Method for producing molecules

Abstract

Methods for producing a molecule comprising a polypeptide complex formed by interaction between a first polypeptide comprising a pestle modification-containing CH3 region and a second polypeptide comprising a mortar modification-containing CH3 region are disclosed.

Description

Technical Field

[0001] This disclosure relates to the fields of molecular biology and target-binding molecule generation technologies. Background Technology

[0002] Recombinant co-expression of the constituent peptides of the target-binding molecule and subsequent association can produce several possible combinations. To improve the yield of the desired peptide combination in the target-binding molecule during recombinant production, introducing Fc region modifications that promote the association of the desired peptide combination is advantageous. These modifications can promote… For example Hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Such modifications are described in... For example Ha et al. In Front Immunol. (2016) 7:394, it is incorporated here in its entirety by reference.

[0003] One such modification is "knobs-into-holes" or "KiH", which is described in For example US 7,695,936, Atwell et al. In J Mol Biol. (1997) 270(1):26-35 and Carter, J ImmunolMeth. (2001) 248(1-2):7-15. In such molecules, one of the CH3 regions of the Fc region contains a "pestle" modification, and the other CH3 region contains a "mortar" modification. The "pestle" and "mortar" modifications are located within the corresponding CH3 regions, allowing the "pestle" to be located within the "mortar" in order to promote heterodimerization of the polypeptide (and inhibit homodimerization) and / or stabilize the heterodimer. By using amino acids with larger side chains ( For example The pestle is constructed by replacing amino acids with smaller chains (such as tyrosine or tryptophan). For example Atwell uses amino acids with larger side chains (such as alanine or threonine) to create mortar. et al. J Mol Biol. (1997) 270(1):26-35 describes the conventional "pestle" modification T366W and the "mortar" modification Y407V:T366S:L368A. It also describes the KiH modification containing additional substitutions. For example Used to form stabilizing interchain disulfide bonds (see Merchant) et al. , Nature Biotech. (1998) 16:677-681).

[0004] Although such modifications are made to facilitate the desired pairing of peptides carrying the CH3 region, undesirable molecules, such as mismatched mordone-mordone homodimers, are often present in significant proportions in the products of cell cultures used to generate KiH molecules. et al. Bioresources and Bioprocessing (2022) 9:72 describes a method for the efficient removal of such undesirable byproducts. There remains a need in the art to increase the production of desired KiH molecules relative to the generation of undesirable byproducts, such as mismatched mordone-mordone homodimers. Summary of the Invention

[0005] In a first aspect, this disclosure provides a method for generating a molecule comprising a polypeptide complex formed by an interaction between a first polypeptide comprising a CH3 region containing a mordant-modified region and a second polypeptide comprising a CH3 region containing a mordant-modified region, wherein the method comprises culturing cells comprising nucleic acids encoding the first and second polypeptides in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM for a substantial portion of the culture period.

[0006] In some embodiments, the cell culture medium has an osmolar concentration greater than 300 mOsmol / kg.

[0007] In some embodiments, the method includes seeding cells in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM, optionally wherein the method includes seeding cells in a cell culture medium containing cysteine ​​at a concentration of 1 mM to 5 mM.

[0008] In some embodiments, the method includes seeding cells in a cell culture medium containing cysteine ​​at a concentration of 2.5 mM to 4.75 mM.

[0009] In some embodiments, the method includes seeding cells in a cell culture medium having an osmolar concentration greater than 300 mOsmol / kg, optionally wherein the method includes seeding cells in a cell culture medium having an osmolar concentration of 320 mOsmol / kg to 420 mOsmol / kg.

[0010] In some embodiments, the method includes seeding cells in a cell culture medium having an osmolar concentration of about 370 mOsmol / kg.

[0011] In some embodiments, the incubation period is at least 3 days, optionally at least 7 days or at least 14 days.

[0012] In some embodiments, the cell culture medium has a pH of 6.8 to 7.4, optionally wherein the cell culture medium has a pH of about 7.2.

[0013] In some embodiments, the first polypeptide or the second polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the extracellular domain of the ligand used for co-stimulatory molecules.

[0014] In some embodiments: (i) The first polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the extracellular domain of the ligand used for co-stimulatory molecules, and the second polypeptide comprises all or part of the amino acid sequence forming the antigen-binding moiety that binds to the target antigen; or (ii) The first polypeptide comprises all or part of an amino acid sequence forming an antigen-binding portion that binds to the target antigen, and the second polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the extracellular domain of the ligand used for co-stimulatory molecules.

[0015] illustrate

[0016] The resulting molecule comprises a polypeptide complex formed through the interaction between a first polypeptide containing a CH3 region modified with mortar and a second polypeptide containing a CH3 region modified with mortar.

[0017] The aspects and embodiments of this disclosure relate to the generation of molecules comprising polypeptide complexes formed by the interaction between a first polypeptide comprising a CH3 region containing a mortar-modified region and a second polypeptide comprising a CH3 region containing a mortar-modified region.

[0018] In this paper, for the sake of brevity, a molecule comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a CH3 region containing a mortar-modified region and a second polypeptide comprising a CH3 region containing a mortar-modified region can be simply referred to as a "KiH molecule". Similarly, a molecule comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a CH3 region containing a mortar-modified region and a second polypeptide comprising a CH3 region containing a mortar-modified region can be simply referred to as a "KiH molecule".

[0019] As used herein, a "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides typically have a length of about 2 to about 50 amino acids. A "polypeptide" contains more than one peptide bond and comprises 3 or more amino acids.

[0020] A “peptide complex” is characterized by protein-protein interactions between its constituent peptides. In some embodiments, protein-protein interactions include non-covalent interactions. For example electrostatic interaction ( For example Ionic bonding, hydrogen bonding) and / or van der Waals forces. In some embodiments, protein:protein interactions include covalent interactions ( example like (Disulfide bonding, electron sharing). For example, a polypeptide complex contemplated according to this disclosure includes a molecule comprising a polypeptide complex formed by the interaction between a first polypeptide containing a CH3 region and a second polypeptide containing a CH3 region.

[0021] Immunoglobulins and their structures are described in For example Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202):S41-S52, which are hereby incorporated in their entirety by reference. G-type immunoglobulins (i.e. Immunoglobulin IgG (IgG) is a glycoprotein of approximately 150 kDa, consisting of two heavy chains and two light chains. From the N-terminus to the C-terminus, the immunoglobulin heavy chain contains a heavy chain variable region (VH), followed by a heavy chain constant region containing three constant regions (CH1, CH2, and CH3, with the CH1-CH2 hinge region located between CH1 and CH2). The immunoglobulin light chain contains a light chain variable region (VL), followed by a light chain constant region (CL). Based on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG ... IgG1, IgG2, IgG3, IgG4), IgA (e.g. IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0022] The "CH1 region" refers to the amino acid sequence corresponding to the CH1 region of immunoglobulins. According to the EU numbering system described in the following literature, the CH1 region is the region of Ig formed by positions 118 to 215 of the Ig constant region: Edelman. et al. , Proc Natl Acad Sci USA. (1969) 63(1):78-85. The “CH1-CH2 hinge region” refers to the amino acid sequence corresponding to the CH1-CH2 hinge region of immunoglobulin. According to the EU numbering system described in the following literature, the CH1-CH2 hinge region is the region of Ig formed by positions 216 to 230 of the Ig constant region: Edelman et al., Proc Natl Acad Sci USA. (1969) 63(1):78-85. The “CH2 region” refers to the amino acid sequence corresponding to the CH2 region of immunoglobulins. According to the EU numbering system described in the following literature, the CH2 region is the region of Ig formed by positions 231 to 340 of the Ig constant region: Edelman et al. , Proc Natl Acad Sci USA. (1969) 63(1):78-85. The “CH2-CH3 region” refers to the amino acid sequence corresponding to the CH2 and CH3 regions of immunoglobulins. According to the EU numbering system described in the following literature, the CH2-CH3 region is the region of Ig formed by positions 231 to 447 of the Ig constant region: Edelman et al. , Proc Natl Acad Sci USA. (1969) 63(1):78-85.

[0023] The "CH3 region" refers to the amino acid sequence corresponding to the CH3 region of immunoglobulins. According to the EU numbering system described in the following literature, the CH3 region is the region of Ig formed by positions 341 to 447 of the Ig constant region: Edelman et al. , Proc Natl Acad Sci USA.(1969) 63(1):78-85. The CH3 region of human IgG1 (G1m1 allotype) is shown in SEQ ID NO:5. The CH3 region of human IgG1 (G1m3 allotype) is shown in SEQ ID NO:8.

[0024] In some embodiments, the CH3 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:5, or having at least 70% amino acid sequence identity with SEQ ID NO:5. For example An amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the CH3 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:8, or having at least 70% amino acid sequence identity with SEQ ID NO:8. For exampleThe amino acid sequence of one of the following sequences (≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity).

[0025] The CH3 region according to this disclosure includes modifications to facilitate association with another CH3 region. In some embodiments, the CH3 region includes modifications to facilitate heteropolymerization ( Right now Modifications (association between different CH3 regions).

[0026] In the embodiments described herein (and more generally in the art), “modification” may also be referred to as “substitution” or “mutation”.

[0027] In aspects and embodiments of this disclosure, the interactions between the CH3 regions of the constituent polypeptides of the molecules described herein are enhanced using a "mortar and pestle" technique. The mortar and pestle (or "KiH") technique is described in... For example WO 96 / 027011, Ridgway, JB et al. , Protein Eng. (1996) 9(7):617-621, Merchant, AM et al. All these references are incorporated here as a whole by way of citation in NatBiotechnol. (1998) 16(7):677-681, US 7,695,936 and Carter, J Immunol Meth. (2001) 248(1-2):7-15.

[0028] Heterodimerization between peptides is promoted by modifying the interaction surface of the peptide carrying the CH3 region, providing complementary "pestle" and "mortar" modifications in the amino acid sequence of the peptide's CH3 region. The "pestle" and "mortar" modifications are localized within the corresponding CH3 region, allowing the "pestle" to be positioned within the "mortar," thereby promoting heterodimerization (and inhibiting homodimerization) and / or stabilizing the heterodimer. This is achieved by using amino acids with larger side chains ( For example The pestle is constructed by replacing amino acids with smaller side chains (such as tyrosine or tryptophan). For example The amino acid valine, alanine, serine, or threonine is substituted to produce acetylcholine.

[0029] For example, in the embodiments herein, certain CH3 regions contain a pestle modification, which may include a modification at position 366 of the CH3 region (unless otherwise stated, the position or substitution number in the CH3 region herein follows the EU numbering system described in the following literature: Edelman). et al. , Proc Natl Acad Sci USA. (1969) 63(1): 78-85). This modification can provide a tryptophan residue at position 366, which is a threonine residue in the canonical sequence for human IgG1 / IgG2 / IgG3 / IgG4.

[0030] In embodiments and aspects of this disclosure, the CH3 region ( For example According to the present disclosure, the constituent peptide complex (the CH3 region of the peptide) contains paired CH3 regions “KiH” or “KiH”. S-S "Modification".

[0031] In some embodiments, the CH3 region containing the pestle-modified region is located at position 366 ( Right now The CH3 region containing tryptophan residues is located at (366W). In some embodiments, the CH3 region containing guar gum-modified residues is located at (366W). Right now The 366Y region contains a tyrosine residue. In some embodiments, the trehalose modification is T366W or T366Y. In some embodiments, the trehalose-modified CH3 region contains 366W. In some embodiments, the trehalose modification is T366W or T366Y.

[0032] In this article, when referring to the location of the immunoglobulin homeostasis region ( For example When considering EU numbering, corresponding positions in homologous sequences of the constant region of human IgG1 (G1m1 allotype) were also taken into account. Positions corresponding to those identified in the CH3 region of human IgG1 (G1m1 allotype) can be identified through sequence alignment. example likeSequence alignment software such as ClustalOmega is used to perform this (Söding, J. Bioinformatics (2005)21:951-960). For example, 366T in the human IgG1 CH3 region (position 26 of SEQ ID NO:5) corresponds to position 26 of SEQ ID NO:8 (hIgG1 G1m3 allotype CH3 region), position 26 of SEQ ID NO:9 (hIgG2 CH3 region), position 26 of SEQ ID NO:10 (hIgG3 CH3 region), and position 26 of SEQ ID NO:11 (hIgG4 CH3 region).

[0033] In some embodiments, the CH3 region containing the mortar-modified region is located at position 366 ( Right now The CH3 region containing serine, valine, or alanine is located at position 368 (366S, 366V, or 366A). In some embodiments, the CH3 region containing the mortar-modified region is located at position 368 (366S, 366V, or 366A). Right now The CH3 region containing 368A, 368V, 368S, or 368T contains an alanine, valine, serine, or threonine residue. In some embodiments, the mordant-modified CH3 region contains a residue at position 407. Right now The CH3 region containing 407V, 407A, 407S, or 407T contains valine, alanine, serine, or threonine residues. In some embodiments, the mortar-modified CH3 region contains: 366S, 366V, or 366A; 368A, 368V, 368S, or 368T; and 407V, 407A, 407S, or 407T. In some embodiments, the mortar modification is or includes T366S, T366V, or T366A; L368A, L368V, L368S, or L368T; and Y407V, Y407A, Y407S, or Y407T.

[0034] In some embodiments, the CH3 region containing mortar modification includes 366S. In some embodiments, the CH3 region containing mortar modification includes 368A. In some embodiments, the CH3 region containing mortar modification includes 407V. In some embodiments, the CH3 region containing mortar modification includes: 366S, 368A, and 407V. In some embodiments, mortar modification is or includes T366S, L368A, and Y407V.

[0035] In some embodiments, the CH3 region containing the mortar modification includes: 366S, 368A, and 407V. In some embodiments, the mortar modification is or includes Y407V, Y407A, Y407S, or Y407T; T366S, T366V, or T366A; and L368A, L368V, L368S, or L368T.

[0036] In some embodiments, the CH3 region containing the mortar modification includes 407V, 366S, and 368A. In some embodiments, the mortar modification is or includes Y407V, T366S, and L368A.

[0037] Additional interchain disulfide bonds can also be introduced between CH3 regions (such as Merchant, AM). et al. (As described in Nature Biotech. (1998) 16:677-681). For example The modification involves introducing a cysteine ​​residue at position 354 of the CH3 region with a "palm" modification and at position 349 of the CH3 region with a "mortar" modification. The palm-shaped modification, which further includes the introduction of cysteine ​​residues for the formation of interchain disulfide bonds, can be termed a "palm-cys" modification, and similarly, the mortar-shaped modification, which further includes the introduction of cysteine ​​residues for the formation of interchain disulfide bonds, can be termed a "mortar-cys" modification.

[0038] Therefore, in some embodiments, the CH3 region containing the pestle modification includes 354C. In some embodiments, the pestle modification includes S354C. In some embodiments, the CH3 region containing the pestle modification includes 366W or 366Y; and 354C. In some embodiments, the CH3 region containing the pestle modification includes 366W and 354C. In some embodiments, the pestle modification is or includes T366W or T366Y; and S354C. In some embodiments, the pestle modification is or includes T366W and S354C.

