embodiments, the heterodimeric protein complex provided herein comprises a heterodimeric antibody.

[0223]

[0241] As used herein, the term "heterodimeric antibody" refers to an asymmetric antibody having two different subunits associated with each other, in contrast to natural or naturally occurring antibodies, which are essentially homodimers. Such homodimers consist of two identical light chains and two identical heavy chains, which are conformationally grouped into a first pair of light chains and heavy chains and a second pair of identical light chains and heavy chains that constitute two identical subunits, each containing an identical antigen-binding domain as each of the two arms of the Y-shaped antibody. In the context of the present disclosure, a heterodimeric antibody comprises at least two different antigen-binding regions / domains (i.e., a first and a second antigen-binding domain), each of which comprises a constant portion operably linked to a variable domain. Each constant portion comprises an immunoglobulin heavy chain constant region 1 (CH1) and an immunoglobulin light chain constant region (CL). Each antigen-binding domain comprises a different antigen-binding portion that specifically binds to a different epitope, and typically comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). The VH region and the CH1 region are operably linked to each other, and the VL region and the CL region are operably linked to each other. In each antigen-binding domain, the CL region and the CH1 region are associated with each other by at least one non-natural interchain covalent bond (e.g., disulfide bond) and at least one non-natural non-covalent bond (e.g., electrostatic interaction or salt bridge). Further, in the context of the present disclosure, the heterodimeric antibody may optionally further comprise an Fc domain operably linked to the first and second antigen-binding domains. The Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, which comprise, for example, the CH2 and CH3 regions of an immunoglobulin heavy chain, respectively. The Fc domain may be altered (i.e., mutated or modified) to promote heterodimerization. Furthermore, it is possible that two different antigen-binding domains may associate with each other by means other than Fc domains, e.g., polypeptide, chemical covalent bonds, etc.

[0224]

[0242] Heterodimeric antibodies as used herein are essentially modified monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to a type of antibody produced by the same immune cell, all of which are clones belonging to a unique parent cell. Monoclonal antibodies are highly specific and are produced against a single epitope. The modifier "monoclonal" as used herein should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used according to the present invention can be produced by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or can be produced by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0225]

[0243] The heterodimeric antibody or antigen-binding fragment thereof comprises a first antigen-binding domain and a second antigen-binding domain. The first antigen-binding domain and the second antigen-binding domain are two different domains. This configuration is favorable for the formation of the heterodimeric antibody or antigen-binding fragment thereof, since the first CH1 region selectively associates with the first CL region to form the first antigen-binding domain, and there is substantially no binding between the first CH1 region and the second CL region or between the first CL region and the second CH1 region. This then allows the efficient formation of the heterodimeric antibody or antigen-binding fragment thereof, which has two different antigen-binding domains, each potentially having a different antigen-binding portion.

[0226]

[0244] The first antigen-binding domain and second antigen-binding domain described above under section II. Polypeptide Complexes are also applicable to the heterodimeric antibodies described herein.

[0227]

[0245] In certain embodiments, the second antigen-binding domain comprises one or more light chain CDRs and / or light chain framework regions that differ from those of the first antigen-binding domain. In certain embodiments, the second antigen-binding domain comprises one or more heavy chain CDRs and / or heavy chain framework regions that differ from those of the first antigen-binding domain. This configuration configures the first and second antigen-binding domains to specifically target and bind different antigens or different epitopes of the same antigen.

[0228]

[0246] In some embodiments, a heterodimeric antibody is a bispecific antibody. As used herein, a heterodimeric antibody may have two antigen-binding domains that specifically target two different antigens or two different epitopes of a single antigen, respectively.

[0229]

[0247] In some other embodiments, the heterodimeric antibody is a multispecific antibody. In this specification, in addition to the two antigen-binding domains that specifically target two different antigens or two different epitopes of a single antigen, the heterodimeric antibody may further include one or more other antigen-binding moieties that specifically target other antigens or other epitopes. In one non-limiting example, the antigen-binding site can be modified to acquire an additional antigen-binding site by introducing it into the Fc region of an originally bispecific antibody, called Fcab (Wozniak-Knopp G, et al., (2010). Protein Eng Des. 23 (4): 289-297), and the antibody thus obtained can be a multispecific antibody with three antigen-binding sites. Other examples may exist.

[0230]

[0248] In some embodiments, the second antigen-binding domain and the first antigen-binding domain bind to different antigens or bind to different epitopes on the same antigen.

[0231]

[0249] In some embodiments, the antigen may be a tumor-associated antigen, an immune-associated target, or an infectious pathogen-associated target.

[0232]

[0250] In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain binds to a tumor-associated antigen, and the other binds to an immune-related target. In some embodiments, one of the first antigen-binding domain and the second antigen-binding domain binds to a first tumor-associated antigen, and the other binds to a second tumor-associated antigen. For a detailed description of tumor-associated antigens, please refer to Section VIII. Treatment. Immune related targets provided herein include CD2, CD3, CD7, CD16, CD27, CD30, CD70, CD83, CD28, CD80 (B7-1), CD86 (B7-2), CD40, CD40L (CD154), CD47, CD122, CD137, CD137L, OX40 (CD134), OX40L (CD252), NKG2C, 4-1BB, LIGHT, PVRIG, SLAMF7, HVEM, BAFFR, ICAM-1, 2B4, LFA-1, GITR, ICOS (CD278), IC The polypeptide may be selected from the group consisting of OSLG (CD275), LAG3 (CD223), A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), BTLA, CD160, CTLA-4 (CD152), IDO1, IDO2, TDO, KIR, LAIR-1, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC-7 (CD328), TIGIT, PVR (CD155), TGFβ, SIGLEC9 (CD329), and any combination thereof.

[0233]

[0251] In some embodiments, the first CL region or the second CL region is derived from a kappa light chain. In some embodiments, the first CL region or the second CL region is derived from a lambda light chain. In some embodiments, the first CL region or the second CL region is derived from a kappa light chain and a lambda light chain, respectively.

[0234]

[0252] According to various embodiments of the antibody or antigen-binding fragment thereof, the first and / or second antigen-binding domain may be a chimeric, humanized or fully human antigen-binding domain.

[0235]

[0253] As used herein, the term "chimeric" refers to a situation in which portions of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0236]

[0254] As used herein, the term "humanization" refers to the situation in which the sequences of polypeptides (e.g., antibodies) from non-human species are modified to improve their similarity to antibody variants naturally generated in humans, which are a special form of chimeric polypeptide. For example, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's hypervariable region are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, with the desired specificity, affinity, and capacity. In some cases, Fv fragment region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Moreover, humanized antibodies may contain residues that are not found in the recipient antibody or donor antibody. Such modifications are made to further improve the performance of the antibody. Generally, a humanized antibody contains substantially all of at least one, and typically two, variable domains, whereby all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of a human immunoglobulin sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0237]

[0255] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that, upon immunization, can produce a full repertoire or selection of human antibodies without producing endogenous immunoglobulins. Introduction of the human germline immunoglobulin gene array in such germline mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be generated by phage display libraries (Hoogenboom, HR, and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, JD, et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the invention, the term "human antibody", as used herein, also includes antibodies modified in the constant region to provide the properties according to the invention, in particular with regard to Clq binding and / or FcR binding, for example by "class switching", i.e. by changing or mutating the Fc part (e.g. from IgG1 to IgG4 and / or IgG1 / IgG4 mutations).The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant methods, such as antibodies isolated from host cells, e.g., NSO or CHO cells, or animals (e.g., mice) transgenic for human immunoglobulin genes, or expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies according to the invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that are derived and related to human germline VH and VL sequences, but may not naturally occur in the human antibody germline repertoire in vivo.

[0238]

[0256] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc region / domain operably linked to the first and second antigen-binding domains, which may enhance the stability of the antibody or antigen-binding fragment thereof but may also mediate various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), through interactions with cell surface receptors (i.e., Fc receptors) and specific proteins in the complement system.

[0239]

[0257] In certain embodiments, the Fc region is operably linked to both the first and second antigen binding domains by a spacer / linker. Optionally and preferably, such a spacer is the natural hinge region present in the two heavy chains of an immunoglobulin, which includes one or more interchain disulfide bonds linking the respective CH1 and CH2 regions and forming an interchain bridge connecting the two heavy chains. Alternatively, the spacer may be an artificial polypeptide liner comprising approximately 10-40 amino acid residues, which is flexible to allow the Fc region to bind to both the first and second antigen binding domains without interfering with their respective functionality. Alternatively still, the spacer may simply be a chemical bridge.

[0240]

[0258] Optionally, the Fc region is derived from IgG, IgA, IgM, IgE or IgD, preferably from IgG1, IgG2, IgG3 or IgG4, more preferably from IgG1.

[0241]

[0259] As used herein, the Fc region may be a wild-type Fc region or a variant Fc region.

[0242]

[0260] In certain preferred embodiments, the Fc region is a heterodimeric variant Fc region comprising a first Fc polypeptide and a second Fc polypeptide, the variant Fc region further comprising one or more mutations that promote heterodimerization.

[0243]

[0261] It has been shown that modification of multiple amino acid residues of two immunoglobulin heavy chains at the interface in the Fc region, most notably in the CH3 region, can increase the percentage of heterodimers recovered from recombinant cell culture. One strategy thus employed is sometimes referred to as the "knob and hole" strategy, where one or more protrusions (i.e., "knobs") can be generated by replacing one or more amino acid residues with small side chains (e.g., glycine, alanine, threonine) on the interface of a first heavy chain with amino acid residues with large side chains (tyrosine or tryptophan), and a compensatory "cavity" (i.e., "hole") of similar size to the large side chain(s) is simultaneously generated on the interface of a second heavy chain by replacing the amino acid residues with large side chains with amino acid residues with small side chains (e.g., alanine or threonine). In another strategy, the CH3 region can be modified to include a mutation that introduces a cysteine ​​residue capable of forming a disulfide bond. Such modifications provide a mechanism for increasing the yield of heterodimer over other unwanted end products, such as homodimer.CH3 modifications that enhance heterodimerization include, for example, Y407V / T366S / L368A on one heavy chain and T366W on the other heavy chain; S354C, T366W on one heavy chain and Y349C / Y407V / T366S / L368A on the other heavy chain.Further modifications that create protrusions on one chain and cavities on the other are described in U.S. Patent No. 7,183,076 and U.S. Patent No. 9,527,927 and Merchant et al., 1998, Nat.Biotech 16:677-681. Another strategy for modifying amino acid residues to promote heterodimer formation involves altering the charge polarity across the Fc dimer interface to induce heterodimerization upon co-expression of electrostatically compatible Fc regions.One such charge pair mutation includes T366K+L351D and L351K+Y349E / Y349D / L368E (WO 2013 / 157953), and other such charge pair mutations are described in WO 2007 / 147901, WO 2012 / 058768, U.S. Pat. No. 9,527,927, WO 96 / 27011, WO 98 / 050431, European Patent Application Publication No. 2009 / 023662, and the like. No. 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, WO 2013 / 096291 and Gunasekaran et al., 2010, JBC 285:19637-46. Yet another strategy to promote heterodimeric Fc formation is described in WO 2007 / 110205 and Davis et al. (2010), Prot. Eng. Design & Selection 23:195-202, in this case using a strand-exchange engineered domain (SEED) CH3 region, a derivative of the human IgG and IgA CH3 domains.

[0244]

[0262] As used herein, optionally the variant Fc region comprises a first Fc mutation in a first Fc polypeptide and / or a second Fc mutation in a second Fc polypeptide.

[0245]

[0263] Further optionally, the first Fc mutation and the second Fc mutation are selected from any one of the following combinations:

[0246]

[0264] a) a first Fc mutation comprising T366W or S354C, and a second Fc mutation comprising Y349C, T366S, L368A, or Y407V;

[0247]

[0265] b) a first Fc mutation comprising D399K or E356K, and a second mutation comprising K392D or K409D;

[0248]

[0266] c) a first Fc mutation comprising E356K, E357K, or D399K, and a second Fc mutation comprising K370E, K409D, or K439E;

[0249]

[0267] d) a first Fc mutation comprising S364H or F405A, and a second Fc mutation comprising Y349T or T394F;

[0250]

[0268] e) a first Fc mutation comprising S364H or T394F, and a second Fc mutation comprising Y394T or F405A;

[0251]

[0269] f) a first Fc mutation comprising K370D or K409D, and a second Fc mutation comprising E357K or D399K, or

[0252]

[0270] g) A first Fc mutation comprising L351D or L368E, and a second Fc mutation comprising L351K or T366K.

[0253]

[0271] In both cases, the numbering follows the EU index.

[0254] IV. Antibody Conjugates

[0272] The polypeptide complexes provided herein can be used in unconjugated or conjugated form.

[0255]

[0273] In the conjugated form, the polypeptide complex is conjugated to one or more desired conjugates, i.e., heterologous moieties, to achieve a particular function, for example, to facilitate target detection or for imaging or therapy.

[0256]

[0274] The present disclosure provides herein a conjugate comprising the polypeptide complex provided herein and a payload conjugated thereto.The payload can be any one of the group consisting of radioactive label, fluorescent label, enzyme substrate label, affinity purification tag, tracer molecule, anticancer drug and cytotoxic molecule.

[0257]

[0275] A variety of conjugates can be linked to the polypeptide complexes provided herein by covalent bonding, affinity binding, intercalation, coordinate binding, complex formation, association, blending or addition, among others (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)).

[0258]

[0276] In certain embodiments, the polypeptide complexes provided herein may be modified to include specific sites on the exterior of the epitope-binding moiety, which can be specifically utilized for linking to one or more conjugates.For example, such sites may include one or more reactive amino acid residues, such as, for example, cysteine ​​or histidine residues, that facilitate covalent linking to the conjugate.

[0259]

[0277] In certain embodiments, the N-terminus and / or C-terminus of the polypeptide complexes provided herein may also serve to provide reactive groups for conjugation. For example, the N-terminus may be conjugated to one moiety (e.g., polyethylene glycol (PEG) or the like) and the C-terminus is conjugated to another moiety (e.g., biotin or the like).

[0260]

[0278] In certain embodiments, the polypeptide complexes provided herein can be linked to a conjugate, either directly or indirectly, for example, by another conjugate or linker.

[0261]

[0279] For example, a polypeptide conjugate provided herein having a reactive residue, e.g., cysteine, can be linked to a thiol reactant, where the reactive group is, for example, maleimide, iodoacetamide, pyridyl disulfide, or other thiol-reactive conjugation partner (Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671).

[0262]

[0280] In another example, the polypeptide complex provided herein can be conjugated to biotin and then indirectly conjugated to a second conjugate that is conjugated to avidin. In yet another example, the polypeptide complex can be linked to a linker, which can be further linked to a conjugate. Examples of linkers include bifunctional coupling agents such as N-succinimidyl 1-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and his-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), which result in a disulfide linkage.

[0263]

[0281] The conjugate can be a detectable label, a pharmacokinetic-modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S,3 H、 111 Through, 112 Through, 14 C、 64 Cu、 67 Cu、 86 Y、 88 Y、 90 Y、 177 Head, 211 And、 186 Re、 188 Re、 153 Sm、 212 Biおよび 32P, other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection. In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety, such as PEG, which serves to extend antibody half-life. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. In certain embodiments, the conjugate may be a purification moiety, such as a magnetic bead. A "cytotoxic moiety" may be any substance that is harmful to cells or that can damage or kill cells. Examples of cytotoxic moieties include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethate, These include, but are not limited to, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

[0264]

[0282] In certain embodiments, the polypeptide complexes provided herein may be conjugated to a signal peptide. Signal peptides (sometimes referred to as signal sequences, leader sequences or leader peptides) may be used to facilitate secretion and isolation of the polypeptide complexes provided herein. Signal peptides are typically characterized by a core of hydrophobic amino acids that are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow for cleavage of the signal sequence from the mature protein as it passes through the secretory pathway. Thus, the present invention relates to the described polypeptides having a signal sequence, as well as polypeptides in which the signal sequence has been proteolytically cleaved (i.e., cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence may be operably linked to a protein of interest in an expression vector, for example, a protein that is not normally secreted or is otherwise difficult to isolate. The signal sequence directs the secretion of the protein, for example, from a eukaryotic host transformed with the expression vector, and the signal sequence is subsequently or simultaneously cleaved. The protein can then be readily purified from the extracellular medium by art-recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using sequences that facilitate purification, for example, a GST domain.

[0265]

[0283] Methods for conjugating the conjugates to proteins, such as antibodies, immunoglobulins or fragments thereof, can be found, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,4411,163, International Publication No. WO2005037992, International Publication No. WO2005081711 and International Publication No. WO2006 / 034488, which are incorporated by reference in their entireties.

[0266] V. Pharmaceutical Compositions

[0284] The present disclosure also provides a pharmaceutical composition. In addition to the above polypeptide complex, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier.

[0267]

[0285] As used herein, the term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s), salt and / or medium is generally chemically and / or physiologically compatible with other ingredients, e.g., the active ingredient (i.e., the polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof) that comprises the formulation, and physiologically compatible with the subject that ingests the pharmaceutical composition.

[0268]

[0286] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical preparation other than active ingredient, whose biological activity is acceptable to subjects and non-toxic.In the context of the present disclosure, the pharmaceutically acceptable carrier for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antibacterial agent, isotonic agent, buffer, antioxidant, anesthetic, suspending / dispersing agent, sequencing or chelating agent, diluent, adjuvant, excipient, or other non-toxic auxiliary material, or various combinations thereof known in the art.

[0269]

[0287] As used herein, suitable "ingredients" may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorings, emulsifiers or stabilizers, such as sugars and cyclodextrins.Suitable "antioxidants" may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylhydroxyanisole, butylhydroxytoluene and / or propyl gallate.As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces the oxidation of the polypeptide complex or heterodimeric antibody or its antigen-binding fragment.This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving protein stability and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that contain, in addition to the active ingredient (i.e., a polypeptide complex or heterodimeric antibody or antigen-binding fragment thereof disclosed herein), one or more antioxidants, e.g., methionine.

[0270]

[0288] Pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection, non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, antibacterial agents in bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequencing or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antibacterial agent used as carrier may be added to the pharmaceutical composition in a multi-dose container, and includes phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol.Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers or substances such as sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrin.

[0271]

[0289] Pharmaceutically acceptable "diluents" may include saline and aqueous buffer solutions.

[0272]

[0290] Pharmaceutically acceptable "adjuvants" may include 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 also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. In addition, sustained absorption of the injectable pharmaceutical dosage form can be brought about by the inclusion of substances that delay absorption, such as aluminum monostearate and gelatin.

[0273]

[0291] The pharmaceutical compositions may be solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations or powders. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0274]

[0292] In an embodiment, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition can be prepared into any conventional dosage form suitable for producing a solution, suspension or emulsion, such as a solution, suspension, emulsion or solid dosage form.The injectable preparation can include a sterile and / or non-pyrogenic solution for injection, a sterile dry soluble preparation for mixing with a solvent immediately before use, including a subcutaneous tablet, such as a freeze-dried powder, a sterile suspension for injection, a sterile dry insoluble preparation for mixing with a medium immediately before use, and a sterile and / or non-pyrogenic emulsion.The solution can be either aqueous or non-aqueous.

[0275]

[0293] In certain embodiments, the unit dose parenteral preparations are filled into ampoules, vials or syringes with needles. All preparations for parenteral administration should be sterile and non-pyrogenic, as known and practiced in the art.

[0276]

[0294] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the polypeptide complex disclosed herein in a suitable solvent. The solvent may include excipients that improve stability or other pharmacological components of the powder or a reconstitution solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable substances. The solvent may include a buffer, such as a citric acid, sodium or potassium phosphate buffer, or other buffer known to those skilled in the art, in one embodiment, a buffer at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art results in the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of the polypeptide complex. High-fill vials, having a small amount (e.g., about 10%) in excess of that required for a dose or set of doses, are acceptable to facilitate accurate withdrawal of samples and accurate dosing. The lyophilized powder can be stored under appropriate conditions, for example, at about 4° C. to room temperature.

[0277]

[0295] Reconstitution of the lyophilized powder with water for injection produces a formulation for use in parenteral administration. In one embodiment, in reconstitution, sterile and / or non-pyrogenic water or other liquid suitable for carrier is added to the lyophilized powder. The exact amount depends on the selected therapy to be administered and can be empirically determined.

[0278]

[0296] In certain embodiments, there is further provided a composition comprising a pharma- ceutically acceptable carrier, diluent or adjuvant and an active ingredient, which may be a polypeptide complex as disclosed herein, an antibody or antigen-binding fragment thereof, or an antibody conjugate as disclosed herein.

[0279] VI. Preparation method

[0297] The present disclosure provides methods for preparing the polypeptide complexes provided herein.

[0280]

[0298] The method generally includes the following steps:

[0299] (1) providing a nucleic acid encoding a polypeptide complex;

[0300] (2) constructing a vector containing the nucleic acid;

[0301] (3) introducing the vector into a host cell to express the polypeptide complex; and

[0302] (4) isolating the polypeptide complex from the host cell.