[0039] The pestle modification described in the previous paragraphs can be called the "pestle-cys" modification.

[0040] Therefore, in some embodiments, the CH3 region containing the mortar modification includes 349C. In some embodiments, the mortar modification includes Y349C. In some embodiments, the CH3 region containing the mortar modification includes Y407V, Y407A, Y407S, or Y407T; T366S, T366V, or T366A; L368A, L368V, L368S, or L368T; and Y349C. In some embodiments, the CH3 region containing the mortar modification includes 366S, 368A, 407V, and Y349C.

[0041] The mortar modification described in the previous paragraphs can be called the "mortar-cys" modification.

[0042] In some embodiments, the pestle is modified to include T366W or T366Y; and S354C. In some embodiments, the mortar is modified to include T366S, L368A, Y407V, and Y349C.

[0043] In some embodiments, the CH3 region containing the pestle modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:12, or having at least 70% amino acid sequence identity with SEQ ID NO:12. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:13, or having at least 70% amino acid sequence identity with SEQ ID NO:13. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:14, or having at least 70% amino acid sequence identity with SEQ ID NO:14. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:15, or having at least 70% amino acid sequence identity with SEQ ID NO:15. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:16, or having at least 70% amino acid sequence identity with SEQ ID NO:16. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:17, or having at least 70% amino acid sequence identity with SEQ ID NO:17. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:18, or having at least 70% amino acid sequence identity with SEQ ID NO:18. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:19, or having at least 70% amino acid sequence identity with SEQ ID NO:19. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:20, or having at least 70% amino acid sequence identity with SEQ ID NO:20. For exampleThe amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:21, or having at least 70% amino acid sequence identity with SEQ ID NO:21. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence contains 366W.

[0044] In some embodiments, the CH3 region comprising mortar modification includes or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:22, or having at least 70% amino acid sequence identity with SEQ ID NO:22. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:23, or having at least 70% amino acid sequence identity with SEQ ID NO:23. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:24, or having at least 70% amino acid sequence identity with SEQ ID NO:24. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:25, or having at least 70% amino acid sequence identity with SEQ ID NO:25. For exampleThe amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:26, or having at least 70% amino acid sequence identity with SEQ ID NO:26. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:27, or having at least 70% amino acid sequence identity with SEQ ID NO:27. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:28, or having at least 70% amino acid sequence identity with SEQ ID NO:28. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:29, or having at least 70% amino acid sequence identity with SEQ ID NO:29. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:30, or having at least 70% amino acid sequence identity with SEQ ID NO:30. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:31, or having at least 70% amino acid sequence identity with SEQ ID NO:31. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence includes 366S, 368A, and 407V.

[0045] In some embodiments, the CH3 region containing the pestle modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:17, or having at least 70% amino acid sequence identity with SEQ ID NO:17. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:18, or having at least 70% amino acid sequence identity with SEQ ID NO:18. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:19, or having at least 70% amino acid sequence identity with SEQ ID NO:19. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:20, or having at least 70% amino acid sequence identity with SEQ ID NO:20. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:21, or having at least 70% amino acid sequence identity with SEQ ID NO:21. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence contains 366W and 354C.

[0046] In some embodiments, the CH3 region comprising mortar modification includes or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:27, or having at least 70% amino acid sequence identity with SEQ ID NO:27. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:28, or having at least 70% amino acid sequence identity with SEQ ID NO:28. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:29, or having at least 70% amino acid sequence identity with SEQ ID NO:29. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:30, or having at least 70% amino acid sequence identity with SEQ ID NO:30. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:31, or having at least 70% amino acid sequence identity with SEQ ID NO:31. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence includes 366S, 368A, 407V, and 349C.

[0047] In some embodiments, the polypeptide according to this disclosure that includes a CH3 region includes a CH2 region. The CH2 region is preferably adjacent to and immediately upstream of the CH3 region in the amino acid sequence of the polypeptide. Right now (in the direction of the N end of the CH3 region).

[0048] In some embodiments, the CH3 region according to this disclosure is contained within the CH2-CH3 region. That is, in some embodiments, the polypeptide according to this disclosure containing the CH3 region contains the CH2-CH3 region.

[0049] In some embodiments, the CH2 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:4, or having at least 70% amino acid sequence identity with SEQ ID NO:4. For example An amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the CH2 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:32, or having at least 70% amino acid sequence identity with SEQ ID NO:32. For example An amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the CH2 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:33, or having at least 70% amino acid sequence identity with SEQ ID NO:33. For exampleAn amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the CH2 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:34, or having at least 70% amino acid sequence identity with SEQ ID NO:34. For example The amino acid sequence of one of the following sequences (≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity).

[0050] In some embodiments, the CH2-CH3 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:35, or having at least 70% amino acid sequence identity with SEQ ID NO:35. For example An amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the CH2-CH3 region comprises or consists of the following: an amino acid sequence having the amino acid sequence of SEQ ID NO:36, or having at least 70% amino acid sequence identity with SEQ ID NO:36. For example The amino acid sequence of one of the following sequences (≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity).

[0051] It should be understood that this disclosure provides CH2-CH3 regions comprising CH3 regions containing pestle-modified regions. Such CH2-CH3 regions can be simply described as CH2-CH3 regions containing pestle-modified regions.

[0052] Similarly, this disclosure provides CH2-CH3 regions comprising CH3 regions containing mortar modifications. Such CH2-CH3 regions can be simply described as CH2-CH3 regions comprising mortar modifications.

[0053] In some embodiments, the CH2-CH3 region containing the pestle modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:37, or having at least 70% amino acid sequence identity with SEQ ID NO:37. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:38, or having at least 70% amino acid sequence identity with SEQ ID NO:38. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:39, or having at least 70% amino acid sequence identity with SEQ ID NO:39. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:40, or having at least 70% amino acid sequence identity with SEQ ID NO:40. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:41, or having at least 70% amino acid sequence identity with SEQ ID NO:41. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:42, or having at least 70% amino acid sequence identity with SEQ ID NO:42. For exampleThe amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:43, or having at least 70% amino acid sequence identity with SEQ ID NO:43. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:44, or having at least 70% amino acid sequence identity with SEQ ID NO:44. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:45, or having at least 70% amino acid sequence identity with SEQ ID NO:45. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:46, or having at least 70% amino acid sequence identity with SEQ ID NO:46. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence contains 366W.

[0054] In some embodiments, the CH2-CH3 region containing mortar modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:47, or having at least 70% amino acid sequence identity with SEQ ID NO:47. For exampleThe amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:48, or having at least 70% amino acid sequence identity with SEQ ID NO:48. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:49, or having at least 70% amino acid sequence identity with SEQ ID NO:49. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:50, or having at least 70% amino acid sequence identity with SEQ ID NO:50. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:51, or having at least 70% amino acid sequence identity with SEQ ID NO:51. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:52, or having at least 70% amino acid sequence identity with SEQ ID NO:52. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:53, or having at least 70% amino acid sequence identity with SEQ ID NO:53. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:54, or having at least 70% amino acid sequence identity with SEQ ID NO:54. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:55, or having at least 70% amino acid sequence identity with SEQ ID NO:55. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:56, or having at least 70% amino acid sequence identity with SEQ ID NO:56. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence includes 366S, 368A, and 407V.

[0055] In some embodiments, the CH2-CH3 region containing the pestle modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:42, or having at least 70% amino acid sequence identity with SEQ ID NO:42. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:43, or having at least 70% amino acid sequence identity with SEQ ID NO:43. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:44, or having at least 70% amino acid sequence identity with SEQ ID NO:44. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:45, or having at least 70% amino acid sequence identity with SEQ ID NO:45. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:46, or having at least 70% amino acid sequence identity with SEQ ID NO:46. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence contains 366W and 354C.

[0056] In some embodiments, the CH2-CH3 region containing mortar modification comprises or consists of the following: An amino acid sequence having the amino acid sequence of SEQ ID NO:52, or having at least 70% amino acid sequence identity with SEQ ID NO:52. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:53, or having at least 70% amino acid sequence identity with SEQ ID NO:53. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:54, or having at least 70% amino acid sequence identity with SEQ ID NO:54. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:55, or having at least 70% amino acid sequence identity with SEQ ID NO:55. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; or An amino acid sequence having the amino acid sequence of SEQ ID NO:56, or having at least 70% amino acid sequence identity with SEQ ID NO:56. For example The amino acid sequence of one of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity. The amino acid sequence includes 366S, 368A, 407V, and 349C.

[0057] In some embodiments, the KiH molecule according to this disclosure includes an Fc moiety. As used herein, an "Fc moiety" refers to a polypeptide complex formed by the interaction between two polypeptides, wherein each polypeptide contains a CH2-CH3 region.

[0058] In some embodiments, the Fc portion according to this disclosure includes (i) a polypeptide containing a CH2-CH3 region modified with mortar; and (ii) a polypeptide containing a CH2-CH3 region modified with mortar.

[0059] In some embodiments, the KiH molecule or Fc portion according to this disclosure comprises: (a) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:37, or having at least 70% amino acid sequence identity with SEQ ID NO:37. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:47, or having at least 70% amino acid sequence identity with SEQ ID NO:47. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (b) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:38, or having at least 70% amino acid sequence identity with SEQ ID NO:38. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:48, or having at least 70% amino acid sequence identity with SEQ ID NO:48. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (c) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:39, or having at least 70% amino acid sequence identity with SEQ ID NO:39. For example(ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:49, or having at least 70% amino acid sequence identity with SEQ ID NO:49. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (d) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:40, or having at least 70% amino acid sequence identity with SEQ ID NO:40. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence contains ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:50, or having at least 70% amino acid sequence identity with SEQ ID NO:50. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (e) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:41, or having at least 70% amino acid sequence identity with SEQ ID NO:41. For example(ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:51, or having at least 70% amino acid sequence identity with SEQ ID NO:51. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (f) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:42, or having at least 70% amino acid sequence identity with SEQ ID NO:42. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:52, or having at least 70% amino acid sequence identity with SEQ ID NO:52. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (g) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:43, or having at least 70% amino acid sequence identity with SEQ ID NO:43. For example(ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:53, or having at least 70% amino acid sequence identity with SEQ ID NO:53. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (h) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:44, or having at least 70% amino acid sequence identity with SEQ ID NO:44. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:54, or having at least 70% amino acid sequence identity with SEQ ID NO:54. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:45, or having at least 70% amino acid sequence identity with SEQ ID NO:45. For example(ii) an amino acid sequence comprising 366W, wherein the amino acid sequence contains ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:55, or having at least 70% amino acid sequence identity with SEQ ID NO:55. example like An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V; or (j) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:46, or having at least 70% amino acid sequence identity with SEQ ID NO:46. For example (ii) an amino acid sequence comprising 366W, wherein the amino acid sequence comprises an amino acid sequence having the amino acid sequence of SEQ ID NO:56, or having at least 70% amino acid sequence identity with SEQ ID NO:56. example like The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, and 407V.

[0060] In some embodiments, the KiH molecule or Fc portion according to this disclosure comprises: (a) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:42, or having at least 70% amino acid sequence identity with SEQ ID NO:42. For example(ii) an amino acid sequence comprising 366W and 354C, wherein the amino acid sequence comprises 366W and 354C; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:52, or having at least 70% amino acid sequence identity with SEQ ID NO:52. For example An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, 407V, and 349C; or (b) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:43, or having at least 70% amino acid sequence identity with SEQ ID NO:43. For example (ii) an amino acid sequence comprising 366W and 354C, wherein the amino acid sequence comprises 366W and 354C; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:53, or having at least 70% amino acid sequence identity with SEQ ID NO:53. For example An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, 407V, and 349C; or (c) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:44, or having at least 70% amino acid sequence identity with SEQ ID NO:44. For example(ii) an amino acid sequence comprising 366W and 354C, wherein the amino acid sequence comprises 366W and 354C; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:54, or having at least 70% amino acid sequence identity with SEQ ID NO:54. For example An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, 407V, and 349C; or (d) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:45, or having at least 70% amino acid sequence identity with SEQ ID NO:45. For example (ii) an amino acid sequence comprising 366W and 354C, wherein the amino acid sequence comprises the amino acid sequence having the amino acid sequence of SEQ ID NO:55, or having at least 70% amino acid sequence identity with SEQ ID NO:55. For example An amino acid sequence that exhibits ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, 407V, and 349C; or (e) (i) A polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:46, or having at least 70% amino acid sequence identity with SEQ ID NO:46. For example(ii) an amino acid sequence comprising 366W and 354C, wherein the amino acid sequence comprises 366W and 354C; and (ii) a polypeptide comprising an amino acid sequence having the amino acid sequence of SEQ ID NO:56, or having at least 70% amino acid sequence identity with SEQ ID NO:56. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity, wherein the amino acid sequence contains 366S, 368A, 407V, and 349C.

[0061] In some embodiments, the KiH molecule according to this disclosure further includes a target-binding portion. In some embodiments, the KiH molecule according to this disclosure is a target-binding molecule.

[0062] A "target-binding molecule" is a molecule that binds to a given target molecule. Target-binding molecules include antigen-binding molecules, such as antibodies (…). Right now Immunoglobulins (Ig) and their antigen-binding fragments.

[0063] As used in this article, "antibody" includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies. For example Bispecific and trispecific wait Antibodies, and antibody-derived antigen-binding molecules, such as scFv, scFab, bisomal antibodies, trisomal antibodies, scFv-Fc, microantibodies, and single-domain antibodies. For example VhH wait Antibody antigen-binding fragments include... For example Fv, Fab, F(ab')2, and F(ab') fragments. Antigen-binding molecules also include antibody-derived molecules. For example A molecule containing an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules may contain an antigen-binding region / domain that includes the antigen-binding region of the antibody. For example The antigen-binding fragment of the antibody (or a domain thereof). In some embodiments, the antigen-binding region / domain of the antibody-derived antigen-binding molecule may be or contain the Fv (antibody). example like (provided as scFv) or Fab region, or the entire antibody.

[0064] The target-binding molecule disclosed herein comprises one or more target-binding moieties. The target-binding moieties are the portions capable of binding to the target molecule. The binding of the target-binding moieties to the target-binding molecule can be characterized by non-covalent interactions, electrostatic interactions, etc. For example (ionic bonding, hydrogen bonding) and / or van der Waals forces.

[0065] Consideration is given to target-binding portions related to this disclosure, including antigen-binding portions and peptides / polypeptides / complexes derived from interacting partners for target molecules.

[0066] In some embodiments, the target-binding molecule of this disclosure comprises one or more antigen-binding portions. An antigen-binding portion is a portion capable of binding to a given target antigen.

[0067] The antigen-binding moiety can be derived from the antibody. Antibody-derived antigen-binding moieties can contain the antigen-binding region of the antibody. For example The antigen-binding portion may be or comprise an Fv (antibody antigen-binding fragment) of an antibody that binds to a given target antigen. In some embodiments, the antigen-binding portion may be or comprise an Fv (antibody antigen-binding fragment) of an antibody that binds to a given target antigen. For example (provided as scFv) or Fab region, or the entire antibody.