[0281]

[0303] In a first aspect of this section, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide complex as disclosed herein.

[0282]

[0304] Nucleic acids encoding the polypeptide complexes disclosed herein can be obtained by one of the following techniques.

[0283]

[0305] In the first approach, a nucleic acid encoding a polypeptide complex as disclosed herein may be generated from another available nucleic acid (hereinafter referred to as a "parent antibody") that encodes a polypeptide having a sequence homologous to the polypeptide of the polypeptide complex as disclosed herein. DNA engineering processes can then be applied to manipulate the sequence of the nucleic acid encoding the parent antibody, for example by introducing mutations, insertions, deletions, etc., to obtain a nucleic acid encoding a polypeptide complex as disclosed herein.

[0284]

[0306] A "parent antibody" is defined herein as an antibody or fragment thereof that can obtain the polypeptide complex disclosed herein. The parent antibody may have a polypeptide sequence of a heavy chain CH1 region and / or a light chain CL region that is homologous to the heavy chain CH1 region and / or the light chain CL region of the polypeptide complex disclosed herein. The term "homologous" as used herein refers to a situation in which a first sequence has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to a second sequence when aligned. Consensus sequence alignment software such as ClustalW (European Bioinformatics Institute website) is readily available, and DNA manipulation methods are well known in the art, which may include site-directed mutagenesis, recombinant DNA technology, PCR, etc. The parent antibody may be of any type, including, for example, a fully human antibody, a humanized antibody, or an animal antibody (e.g., mouse, rat, rabbit, sheep, cow, dog, etc.). The antibody may be a monoclonal antibody or a polyclonal antibody.

[0285]

[0307] If the nucleic acid encoding the parent antibody is not available, optionally, several different approaches can be applied to obtain the nucleic acid encoding the polypeptide complex disclosed herein.For example, if the host cell (e.g., hybridoma) expressing the parent antibody, or its lysate, including mRNA, is available, reverse transcriptase PCR approach can be applied to obtain cDNA from the host cell, followed by high-fidelity PCR amplification and sequencing confirmation, thereby obtaining the nucleic acid encoding the parent antibody.If the cDNA library from the parent antibody host cell is available, it simply requires high-fidelity PCR and sequencing confirmation.

[0286]

[0308] However, if only the polypeptide sequences of the polypeptide complexes disclosed herein are known and, in addition, the host cells, or their cell lysates or cDNA libraries are available, the nucleic acids encoding them can alternatively be produced by chemical synthesis, which may include translating the polypeptide sequence into a nucleotide sequence. Knowledge of this subject, for example, the nucleotide codons known to code for specific amino acids, is well known in the art.

[0287]

[0309] It is noteworthy that the various techniques described above can be combined to obtain nucleic acids encoding the polypeptide complexes disclosed herein.

[0288]

[0310] In a second aspect of this section, the disclosure also provides a vector comprising a nucleic acid encoding a polypeptide complex as disclosed herein.

[0289]

[0311] In this regard, a nucleic acid encoding a polypeptide complex disclosed herein is operably inserted into a vector.

[0290]

[0312] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted to effect expression of the protein (i.e., referred to as an expression vector) and / or which upon introduction can effect replication and amplification of the polynucleotide (i.e., referred to as a cloning vector). Depending on the various expression systems, the vector may optionally contain one or more regulatory sequences, including a promoter, an enhancer element, a terminator element, an origin of replication, or one or more other regulatory elements.

[0291]

[0313] "Promoter" as used herein is typically a regulatory sequence upstream of a nucleotide sequence encoding a polypeptide in a vector, and acts to promote the transcription of a target nucleotide sequence by recognition by a host cell carrying the vector. The promoter of a vector is typically compatible with a host cell. The promoter can be a bacterial promoter when the host cell is a bacterial expression system, or a eukaryotic promoter when the host cell is a eukaryotic expression system. Commonly used promoters are well known in the art.

[0292]

[0314] "Enhancer element" as used herein refers to a special sequence in a vector that can enhance the transcription of a target nucleotide sequence when expressed in a host cell. Examples of enhancer elements can include those obtained from mammalian genes (e.g., globulin, elastase, albumin, insulin, etc.), but can also include eukaryotic cell virus enhancers, such as SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer (see also Yaniv, Nature, 297:17-18 (1982)). Enhancer elements can be located at either the 5' or 3' end of a polypeptide coding sequence, but are preferably located at its 5' end.

[0293]

[0315] "Terminator sequence" as used herein refers to a sequence in a vector that is required to terminate transcription and stabilize mRNA. Such sequences can generally be obtained from the 5', sometimes 3', or untranslated regions of eukaryotic or viral DNA or cDNA. A specific example of a terminator sequence is from the bovine growth hormone polyadenylation region (see, for example, WO 94 / 11026).

[0294]

[0316] Many vectors also often contain an "origin of replication", which is a special nucleic acid sequence that allows such vectors to replicate independently of the host chromosomal DNA in a host cell. Replication origin sequences are well known for many bacteria, yeasts and viruses. Examples of replication origins include the plasmid pBR322 origin suitable for most gram-negative bacteria, the 2μ plasmid origin suitable for yeasts, and various viral origins (SV40, polyoma, adenovirus, VSV, BPV, etc.) for cloning vectors in mammalian cells. Generally, mammalian expression vectors do not require an origin of replication (indeed, the SV40 origin is typically often used as a promoter).

[0295]

[0317] Optionally, some other regulatory elements are also present.For example, the 3' end of most eukaryotic genes has the AATAAA sequence, which is the signal to add poly A to the 3' end of mRNA, and therefore this sequence is generally found to be inserted into eukaryotic expression vectors.Others include signal sequences, transcription initiation sequences, selectable markers and reporter genes, etc.

[0296]

[0318] Typically, the polynucleotide sequence encoding the target polypeptide must be inserted at an appropriate locus so that it is operably linked to regulatory sequences such that expression of the target polypeptide is feasible and under appropriate control.

[0297]

[0319] Vectors may be used to transform, transduce or transfect host cells to bring about the expression of genetic elements that they carry in the host cell (i.e., an "expression vector"), and / or to bring about the replication of the vector (a "cloning vector").

[0298]

[0320] Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), bacteriophages, such as lambda phage or M13 phage, and animal viruses.Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses and papovaviruses (e.g., SV40).

[0299]

[0321] The encoding polynucleotide sequence(s) can be inserted into a vector for further cloning (amplification of DNA) or expression using recombinant techniques known in the art. In another embodiment, the polypeptide complexes and bispecific polypeptide complexes provided herein can be produced by homologous recombination, as known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0300]

[0322] In some embodiments, vector systems include mammalian, bacterial, yeast systems, etc., including, but not limited to, plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses).

[0301]

[0323] In a third aspect of this section, the disclosure also provides a host cell that contains the above-described nucleic acid and expresses a polypeptide complex, antibody or antigen-binding fragment thereof disclosed herein upon induction of the nucleic acid.

[0302]

[0324] As used herein, the term "host cell" refers to a cell into which an exogenous polypeptide (such as the above-mentioned vector) is introduced. Suitable host cells for cloning or expressing DNA in a vector can be prokaryote, yeast or higher eukaryote cells as mentioned above.

[0303]

[0325] Suitable prokaryotes for this purpose include eubacteria, either gram-negative or gram-positive, such as Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, etc.

[0304]

[0326] Eukaryotic microbes, including filamentous fungi or yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces, etc.), are suitable cloning or expression hosts for the vectors provided herein.

[0305]

[0327] Examples of invertebrate host cells suitable for the expression of vectors provided herein include plant and insect cells.Identify a large number of baculovirus strains and variants and corresponding insect permissive host cells from hosts such as caterpillars, mosquitoes, fruit flies (fruiffly) and silkworms (Bombyx mori).Also, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can be used as hosts.

[0306]

[0328] Examples of vertebrate host cells, particularly mammalian host cell lines, suitable for expression of the vectors provided herein include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), such as Expi293; baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat liver cells (BRL3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (HepG2, HB8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (HepG2). Vertebrate cells and the propagation of vertebrate cells in culture (tissue culture) have become routine procedures.

[0307]

[0329] In certain embodiments, the expression vector containing the nucleic acid encoding the polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof provided herein is introduced into the host cell by transient transfection. Thus, the expression and production of the polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof provided herein in the host cell is transient and does not persist for a long period of time. As used herein, the term "transient transfection" refers to a process in which the exogenous nucleic acid introduced into the host cell is not integrated into the genome or chromosomal DNA of the host cell. After transient transfection, the nucleic acid thus introduced is maintained as an extrachromosomal element (e.g., episome) in the host cell, which can still provide a template for transcription of the nucleic acid into messenger RNA (mRNA) and subsequent translation of the mRNA into the polypeptide encoded by the nucleic acid in the host cell. Thus, by transient transfection, the production of the polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof provided herein is merely transient and not stable, and usually does not persist for a long period of time.

[0308]

[0330] In certain embodiments, to achieve long-term and high-yield production of the polypeptide complexes, heterodimeric antibodies or antigen-binding fragments thereof provided herein, cell lines that stably express the nucleic acid encoding the polypeptide complexes, heterodimeric antibodies or antigen-binding fragments thereof provided herein are modified.For this purpose, host cells can be transformed with an expression vector that contains a selectable marker.As used herein, the term "selectable marker" refers to a polynucleotide sequence in a vector that encodes a functional polypeptide, e.g., an enzyme, that provides a specific selectivity for cells that stably express the expression vector.

[0309]

[0331] In some embodiments, the selectable marker encodes an enzyme that confers resistance to an antibiotic or another toxin (e.g., ampicillin, neomycin, methotrexate, or tetracycline, etc.), which is used in the selection medium to select cells expressing the expression vector. More specifically, after transfection of the expression vector, the host cells may be grown in a first culture medium for several days, and then the medium is replaced with a selection medium containing the corresponding antibiotic / toxin. The selectable marker in the expression vector confers resistance to selection, allowing the cells to stably integrate the vector into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method is commonly used in the art to engineer cell lines that stably express foreign proteins, and may be useful for screening cell lines that stably express the polypeptide complexes, heterodimeric antibodies, or antigen-binding fragments thereof, provided herein.

[0310]

[0332] Optionally, the selectable marker encodes a gene that complements an auxotrophic deficiency exhibited by the host cell and can thus be used to select for cells expressing the expression vector. Further optionally, the selectable marker encodes a gene that supplies a vital nutrient that cannot be obtained from a given (particular) medium.

[0311]

[0333] Host cells transformed or transfected with the above-described vectors can be cultured in conventional nutrient media to express the polypeptide complexes, antibodies or antigen-binding fragments thereof provided herein, or to amplify the vector itself.

[0312]

[0334] In a fourth aspect of this section, the disclosure provides a method for expressing a polypeptide complex disclosed herein.

[0313]

[0335] Generally, the methods include culturing a host cell provided herein under suitable culture conditions such that the polypeptide complexes disclosed herein are expressed.

[0314]

[0336] In some embodiments, a transient expression system is applied, thus the method comprises the steps of:

[0337] (a) transfecting a host cell with an expression vector configured to express each polypeptide of the polypeptide complex; and

[0338] (b) culturing the host cells to express each polypeptide and allow for the production of the polypeptide complex.

[0315]

[0339] In some other embodiments, a stable expression system is applied, and thus the method comprises the steps of:

[0340] (a) transfecting a host cell with an expression vector configured to express each polypeptide of the polypeptide complex, wherein the expression vector comprises a selectable marker;

[0341] (b) obtaining a stable cell line by culturing the transfected host cells in a selective medium corresponding to the selection marker; and

[0342] (c) culturing the stable cell lines to express each polypeptide and allow for the production of the polypeptide complex.

[0316]

[0343] In any of the above embodiments, the host cells transformed with the expression vectors described above or the stable host cell lines stably expressing the polypeptide complexes provided herein may be cultured in a variety of media. Examples of commercially available media include Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle Medium (DMEM, Sigma), suitable for culturing the host cells. Alternatively, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 or 5,122,469, WO 90 / 03430, WO 87 / 00195 or U.S. Reissue Application No. 30,985 may be used as a culture medium for the host cells.

[0317]

[0344] During the cultivation, any of the above-mentioned media may further be supplemented with one or more supplements as appropriate or necessary. Non-limiting examples of such supplements include, among others, salts (e.g., sodium chloride, calcium, magnesium and phosphate, etc.), glucose or equivalent energy source, buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine, etc.), selective antibiotics (e.g., GENTAMYCIN™ drug), hormones and / or growth factors (e.g., insulin, transferrin or epidermal growth factor, etc.). Such supplements should be added at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cell selected for expression, and will be clear to those skilled in the art.

[0318]

[0345] In a fifth aspect of this section, the disclosure provides a method for isolating a polypeptide complex disclosed herein.

[0319]

[0346] In certain embodiments, the method comprises recovering the polypeptide complex disclosed herein.

[0320]

[0347] As used herein, the term "recovery" is considered equivalent to "purification," "separation," "isolation," and the like, and generally refers to a situation in which a molecule of interest (i.e., a polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof disclosed herein) is concentrated, recovered, or separated from a mixture containing the molecule and other associated components in its environment.

[0321]

[0348] Depending on the expression system used (i.e., expression vector and host cell), the polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof (hereafter referred to as the "desired molecule") may be produced in the periplasmic space within the host cell or directly secreted into the medium, and the recovery methods involve different first steps, as follows:

[0322]

[0349] In certain embodiments that produce the desired molecule within the cell or intracellularly, the recovery methods disclosed herein include as a first step a centrifugation or ultrafiltration process, which acts to remove cellular debris, including fragments or other unwanted material.

[0323]

[0350] In some other embodiments, where the desired molecule is secreted into the periplasmic space of the host cell, for example, E. Coli, the method by Carter et al., Bio / Technology (NY) 10:163-167 (1992) can be applied as a first step. Briefly, the cell paste is cooled and thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can then be removed by centrifugation.

[0324]

[0351] In some other embodiments where the desired molecule is secreted into the medium, the recovery process may include in a first step concentrating the supernatant of such expression systems using a protein concentration filter (e.g., an Amicon or Pellicon ultrafiltration membrane), and this first and other steps may require the presence of protease inhibitors (e.g., PMSF) to inhibit degradation of the antibody and antibiotics to inhibit the growth of adventitious contaminating organisms.

[0325]

[0352] After the first step above, the composition prepared from the host cells may be further purified, which can be achieved using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0326]

[0353] In certain embodiments where the desired molecule (i.e., a polypeptide complex, heterodimeric antibody or antigen-binding fragment thereof disclosed herein) comprises an immunoglobulin Fc domain, depending on the species and isotype of the immunoglobulin Fc region present in the molecule, Protein A and / or Protein G can be used as affinity ligands in affinity chromatography. Protein A can be used for affinity purification of the desired molecule based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)), while Protein G can be used for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)).

[0327]

[0354] In other embodiments where the immunoglobulin Fc region is not present in the desired molecule to be purified, other affinity ligands capable of specifically targeting other epitopes on the desired molecule can also be used for affinity chromatography. For example, if the desired molecule contains a CH3 domain, Bakerbond® ABX resin (JT Baker, Phillipsburg, NJ) can be applied for purification.

[0328]

[0355] In certain embodiments, where the desired molecule comprises an immunoglobulin kappa or lambda type light chain, specific affinity chromatography matrices (e.g., resins) (e.g., CaptureSelect Kappa and CaptureSelect Lambda affinity matrices (BAC BV, The Netherlands)) can be used for purification.

[0329]

[0356] As used herein, the matrix to which the affinity ligand is attached typically comprises agarose, but may also comprise other materials. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose.

[0330]

[0357] Other techniques for protein purification are also available depending on the antibody being recovered, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

[0331]

[0358] After any of the above preliminary purification step(s), the mixture containing the desired molecule may be further subjected to low pH hydrophobic interaction chromatography using an elution buffer of about pH 2.5-4.5, preferably performed at a low salt concentration (e.g., about 0-0.25 M NaCl).

[0332]

[0359] One advantage of the desired molecules (i.e., polypeptide complexes and heterodimeric antibodies or antigen-binding fragments thereof) is that unwanted mispairing between heavy and light chains can be significantly reduced, and thus the generation of unwanted by-products can typically be minimized even using relatively simple purification processes such as those described above. Thus, in certain embodiments, it is feasible to obtain high yields and high purity products.

[0333] VII. Screening and Diagnosis

[0360] The polypeptide complexes disclosed herein can be used in vivo and / or in vitro for the diagnosis or screening of diseases associated with the targeted antigen(s).

[0334]

[0361] In one aspect, a method of detecting the presence or level of an antigen is provided. The method includes:

[0362] (1) contacting a sample suspected of containing an antigen with a polypeptide complex disclosed herein; and

[0363] (2) determining complex formation between the antigen and the polypeptide complex.

[0335]

[0364] In some embodiments, step (1) is typically performed under conditions that allow complex formation between the antigen and the polypeptide complex, and in step (2), detection of complex formation can be accomplished using a wide variety of known methods, such as ELISA, Western blot, FISH assay, immunofluorescence, etc. The sample can be a biological sample, such as plasma, serum, urine, cell lysate, biopsy sample, etc., of a subject suspected of having a disease of interest. The polypeptide complexes provided herein are configured to target one or more antigens in the sample that are associated with the disease.

[0336]

[0365] In certain embodiments, complex formation may be quantified. If the amount of target antigen molecule in the sample increases or decreases above a pre-set threshold, it is determined that the subject may be in contact with the disease.

[0337]

[0366] In certain embodiments, when a control sample is used along with the test sample, determining complex formation in step (2) may require statistical analysis, where complex formation is detected and compared in both samples, and a statistically significant difference (e.g., P<0.05) in complex formation between the samples indicates the presence of the molecule of interest in the test sample. As used herein, the control sample may be from a subject that does not have the disease, while the test sample is suspected of carrying the disease.

[0338] VIII. Treatment

[0367] Depending on the antigen that is specifically targeted, the polypeptide complexes disclosed herein can be used as therapeutic agents to treat a wide range of diseases, conditions, or symptoms.

[0339]

[0368] In one aspect, the disclosure provides a method for treating or preventing a disease, condition or symptom, the method comprising administering to a subject in need thereof a therapeutically effective amount of the above provided polypeptide complex.

[0340]

[0369] As used herein, the polypeptide complexes provided herein may be present in a pharmaceutical composition as provided above, or may be conjugated to a conjugate as provided above, or may be present in a composition as provided above in the form of a therapeutic formulation.

[0341]

[0370] As used herein, the terms "subject" or "individual" or "animal" or "patient" refer to a human or non-human animal, including a mammal or primate in need of diagnosis, prognosis, amelioration, prevention and / or treatment of a disease or disorder. Mammalian subjects include humans, farm animals, livestock and zoo, sport or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0342]

[0371] As used herein, the terms "disorder," "disease," "condition," and the like refer to a condition that affects a subject who is afflicted but would benefit from treatment with a polypeptide complex. The term "symptom" refers to physical, mental, or physiological characteristics of a patient with a disease that are considered to be indicative of such disease symptoms.

[0343]

[0372] The term "treatment" is intended to encompass both therapeutic and prophylactic treatment, such that those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. As used herein, "treatment" of a condition can include preventing or alleviating the condition, slowing the onset or rate of onset of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0344]

[0373] As used herein, the term "therapeutically effective amount" of a therapeutic agent refers to the amount of the therapeutic agent that can produce sufficient therapeutic effect on the subject when taken by the subject in an appropriate manner.Just like other therapeutic agents, it should be understood that the polypeptide complex provided above a therapeutically effective amount is influenced by various factors known in the art, such as the subject's weight, age, past medical history, current medication, health condition and potential cross-reactions, allergies, sensitivity and adverse side effects, as well as the route of administration and the degree of disease development.Dosage amount can be proportionally reduced or increased by a person skilled in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0345]

[0374] In certain embodiments, the polypeptide complexes provided herein may be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain such embodiments, the polypeptide complex or bispecific polypeptide complex provided herein is administered at a dosage of about 50 mg / kg or less, and in certain such embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage may be adjusted during treatment. For example, in certain embodiments, the initial dosage may be higher than subsequent dosages. In certain embodiments, the dosage may vary during treatment depending on the subject's response.

[0346]

[0375] Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0347]

[0376] The polypeptide complexes provided herein may be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-oral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0348]

[0377] The polypeptide complexes provided herein can be administered alone or in combination with one or more additional therapeutic methods or agents.

[0349]

[0378] The polypeptide complexes and heterodimeric antibodies or antigen-binding fragments thereof and the methods disclosed herein can be applied to treat a wide variety of diseases. Diseases contemplated as treatable by the polypeptide complexes and heterodimeric antibodies or antigen-binding fragments thereof and the methods disclosed herein, both in humans and other primates alike, may include:

[0350]

[0379] (1) Cancer and other hyperproliferative disorders, including both benign and malignant tumors, leukemias and lymphatic tumors. Depending on the cell type having the cancer or hyperproliferative disorder, examples include neuronal, glial, astrocyte, hypothalamic, glandular, macrophage, epithelial, endothelial and stromal malignancies. Depending on the organ / location affected by the cancer or hyperproliferative disorder, examples include cancer of the head, neck, eye, oral cavity, throat, esophagus, breast, skin, bone, lung, colon, rectum, large intestine, stomach, spleen, kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma, endometrium, prostate, breast, ovary, testis, thyroid, blood, lymph node, kidney, liver, pancreas, brain or central nervous system.