[0068] In some embodiments, the antigen-binding portion comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) capable of specifically binding to a target antigen. In some embodiments, the antigen-binding portion comprises or consists of an aptamer capable of binding to a target antigen. For example Nucleic acid aptamers (reviewed in, for example, Zhou and Rossi Nat RevDrug Discov. (2017) 16(3):181-202).

[0069] The antigen-binding moiety can be formed from an antigen-binding peptide / peptide or an antigen-binding peptide / peptide complex. The antigen-binding moiety may contain more than one peptide / peptide, which together form the antigen-binding moiety. Peptides / peptides can associate via protein:protein interactions to form the antigen-binding moiety. For example, in some embodiments described herein, the KiH molecule of this disclosure comprises an antigen-binding moiety (particularly the Fab moiety) formed by association between: (i) a peptide comprising a VH region and a CH1 region (and further comprising a CH2-CH3 region according to this disclosure), and (ii) a peptide comprising a VL region and a CL region.

[0070] In some embodiments, the antigen-binding portion comprises or consists of the following: an antigen-binding peptide / polypeptide, or an antigen-binding peptide / polypeptide complex. The antigen-binding polypeptide may be... For examplePeptide aptamers, thioredoxins, monobodies, anticalins, Kunitz domains, high-affinity polymers (avimers), knottin, fynomers, atrimers, DARPin, affibody, nanobodies ( Right now Single-domain antibody (sdAb), affilin, armadillo repeat protein (ArmRP), OBody, and fibronectin ( For example In Reverdatto et al. A review was conducted in Curr Top Med Chem. (2015) 15(12):1082–1101, which is incorporated herein by reference in its entirety (see also: Curr Top Med Chem. (2015) 15(12):1082–1101). For example Boersma et al. J Biol Chem. (2011) 286:41273-85 and Emanuel et al. , Mabs(2011) 3:38-48)).

[0071] In some embodiments, the antigen-binding portion according to this disclosure comprises or consists of the following: a polypeptide complex formed by protein-protein interactions between the constituent peptides / polypeptides of the antigen-binding portion.

[0072] For example, in some embodiments described herein, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to FAP, and (ii) a polypeptide comprising a VL region of an antibody that binds to FAP. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to BCMA, and (ii) a polypeptide comprising a VL region of an antibody that binds to BCMA. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to CD19, and (ii) a polypeptide comprising a VL region of an antibody that binds to CD19. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to CEA, and (ii) a polypeptide comprising a VL region of an antibody that binds to CEA. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to HER2, and (ii) a polypeptide comprising a VL region of an antibody that binds to HER2. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region of an antibody that binds to PD-L1, and (ii) a polypeptide comprising a VL region of an antibody that binds to PD-L1.

[0073] It should be understood that, in some embodiments, the constituent polypeptides of the KiH molecule according to this disclosure contain all or part of the amino acid sequence that forms the antigen-binding portion.

[0074] In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure comprises an antigen-binding moiety. In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure comprises an antigen-binding peptide / peptide.

[0075] In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a portion of the antigen-binding portion. In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a component / fragment of the antigen-binding portion. For example, in some embodiments described herein, the KiH molecule includes a polypeptide containing a VH having an antigen-binding portion specific to the target antigen, and such molecules further include a polypeptide containing a VL having an antigen-binding portion.

[0076] In some embodiments, the target-binding portion according to this disclosure may comprise or consist of an antigenic peptide / peptide or peptide / peptide complex, which is or is derived from an interacting partner for binding to a target molecule. For example, the target-binding portion may comprise or consist of a peptide / peptide having an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the target interaction region of the interacting partner for binding to the target molecule. For example ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0077] The interaction partner used for the target molecule can be the interaction between the target molecule and it. For example Peptides or peptides / peptides or peptide / peptide complexes (via protein:protein interactions). In some embodiments, the interacting partner for the target molecule may be the target molecule interacting with it to form a peptide / peptide complex (…). Right now A peptide / peptide complex containing a target molecule and a peptide / peptide complex that interacts with the target molecule.

[0078] The "target interaction region" of an interacting chaperone for a target molecule refers to the region / domain of the molecule through which the interacting chaperone interacts with the target molecule. For example Through protein-protein interactions. This region may contain amino acids that contact the target molecule. The target interaction region for the interaction chaperone of the target molecule can also be called the "target binding region". For example, the "target interaction region" of 4-1BBL is the extracellular domain of 4-1BBL, which is formed by the following from position 24 to position 186: UniProtKB: Q07011-1, v1.

[0079] In some embodiments, the interacting partner for the target molecule and the target molecule interact with each other in a receptor:ligand interaction manner. In some embodiments, the target-binding portion may comprise or may consist of a peptide / polypeptide having an amino acid sequence that has at least 70% amino acid sequence identity with the amino acid sequence of the target interaction region of the ligand or receptor for binding to the antigen thereto. For example (≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity). For example, in some embodiments described herein, the KiH molecule of this disclosure comprises a target-binding peptide / peptide or a target-binding peptide / peptide complex containing an amino acid sequence corresponding to an extracellular domain or a fragment thereof of 4-1BBL (which is a ligand for 4-1BB).

[0080] In some embodiments, the target binding portion according to this disclosure includes a plurality of ( For example A target-binding peptide / peptide or target-binding peptide / peptide complex or composed of two, three or more (non-overlapping amino acid sequences), each non-overlapping amino acid sequence having a target interaction region with an interaction chaperone for binding to the target molecule. For example The amino acid sequence of the ligand or receptor target binding region of the molecule to which it binds has at least 70% amino acid sequence identity. For example ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0081] In some embodiments, the plurality of non-overlapping amino acid sequences are provided in tandem in the amino acid sequences of: target-binding peptides / peptides, or peptides / peptides of target-binding peptide / peptide complexes. In some embodiments, the plurality of non-overlapping amino acid sequences are linked to each other via adapter sequences.

[0082] The connector sequence is known to those skilled in the art, and for example in Chen et al. As described in Adv Drug Deliv Rev. (2013) 65(10):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of amino acid sequences linked by the linker sequence. Flexible linkers are known to those skilled in the art and are described in Chen et al.Several flexible linkers were identified in Adv Drug Deliv Rev. (2013) 65(10):1357-1369. Flexible linker sequences typically contain a high proportion of glycine and / or serine residues. In some embodiments, the linker sequence contains at least one glycine residue and / or at least one serine residue. In some embodiments, the linker contains or consists of glycine and serine residues. In some embodiments, the linker sequence has the following structure: (G x S)n or (G x S) n G m Where G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1, 2 or 3.

[0083] In some embodiments, the connector sequence comprises one or more of the sequence motif G4S (SEQ ID NO: 66). For example 1, 2, 3, 4, 5, or 6 copies For example (In tandem). In some embodiments, the linker sequence comprises or consists of the following: (G4S)2 (SEQ ID NO:67), (G4S)3 (SEQ ID NO:68), or (G4S)4 (SEQ ID NO:69). In one embodiment, the length of the linker sequence is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, or 1 to 30 amino acids.

[0084] In some embodiments, the target-binding portion according to this disclosure includes or is composed of a ligand for a co-stimulatory molecule. Therefore, in some embodiments, the KiH molecule according to this disclosure includes a ligand for a co-stimulatory molecule. The co-stimulatory molecule includes... For example 4-1BB, CD28, OX40, ICOS, and CD27, and ligands used for co-stimulatory molecules include For example 4-1BBL, CD86, CD80, OX40L ICOSL, and CD70. As used herein, “ligands for co-stimulatory molecules” refers to any entity to which the co-stimulatory molecule binds.

[0085] In some embodiments, the target-binding portion according to this disclosure comprises or is composed of a ligand for 4-1BB, comprising an amino acid sequence corresponding to an extracellular domain of 4-1BBL or a fragment thereof. The extracellular domain of 4-1BBL is formed by positions 24 to 186 of the following: UniProtKB: Q07011-1, v1.

[0086] 4-1BBL (or “4-1BB ligand” or “CD137L”) is a member of the costimulatory TNF ligand family that costimulates T cell proliferation and cytokine production. Costimulatory TNF family ligands can costimulate TCR signaling upon interaction with their corresponding TNF receptors, and this interaction leads to the recruitment of TNFR-associated factor (TRAF), thereby initiating a signaling cascade that leads to T cell activation. 4-1BBL is a type II transmembrane protein. Full-length or complete-length 4-1BBL with the amino acid sequence shown in UniProt accession number P41273 (entry version 153) has been described forming a trimer on the cell surface. Trimer formation is facilitated by a specific motif of the 4-1BBL extracellular domain. This motif is designated herein as the “trimerizing region”. Amino acids 50-254 of the human 4-1BBL sequence (SEQ ID NO:57) form the extracellular domain of 4-1BBL, but even fragments of it can form trimers.

[0087] "Extracellular domains" are extensions into the extracellular space. Right now The extracellular domain (the domain of a membrane protein in the extracellular space) is also called the "extracellular domain". As defined herein, the extracellular domain of 4-1BBL refers to the portion of the 4-1BBL protein, particularly the human 4-1BBL protein (UniProt accession number P41273 (entry version 153)), that extends into the extracellular space, but also includes shorter portions or fragments thereof responsible for trimerization and binding to the corresponding receptor 4-1BB. In a specific aspect of this disclosure, the extracellular domain of 4-1BBL is formed by positions 24 to 186 of the following: UniProtKB: Q07011-1, v1.

[0088] Therefore, the term "extracellular domain of 4-1BBL or a fragment thereof" refers to the extracellular domain of 4-1BBL, or the portion that can bind to 4-1BB and is capable of trimerization. In a specific aspect of this disclosure, the term "extracellular domain of 4-1BBL or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO:57 (amino acids 50-254 of human 4-1BBL), SEQ ID NO:58 (amino acids 71-254 of human 4-1BBL), SEQ ID NO:59 (amino acids 85-254 of human 4-1BBL), SEQ ID NO:60 (amino acids 80-254 of human 4-1BBL), SEQ ID NO:61 (amino acids 52-254 of human 4-1BBL), SEQ ID NO:62 (amino acids 71-248 of human 4-1BBL), SEQ ID NO:63 (amino acids 85-248 of human 4-1BBL), SEQ ID NO:64 (amino acids 80-248 of human 4-1BBL), and SEQ ID NO:65 (amino acids 52-248 of human 4-1BBL).

[0089] In some embodiments, the target-binding portion according to this disclosure comprises or is composed of a polypeptide having an amino acid sequence having at least 70% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65. For example ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0090] In some embodiments, the target binding portion according to this disclosure includes having a plurality of ( For example A polypeptide or composed of two, three or more non-overlapping amino acid sequences, each non-overlapping amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the target interaction region of the interacting chaperone used for the target molecule. For example(≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity). For example, in some embodiments described herein, the KiH molecule of this disclosure comprises a 4-1BB binding moiety containing three amino acid sequences, each corresponding to an amino acid sequence of the extracellular domain of 4-1BBL or a fragment thereof.

[0091] According to this disclosure, the target binding portion includes having multiple ( For example A polypeptide or composed of two, three or more non-overlapping amino acid sequences, each non-overlapping amino acid sequence having at least 70% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65. For example (One of the following: ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity).

[0092] In some embodiments, the plurality of non-overlapping amino acid sequences are provided in tandem in the amino acid sequence of the peptide of the target-binding portion. In some embodiments, the plurality of non-overlapping amino acid sequences are linked to each other via a linker sequence. For example, in some embodiments described herein, the KiH molecule of this disclosure comprises a 4-1BB binding portion of a peptide comprising two amino acid sequences corresponding to an extracellular domain or fragment thereof of 4-1BBL linked by a linker sequence.

[0093] In some embodiments, the target-binding portion according to this disclosure comprises or is composed of a polypeptide containing an amino acid sequence having at least 70% amino acid sequence identity with an amino acid sequence selected from SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76 and SEQ ID NO:77. For example≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0094] In some embodiments, the target-binding moiety according to this disclosure comprises or consists of: a polypeptide complex formed by protein:protein interactions between the constituent peptides / peptides of the target-binding moiety. For example, in some embodiments described herein, the KiH molecule of this disclosure comprises a target-binding moiety consisting of: (i) a polypeptide comprising two amino acid sequences corresponding to an extracellular domain of 4-1BBL or a fragment thereof linked by a linker sequence, and (ii) a polypeptide comprising an extracellular domain of 4-1BBL or a fragment thereof. In such embodiments, polypeptides (i) and (ii) associate to form the complete target-binding moiety.

[0095] It should be understood that, in some embodiments, the constituent polypeptides of the KiH molecule according to this disclosure contain all or part of the amino acid sequence that forms the target-binding portion.

[0096] In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a target-binding portion. In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a target-binding peptide / peptide. For example, in some embodiments described herein, the KiH molecule includes a polypeptide containing a ligand for 4-1BB, comprising an amino acid sequence corresponding to the amino acid sequence of the extracellular domain of 4-1BBL.

[0097] In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a portion of the target-binding portion. In some embodiments, the constituent polypeptide of the KiH molecule according to this disclosure includes a component / fragment of the target-binding portion.

[0098] In some embodiments, the constituent polypeptides forming the target-binding portion of the polypeptide complex according to this disclosure include one or more regions that promote / enhance protein-protein interactions between the polypeptides. For example, in some embodiments, one polypeptide may include a CH1 region and another polypeptide may include a CL region.

[0099] For example, in some embodiments described herein, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to FAP, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to FAP. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to BCMA, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to BCMA. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion consisting of an antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to CD19, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to CD19. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion composed of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to CEA, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to CEA. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion composed of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to HER2, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to HER2. In some embodiments, the KiH molecule of this disclosure comprises an antigen-binding portion composed of an antigen-binding polypeptide, the antigen-binding polypeptide comprising (i) a polypeptide comprising a VH region and a CH1 region of an antibody that binds to PD-L1, and (ii) a polypeptide comprising a VL region and a CL region of an antibody that binds to PD-L1.

[0100] As further examples, in some embodiments described herein, the KiH molecule of this disclosure comprises a 4-1BB binding moiety consisting of: (i) a polypeptide comprising two amino acid sequences corresponding to an extracellular domain or a fragment thereof of 4-1BBL linked by a linker sequence, followed by a CL region; and (ii) a polypeptide comprising an extracellular domain or a fragment thereof of 4-1BBL, followed by a CH1 region.

[0101] In some embodiments, the CL region and / or CH1 region may contain modifications to promote their association. In some embodiments, the CL region contains modifications to promote association with the CH1 region. In some embodiments, the CH1 region contains modifications to promote association with the CL region. In some embodiments, the CH1 region and CL region contain paired modifications to promote their association. Such modifications include modifications to promote electrostatic interactions between the CH1 region and the CL region. For example, modifications may introduce amino acid residues that participate in the formation of a salt bridge between the polypeptide containing the CH1 region and the polypeptide containing the CL region.