[0351]

[0380] (2) Alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's syndrome, psoriasis, atherosclerosis, diabetic or other retinopathies, retrolental fibroplasia, age-related macular degeneration, neovascular glaucoma, hemangiomas, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants and chronic inflammation, sepsis, rheumatoid arthritis, peritonitis, Crohn's disease, reperfusion injury, sepsis, endotoxic shock, cystic fibrosis, endocarditis, psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic shock, organ ischemia, reperfusion injury, spinal cord injury and allograft rejection, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, mycoses, celiac dermatitis, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia and fibromyositis, glomerulonephritis, Guillain-Barré, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon autoimmune and / or inflammatory disorders including rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteristis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as cutaneous vasculitis herpetiformis, leukoplakia, and Wegener's granulomatosis.Inflammatory disorders may further include, but are not limited to, asthma, encephilitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infections.

[0352]

[0381] (3) Infectious and parasitic diseases, such as viruses (e.g., HBV, HCV, HIV, RSV, hMPV, PIV, coronaviruses or influenza viruses, etc.), fungi (e.g., Naegleria, Aspergillus, Blastomyces, Histoplasma, Candida or Tinea, etc.), eukaryotic microorganisms (e.g., Giardia, Toxoplasma) , Plasmodium, Trypanosoma, and Entamoeba) and those caused by bacteria (Staphylococcus, Streptococcus, Pseudomonas, Clostridium, Borrelia, Vibro / Vibrio, and Neiserria / Neisseria).

[0353]

[0382] (4) Other diseases or disorders including those not encompassed by the above (1) to (3), such as cardiovascular disease, neurological disorders, psychoneurological symptoms, injuries or blood coagulation disorders.

[0354]

[0383] Antigens associated with the above-listed diseases treatable by the above polypeptide complexes or methods of treatment disclosed herein include the following:

[0355]

[0384] (1) Tumor-associated antigens. These refer to antigens that are presented on the surface of tumor cells, that are located on or within tumor cells, that are presented only by tumor cells and not by normal cells, i.e., non-tumor cells, that represent proteins that harbor one or more tumor-specific mutations compared to non-tumor cells, that are highly expressed in tumor cells compared to non-tumor cells, that are accessible for antibody binding in tumor cells due to the less dense tumor tissue structure compared to non-tumor tissue, that are presented on the tumor vasculature, etc. Examples include, but are not limited to, CD19, CD20, CD38, CD30, Her2 / neu / ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor 2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, etc.In addition, cancer-associated antigens include, for example, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate cancer cells, RANKL, RON, ROR1, SCH900105, SDC1, SLAMF7, TAG-72, tenascin-C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2 and vimentin, etc.

[0356]

[0385] (2) including, but not limited to, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (IL-2 receptor chain), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE Antigens associated with autoimmune or inflammatory diseases, including the Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, LFA-1 (CD11a), myostatin, OX-40, sclerostin, SOST, TGF beta 1, TNF-α, and VEGF-A.

[0357]

[0386] The heterodimeric antibodies or antigen-binding fragments thereof disclosed herein are capable of specifically targeting two or more antigens simultaneously, which can be used for the effective treatment of certain diseases.

[0358]

[0387] In some preferred embodiments of the heterodimeric antibody or antigen-binding fragment thereof provided herein, one antigen-binding portion targets a receptor (e.g., CD3) on cytotoxic T lymphocytes, and the other antigen-binding portion targets one of the following tumor cell-expressed antigens: CD19, CD20, CD33, CD123, HER1, HER2, CEA, disialoganglioside GD2, PSMA, gpA33, EpCAM, P-cadherin, and B7H3 (Sedykh SE, et al., Drug Des Devel Ther. 2018; 12: 195-208). Thus, bispecific antibodies or antigen-binding fragments thereof can be used to form a link between T cells and tumor cells, which can allow T cells to exert cytotoxic activity against tumor cells.

[0359]

[0388] Other examples of antigen pairs that may have potential therapeutic effects when targeted by heterodimeric antibodies or antigen-binding fragments thereof include PD-L1:TGFβ, CD38:EGFR, HER2:VEGF, HER2:EGFR, PD-1:CTLA-4, PD-1:TIM3, OX40:PD-L1, FIXa:FX, CD32B:CD79B, Angiopoietin 2:VEGF, IL13:IL4, TNF These may include, but are not limited to, IL17A, DLL4:VEGF, IL1α:IL1β, FAP:DR5, CD30:gpA33, TNF:HSA, IL6R:HSA, IL17A / F:HSA, RANKL:HSA, Aβ40:Aβ42, IL13:IL17, FGFR1:KLB, PsI:PcrV, BAFF:B7RP1, NGF:TNF and TNF:IL17A (Sedykh SE, et al., Drug Des Devel Ther. 2018;12:195-208).

[0360]

[0389] The following examples are provided to better illustrate the claimed invention and should not be interpreted as limiting the scope of the invention. All specific compositions, materials and methods described below are wholly or partially within the scope of the invention. Such specific compositions, materials and methods are not intended to limit the invention, but merely to illustrate specific embodiments within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials and methods without using their inventive abilities and without departing from the scope of the invention. It is understood that many variations can be made in the procedures described herein while still remaining bound by the invention. The inventors intend such variations to be within the scope of the invention. EXAMPLES

[0361] Example 1: CH-CL interface test

[0390] The disulfide bond formed by C220 of CH1 (C131 in IgG2 and IgG4) and C214 of CL (kappa or lambda chain) is the basis for the association between CH1 and CL regions. Therefore, by changing the association mode of the disulfide bond between CH1 and CL regions of LC(B)-HC(B), it is possible to avoid the mispairing of LC(A)-HC(B) and LC(B)-HC(A), which allows the four chains, i.e., LC(A), HC(A), HC(B) and LC(B), to be expressed in one cell line, thereby simplifying the expression and purification process.

[0362]

[0391] To alter the association mode of LC(B)-HC(B), we examined the CH-CL interface and enumerated the relevant amino acids and analyzed their respective ratios at the interface (see FIG. 14), which we use as a basis for subsequent constructions.

[0363] Example 2: CH1-VH and CL-VL interface studies

[0392] Since CH1 and CL regions are associated with VH and VL regions respectively, there are also CH1-VH and CL-VL interfaces. In order to maintain the spatial structure of the original VH and VL regions as much as possible to maintain stability, and to avoid the mutation sites being exposed to the hydrophilic surface of CH1 and CL regions (which may cause changes in druggability and immunogenicity), the inventors analyzed the CH1-VH and CL-VL interfaces, and the results are shown in Figure 15. The binding rate of related amino acids was also analyzed, and some amino acids included in the interface of constant region and variable region were excluded.

[0364] Example 3: Distance measurement

[0393] Residues available for mutation testing were selected by analyzing Figure 14 and Figure 15. The results are shown in Figure 16. Residues with binding areas greater than 40% were analyzed and the distances of such residues on the CH1 and CL regions were measured using the software. The results are shown in Table 1 below.

[0365] [Table 1]

[0366] Example 4: Calculations

[0394] To avoid the formation of disulfide bonds between the mutated residues and C220 (CH1) and C214 (CL), L128-F118 and V173-Q160 were selected for mutation by comprehensive analysis, based on which a novel CH1-CL binding mode was constructed to avoid the formation of mispairs with the wild-type CH1 / CL.

[0367] Example 5: Expression of bispecific antibodies based on the L128C-F118C or V173C-Q160C constructs

[0395] The sequences shown in Table 2 below were utilized as templates to construct bispecific antibodies. In Table 1 below, "Antibody A" refers to the "anti-PD1 antibody," "Antibody B" refers to the "anti-TGFβR antibody," "HC(A)" refers to the heavy chain of Antibody A, "LC(A)" refers to the light chain of Antibody A, "HC(B)" refers to the heavy chain of Antibody B, and "LC(B)" refers to the light chain of Antibody B.

[0368] [Table 2]

[0369]

[0396] To prevent homodimer formation of HC(A) and HC(B), K392D and K409D mutations were introduced into CH3 of HC(A), and D399K and E356K mutations were introduced into CH3 of HC(B). The sequences of HC(A) and HC(B) with mutations in the CH3 domain are shown in Table 3 below, with the mutated amino acids underlined.

[0370] [Table 3]

[0371]

[0397] To evaluate the effect of various CH1-CL interface residue mutations on mispairing, antibodies DIC014, DIC002, and DIC007 were constructed. As shown in Table 4 below, DIC014 had no mutations in the CH1 and CL regions, DIC002 had L128C and C220S mutations in the CH1(A) region, F118C and C214S mutations in the LC(A) region, and no mutations in the CH1(B) and LC(B) regions, and DIC007 had V173C and C220S mutations in the CH1(A) region, Q160C and C214S mutations in the LC(A) region, and no mutations in the CH1(B) and LC(B) regions.

[0372] [Table 4]

[0373]

[0398] The sequence information of the LC(A), HC(A), LC(B) and HC(B) regions of DIC014, DIC002 and DIC007 is shown below in Table 5. As shown in Table 5, DIC014 contains the LC(A), HC(A), LC(B) and HC(B) regions set forth in SEQ ID NOs: 2, 7, 4 and 8, respectively, DIC002 contains the LC(A), HC(A), LC(B) and HC(B) regions set forth in SEQ ID NOs: 9, 10, 4 and 8, respectively, and DIC007 contains the LC(A), HC(A), LC(B) and HC(B) regions set forth in SEQ ID NOs: 30, 11, 4 and 8, respectively.

[0374] [Table 5-1]

[0375] [Table 5-2]

[0376] [Table 5-3]

[0377]

[0399] a) Expression

[0400] The DNA sequences encoding the four chains of DIC014, DIC002 and DIC007 were cloned into vectors (in a 1:1:1:1 ratio), transfected into ExpiCHO cells for transient expression, and then analyzed after affinity chromatography purification.

[0378]

[0401] b) SDS-PAGE and SEC-HPLC analysis

[0402] The above purified antibodies were detected by SDS-PAGE and SEC-HPLC, respectively. The SDS-PAGE results are shown in Figure 1A (DIC002), Figure 1B (DIC007) and Figure 1C (DIC014). The SEC-HPLC results are shown in Figure 2A (DIC002), Figure 2B (DIC007) and Figure 2C (DIC014).

[0379]

[0403] Because the left and right CL and CH associate in different ways, if HC(A)-LC(B) and / or HC(B)-LC(A) mispairing occurs during the construction of the above asymmetric antibody, such mispairing cannot form a stable disulfide bond linkage, which not only results in low molecular weight bands of 125 KDa and 100 KDa on SDS-PAGE, but also in shoulder peaks and / or small peaks to the right of the main peak on SEC-HPLC.

[0380]

[0404] For DIC014, both HC(A)-LC(A) and HC(B)-LC(B) are linked by C220(CH1)-C214(CL) to form a disulfide bond linkage, so that the HC(A)-LC(B) and HC(B)-LC(A) mispairs formed in the expression product were also covalently linked, which made the performance of DIC014 similar to that of a normal antibody in SDS-PAGE and SEC-HPLC. Therefore, DIC014 was analyzed by LC-MS, which identified a high rate of mispairs. The main type of mispair is HC(B)-HC(B)-LC(A)-LC(B). The LC-MS results of intact DIC014 and deglycosylated DIC014 are shown in Figure 3A (intact DIC014) and Figure 3B (deglycosylated DIC014). The results showed that HC(A)-LC(B) and / or HC(B)-LC(A) mispairings occurred during the construction of DIC002, DIC007 and DIC014.

[0381] Example 6: Charges were introduced to reduce mispairing in bispecific antibodies constructed based on L128C-F118C or V173C-Q160C

[0405] HC(A)-LC(B) and / or HC(B)-LC(A) mispairing still occurs in bispecific antibodies constructed based on L128C-F118C or V173C-Q160C. To reduce the mispairing rate, the inventors adjusted the charge between the CH1(A)-LC(A) and CH1(B)-LC(B) interfaces in addition to the L128C-F118C or V173C-Q160C mutations.

[0382]

[0406] By modeling the protein structure of CH1-CL, charge mutations were introduced in addition to the L128C-F118C or V173C-Q160C mutations. As shown in Table 6 below, when a positively charged amino acid (e.g., R, H, or K) was introduced into a residue, a negatively charged amino acid (e.g., D or E) was introduced into the other residue. For example, in No. 1 of Table 6, when a positively charged amino acid (e.g., R, H, or K) was introduced into S183 of CH1(A), a negatively charged amino acid (e.g., D or E) was introduced into S176 of LC(A).

[0383] [Table 6]

[0384]

[0407] Because some residue mutations affect the linkage of CH1-LC disulfide bond, it was found after calculation that the V173C-Q160C mutation may not increase the F126-S121 pairing mutation. Therefore, the combinations of mutations shown in Table 7 below were analyzed (positively and negatively charged residues can be interchanged). As shown in Table 7, no mutations were introduced in the CH1(B) and LC(B) regions of each antibody. In DIC003, L128C, C220S and S183D mutations were introduced into the CH1(A) region, and F118C, C214S and S176K mutations were introduced into the LC(A) region. In DIC004, L128C, C220S and F126D mutations were introduced into the CH1(A) region, and F118C, C214S and S121K mutations were introduced into the LC(A) region. In DIC005, L128C, C220S and A141D mutations were introduced into the CH1(A) region, and F118C, C214S and F116K mutations were introduced into the LC(A) region. In IC006, L128C, C220S and K218D mutations were introduced into the CH1(A) region and F118C, C214S and D122K mutations were introduced into the LC(A) region; in DIC009, V173C, C220S and S183D mutations were introduced into the CH1(A) region and Q160C, C214S and S176K mutations were introduced into the LC(A) region; and in DIC010, V173C, C220S and A141D mutations were introduced into the CH1(A) region and Q160C, C214S and F116K mutations were introduced into the LC(A) region.

[0385] [Table 7]

[0386]

[0408] The sequence information for the LC(A), HC(A), LC(B) and HC(B) regions of DIC003, DIC004, DIC005, DIC006, DIC009 and DIC010 is shown in Table 8 below.

[0387] [Table 8-1]

[0388] [Table 8-2]

[0389] [Table 8-3]

[0390] [Table 8-4]

[0391] [Table 8-5]

[0392]

[0409] a) Expression

[0410] The DNA sequences encoding the four chains of DIC003, DIC004, DIC005, DIC006, DIC009 and DIC010 were cloned into vectors (at a ratio of 1:1:1:1), transfected into ExpiCHO cells for transient expression, and then analyzed after AC purification.

[0393]

[0411] b) SDS-PAGE and SEC-HPLC analysis

[0412] The above purified antibodies were detected by SDS-PAGE and SEC-HPLC, respectively. The SDS-PAGE results are shown in Figure 4A (DIC003), Figure 4B (DIC004), Figure 4C (DIC005), Figure 4D (DIC006), Figure 4E (DIC009) and Figure 4F (DIC010). The SEC-HPLC results are shown in Figure 5A (DIC003), Figure 5B (DIC004), Figure 5C (DIC005), Figure 5D (DIC006), Figure 5E (DIC009) and Figure 5F (DIC010).

[0394]

[0413] The SDS-PAGE and SEC-HPLC analysis results of the above antibodies showed that DIC006, DIC009 and DIC010 showed significantly higher purity than DIC007 and DIC002. However, the above methods have obvious limitations in quantifying the content and type of mismatches. For example, homodimers and CH1-LC mismatches are indistinguishable from the target product by SDS-PAGE and SEC-HPLC because their molecular weights are close to those of the target product. Therefore, LC-MS methods were used to distinguish and identify them.

[0395]

[0414] The left and right sides of DIC014 are bound by C220-C214, which makes the performance of SDS-PAGE and SEC-HPLC similar to that of normal antibodies. Therefore, it is impossible to observe whether there is any mismatching. In order to identify the actual molecular weight of each mismatching product and test the composition of various mismatching products in DIC009 products, DIC015 expressing three chains of HC(A)-HC(B)-LC(B) and DIC016 expressing three chains of LC(A)-HC(A)-HC(B) were constructed as controls based on the four chains of DIC009 (LC(A)-HC(A)-HC(B)-LC(B). As shown in Table 9 below, DIC016 does not have an LC(A) region, and DIC016 does not have an LC(B) region. In DIC015, V173C, C220S and S183D were introduced into the CH1(A) region, and in DIC016, V173C, C220S and S183D were introduced into the CH1(A) region and Q160C, C214S and S176K were introduced into the LC(A) region.

[0396] [Table 9]

[0397]

[0415] The SDS-PAGE results of DIC015 and DIC016 are shown in Figure 6A (DIC015) and Figure 6B (DIC016), respectively. As shown in Figures 6A and 6B, both DIC015 and DIC016 had bands of 150 KDa, 125 KDa and 100 KDa. Since the 150 KDa products are the mispairing products of HC(A)-LC(B) and HC(B)-LC(A) of DIC015 and DIC016, respectively, their amounts were less as shown in SDS-PAGE.

[0398]

[0416] The SEC-HPLC results of DIC015 and DIC016 are shown in Figure 7A (DIC015) and Figure 7B (DIC016), respectively. As shown in Figures 7A and 7B, obvious dimers were observed for DIC015 and DIC016. DIC015 showed obvious shoulder and main peaks around the target molecular weight, and a part of the HC(A)-LC(B) mismatch showed a protein affinity binding form without forming a covalent bond, showing a molecular weight of about 150 KDa (12.2 min), while the part of HC(A) that did not bind to LC(B) formed a product of 125 KDa (12.8 min) and had a small amount of 100 KDa product of HC(A)-HC(B) (13.5 min). DIC016 showed a single peak, indicating that a large amount of HC(B)-LC(A) mismatch formed a mismatch product of 150 KDa (12.2 min) by protein affinity binding. The results of SDS-PAGE and SEC-HPLC methods showed that the above methods could not efficiently separate the non-covalent mismatch binding product from the target final product. Based on this, LC-MS analysis of DIC015 and DIC016 may be useful for determining the molecular weight of various mismatches and identifying the content of mismatch products in DIC009.

[0399]

[0417] The molecular weights of various possible mismatches were calculated based on the compositions of DIC009, DIC015 and DIC016 to identify the composition of the final product. The molecular weights of each possible mismatch are shown in Table 10 below. The LC-MS results of intact DIC015 and deglycosylated DIC015 are shown in Figure 8A (intact DIC015) and Figure 8B (deglycosylated DIC015), respectively. The LC-MS results of intact DIC016 and deglycosylated DIC016 are shown in Figure 9A (intact DIC015) and Figure 9B (deglycosylated DIC016), respectively.

[0400] [Table 10]

[0401]

[0418] According to the above data, LC-MS analysis was performed on DIC009 expressing four chains to identify the mispairing rate of DIC009 product. The LC-MS results are shown in Figure 10A (intact DIC009), Figure 10B (deglycosylated DIC009) and Figure 10C (partial enlargement of Figure 10B). As shown in Figure 10, in addition to HC(A)-HC(B)-LC(A)-LC(B), the final product contained some other by-products including HC(A)-HC(B)-LC(B)-LC(B) and HC(B)-HC(B)-LC(B)-LC(B).

[0402]

[0419] DIC010 was also detected by LC-MS, and the composition of the final product was analyzed by using a similar method as above. The LC-MS results are shown in Figure 11A (intact DIC010) and Figure 11B (deglycosylated DIC010) and Figure 11C (partial enlargement of Figure 11B). As shown in Figure 11, the final product of DIC010 also contained small amounts of mismatched products of HC(A)-HC(B)-LC(B)-LC(B) and HC(B)-HC(B)-LC(B)-LC(B).

[0403]

[0420] It can be seen that the purity of DIC009 and DIC010 is significantly increased and the amount of mismatched products is reduced compared to DIC014 (no mutation). Therefore, the construction method of DIC009 and DIC010 can significantly reduce the mismatches of HC(A)-LC(B) and HC(B)-LC(A).

[0404] Example 7: Biological activity of DIC010

[0421] DIC010 constructed by the above method was expressed and purified, and the biological activity of the constructed product was evaluated.

[0405]

[0422] a) Affinity

[0423] SPR method was used to detect the binding affinity of DIC010 to human PD1, TGFβR2 and TGFβR3 to observe whether the constructed bispecific product still retained the affinity of the original monoclonal antibody to each target. The SPR results were shown in Figure 12A (human PD1), Figure 12B (human TGFβR2) and Figure 12C (human TGFβR3). As shown in Figure 12, DIC010 still showed good affinity to human PD1, human TGFβR2 and human TGFβR3.