[0102] Paired modifications that promote association between the CH1 and CL regions are described in For example WO 2015 / 150447 A1 is hereby incorporated in its entirety by reference. Such modifications include CH1-block modifications K147E and K213E, and CL-block modifications E123R and Q124K (EU numbers). In this example, the E147 COO- group is associated with the K124 NH3 group. + Salt bridges are formed between the groups, and the E213 COO- group interacts with the R123 NH3 group. + Another salt bridge is formed between the groups, thereby stabilizing the interaction between the CH1 and CL regions.

[0103] In some embodiments, the CH1 region comprising modifications to promote association with the CL region has at least 70% amino acid sequence identity with SEQ ID NO:81. For example The amino acid sequence has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and includes K147E and K213E. In some embodiments, the CH1 region has at least 70% amino acid sequence identity with SEQ ID NO:82. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity, and containing E147 and E213.

[0104] In some embodiments, the CL region comprising modifications to promote association with the CH1 region has at least 70% amino acid sequence identity with SEQ ID NO:82. For exampleThe amino acid sequence has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, and includes E123R and Q124K. In some embodiments, the CL region has at least 70% amino acid sequence identity with SEQ ID NO:82. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity, and containing R123 and K124.

[0105] In some embodiments, the target-binding portion according to this disclosure comprises: (i) a polypeptide containing at least 70% amino acid sequence identity with SEQ ID NO:81. For example The amino acid sequence comprises (≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity) and includes E147 and E213, and (ii) a polypeptide comprising at least 70% amino acid sequence identity with SEQ ID NO:82. For example The amino acid sequence of ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity, and containing R123 and K124.

[0106] In some embodiments, the target binding portion according to this disclosure comprises or consists of the following: (i) having at least 70% amino acid sequence identity with SEQ ID NO:78 ( For example (i) a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:80; and (ii) a polypeptide having at least 70% amino acid sequence identity with SEQ ID NO:80. For example A polypeptide with ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0107] In some embodiments, the KiH molecule according to this disclosure comprises: (i) having at least 70% amino acid sequence identity with SEQ ID NO:78 ( For example (i) a polypeptide having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity; and (ii) a polypeptide having at least 70% amino acid sequence identity with SEQ ID NO:79. For example A polypeptide with ≥75%, ≥80%, ≥85%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100% sequence identity.

[0108] The target molecule used for the target binding moiety according to this disclosure can be any target molecule. In some embodiments, the target molecule can be a peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. The target molecule can be expressed at the cell surface of a cell expressing the target molecule.

[0109] In some embodiments, the target molecule is an antigen. As used herein, an antigen is a molecule capable of evoking an immune response in a subject.

[0110] In some embodiments, the target molecule is a disease-associated antigen. A “disease-associated antigen” is an antigen whose presence indicates a given disease / disease state, or whose elevated levels are positively correlated with a given disease / disease state. A disease-associated antigen may be an antigen whose expression is associated with the development, progression, or severity of symptoms of a given disease. Disease-associated antigens may be associated with the etiology or pathology of a disease, or may be abnormally expressed due to the disease. Disease-associated antigens can be antigens of infectious agents or pathogens, cancer-associated antigens, or antigens associated with autoimmune diseases.

[0111] In some embodiments, disease-associated antigens are antigens of pathogens. Pathogens can be prokaryotes (bacteria), eukaryotes (…), or… For example Protozoa, worms, fungi), viruses, or prions. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus. For example As described above, the virus is a pathogen. In some embodiments, the pathogen is bacteria.

[0112] In some embodiments, the target molecule is a cancer-associated antigen. A cancer-associated antigen is an antigen that is expressed or overexpressed and associated with cancer. In some embodiments, the cancer-associated antigen is a receptor molecule. For exampleCell surface receptors. In some embodiments, cancer-associated antigens are cell signaling molecules. For example Cytokines, chemokines, interferons, interleukins, or lymphokines. In some embodiments, cancer-associated antigens are growth factors or hormones. In some embodiments, cancer-associated antigens are viral antigens. Cancer cell antigens can be abnormally expressed by cancer cells ( For example Cancer cell antigens can be expressed at anomalous locations or by cancer cells in anomalous structures. Cancer cell antigens may be able to elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (…). Right now Cancer cell antigens are cancer cell surface antigens. In some embodiments, a portion of the antigen bound by the antigen-binding molecule described herein is displayed on the outer surface of the cancer cell. Right now Extracellularly. Cancer cell antigens can be cancer-associated antigens. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of cancer symptoms. Cancer-associated antigens may be associated with the etiology or pathology of cancer, or may be aberrantly expressed due to cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated by cancer cells (…). For example Antigens at the RNA and / or protein levels, For example Compared with comparable non-cancer cells ( For example The levels expressed by cancer-associated antigens are compared to those expressed by non-cancer cells derived from the same tissue / cell type. In some embodiments, cancer-associated antigens may be preferentially expressed by cancer cells and not by comparable non-cancer cells. For example (Non-cancerous cells derived from the same tissue / cell type) are expressed. In some embodiments, cancer-associated antigens may be products of mutated oncogenes or mutated tumor suppressor genes. In some embodiments, cancer-associated antigens may be products of overexpressed cellular proteins, cancer antigens produced by oncopathogenic viruses, carcinoembryonic antigens, or cell surface glycolipids or glycoproteins.

[0113] Cancer-associated antigens are reviewed in the following literature: Zarour HM, DeLeo A, Finn OJ, et al. Categories of Tumor Antigens.In: Kufe DW, Pollock RE, Weichselbaum RR, Holland-Frei Cancer Medicine, ed., et al., Vol. 6 Hamilton (ON): BC Decker; 2003. Cancer-associated antigens include carcinoembryonic antigens: CEA, immature laminin receptor, TAG-72; tumor virus antigens, such as HPV E6 and E7; and overexpressed proteins: fibroblast activation protein (FAP), B... Cellular maturation antigen (BCMA), CD19, HER2 / neu, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, telomerase, mesothelin, SAP-1, survivin; Cancer testis antigens: BAGE, CAGE, GAGE, MAGE, SAGE, XAGE, CT9, CT10, NY-ESO-1, PRAME, SSX-2; Lineage-restricted antigens: MART1, Gp100, tyrosinase, TRP-½, MC1R, prostate-specific antigen; Mutant antigens: β-catenin, BRCA½, CDK4, CML66, fibronectin, MART-2, p53, Ras, TGF-βRII; Post-translational altered antigens: MUC1; Idiotypic antigens: immunoglobulins, TCR. Other cancer cell antigens include heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), glucose regulatory protein 78 (GRP78), vimentin, nucleolar protein, fetal pancreatic acinar protein (FAPP), alkaline phosphatase placental-like protein 2 (ALPPL-2), siglec-5, stress-induced phosphoprotein 1 (STIP1), protein tyrosine kinase 7 (PTK7), and cyclophilic protein B. In some embodiments, the cancer cell antigen is the cancer cell antigen described in Zhao and Cao, Front Immunol. (2019) 10:2250, which is incorporated herein by reference in its entirety.

[0114] In some embodiments, the target molecule is selected from FAP, BCMA, CD19, CEA, HER2, and PD-L1. In some embodiments, the target molecule is FAP. In some embodiments, the target molecule is BCMA. In some embodiments, the target molecule is CD19. In some embodiments, the target molecule is CEA. In some embodiments, the target molecule is HER2. In some embodiments, the target molecule is PD-L1.

[0115] In some embodiments, the target molecule is an immune cell surface molecule. An immune cell surface molecule is any molecule expressed in or at the cell membrane of an immune cell. In some embodiments, a portion of the immune cell surface molecule bound by the antigen-binding portion is located on the outer surface of the immune cell. Right now(Extracellular). Immune cell surface molecules can be expressed at the cell surface of any immune cell. In some embodiments, immune cells can be hematopoietic cells. For example Neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. Lymphocytes can be... For example T cells, B cells, natural killer (NK) cells, NKT cells, or innate lymphoid cells (ILCs) or their precursors ( For example (Thymocytes or pre-B cells). Immune cells can express CD3 polypeptide (...). For example (CD3γ, CD3ε, CD3ζ, or CD3δ), TCR peptides (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the immune cells are T cells. For example CD3+ T cells. In some embodiments, T cells are CD3+, CD4+ T cells. In some embodiments, T cells are CD3+, CD8+ T cells. In some embodiments, T cells are T helper cells (TH cells). In some embodiments, T cells are cytotoxic T cells (Cytotoxic T cells). For example Cytotoxic T lymphocytes (CTLs). In some embodiments, the immune cells are T cells or NK cells.

[0116] In some embodiments, the surface molecules of immune cells may be CD3-TCR complex peptides. For example TCRα, TCRβ, TCRγ, TCRδ, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, TRDC, CD3ε, CD3δ, CD3γ, CD3ζ, or CD3η. In some embodiments, the immune cell surface molecules are CD3, CD8, CD4, or CD28. In some embodiments, the immune cell surface molecules are checkpoint molecules (… For example PD-1, CTLA-4, LAG-3, TIM-3, VISTA, TIGIT, or BTLA), or ligands for checkpoint molecules ( For example PD-L1, PD-L2, CD80, CD86, MHC class I, MHC class II, galactoglobulin 9, VSIG3, VSIG8, LRIG1, PSGL1, CD155, or HVEM. In some embodiments, the immune cell surface molecules are co-stimulatory molecules (PD-L1, PD-L2, CD80, CD86, MHC class I, MHC class II, galactoglobulin 9, VSIG3, VSIG8, LRIG1, PSGL1, CD155, or HVEM). For example CD28, OX40, 4-1BB, ICOS, or CD27), or ligands for co-stimulatory molecules ( For exampleCD86, CD80, OX40L 4-1BBL, ICOSL, or CD70.

[0117] It should be understood that the KiH molecule of this disclosure is preferably multispecific. "Multispecific" means that the molecule binds to more than one target molecule. The multispecific KiH molecule of this disclosure contains at least two different target-binding moieties.

[0118] For example, the multispecific KiH molecule described herein comprises an FAP-binding moiety (specifically, the Fab moiety that binds FAP) and a 4-1BB-binding moiety (specifically, the 4-1BB-derived 4-1BBL-binding moiety). In some embodiments, the multispecific KiH molecule comprises an FAP-binding moiety ( For example The Fab section of the FAP assembly and the 4-1BB assembly section ( For example 4-1BBL-derived 4-1BB binding moiety). In some embodiments, the multispecific KiH molecule includes a BCMA binding moiety ( For example Combining the Fab section of BCMA and the 4-1BB section ( For example 4-1BBL-derived 4-1BB binding moiety). In some embodiments, the multispecific KiH molecule includes a CD19 binding moiety ( For example (Combined with the Fab section of CD19) and the 4-1BB section ( For example 4-1BBL-derived 4-1BB binding moiety). In some embodiments, the multispecific KiH molecule includes a CEA binding moiety ( For example (Combining the Fab portion of CEA) and the 4-1BB portion ( For example 4-1BBL-derived 4-1BB binding moiety). In some embodiments, the multispecific KiH molecule includes a HER2 binding moiety ( For example (Combining the Fab portion of HER2) and the 4-1BB binding portion ( For example 4-1BBL-derived 4-1BB binding moiety). In some embodiments, the multispecific KiH molecule includes a HER2 binding moiety ( For example (combining the Fab portion of PD-L1) and the PD-L1 bonding portion ( For example (4-1BBL derived from 4-1BB combined part).

[0119] It should be understood that multispecific KiH molecules are at least bispecific. The term "bispecific" means that a KiH molecule binds to at least two distinct targets. In some embodiments, the KiH molecule is bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, heptaspecific, octaspecific, ninaspecific, or decaspecific.

[0120] According to this disclosure, the multispecific KiH molecule exhibits at least monovalent binding with a first target molecule and also at least monovalent binding with a second target molecule. Binding valence refers to the number of binding sites in the KiH molecule for a given target molecule.

[0121] In some aspects and embodiments, the KiH molecule according to this disclosure may comprise a polypeptide complex formed by protein-protein interactions between polypeptides having structures (a) and (b) according to one of (1) to (7) below: (1) (a) N-terminus-[...]-[CH3 region containing pestle modification]-[...]-C-terminus (b) N-terminus-[...]-[CH3 region containing mortar modification]-[...]-C-terminus (2) (a) N-terminus-[...]-[CH2 region]-[CH3 region containing pestle modification]-[...]-C-terminus (b) N-terminus-[...]-[CH2 region]-[CH3 region containing mortar modification]-[...]-C-terminus (3) (a) N-terminus-[...]-[target-binding region / fragment of target-binding region]-[...]-[CH3 region containing pestle modification]-[...]-C-terminus (b) N-terminus-[...]-[target-binding region / fragment of target-binding region]-[...]-[CH3 region containing mortar modification]-[...]-C-terminus (4) (a) N-terminus-[...]-[target-binding region / fragment of target-binding region]-[...]-[CH3 region containing pestle modification]-[...]-C-terminus (b) N-terminus-[...]-[target-binding region / fragment of target-binding region]-[...]-[CH3 region containing mortar modification]-[...]-C-terminus (5) (a) N-terminus-[...]-[antigen-binding peptide / polypeptide]-[...]-[CH3 region containing acetoside modification]-[...]-C-terminus (b) N-terminus-[...]-[antigen-binding region / fragment of antigen-binding region]-[...]-[CH3 region containing club-modified region]-[...]-C-terminus (6) (a) N-terminus-[...]-[target-binding peptide / peptide containing an amino acid sequence corresponding to the amino acid sequence of the target interaction region for the target chaperone]-[...]-[containing a gluten-modified CH3 region]-[...]-C-terminus (b) N-terminus - [VH / VL region of antibody binding to target antigen] - [...] - [CH3 region containing acetonitrile modification] - [...] - C-terminus (7) (a) N-terminus-[...]-[target-binding peptide / peptide containing an amino acid sequence corresponding to the amino acid sequence of the target interaction region for the target chaperone]-[...]-[CH3 region containing acetochlor modification]-[...]-C-terminus (b) N-terminus-[...]-[VH / VL region of antibody binding to target antigen]-[...]-[CH3 region containing thallium modification]-[...]-C-terminus As used in the representation of polypeptide structures in this article, "[...]" indicates the optional presence of other amino acid sequences / protein domains. For example, in the structure above (1)(a), upstream of the amino acid sequence in the CH3 region ( Right now (In the N-terminal direction of the CH3 region), prior to the N-terminus of the polypeptide, other amino acid sequences / protein domains may optionally be present. Furthermore, as used in the representation of polypeptide structures herein, "-" indicates an optional linker sequence (…). For example (e.g., the linker sequence described above). For example, protein domains can be linked to each other via linker sequences.

[0122] It should be understood that the KiH molecule according to this disclosure may contain other polypeptides in addition to those polypeptides that conform to (a) and (b) of (1) to (7) above. For example, in conjunction with (7) above, the KiH molecule may further contain polypeptides that provide the relevant antibody VH or VL region (which is not provided in the polypeptide of (b)) to form a complete antigen-binding Fv moiety.

[0123] Cells containing nucleic acids encoding polypeptides according to this disclosure

[0124] Methods for producing KiH molecules involve culturing cells containing nucleic acids that encode the constituent polypeptides of the KiH molecule.

[0125] This disclosure provides a method for culturing cells containing a polypeptide encoded according to this disclosure. For example The KiH molecule described herein comprises a polypeptide or a variety of nucleic acids. In some embodiments, the nucleic acid comprises or is composed of DNA and / or RNA.

[0126] The disclosed polypeptides can be produced intracellularly via the translation of RNA encoding the polypeptide. The disclosed polypeptides can be produced intracellularly via transcription from nucleic acids encoding the polypeptide and subsequent translation of the transcribed RNA.

[0127] In some embodiments, the nucleic acid may be a vector or multiple vectors, or may be contained in or contained within a vector or multiple vectors. As used herein, a "vector" is a nucleic acid molecule used as a medium for transferring exogenous nucleic acids into cells. Thus, a cell according to this disclosure may contain a vector or multiple vectors that contain nucleic acids or multiple nucleic acids according to this disclosure. Constituent polypeptides of molecules of this disclosure may be encoded by nucleic acids provided in different vectors. For example, one or more constituent polypeptides of a KiH molecule according to this disclosure may be encoded by nucleic acids of a first vector, and one or more constituent polypeptides of a KiH molecule may be encoded by nucleic acids of a second vector. In some embodiments, a vector may contain multiple non-overlapping copies of a nucleic acid encoding a constituent polypeptide of a KiH molecule according to this disclosure.

[0128] The vector can facilitate the delivery of nucleic acids encoding polypeptides according to the present disclosure into cells. The vector can be an expression vector containing desired elements for expressing the polypeptide according to the invention. The vector can contain elements that facilitate the integration of nucleic acids into the genomic DNA of the cell in which the vector is introduced.

[0129] A vector can be a carrier used to express nucleic acids in cells. Right now Expression vectors. Such vectors may include a promoter sequence operatively linked to a nucleotide sequence encoding a polypeptide according to the present disclosure. The vector may also include a stop codon ( Right now The nucleotide sequence of the vector is located at the 3' of the nucleotide sequence encoding the recombinant polypeptide and the expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express peptides or polypeptides from vectors according to this disclosure.

[0130] The term "operably linked" can include cases where a nucleic acid encoding a polypeptide according to this disclosure and a regulatory nucleic acid sequence ( For example Promoters and / or enhancers are covalently linked in this manner to place the expression of the nucleic acid encoding the polypeptide under the influence or control of a regulatory nucleic acid sequence (thus forming an expression cassette). Therefore, if the regulatory sequence can influence the transcription of a selected nucleic acid sequence, the regulatory sequence is operatively linked to that nucleic acid sequence. The resulting transcript can then be translated into the desired polypeptide.

[0131] Vectors considered relevant to this disclosure include DNA vectors, RNA vectors, and plasmids. For example Conjugation plasmid ( For example F plasmid, non-conjugating plasmid, R plasmid, col plasmid, episome, viral vector ( For example Retroviral vectors, For example γ-retroviral vector ( For example Vectors derived from murine leukemia virus (MLV) For example SFG vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors, and herpesvirus vectors, transposon-based vectors, and artificial chromosomes ( For example Yeast artificial chromosomes). For example such as Maus et al. As described in Annu Rev Immunol. (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entirety. In some embodiments, the vector according to this disclosure is a lentiviral vector.

[0132] In some embodiments, the vector may be a eukaryotic vector. Right now A vector containing elements necessary for protein expression from a vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, which For example It contains a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0133] It should be understood that when a cell is mentioned in the singular in this article ( Right now "One / the cell") also considers multiple cells / populations of this type of cell.

[0134] According to this disclosure, the cells can be eukaryotic cells. For example Mammalian cells. Mammals can be primates (rhesus monkeys, cynomolgus monkeys, non-human primates, or humans) or non-human mammals (…). For example Rabbits, guinea pigs, rats, mice or other rodents (including any animal in the order Rodentia), cats, dogs, pigs, sheep, goats, and cattle (including dairy cows). For exampleThe cells can be any animal in the order Bos (dairy cow or Bos), horses (including any animal in the family Equidae), donkeys, or non-human primates. In a preferred embodiment, the cells are human cells.

[0135] In some embodiments, the cells are or are derived from cell types commonly used to express peptides for use in human therapies. Exemplary cells are described in For example Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100:3451–3461 (which are incorporated herein by reference in their entirety), and include For example CHO, HEK 293, PER.C6, NSO, and BHK cells. In a preferred embodiment, the cells are or are derived from CHO cells.

[0136] Any suitable method may be used to generate cells to be used in the methods according to this disclosure. Such methods may include nucleic acid transfer for permanent (or permanent) of the transferred nucleic acids. Right now (Stable) or transient expression. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may integrate into the cell's genomic DNA or form part of the cell's genomic DNA. In some embodiments, after introduction into a cell, the nucleic acid encoding the polypeptide of interest may be maintained extrachromosomally.

[0137] Any suitable genetic engineering platform can be used, including gamma retroviral vectors, lentiviral vectors, adenoviral vectors, DNA transfection, transposon-based gene delivery, and RNA transfection, such as Maus. et al. As described in Annu Rev Immunol (2014) 32:189-225, which is hereby incorporated in its entirety by reference. The method also includes... For example The entire contents of Wang and Rivière Mol Ther Oncolytics (2016) 3:16015 are incorporated herein by reference. Suitable methods for introducing nucleic acids / vectors into cells include transduction, transfection, and electroporation.

[0138] Methods for generating KiH molecules

[0139] Aspects and embodiments of this disclosure relate to methods for generating KiH molecules.

[0140] The method involves culturing cells containing nucleic acids encoding constituent peptides of KiH molecules under conditions suitable for expressing peptides and assembling KiH molecules.

[0141] Suitable culture conditions will be apparent to those skilled in the art. Culture conditions for expressing antibodies derived from mammalian cells in culture are described in [the relevant section]. For example Birch and Racher, Adv Drug Deliv Rev. (2006) 58(5-6):671-85 and Li et al. MAbs (2010) 2(5):466-477, these two articles are included here in their entirety by citation. Suitable culture conditions include those applicable to... in vitro Conditions for maintaining the CHO-K1 cell line (ATCC, catalog number CCL-61) in culture.

[0142] Cells are cultured in a cell culture medium containing amino acids, vitamins, inorganic salts, and sugars. In some embodiments, the cell culture medium contains amino acids selected from the following: L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine / L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine. In some embodiments, the cell culture medium contains vitamins and / or vitamin-like substances selected from the following: D-biotin, choline chloride, D-calcium pantothenate, folic acid, inositol, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, and vitamins. B12. In some embodiments, the cell culture medium contains other components selected from the group consisting of calcium chloride, hypoxanthine, ferric nitrate, linoleic acid, putrescine hydrochloride, pyruvate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate, lipoic acid, and thymidine. In some embodiments, the cell culture medium contains D-glucose.

[0143] In some embodiments, the cell culture medium is a cell culture medium suitable for the culture of mammalian cells. Such cell culture media include Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's Modified Eagle medium (DMEM), F-12 medium, DMEM / F12, CD-CHO medium, and PowerCHO medium.

[0144] In a preferred embodiment, the cell culture medium is a suitable cell culture medium for culturing cells used to produce molecules to be used in human therapies. Such culture media include… For example EX-CELL Advanced CHO Fed-Batch culture medium.

[0145] It should be understood that cells are cultured under suitable environmental conditions.

[0146] Cells can be kept at 35.5℃ to 37.5℃. For example Incubate at one of the following temperatures: approximately 35.5°C, approximately 36°C, approximately 36.5°C, approximately 37°C, or approximately 37.5°C.

[0147] Cells can exist in 4% to 10% CO2. For example Incubate in 5% to 8% CO2.

[0148] Cells can For example ≥90% humidity For example Cultivate at approximately 95% humidity.

[0149] Cells can be cultured with or without agitation. Agitation can be performed at 75 rpm to 175 rpm. For example 90 rpm to 130 rpm, For example At approximately 110 rpm.

[0150] The pH of cell cultures can be between 6.8 and 7.4. For example The pH is one of about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, or 7.4. In some embodiments, the pH of the cell culture is about 7.0. In some embodiments, the pH of the cell culture is about 7.2.

[0151] Cell culture can be performed in a bioreactor that provides an appropriate supply of nutrients, air / oxygen, and / or growth factors. Environmental conditions, such as pH, oxygen, inflow / outflow rates, and agitation within the vessel, can be monitored and controlled in the bioreactor to provide optimal conditions for the cultured cells.

[0152] The culture can be a continuous culture, wherein cell culture medium is continuously fed into the cell culture vessel and the cultured cells are continuously discharged from the cell culture vessel. In some embodiments, the culture can be a batch culture using a closed system and a limited amount of cell culture medium. In some embodiments, the culture can be a fed batch culture, wherein cell culture medium is supplied to the cell culture vessel during the culture, but unlike continuous culture, no material is removed from the cell culture vessel during the cell culture process.

[0153] In some embodiments, the method includes adding one or more components to the cell culture during the culture period. In some embodiments, the method includes adding cell culture medium to the cell culture during the culture period. In some embodiments, the method includes adding nutrients to the cell culture during the culture period. In some embodiments, the method includes adding amino acids to the cell culture during the culture period.

[0154] In some embodiments, as described in the preceding paragraphs, the components added to the cell culture during the culture period do not contain cysteine ​​( Right now It does not contain substances having the following formula: HOOC-CH(-NH2)-CH2-SH - OOC-CH(-N + H3)-CH2-SH、 - OOC-CH(-NH2)-CH2-SH (or its salt) or HOOC-CH(-N + H3)-CH2-SH (or its salts)). That is, in some embodiments, the cell culture medium, nutrients and / or amino acids added to the cell culture during the culture period do not contain cysteine ​​(H3)-CH2-SH (or its salts). Right now (Does not contain cysteine).

[0155] After the cell culture period, KiH molecules can... For example Separated / isolated / purified from cell cultures or cell culture media.

[0156] KiH molecules are typically secreted from cells in a culture into the cell culture medium. The secreted KiH molecules can be separated from the cells by the culture medium (…). For example The secreted KiH molecules are collected by centrifugation and then isolated / purified from the culture medium.

[0157] Techniques for isolating / purifying antigen-binding molecules from compositions containing heterogeneous populations of proteins are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Birch and Racher, Adv Drug Deliv Rev. (2006) 58(5-6):671-85, and Murphy et al. All of these references are included here in their entirety by way of citation in Antibody Technology Journal (2016) 6:17-32.

[0158] Large-scale purification of antigen-binding molecules is typically based on affinity chromatography, and in many cases, protein A or G affinity purification is used. Purification may alternatively or additionally include purification by anion / cation exchange chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography, as is well known to those skilled in the art.

[0159] Various purification steps are designed to remove contaminant proteins from cells or culture media to ppm levels and to reduce DNA to ppb levels. Depending on the process used, there may be additional specific contaminants to be removed. For example (Leachable protein A / G). Purification may include filtration ( For example (Using a 0.22 µm filter) to remove potential biocontaminants.

[0160] In addition to contaminants, it may be necessary or required to remove undesirable products such as mortar-mortar molecules, pestle-pepper molecules, half-mortar molecules and half-pepper molecules, as well as other undesirable products such as aggregates and degradation products.

[0161] As used herein, a “mortar-mortar molecule” is a molecule comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a CH3 region containing a mortar-modified region and a second polypeptide comprising a CH3 region containing a mortar-modified region. In some embodiments, the first polypeptide and the second polypeptide are identical, such that the “mortar-mortar molecule” is a homologous polypeptide complex. For example Homodimer). Similarly, a "pestle-pestle molecule" is a molecule comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a pestle-modified CH3 region and a second polypeptide comprising a pestle-modified CH3 region. In some embodiments, the first and second polypeptides are identical, such that the "pestle-pestle molecule" is a homologous polypeptide complex ( For example (Homodimer). A "half-mortar molecule" refers to a polypeptide / polypeptide complex containing a mortar-modified CH3 region that does not associate with another polypeptide containing a mortar-modified CH3 region to form a KiH molecule, and the polypeptide / polypeptide complex is not a mortar-mortar molecule. Similarly, a "half-mortar molecule" refers to a polypeptide / polypeptide complex containing a mortar-modified CH3 region that does not associate with another polypeptide containing a mortar-modified CH3 region to form a KiH molecule, and the polypeptide / polypeptide complex is not a mortar-mortar molecule.

[0162] In conjunction with various aspects and embodiments of this disclosure, the “undesired product” may be selected from mortar-mortar molecules, pestle-pepper molecules, half-mortar molecules, and half-pepper molecules.

[0163] In some embodiments, the separation or purification of KiH molecules according to this disclosure includes separation / purification performed by one or more of the following: affinity chromatography ( For example Methods disclosed herein include protein G chromatography or protein A chromatography, size exclusion chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography, and ion exchange chromatography. In some embodiments, the methods of this disclosure include separating / isolating / purifying polypeptide complexes by size exclusion chromatography and / or capillary electrophoresis.

[0164] After separation / purification, KiH molecules can be provided in a suitable buffer. For example For storage purposes. As used herein, "buffer solution" refers to a buffer solution that is resistant to pH changes by the action of its acid-base conjugate components. Compositions containing KiH molecules according to this disclosure may contain KiH molecules in the buffer solution.

[0165] Examples of suitable buffers include acetate, histidine, histidine-arginine, histidine-methionine, and other organic acid buffers. KiH molecules can be exchanged for the buffer of interest via buffer dialysis.

[0166] In some embodiments, the method of this disclosure includes formulating a composition according to the KiH molecule of this disclosure. example like Pharmaceutical composition. In some embodiments, the method includes mixing a KiH molecule or a composition containing a KiH molecule with a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.

[0167] Cysteine ​​concentration

[0168] Aspects and embodiments of this disclosure relate to a method for generating KiH molecules, the method comprising culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules in the presence of a substance containing thiol / selenool groups at certain concentrations.

[0169] This disclosure is partly based on the inventors' discovery that substances containing relatively low concentrations of thiol / selenool groups ( For example Cells cultured in a cell culture medium containing cysteine ​​(a type of cysteine) containing nucleic acids encoding the constituent polypeptide of the KiH molecule showed a higher yield than undesirable products ( For example Morphine-morchine molecules, ½-morchine molecules, and / or ½-morchine molecules are more conducive to the production of such molecules. As experimental examples have shown, maintaining relatively low levels of cysteine ​​in cell cultures increases the proportion of KiH molecules among molecules expressed from cells.

[0170] Not wanting to be bound by any particular theory, the inventors believe that the cell culture medium contains a relatively low concentration of substances containing thiol groups. For example Under the condition of cysteine, the increased formation of KiH molecules may be due to the increased availability of intermolecular disulfide bonds formed by the thiol groups of cysteine ​​residues in the CH3 mortise and CH3 saccharide regions.