[0406]

[0424] b) In vivo activity

[0425] MC38 / H-11 mouse colon cancer cells 1×10 6 The cells were injected into the left armpit of one PD-1-humanized mouse. The tumors were 50–100 mm 3 After tumor growth to an average volume of 1000 mg / kg, the animals were randomly grouped according to tumor burden. Mice were divided into 4 groups (6 mice / group): negative control group, DIC010 (1 mg / kg) group, DIC010 (3 mg / kg) group and DIC010 (10 mg / kg) group. Animals in each group were intraperitoneally injected with the corresponding concentration of the test product at a dose of 10 ml / kg twice a week for a total of 4 doses, with a dosing cycle of 15 days.

[0407]

[0426] Mice were weighed and measured for tumor burden twice a week. On day 15, mice were weighed and tumor burden was measured to calculate relative tumor burden (RTV), relative tumor growth rate (T / C) and tumor growth inhibition rate (TGI). The results were shown in Figure 13. As shown in Figure 13, DIC010 at 10 mg / kg significantly inhibited tumor growth in the MC38 mouse model and had good biological activity in vivo.

Claims

1. A polypeptide complex comprising a first target binding domain comprising a first target binding moiety operably linked to a first constant portion, said first constant portion comprising a first heavy chain constant region 1 (CH1) associated with a first light chain constant region (CL); a) the first CH1 region comprises a first amino acid residue at EU position n1 and the first CL region comprises a second amino acid residue at EU position n2, the n1:n2 position pair being selected from the group consisting of 128:118 and 173:160, and the first amino acid residue and the second amino acid residue form a covalent bond; b) the first CH1 region further comprises a third amino acid residue at EU position n3, and the first CL region further comprises a fourth amino acid residue at EU position n4, the n3:n4 position pair being selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, and wherein the third amino acid residue and the fourth amino acid residue form a non-covalent bond.

2. 2. The polypeptide complex of claim 1, wherein the third amino acid residue at EU position n3 and the fourth amino acid residue at EU position n4 are oppositely charged, Optionally, (i) a second target binding domain comprising a second target binding moiety operably linked to a second constant portion, said second constant portion comprising a second CH1 region associated with a second CL region, said first CH1 region does not substantially bind to said second CL region, and said second CH1 region does not substantially bind to said first CL region; (ii) the first or second target binding domain comprises or is an antigen-binding domain, and / or the first or second target binding moiety comprises or is an antigen-binding moiety; or (iii) said second CH1 region comprises a first corresponding amino acid residue at EU position n1' and said second CL region comprises a second corresponding amino acid residue at EU position n2', wherein the n1':n2' position pair is identical to said n1:n2 position pair, and wherein said first corresponding amino acid residue at EU position n1' does not form a covalent bond with said second amino acid residue at EU position n2, and / or said second corresponding amino acid residue at EU position n2' does not form a covalent bond with said first amino acid residue at EU position n1. The polypeptide complex. (i) the first corresponding amino acid residue at EU position n1' and the second corresponding amino acid residue at EU position n2' do not form a covalent bond; and / or (ii) said second CH1 region further comprises a third corresponding amino acid residue at EU position n3' and said second CL region further comprises a fourth corresponding amino acid residue at EU position n4', wherein the n3':n4' position pair is identical to said n3:n4 position pair, and wherein (a) said fourth corresponding amino acid residue at EU position n4' and said third amino acid residue at EU position n3 are unoppositely charged or similarly charged, and / or (b) said third corresponding amino acid residue at EU position n3' and said fourth amino acid residue at EU position n4 are unoppositely charged or similarly charged, optionally wherein said third corresponding amino acid residue at EU position n3' and / or said fourth corresponding amino acid residue at EU position n4' are uncharged. The polypeptide complex of claim 2.

4. 3. The polypeptide complex of claim 2, wherein the second CH1 region further comprises a fifth corresponding amino acid residue at EU position n5' and the second CL region further comprises a sixth corresponding amino acid residue at EU position n6', the fifth corresponding amino acid residue and the sixth corresponding amino acid residue form a covalent bond, and the n5':n6' position pair is different from the n1:n2 position pair, Optionally, (i) the n5':n6' position pair is selected from the group consisting of 220:214 (IgG1) or 131:214 (IgG2 and IgG4), 128:118, and 173:160; (ii) the n5':n6' position pair is 220:214 (IgG1) or 131:214 (IgG2 and IgG4); or (iii) the n5':n6' position pair is 128:118 and the n1:n2 position pair is 173:160, or the n5':n6' position pair is 173:160 and the n1:n2 position pair is 128:118; (iv) (a) at least one of the first CH1 region and the second CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4), and / or at least one of the first CL region and the second CL region has an amino acid residue other than cysteine ​​at EU position 214; or (b) neither the first nor the second CH1 region has a cysteine ​​residue at EU position 220 (IgG1) or 131 (IgG2 and IgG4), and / or neither the first nor the second CL region has a cysteine ​​residue at EU position 214; (v) the first CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4) and the first CL region has an amino acid residue other than cysteine ​​at EU position 214; or (vi) the second CH1 region has an amino acid residue other than cysteine ​​at EU position 220 (IgG1) or 131 (IgG2 and IgG4), and the second CL region has an amino acid residue other than cysteine ​​at EU position 214; The polypeptide complex.

5. 5. The polypeptide complex of claim 4, wherein the first CH1 region further comprises a fifth amino acid residue at EU position n5 and the first CL region further comprises a sixth amino acid residue at EU position n6, the n5:n6 position pair being identical to the n5':n6' position pair, and wherein the fifth corresponding amino acid residue at EU position n5' does not form a covalent bond with the sixth amino acid residue at EU position n6 and / or the sixth corresponding amino acid residue at EU position n6' does not form a covalent bond with the fifth amino acid residue at EU position n5, Optionally, the fifth amino acid residue at EU position n5 and the sixth amino acid residue at EU position n6 do not form a covalent bond. The polypeptide complex.

6. 3. The polypeptide complex of claim 2, wherein the second CH1 region further comprises a seventh corresponding amino acid residue at EU position n7' and the second CL region further comprises an eighth corresponding amino acid residue at EU position n8', the n7':n8' position pair being selected from the group consisting of 183:176, 141:116, 126:121 and 218:122, the seventh corresponding amino acid residue and the eighth corresponding amino acid residue are oppositely charged, and the n7':n8' position pair is different from the n3:n4 position pair, Optionally, (a) the n7':n8' position pair is 183:176 and the n3:n4 position pair is selected from the group consisting of 141:116, 126:121 and 218:122; (b) the n7':n8' position pair is 141:116 and the n3:n4 position pair is selected from the group consisting of 183:176, 126:121 and 218:122; (c) the n7':n8' position pair is 126:121 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 218:122; or (d) the n7':n8' position pair is 218:122 and the n3:n4 position pair is selected from the group consisting of 183:176, 141:116 and 126:121; The polypeptide complex.

7. 7. The polypeptide complex of claim 6, wherein the first CH1 region further comprises a seventh amino acid residue at EU position n7 and the second CL region further comprises an eighth amino acid residue at EU position n8, wherein the n7:n8 position pair is identical to the n7':n8' position pair, and wherein the seventh corresponding amino acid residue at EU position n7' and the eighth amino acid residue at EU position n8 are not oppositely charged or are similarly charged, and / or the eighth corresponding amino acid residue at EU position n8' and the seventh amino acid residue at EU position n7 are not oppositely charged or are similarly charged, Optionally, the seventh amino acid residue at EU position n7 and / or the eighth amino acid residue at EU position n8 is uncharged; The polypeptide complex.

8. 2. The polypeptide complex of claim 1, wherein the covalent bond is a disulfide bond. Optionally, (i) the disulfide bond is formed between two cysteine ​​residues; (ii) the first amino acid residue at EU position n1 and the second amino acid residue at EU position n2 are both cysteine ​​residues, and / or the fifth corresponding amino acid residue at EU position n5' and the sixth corresponding amino acid residue at EU position n6' are both cysteine ​​residues; (iii) the first CH1 region comprises a substitution at L128C (EU position n1) and the first CL region comprises a substitution at F118C (EU position n2); or (iv) the second CH1 region comprises a substitution of V173C (EU position n5') and the second CL region comprises a substitution of Q160C (EU position n6') of a kappa light chain or E160C (EU position n6') of a lambda light chain; The polypeptide complex.

9. (a) the third amino acid residue at EU position n3 is a positively charged amino acid residue and the fourth amino acid residue at EU position n4 is a negatively charged amino acid residue; or (b) the third amino acid residue at EU position n3 is a negatively charged amino acid residue and the fourth amino acid residue at EU position n4 is a positively charged amino acid residue; 2. The polypeptide complex of claim 1, Optionally, the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E). The polypeptide complex.

10. (c) the seventh corresponding amino acid residue at EU position n7' is a positively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a negatively charged amino acid residue; or (d) the seventh corresponding amino acid residue at EU position n7' is a negatively charged amino acid residue and the eighth corresponding amino acid residue at EU position n8' is a positively charged amino acid residue; 7. The polypeptide complex of claim 6, Optionally, the positively charged amino acid residue is selected from the group consisting of lysine (K), histidine (H) and arginine (R), and / or the negatively charged amino acid residue is selected from the group consisting of aspartic acid (D) and glutamic acid (E). The polypeptide complex.

11. 2. The polypeptide complex of claim 1, wherein at least one, two, three or four of the first amino acid residue at EU position n1, the second amino acid residue at EU position n2, the third amino acid residue at EU position n3 and the fourth amino acid residue at EU position n4 are introduced by substitution, Optionally, the third amino acid residue and the fourth amino acid residue of the n3:n4 position pair are selected from the group consisting of S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K, S183D:S176R, S18 3D: S176H, S183E: S176K, S183E: S176R, S183E: S176H, A141K: F116D, A141K: F116E, A1 41R:F116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141D:F116R, A 141D: F116H, A141E: F116K, A141E: F116R, A141E: F116H, F126K: S121D, F126K: S121E , F126R: S121D, F126R: S121E, F126H: S121D, F126H: S121E, F126D: S121K, F126D: S121 R, F126D:S121H, F126E:S121K, F126E:S121R, F126E:S121H, K218D:D122K, K218D:D122H, K218D:D122R, K218E:D122K, K218E:D122H, and K218E:D122R; The polypeptide complex.