[0171] Therefore, this disclosure provides for the production of KiH molecules in cell culture from cells containing nucleic acids encoding constituent polypeptides of KiH molecules, wherein substances containing thiol / selenool groups are provided and / or maintained at low levels. For example The concentration of cysteine.

[0172] As used herein, "thiol group" refers to a group having the structure "R-SH", and "selenool group" refers to a group having the structure "R-SeH". In both cases, "R" represents an alkyl or other organic substituent. Substances containing thiol groups include... For example Cysteine, N-acetylcysteine, N-acetyl-L-cysteine ​​amide (NACA), S-sulfocysteine, N,N′-diacetyl-L-cysteine, N,N′-diacetyl-L-cysteine ​​dimethyl ester (DACDM), glutathione, dithiothreitol, and 2-mercaptoethanol. Substances containing selenoyl groups include... For example Selenocysteine.

[0173] More specifically, aspects and embodiments of this disclosure relate to a method for generating KiH molecules, the method comprising culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules in the presence of certain concentrations of cysteine.

[0174] In this article, "cysteine" refers to a substance having a structure of (1) or (2):

[0175] Where “X” represents O or N.

[0176] Substances having a structure of (1) or (2) For example They are contained in cysteine ​​in their L and D enantiomers ( Right now , HOOC-CH(-NH2)-CH2-SH), OOC-CH(-N + H3)-CH2-SH、 - OOC-CH(-NH2)-CH2-SH (and its salts) and HOOC-CH(-N + H3)-CH2-SH (and its salts). This substance also contains cystine ( Right nowIn the case of L-cysteine ​​and D-cysteine, the cysteine ​​is a dimer of cysteine ​​formed by a disulfide bond between the thiol groups of two cysteine ​​monomers. Cystine can be introduced into cells via the xCT transporter and is subsequently reduced to cysteine ​​by cysteine ​​reductase.

[0177] According to the solution of this disclosure ( For example The concentration of cysteine ​​in cell culture medium can be calculated as the concentration of substances having the structure described in (1) or (2) above. For example, the calculation of cysteine ​​concentration also takes into account cystine ( For example The concentration of L-cysteine.

[0178] Therefore, a given concentration of cystine is equivalent to a cysteine ​​concentration that is twice the concentration of cystine. For example, according to this disclosure, a cell culture medium containing 1 mM cystine (and lacking any other form of cysteine) is considered to have a cysteine ​​concentration of 2 mM.

[0179] Cell cultures according to this disclosure may contain both monomeric cysteine ​​and cysteine. In such embodiments, the concentration of cysteine ​​in the cell culture is determined by combining the concentrations of monomeric cysteine ​​and cysteine. For example, according to this disclosure, a cell culture medium containing cysteine ​​at a concentration of 1 mM and monomeric cysteine ​​at a concentration of 2 mM is considered to have a cumulative concentration of 4 mM cysteine ​​molar equivalents.

[0180] In aspects and embodiments of this disclosure, a method for generating KiH molecules includes culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules in a cell culture medium containing cysteine ​​at a concentration of less than 8 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is >0 mM and <8 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥0.5 mM and <8 mM. In a preferred embodiment, the concentration of cysteine ​​in the cell culture medium is less than 6 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is >0 mM and <6 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥1 mM and <6 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥2 mM and ≤5 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥2.5 mM and ≤4.75 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is about 3 mM. In some embodiments, the concentration of cysteine ​​in the cell culture medium is about 4.5 mM.

[0181] In aspects and embodiments of this disclosure, the method includes culturing cells containing nucleic acids encoding constituent polypeptides of the KiH molecule in a cell culture medium containing cysteine ​​at a concentration according to one embodiment of the preceding paragraph for a substantial portion of the culture period.

[0182] That is, in some embodiments, the method includes culturing cells containing nucleic acids encoding constituent polypeptides of the KiH molecule in a cell culture medium containing cysteine ​​at a concentration of less than 8 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is >0 mM and <8 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥0.5 mM and <8 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is less than 6 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is >0 mM and <6 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥1 mM and <6 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥2 mM and ≤5 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is ≥2.5 mM and ≤4.75 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is about 3 mM for most of the culture period. In some embodiments, the concentration of cysteine ​​in the cell culture medium is about 4.5 mM for most of the culture period.

[0183] In this document, "most of the culture period" means >50% of the culture period. For example, in the case of a 14-day culture period, "most of the culture period" means >7 days of the 14-day culture period. In some embodiments, the concentration of cysteine ​​in the culture is maintained at the relevant concentration for most of the culture period.

[0184] In some embodiments, the method includes >50% of the cell culture medium containing cysteine ​​at a concentration of less than 8 mM during the culture period. For example Cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule are cultured within one of the following concentrations: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or within the duration of the culture period). In some embodiments, cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule are cultured within one of the following concentrations: >50% (or within the duration of the culture period). For exampleWithin one of ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or during the duration of the culture period), the concentration of cysteine ​​in the cell culture medium is >0 mM and <8 mM. In some embodiments, within >50% ( For example Within one of ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or during the duration of the culture period), the concentration of cysteine ​​in the cell culture medium is ≥0.5 mM and <8 mM. In some embodiments, within >50% ( For example The cysteine ​​concentration in the cell culture medium is less than 6 mM within one of the following ranges: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%, or throughout the duration of the culture period. In some embodiments, the concentration is less than 6 mM within >50% of the culture period. For example Within one of ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or during the duration of the culture period), the concentration of cysteine ​​in the cell culture medium is >0 mM and <6 mM. In some embodiments, within >50% ( For example Within one of ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or during the duration of the culture period), the concentration of cysteine ​​in the cell culture medium is ≥1 mM and <6 mM. In some embodiments, within >50% ( For example Within one of ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or during the duration of the culture period), the concentration of cysteine ​​in the cell culture medium is ≥2 mM and ≤5 mM. In some embodiments, within >50% ( For example Within one of the following ranges (≥60%, ≥70%, ≥80%, ≥90%, or ≥95%) or during the duration of the culture period, the concentration of cysteine ​​in the cell culture medium is ≥2.5 mM and ≤4.75 mM. In some embodiments, during >50% ( For example The cysteine ​​concentration in the cell culture medium is approximately 3 mM within one of the following ranges: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%, or throughout the duration of the culture period. In some embodiments, during the >50% ( For example The concentration of cysteine ​​in the cell culture medium is approximately 4.5 mM (within one of the following ranges: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%) or during the duration of the culture period.

[0185] In some aspects and embodiments of this disclosure, cell cultures can be established ( Right nowThe method involves "establishing" or "seeding" cells in a cell culture medium containing cysteine ​​at a concentration of less than 8 mM. It should be understood that "establishing" a cell culture or "seeding" cells includes contacting the cells with the cell culture medium. For example In a suitable cell culture container.

[0186] Therefore, in some aspects and embodiments of this disclosure, the method includes seeding cells containing nucleic acids encoding a constituent polypeptide of a KiH molecule in a cell culture medium containing cysteine ​​at a concentration of less than 8 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of ≥0.5 mM and <8 mM. In a preferred embodiment, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of ≥1 mM and <6 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of ≥2 mM and ≤5 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of ≥2.5 mM and ≤4.75 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of about 3 mM. In some embodiments, cells are seeded in a cell culture medium containing cysteine ​​at a concentration of about 4.5 mM.

[0187] In some aspects and embodiments according to this disclosure, the method includes the use of the culture period for most of the time. example like Within one of the following ranges during the culture period (≥60%, ≥70%, ≥80%, ≥90%, or ≥95%), or throughout the duration of the culture period, maintain the cysteine ​​concentration in the cell culture at less than 8 mM. For example ≥0.5 mM and <8 mM, example like Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example At concentrations of approximately 3 mM or 4.5 mM.

[0188] Due to the metabolism and / or degradation of cysteine ​​by cells in the culture, the concentration of cysteine ​​in the cell culture naturally decreases over time. Right now(without adding further cysteine ​​to the culture).

[0189] The concentration of cysteine ​​in a given cell culture can be maintained below a certain threshold by either not adding cysteine ​​to the cell culture during the culture period, or by introducing cysteine ​​into the cell culture only at a concentration not exceeding a relevant threshold during the culture period. For example <8 mM, <6 mM, <5 mM, <4.75 mM). For example, in embodiments where the culture is fed-batch or continuous, the cell culture medium added to the cell culture during the culture period may be deficient in cysteine ​​(…). Right now (may not contain cysteine), or may contain cysteine ​​at a concentration not exceeding a relevant threshold, or may contain cysteine ​​at a concentration such that, after cell culture medium is added to the culture, the concentration of cysteine ​​in the cell culture does not exceed a relevant threshold. Right now , <8mM, <6mM, <5mM or <4.75mM).

[0190] Conversely, by introducing cysteine ​​into the cell culture at a certain concentration during the culture period, the concentration of cysteine ​​in the cell culture after its addition is at or above a relevant threshold or at a certain value, thus maintaining the concentration of cysteine ​​in a given cell culture at or above a certain threshold. For example ≥0.5 mM, ≥1 mM, ≥2 mM) or at a certain value ( For example (approximately 3 mM or 4.5 mM). For example, in embodiments where the culture is fed-batch or continuous, the cell culture medium added to the cell culture during the culture period may contain cysteine ​​at a concentration such that, after the cell culture medium is added to the culture, the concentration of cysteine ​​in the cell culture is one of ≥0.5 mM, ≥1 mM, ≥2 mM, or approximately 3 mM or 4.5 mM.

[0191] When cysteine ​​is provided to cell cultures according to the present disclosure, it can be provided in the form of cell culture medium or cell culture medium supplement or additive, the cysteine ​​comprising monomeric cysteine, cystine, or a cysteine / cystine metabolic precursor or derivative capable of being metabolized to cysteine ​​by cells in the culture. The cysteine / cystine metabolic precursor or derivative according to the present disclosure can be a molecule comprising the structure of (1) or (2):

[0192] Where “X” represents O or N.

[0193] Metabolic precursors and derivatives of cysteine / cystine considered according to this disclosure include N-acetyl-cysteine ​​(NAC), N-acetyl-L-cysteine ​​amide (NACA), S-sulfocysteine, N,N′-diacetyl-L-cysteine ​​and N,N′-diacetyl-L-cysteine ​​dimethyl ester (DACDM).

[0194] osmolar concentration

[0195] Aspects and embodiments of this disclosure relate to a method for generating KiH molecules, the method comprising culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules at a certain osmolar concentration.

[0196] Osmotic molar concentration refers to the number of solute particles per kilogram of solvent. It is expressed as the osmotic molar concentration of the solute per kilogram of solvent. For example The number of milliosmol (mOsmol) For example (mOsmol / kg). Osmotic molar concentration is different from isotonic molar concentration, which refers to the number of solute particles per liter of solvent.

[0197] This disclosure is partly based on the inventors' discovery that culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules in a cell culture medium at a certain osmolar concentration increases the yield of such molecules. As experimental examples show, maintaining a relatively high osmolar concentration increases the production of KiH molecules.

[0198] Therefore, in aspects and embodiments of this disclosure, the method for generating KiH molecules includes having a concentration greater than 280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example Cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule are cultured in a cell culture medium with an osmolar concentration of ≥370 mOsmol / kg.

[0199] Based on the various aspects and embodiments described herein (where lower threshold osmolality values ​​are specified), it should be understood that the osmolality values ​​are not high enough to significantly impair cell growth in cell cultures and / or the expression of KiH molecules from such cells according to this disclosure. For example, in some embodiments, the osmolality of the cell culture medium for cell cultures according to this disclosure is <600 mOsmol / kg. For example <550 mOsmol / kg, For example <500 mOsmol / kg, For example <475 mOsmol / kg.

[0200] In some embodiments, the osmolality of the cell culture medium is >280 mOsmol / kg and <450 mOsmol / kg. In a preferred embodiment, the osmolality of the cell culture medium is >300 mOsmol / kg. In some embodiments, the osmolality of the cell culture medium is >300 mOsmol / kg and <420 mOsmol / kg. In some embodiments, the osmolality of the cell culture medium is ≥320 mOsmol / kg and ≤400 mOsmol / kg. In some embodiments, the osmolality of the cell culture medium is ≥350 mOsmol / kg and ≤390 mOsmol / kg. In some embodiments, the osmolality of the cell culture medium is about 370 mOsmol / kg.

[0201] In aspects and embodiments of this disclosure, the method includes culturing cells containing nucleic acids encoding constituent polypeptides of KiH molecules in a cell culture medium having an osmolar concentration according to an embodiment of the preceding paragraph for a substantial portion of the culture period.

[0202] That is, in some embodiments, the method includes culturing cells containing nucleic acids encoding constituent polypeptides of the KiH molecule in a cell culture medium having an osmolality greater than 280 mOsmol / kg for most of the culture period. In some embodiments, the osmolality of the cell culture medium is greater than 300 mOsmol / kg for most of the culture period. In some embodiments, the osmolality of the cell culture medium is ≥320 mOsmol / kg for most of the culture period. In some embodiments, the osmolality of the cell culture medium is ≥350 mOsmol / kg for most of the culture period. In some embodiments, the osmolality of the cell culture medium is ≥370 mOsmol / kg for most of the culture period.

[0203] In some embodiments, the method includes >50% of the cell culture medium having an osmolar concentration greater than 280 mOsmol / kg during the culture period ( For example Cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule are cultured within one of the following concentrations: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95% (or within the duration of the culture period). In some embodiments, cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule are cultured within one of the following concentrations: >50% (or within the duration of the culture period). For exampleThe osmolar concentration of the cell culture medium is greater than 300 mOsmol / kg within one of the following ranges: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%, or throughout the duration of the culture period. In some embodiments, the osmolar concentration of the cell culture medium is greater than 300 mOsmol / kg during the >50% ( For example Within one of the following ranges (≥60%, ≥70%, ≥80%, ≥90%, or ≥95%) or during the duration of the culture period, the osmolar concentration of the cell culture medium is ≥320 mOsmol / kg. In some embodiments, during the >50% ( For example Within one of the following ranges (≥60%, ≥70%, ≥80%, ≥90%, or ≥95%) or during the duration of the culture period, the osmolar concentration of the cell culture medium is ≥350 mOsmol / kg. In some embodiments, during the >50% ( For example The osmolar concentration of the cell culture medium is ≥370 mOsmol / kg within one of the following ranges (≥60%, ≥70%, ≥80%, ≥90%, or ≥95%) or during the duration of the culture period.

[0204] In some aspects and embodiments of this disclosure, cell cultures can be established ( Right now The method may include "seeding" cells in a cell culture medium having an osmolar concentration greater than 280 mOsmol / kg.

[0205] Therefore, in some aspects and embodiments of this disclosure, the method includes seeding cells containing nucleic acids encoding a constituent polypeptide of a KiH molecule in a cell culture medium having an osmolality greater than 280 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality greater than 280 mOsmol / kg and less than 450 mOsmol / kg. In a preferred embodiment, cells are seeded in a cell culture medium having an osmolality greater than 300 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality greater than 300 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality greater than 300 mOsmol / kg and less than 420 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality greater than 320 mOsmol / kg and less than 400 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality greater than 320 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality of ≥350 mOsmol / kg and ≤390 mOsmol / kg. In some embodiments, cells are seeded in a cell culture medium having an osmolality of about 370 mOsmol / kg.