12. 6. The polypeptide complex of claim 5, wherein at least one, two, three or four of the fifth corresponding amino acid residue at EU position n5', the sixth corresponding amino acid residue at EU position n6', the seventh corresponding amino acid residue at EU position n7' and the eighth corresponding amino acid residue at EU position n8' are introduced by substitution, Optionally, the seventh corresponding amino acid residue and the eighth corresponding amino acid residue of the n7':n8' position pair are selected from the group consisting of S183K:S176D, S183K:S176E, S183R:S176D, S183R:S176E, S183H:S176D, S183H:S176E, S183D:S176K, S183D:S176R, S18 3D: S176H, S183E: S176K, S183E: S176R, S183E: S176H, A141K: F116D, A141K: F116E, A141R: F 116D, A141R:F116E, A141H:F116D, A141H:F116E, A141D:F116K, A141D:F116R, A141D:F116H , A141E: F116K, A141E: F116R, A141E: F116H, F126K: S121D, F126K: S121E, F126R: S121D, F1 26R: S121E, F126H: S121D, F126H: S121E, F126D: S121K, F126D: S121R, F126D: S121H, F126E: and wherein the n7':n8' position pair is different from the n3:n4 position pair. The polypeptide complex.

13. and / or the second target binding domain comprises a first combination of substitutions at positions (n1+n2):(n3+n4) and / or a second combination of substitutions at positions (n5'+n6'):(n7'+n8'), and the first and / or second combination of substitutions are selected from the group consisting of (L128C+S183K):(F118C+S176D), (L128C+S183K):(F118C+S176E), (L128C+S183R):(F118C+S176D), (L128C+S183R ... 8C+S176E), (L128C+S183H): (F118C+S176D), (L128C+S183H): (F118C+S 176E), (L128C+S183D): (F118C+S176K), (L128C+S183D): (F118C+S176R) , (L128C+S183D): (F118C+S176H), (L128C+S183E): (F118C+S176K), (L12 8C+S183E): (F118C+S176R), (L128C+S183E): (F118C+S176H), (V173C+A1 41K): (Q160C (or E160C) + F116D), (V173C + A141K): (Q160C (or E160C) + F116E), (V173C + A141R): (Q160C (or E160C) + F116D), (V173C + A141R): (Q160C (or E160C) + F116E), (V173C + A141H): (Q160C (or E160C) + F116D), (V173C + A141H): (Q160C (or E160C) + F116E), (V173C + A141D): (Q160C (or is E160C) + F116K), (V173C + A141D): (Q160C (or E160C) + F116R), (V173C + A141D): (Q160C (or E160C) + F116H), (V173C + A141E): (Q160C (or E160C) + F116K), (V173C + A141E): (Q160C (or E160C) + F116R), (V173C + A141E): (Q160C (or E160C) + F116H), (V173C + S183K): (Q160C (or E160C) + S176D),(V173C+S183K): (Q160C(or E160C)+S176E), (V173C+S183R): (Q160C(or E160C)+S176D), (V173C+S183R): (Q160C(or E160C)+S176E), (V173C+S183H): (Q160C(or E160C)+S176D), (V173C+S183H): (Q160C(or E160C)+S176E), (V173C+S183D): (Q160C(or E160C)+S176K), (V173C+S183D): (Q1 60C (or E160C) + S176R), (V173C + S183D): (Q160C (or E160C) + S176H), (V173C + S183E): (Q160C (or E160C) + S176K), (V173C + S183E): (Q160C (or E160C) + S176R), (V173C + S183E): (Q160C (or E160C) + S176H), (L128C + F126K): (F118C + S121D), (L128C + F126K): (F118C + S121E), (L128C + F126R): (F1 18C+S121D), (L128C+F126R): (F118C+S121E), (L128C+F126H): (F118C+S 121D), (L128C+F126H): (F118C+S121E), (L128C+F126D): (F118C+S121K) , (L128C+F126D): (F118C+S121R), (L128C+F126D): (F118C+S121H), (L12 8C+F126E): (F118C+S121K), (L128C+F126E): (F118C+S121R), (L128C+F12 6E): (F118C + S121H), (V173C + F126K): (Q160C (or E160C) + S121D), (V173C + F126K): (Q160C (or E160C) + S121E), (V173C + F126R): (Q160C (or E160C) + S121D), (V173C + F126R): (Q160C (or E160C) + S121E), (V173C + F126H): (Q160C (or E160C) + S121D), (V173C + F126H): (Q160C (or E160C) + S121E),(V173C+F126D):(Q160C(or E160C)+S121K), (V173C+F126D):(Q160C(or E160C)+S121R), (V173C+F126D):(Q160C(or E160C)+S121H), (V173C+F126E):(Q160C(or E160C)+S121K), (V173C+F126E):(Q160C(or E160C)+S121R) , (V173C+F126E): (Q160C (or E160C)+S121H), (L128C+K218D): (F118C+D122K), (L128C+K218D): (F118C+D122H), (L128C+K218D): (F118C+D122R), (L128C+K218E): (F118C+D122K), (L128C+K218E): (F118C+D122H), (L128C+K218 E): (F118C + D122R), (V173C + K218D): (Q160C (or E160C) + D122K), (V173C + K218D): (Q160C (or E160C) + D122H), (V173C + K218D): (Q160C (or E160C) + D122R), (V173C + K218E): (Q160C (or E160C) + D122K), (V173C + K218E): (Q160C 2. The polypeptide complex of claim 1, wherein the n5':n6' position pair is different from the n1:n2 position pair and the n7':n8' position pair is different from the n3:n4 position pair, when both the first substitution combination and the second substitution combination are selected.

14. 2. The polypeptide complex of claim 1, wherein the first target binding moiety comprises a first polypeptide fragment operably linked to the first CL region and / or the second target binding moiety comprises a second polypeptide fragment operably linked to the second CL region, and the first polypeptide fragment has an amino acid sequence different from the second polypeptide fragment, or either the first polypeptide fragment or the second polypeptide fragment is not present in the polypeptide complex.

15. 15. The polypeptide complex of claim 14, wherein the first target binding moiety further comprises a third polypeptide fragment operably linked to the first CH1 domain and / or the second target binding moiety comprises a fourth polypeptide fragment operably linked to the second CH1 domain, Optionally, (i) the third polypeptide fragment has a different amino acid sequence from the fourth polypeptide fragment, or either the third polypeptide fragment or the fourth polypeptide fragment is absent from the polypeptide complex; (ii) the first polypeptide fragment and the third polypeptide fragment each comprise a first target binding site or associate with each other to form a first target binding site, and / or the second polypeptide fragment and the fourth polypeptide fragment each comprise a second target binding site or associate with each other to form a second target binding site; and / or (iii) the first target binding site and the second target binding site can bind to the same target molecule, or to different moieties on the same target molecule, or to different target molecules; The polypeptide complex.

16. (i) the first antigen-binding domain and / or the second antigen-binding domain are comprised within an antibody, optionally a bispecific or multispecific antibody; and / or (ii) the second antigen-binding domain and the first antigen-binding domain bind different antigens or different epitopes on the same antigen, optionally wherein the antigen may be a tumor-associated antigen, an immune-associated target or an infectious pathogen-associated target; The polypeptide complex of claim 2.

17. (i) the first and / or second antigen-binding domains are chimeric, humanized or fully human antigen-binding domains; (ii) the first and / or the second antigen-binding portion is selected from the group consisting of a nanobody, an Fv fragment, an scFv, a disulfide-stabilized Fv fragment, a (dsFv) 2 , a bispecific dsFv and a diabody; (iii) the first and / or the second antigen-binding domain is selected from the group consisting of a Fab domain, a Fab' and an F(ab')2, and optionally the first antigen-binding domain and / or the second antigen-binding domain comprises one or more CDRs operably linked to a CH1 domain and a CL domain; and / or (iv) the first antigen-binding domain is a first Fab domain and / or the second antigen-binding domain is a second Fab domain, optionally wherein the second Fab domain comprises (a) one or more light chain CDRs and / or light chain framework regions that differ from the first Fab domain, and optionally (b) one or more heavy chain CDRs and / or heavy chain framework regions that differ from the first Fab domain; The polypeptide complex of claim 2.

18. 2. The polypeptide complex of claim 1, further comprising an Fc region operably linked to the first target binding domain and the second target binding domain, Optionally, (i) the Fc region is derived from IgG1, IgG2, IgG3, or IgG4; (ii) the Fc region is a heterodimer; or (iii) the heterodimeric Fc region comprises one or more mutations that promote heterodimerization; The polypeptide complex.

19. the heterodimeric Fc region comprises a first Fc polypeptide comprising a first Fc mutation and / or a second Fc polypeptide comprising a second Fc mutation; a) the first Fc mutation comprises T366W or S354C and the second Fc mutation comprises Y349C, T366S, L368A or Y407V; b) the first Fc mutation comprises D399K or E356K and the second Fc mutation comprises K392D or K409D; c) the first Fc mutation comprises E356K, E357K or D399K and the second Fc mutation comprises K370E, K409D or K439E; or d) the first Fc mutation comprises S364H or F405A and the second Fc mutation comprises Y349T or T394F; e) the first Fc mutation comprises S364H or T394F and the second Fc mutation comprises Y394T or F405A; or f) the first Fc mutation comprises K370D or K409D and the second Fc mutation comprises E357K or D399K; or g) the first Fc mutation comprises L351D or L368E and the second Fc mutation comprises L351K or T366K; 19. The polypeptide complex of claim 18, wherein the numbering is according to the EU index.

20. A nucleic acid comprising a nucleotide sequence encoding the polypeptide complex of claim 1 or a portion thereof.

21. A vector comprising the nucleic acid of claim 20.

22. 22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21.

23. A pharmaceutical composition comprising the polypeptide complex of claim 1 and a pharma- ceutically acceptable carrier.

24. A conjugate comprising the polypeptide complex of claim 1 and a payload conjugated thereto, wherein the payload is selected from the group consisting of a radioactive label, a fluorescent label, an enzyme substrate label, an affinity purification tag, a tracer molecule, an anticancer drug and a cytotoxic molecule.

25. 25. A composition comprising the polypeptide complex of claim 1 or the conjugate of claim 24 and a pharma- ceutically acceptable carrier.

26. 1. A method for treating or preventing a disease, condition or symptom, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide complex of claim 1, a pharmaceutical composition comprising the polypeptide complex of claim 1, a conjugate comprising the polypeptide complex of claim 1, or a composition comprising the polypeptide complex of claim 1, Optionally, the disease is selected from the group consisting of cancer, an inflammatory disease, an infectious or parasitic disease, a cardiovascular disease, a neurological disorder, a neuropsychiatric condition, an injury, an autoimmune disease, a metabolic disease, a neurodegenerative disease, or a blood clotting disorder. The method.

27. A method for detecting the presence or level of an antigen, comprising contacting a sample suspected of containing the antigen with a polypeptide complex described in claim 1 and determining complex formation between the antigen and the polypeptide complex.

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