[0206] In some aspects and embodiments according to this disclosure, the method includes the use of the culture period for most of the time. example like Within one of the following ranges during the culture period: ≥60%, ≥70%, ≥80%, ≥90%, or ≥95%, or throughout the duration of the culture period, maintain the osmolality of the cell culture at a level greater than 280 mOsmol / kg. For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example ≥370mOsmol / kg)

[0207] Due to the addition of alkaline substances (usually in the form of sodium salts) for pH control, salt metabolism in the cell culture medium, and / or modification of the product from the osmolar concentration in the cells ( For example The secretion of waste products allows the osmolar concentration of cell cultures to change naturally over time. Right now (In the absence of interventions to manipulate the osmolar concentration of cell cultures).

[0208] An osmolar concentration enhancer can be introduced into the cell culture at a certain amount during the culture period, such that after its addition, the osmolar concentration of the cell culture medium is at or above a relevant threshold or at a certain value, thereby maintaining the osmolar concentration of a given cell culture at or above a certain threshold. For example (>280 mOsmol / kg, >300 mOsmol / kg, ≥320 mOsmol / kg, ≥350 mOsmol / kg, ≥370 mOsmol / kg). For example, in embodiments where the culture is fed-batch or continuous, the cell culture medium added to the cell culture during the culture period may contain an amount of an osmolality enhancer such that, after the cell culture medium is added to the culture, the concentration of the cell culture medium in the cell culture is one of >280 mOsmol / kg, >300 mOsmol / kg, ≥320 mOsmol / kg, ≥350 mOsmol / kg, or ≥370 mOsmol / kg.

[0209] Osmolality enhancers can be any agent that contributes to the osmotic pressure of a solution. Osmolality enhancers include organic compounds (…). For example carbohydrate, For example Glucose, trehalose, galactose wait ) and inorganic compounds, For example Inorganic salts. In some embodiments, the osmolar concentration enhancer is a salt. For example Inorganic salts. In some embodiments, the salt may be selected from: sodium chloride, potassium chloride, sodium bicarbonate, calcium chloride, ferric nitrate, magnesium sulfate, and sodium dihydrogen phosphate. In a preferred embodiment, the osmolality increaser is sodium chloride. When the osmolality increaser is provided to cell cultures according to this disclosure, the osmolality increaser may be provided in the form of a cell culture medium containing the relevant agent, or a cell culture medium supplement or additive containing the relevant agent.

[0210] Conversely, the cell culture medium can be diluted during the culture period ( For example Using a cell culture medium with an osmolality lower than that of the cell culture medium to which it is added, or using buffer or water, such that after dilution, the osmolality of the cell culture medium in the cell culture is at or above a relevant threshold or at a certain value, maintaining the osmolality of a given cell culture at or below a certain threshold. For example <600mOsmol / kg, For example <550 mOsmol / kg, For example <500 mOsmol / kg, For example <475 mOsmol / kg). For example, in embodiments where the culture is performed as a fed-batch or continuous culture, the cell culture medium added to the cell culture during the culture period may have a certain osmolal concentration such that, after the cell culture medium is added to the culture, the osmolal concentration of the cell culture medium is <600 mOsmol / kg. For example <550 mOsmol / kg, For example <500mOsmol / kg, For example <475 mOsmol / kg.

[0211] Further methods and uses

[0212] This disclosure provides further methods related to the generation of KiH molecules.

[0213] A method is provided for increasing the yield of KiH molecules produced in a cell culture from cells containing nucleic acids encoding a constituent polypeptide of KiH molecules, wherein the method includes maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 8 mM. For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example Approximately 3 mM or 4.5 mM). In some embodiments, the method further includes maintaining or adjusting the osmolality of the cell culture medium to greater than 280 mOsmol / kg (approximately 3 mM or 4.5 mM). For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example ≥370 mOsmol / kg).

[0214] A method is also provided for increasing the proportion of KiH molecules in molecules produced by cells containing nucleic acids encoding constituent polypeptides of KiH molecules in cell cultures, wherein the method includes maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 8 mM. For example≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example Approximately 3 mM or 4.5 mM). In some embodiments, the method further includes maintaining or adjusting the osmolar concentration of the cell culture medium to greater than 280 mOsmol / kg (approximately 3 mM or 4.5 mM). For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example ≥370 mOsmol / kg).

[0215] Also provided is a method for reducing undesirable products generated in cell cultures from cells containing nucleic acids encoding constituent polypeptides of the KiH molecule. For example A method for producing cysteine ​​(a molar-to-molar molecule, a 1 / 2 molar molecule, a 1 / 2 molar molecule, or a molar-to-molar molecule), wherein the method comprises maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 8 mM ( For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM For example ≥2.5 mM and <4.75 mM, For example Approximately 3 mM or 4.5 mM). In some embodiments, the method further includes maintaining or adjusting the osmolar concentration of the cell culture medium to greater than 280 mOsmol / kg. For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example ≥370 mOsmol / kg).

[0216] Also provided is a method for reducing unwanted products in cell cultures produced by cells containing nucleic acids encoding constituent polypeptides of KiH molecules. For exampleA method for a ratio of 1 / 2 mol ... For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, example like ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example Approximately 3 mM or 4.5 mM). In some embodiments, the method further includes maintaining or adjusting the osmolar concentration of the cell culture medium to greater than 280 mOsmol / kg (approximately 3 mM or 4.5 mM). For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320mOsmol / kg For example ≥350 mOsmol / kg For example ≥370 mOsmol / kg).

[0217] In some embodiments, "maintaining or regulating the concentration of cysteine ​​in the cell culture medium" may include increasing the concentration of cysteine ​​in the cell culture. In such embodiments, the method may include adding cysteine ​​(… example like It exists in the form of monomeric cysteine, cystine, or metabolic precursors or derivatives of cysteine / cystine that can be metabolized to cysteine. For example As mentioned above, For example N-acetylcysteine ​​(NAC), N-acetyl-L-cysteine ​​amide (NACA), S-sulfocysteine, N,N′-diacetyl-L-cysteine, and N,N′-diacetyl-L-cysteine ​​dimethyl ester can be added to cell cultures. Cysteine ​​can be added to cell cultures by adding a cell culture medium containing cysteine, or by adding a cell culture medium additive containing cysteine.

[0218] In some embodiments, "maintaining or regulating the concentration of cysteine ​​in the cell culture medium" may include reducing the concentration of cysteine ​​in the cell culture. In such embodiments, the method may include using a concentration of cysteine ​​lower than that in the cell culture medium. For example It exists in the form of monomeric cysteine, cystine, or metabolic precursors or derivatives of cysteine / cystine that can be metabolized to cysteine. For example As mentioned above, For example The concentrations of N-acetyl-cysteine ​​(NAC), N-acetyl-L-cysteine ​​amide (NACA), S-sulfocysteine, N,N′-diacetyl-L-cysteine, and N,N′-diacetyl-L-cysteine ​​dimethyl ester are added to the cell culture medium containing cysteine, or to the cell culture medium lacking cysteine ​​(or a suitable buffer or water) (thereby diluting the cysteine ​​in the cell culture medium and thus reducing its concentration).

[0219] In some embodiments, "maintaining or adjusting the osmolar concentration of the cell culture medium" may include increasing the osmolar concentration of the cell culture. In such embodiments, the method may include using an osmolar concentration increasing agent ( For example As mentioned above, For example Organic compounds ( For example carbohydrate, For example Glucose, trehalose or galactose), inorganic compounds ( For example Inorganic salts, For example Sodium chloride) can be added to cell cultures. This can be done by adding cell culture medium (or a suitable buffer or water) containing an osmolality enhancer to the cell culture, or by adding a cell culture medium additive containing an osmolality enhancer to the cell culture.

[0220] In some embodiments, “maintaining or adjusting the osmolality of the cell culture medium” may include reducing the osmolality of the cell culture. In such embodiments, the method may include adding cell culture medium (or a suitable buffer or water) having a lower osmolality than that of the cell culture to which it is added.

[0221] This disclosure further provides the use of a cell culture medium containing a certain concentration of cysteine ​​and / or having a certain osmolar concentration in a method involving the generation of KiH molecules.

[0222] This disclosure provides for less than 8 mM ( For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For exampleA cell culture medium containing cysteine ​​at a concentration of approximately 3 mM or 4.5 mM is used to increase the yield of KiH molecules produced in cell cultures by cells containing nucleic acids encoding constituent polypeptides of KiH molecules. In some embodiments, the cell culture medium has a concentration greater than 280 mOsmol / kg. For example >280 mOsmol / kg, For example >300 mOsmol / kg, For example ≥320 mOsmol / kg For example ≥350 mOsmol / kg For example Osmolar concentration (≥370 mOsmol / kg).

[0223] This disclosure also provides for less than 8 mM ( For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example A cell culture medium containing cysteine ​​at a concentration of approximately 3 mM or 4.5 mM is used to increase the proportion of KiH molecules in molecules produced by cells containing nucleic acids encoding constituent polypeptides of KiH molecules in cell cultures. In some embodiments, the cell culture medium has a concentration greater than 280 mOsmol / kg. For example >280 mOsmol / kg and <450 mOsmol / kg For example Greater than 300 mOsmol / kg For example >300 mOsmol / kg and <420 mOsmol / kg For example ≥320 mOsmol / kg and ≤400 mOsmol / kg For example ≥350 mOsmol / kg and ≤390 mOsmol / kg For example The osmolar concentration is approximately 370 mOsmol / kg.

[0224] This disclosure provides for less than 8 mM ( For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example≥2.5 mM and <4.75 mM, For example Cell culture media containing cysteine ​​at a concentration of approximately 3 mM or 4.5 mM are used to reduce undesirable products generated in cell cultures by cells containing nucleic acids encoding constituent polypeptides of the KiH molecule. For example The use of mortar-mortar molecules, ½ mortar molecules, ½ pestle molecules, or pestle-pepper molecules. In some embodiments, the cell culture medium has a yield greater than 280 mOsmol / kg ( For example >280 mOsmol / kg and <450 mOsmol / kg For example Greater than 300 mOsmol / kg For example >300 mOsmol / kg and <420 mOsmol / kg For example ≥320 mOsmol / kg and ≤400 mOsmol / kg For example ≥350 mOsmol / kg and ≤390 mOsmol / kg For example The osmolar concentration is approximately 370 mOsmol / kg.

[0225] This disclosure provides for less than 8 mM ( For example ≥0.5 mM and <8 mM, For example Less than 6 mM For example ≥1 mM and <6 mM, For example ≥2 mM and <5 mM, For example ≥2.5 mM and <4.75 mM, For example Cell culture media containing cysteine ​​at a concentration of approximately 3 mM or 4.5 mM are used to reduce undesirable products in cell cultures produced by cells containing nucleic acids encoding the constituent polypeptide of the KiH molecule. For example The use of ratios of mortar-mortar molecules, ½ mortar molecules, ½ pestle molecules, or pestle-pepper molecules. In some embodiments, the cell culture medium has a concentration greater than 280 mOsmol / kg ( For example >280 mOsmol / kg and <450 mOsmol / kg For example Greater than 300 mOsmol / kg example like >300 mOsmol / kg and <420 mOsmol / kg For example ≥320 mOsmol / kg and ≤400 mOsmol / kg For example≥350 mOsmol / kg and ≤390 mOsmol / kg For example The osmolar concentration is approximately 370 mOsmol / kg.

[0226] Specific exemplary embodiments

[0227] This disclosure provides a method for generating a molecule comprising a polypeptide complex formed by an interaction between a first polypeptide comprising a CH3 region containing a mordant-modified region and a second polypeptide comprising a CH3 region containing a mordant-modified region, wherein the method comprises culturing cells comprising nucleic acids encoding the first and second polypeptides in a cell culture medium for a substantial portion of the culture period, the cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) having an osmolar concentration of greater than 300 mOsmol / kg.

[0228] This disclosure also provides a method for generating a molecule comprising a polypeptide complex formed by an interaction between a first polypeptide comprising a CH3 region containing a mortise-modified region and a second polypeptide comprising a CH3 region containing a mortise-modified region, wherein the method comprises culturing cells comprising nucleic acids encoding the first and second polypeptides in a cell culture medium for a substantial portion of the culture period, the cell culture medium being: (a) 1 mM to 5 mM ( For example (b) Contains cysteine ​​at a concentration of approximately 3 mM or 4.5 mM, and has ≥320 mOsmol / kg ( For example Osmolar concentration (≥370 mOsmol / kg).

[0229] This disclosure also provides a method for generating a molecule comprising a polypeptide complex formed by an interaction between a first polypeptide comprising a CH3 region containing a styrene-modified region and a second polypeptide comprising a CH3 region containing a styrene-modified region, wherein the method comprises culturing cells comprising nucleic acids encoding the first and second polypeptides in a cell culture medium for a substantial portion of the culture period, the cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) having an osmolar concentration greater than 300 mOsmol / kg; and

[0230] The method includes seeding cells in a cell culture medium, wherein the cell culture medium: (c) is at a concentration of 1 mM to 5 mM ( For example The concentration (approximately 3 mM or 4.5 mM) contains cysteine, and (d) has a concentration of 320 mOsmol / kg to 420 mOsmol / kg. For exampleThe osmolar concentration is approximately 370 mOsmol / kg.

[0231] This disclosure also provides a method for increasing the yield of molecules comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a CH3 region containing a styrene-modified region and a second polypeptide comprising a CH3 region containing a styrene-modified region, produced by cells containing nucleic acids encoding a first polypeptide and a second polypeptide, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 6 mM, and (b) maintaining or adjusting the osmolality of the cell culture medium of the cell culture to greater than 300 mOsmol / kg.

[0232] This disclosure also provides a method for increasing the yield of molecules comprising a polypeptide complex formed by the interaction between a first polypeptide containing a CH3 region containing a gluten-modified region and a second polypeptide containing a CH3 region containing a gluten-modified region, produced by cells containing nucleic acids encoding a first polypeptide and a second polypeptide, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to 1 mM to 5 mM ( For example (a) approximately 3 mM or 4.5 mM), and (b) maintaining or adjusting the osmolality of the cell culture medium to ≥320 mOsmol / kg. For example ≥370 mOsmol / kg).

[0233] This disclosure also provides a method for increasing the proportion of molecules containing polypeptide complexes formed by the interaction between a first polypeptide containing a gluten-modified CH3 region and a second polypeptide containing a gluten-modified CH3 region among molecules produced by cells containing nucleic acids encoding a first polypeptide and a second polypeptide, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 6 mM, and (b) maintaining or adjusting the osmolality of the cell culture medium of the cell culture to greater than 300 mOsmol / kg.

[0234] This disclosure also provides a method for increasing the proportion of molecules comprising polypeptide complexes formed by the interaction between a first polypeptide containing a CH3 region containing a gluten-modified region and a second polypeptide containing a CH3 region containing a gluten-modified region, among molecules produced by cells containing nucleic acids encoding a first polypeptide and a second polypeptide, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to 1 mM to 5 mM ( example like(a) approximately 3 mM or 4.5 mM), and (b) maintaining or adjusting the osmolar concentration of the cell culture medium to ≥320 mOsmol / kg. For example ≥370 mOsmol / kg).

[0235] This disclosure also provides a method for reducing the yield of molecules comprising polypeptide complexes formed by the interaction between polypeptides comprising a galvanic-modified CH3 region and a second polypeptide comprising a galvanic-modified CH3 region in a cell culture, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium to less than 6 mM, and (b) maintaining or adjusting the osmolality of the cell culture medium to greater than 300 mOsmol / kg.

[0236] This disclosure also provides a method for reducing the yield of molecules comprising polypeptide complexes formed by interactions between polypeptides comprising acetoylene-modified CH3 regions among molecules produced in cell cultures from cells containing nucleic acids encoding a first polypeptide comprising a acetoylene-modified CH3 region and a second polypeptide comprising a acetoylene-modified CH3 region, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to 1 mM to 5 mM ( For example (a) approximately 3 mM or 4.5 mM), and (b) maintaining or adjusting the osmolar concentration of the cell culture medium to ≥320 mOsmol / kg. For example ≥370 mOsmol / kg).

[0237] This disclosure also provides a method for reducing the proportion of molecules comprising polypeptide complexes formed by interactions between polypeptides comprising acetoylene-modified CH3 regions among molecules produced in cell cultures by cells containing nucleic acids encoding a first polypeptide comprising a acetoylene-modified CH3 region and a second polypeptide comprising a acetoylene-modified CH3 region, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to less than 6 mM, and (b) maintaining or adjusting the osmolality of the cell culture medium of the cell culture to greater than 300 mOsmol / kg.

[0238] This disclosure also provides a method for reducing the proportion of molecules comprising polypeptide complexes formed by interactions between polypeptides comprising acetoylene-modified CH3 regions in a cell culture produced by cells containing nucleic acids encoding a first polypeptide comprising a first polypeptide comprising a acetoylene-modified CH3 region and a second polypeptide comprising a acetoylene-modified CH3 region, wherein the method comprises: (a) maintaining or adjusting the concentration of cysteine ​​in the cell culture medium of the cell culture to 1 mM to 5 mM ( For example (a) approximately 3 mM or 4.5 mM), and (b) maintaining or adjusting the osmolar concentration of the cell culture medium to ≥320 mOsmol / kg. For example ≥370 mOsmol / kg).

[0239] This disclosure also provides the use of a cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) an osmolar concentration of greater than 300 mOsmol / kg, for the purpose of increasing the yield of molecules comprising a polypeptide complex formed by the interaction between a first polypeptide comprising a CH3 region containing a styrene-modified region and a second polypeptide comprising a CH3 region containing a styrene-modified region, produced in a cell culture by cells comprising nucleic acids encoding a first polypeptide and a second polypeptide in a cell culture containing nucleic acids encoding a first polypeptide and a second polypeptide in a cell culture containing a CH3 region containing a styrene-modified region.

[0240] This disclosure also provides the use of a cell culture medium comprising: (a) in 1 mM to 5 mM ( For example (a) cysteine ​​at concentrations of approximately 3 mM or 4.5 mM, and (b) having concentrations of 320 mOsmol / kg to 420 mOsmol / kg. For example The osmolar concentration (approximately 370 mOsmol / kg) is used to increase the yield of molecules in cell cultures containing polypeptide complexes formed by the interaction between a first polypeptide containing a CH3 region with a mortise-and-tenon modification and a second polypeptide containing a CH3 region with a mortise-and-tenon modification.

[0241] This disclosure also provides the use of a cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) having an osmolar concentration of greater than 300 mOsmol / kg, the use being intended to increase the proportion of molecules in a cell culture containing a polypeptide complex formed by the interaction between a first polypeptide containing a CH3 region containing a galvanic-modified region and a second polypeptide containing a CH3 region containing a galvanic-modified region.

[0242] This disclosure also provides the use of a cell culture medium comprising: (a) in 1 mM to 5 mM ( For example (a) cysteine ​​at concentrations of approximately 3 mM or 4.5 mM, and (b) having concentrations of 320 mOsmol / kg to 420 mOsmol / kg. For example The osmolar concentration (approximately 370 mOsmol / kg) is used to increase the proportion of molecules in cell cultures produced by cells containing nucleic acids encoding a first polypeptide and a second polypeptide that form a polypeptide complex through the interaction between a first polypeptide containing a CH3 region containing a mortise-and-tenths modified region and a second polypeptide containing a CH3 region containing a mortise-and-tenths modified region.

[0243] This disclosure also provides the use of a cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) an osmolar concentration of greater than 300 mOsmol / kg, for the purpose of reducing the yield of molecules comprising polypeptide complexes formed by the interaction between polypeptides comprising a galvanic-modified CH3 region and a second polypeptide comprising a galvanic-modified CH3 region produced in a cell culture by cells comprising nucleic acids encoding nucleic acids encoding a first polypeptide comprising a galvanic-modified CH3 region and a second polypeptide comprising a galvanic-modified CH3 region.

[0244] This disclosure also provides the use of a cell culture medium comprising: (a) in 1 mM to 5 mM ( For example (a) cysteine ​​at concentrations of approximately 3 mM or 4.5 mM, and (b) having concentrations of 320 mOsmol / kg to 420 mOsmol / kg. For exampleThe osmolar concentration (approximately 370 mOsmol / kg) is used to reduce the yield of molecules containing polypeptide complexes formed by the interaction between polypeptides containing salami-modified CH3 regions produced in cell cultures by cells containing nucleic acids encoding nucleic acids encoding a first polypeptide containing a salami-modified CH3 region and a second polypeptide containing a salami-modified CH3 region.

[0245] This disclosure also provides the use of a cell culture medium comprising: (a) cysteine ​​at a concentration of less than 6 mM, and (b) an osmolar concentration of greater than 300 mOsmol / kg, the use being intended to reduce the proportion of molecules in a cell culture containing polypeptide complexes formed by interactions between polypeptides containing galvanic-modified CH3 regions, among molecules produced by cells containing nucleic acids encoding a first polypeptide containing a first polypeptide containing a galvanic-modified CH3 region and a second polypeptide containing a second polypeptide containing a galvanic-modified CH3 region.

[0246] This disclosure also provides the use of a cell culture medium comprising: (a) in 1 mM to 5 mM ( For example (a) cysteine ​​at concentrations of approximately 3 mM or 4.5 mM, and (b) having concentrations of 320 mOsmol / kg to 420 mOsmol / kg. For example The osmolar concentration (approximately 370 mOsmol / kg) is used to reduce the proportion of molecules in cell cultures containing molecules of nucleic acids encoding a first polypeptide containing a CH3 region with a mortise-modified region and a second polypeptide containing a CH3 region with a mortise-modified region, which are polypeptide complexes formed by the interaction between polypeptides containing CH3 regions with mortise-modified regions.

[0247] The yield and proportion of KiH molecules increased, while the yield and proportion of undesirable products decreased.

[0248] As explained herein, under the culture conditions described herein, cell culture containing nucleic acids encoding the constituent polypeptides of the KiH molecule increases the yield of KiH molecules and / or increases the proportion of KiH molecules among the molecules produced by cells in the culture, and / or reduces undesirable products. For example The production of mortar-mortar molecules, ½ mortar molecules, ½ pestle molecules, and / or pestle-pepper molecules) and / or the reduction of undesirable products ( For example The proportion of mortar-mortar molecules, half mortar molecules, half pestle molecules and / or pestle-pepper molecules among the molecules produced by cells in the culture.

[0249] It should be understood that the "yield" of a given molecule refers to the number / quantity of the given molecules produced. Similarly, it should be understood that the "proportion" of a given molecule among a variety of different types of molecules refers to the fraction / percentage of the given molecule within the total number / quantity of the different types of molecules produced. For example, in a mixture including KiH molecules and For example Within the molecular populations of mortar-mortar molecules and ½ pestle molecules, the proportion of KiH molecules is the fraction of the total number / quantity of the molecular population of KiH molecules.

[0250] The "increased" yield / proportion of KiH molecules can be increased relative to the yield / proportion of KiH molecules obtained by culture according to a reference method for producing KiH molecules from cells containing nucleic acids encoding component polypeptides of KiH molecules in culture. Similarly, the undesirable product ( For example The "reduced" yield / proportion of mortar-mortar molecules, ½ mortar molecules, ½ pestle molecules, or pestle-pepper molecules can be reduced.

[0251] A reference method for generating KiH molecules in culture from cells containing nucleic acids encoding constituent polypeptides of KiH molecules can be one of the following: No A method for culturing cells in a cell culture medium containing cysteine ​​at a concentration of less than 8 mM for most of the culture period. For example, in the reference method, cells can be cultured in a cell culture medium containing cysteine ​​at a concentration of ≥8 mM for most of the culture period. The reference method can be used in which... No A method for culturing cells in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM for most of the culture period. For example, in the reference method, cells can be cultured in a cell culture medium containing cysteine ​​at a concentration of ≥6 mM for most of the culture period.

[0252] The reference method can be one of them No A method for culturing cells for most of the culture period in a cell culture medium with an osmolar concentration greater than 280 mOsmol / kg. For example, in the reference method, cells can be cultured for most of the culture period in a cell culture medium with an osmolar concentration ≤280 mOsmol / kg. The reference method can be used where... NoA method for culturing cells for most of the culture period in a cell culture medium with an osmolar concentration greater than 300 mOsmol / kg. For example, in the reference method, cells can be cultured for most of the culture period in a cell culture medium with an osmolar concentration ≤300 mOsmol / kg. The reference method can be used where... No A method for seeding cells in a cell culture medium with an osmolar concentration greater than 280 mOsmol / kg. For example, in the reference method, cells can be seeded in a cell culture medium with an osmolar concentration ≤280 mOsmol / kg. The reference method can be used to seed cells in a cell culture medium with an osmolar concentration ≤280 mOsmol / kg. No A method for seeding cells in a cell culture medium with an osmolar concentration greater than 300 mOsmol / kg. For example, in the reference method, cells may be seeded in a cell culture medium with an osmolar concentration ≤300 mOsmol / kg.

[0253] The "increased" yield / proportion of KiH molecules is greater than 1 times that obtained by culture according to a reference method for producing KiH molecules from cells containing nucleic acids encoding constituent polypeptides of KiH molecules in culture. For example (It can be ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times, or ≥10 times).

[0254] Compared to the yield / proportion of related molecules obtained by culture according to a reference method for producing KiH molecules from cells containing constituent polypeptides encoding KiH molecules in culture, the undesirable product ( For example The "reduced" yield / proportion of mortar-mortar molecules, ½ mortar molecules, ½ pestle molecules, or pestle-pepper molecules was less than 1-fold compared to the yield / proportion of the relevant molecules obtained by culture according to the reference method. For exampleThe values ​​can be ≤0.99, ≤0.95, ≤0.9, ≤0.85, ≤0.8, ≤0.75, ≤0.7, ≤0.65, ≤0.6, ≤0.55, ≤0.5, ≤0.45, ≤0.4, ≤0.35, ≤0.3, ≤0.25, ≤0.2, ≤0.15, ≤0.1, ≤0.05, or ≤0.01.

[0255] Sequence identity

[0256] As used herein, “sequence identity” refers to the percentage of nucleotide / amino acid residues in the test sequence that are identical to those in the reference sequence after alignment and, if necessary, cleavage to achieve the maximum percentage of sequence identity between sequences. Pairwise and multiple sequence alignments for the purpose of determining the percentage of sequence identity between two or more amino acid or nucleic acid sequences can be performed in various ways known to those skilled in the art, for example, using publicly available computer software such as ClustalOmega (Söding, J. Bioinformatics (2005) 21:951-960), T-coffee (Notredame). Software such as J Mol Biol (2000) 302:205-217, Kalign (Lassmann and Sonnhammer BMC Bioinformatics (2005) 6(298)), and MAFFT (Katoh and Standley Molecular Biology and Evolution (2013) 30(4):772–780) is preferred when using such software. For example Used for open field penalties and extended penalties.

[0257] sequence

[0258] This disclosure includes combinations of the described aspects and preferred features, unless such combination is obviously not permitted or explicitly avoided.

[0259] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described.

[0260] Aspects and embodiments of this disclosure will now be illustrated by way of example with reference to the accompanying drawings. Other aspects and embodiments will be apparent to those skilled in the art. All documents mentioned herein are incorporated by reference.

[0261] Throughout the specification (including the following claims), unless the context otherwise requires, the word “comprising” and variations such as “including” and “containing” should be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0262] As used herein, the amino acid sequence "corresponding to" a specified reference amino acid sequence, or a region of a polypeptide, or a region of a polypeptide has at least 60% similarity to that amino acid sequence / polypeptide / region. For example Sequence identity must be at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The amino acid sequence / region / position of the polypeptide / amino acid sequence "corresponding" to the specified reference amino acid sequence / region / position can be identified by sequence alignment of the subject sequence with the reference sequence. For example Use sequence alignment software, such as ClustalOmega (Söding, J. 2005, Bioinformatics 21, 951-960).

[0263] In cases where an amino acid (other than glycine) is mentioned herein, both the L enantiomer and the D enantiomer of the relevant amino acid are explicitly considered. In some embodiments, unless otherwise specifically stated, reference to an amino acid herein specifically refers to the L enantiomer, which is the form in which the amino acid occurs in nature.

[0264] It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly specifies otherwise. A range herein may be expressed as “about” a particular value and / or to “about” another particular value. When expressing such a range, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation, the use of the antecedent “about” will be understood to form another embodiment of a particular value.

[0265] When this paper discloses the nucleic acid sequence, its reverse complementary sequence is also explicitly considered.

[0266] The method described herein can preferably be in in vitro To proceed. The term "to carry out". in vitro "Intended to cover procedures performed in cultures using cells, while the term" in vivo "It aims to cover procedures performed on or in whole multicellular organisms."

[0267] A value may be expressed in this document as “about” a specific value. Similarly, a range may be expressed in this document as “about” a specific value and / or to “about” another specific value. The term “about” in relation to numerical values ​​is optional and means, for example, + / - 10%. For example References to "about 10%" will be interpreted as 9% to 11%. In the examples of "about" cited in this article, the preceding value is also given special consideration. For example, For example The reference to "approximately 10%" also takes into account the 10% figure. Attached Figure Description

[0268] Embodiments and experiments illustrating the principles of this disclosure will now be discussed with reference to the accompanying drawings.

[0269] Figures 1A and 1B are schematic diagrams of the structures of (1A) KiH molecules and (1B) mortar-mortar molecules, ½ pestle molecules and ½ mortar molecule products of cell cultures as described in Example 1.

[0270] Figure 2. A graph showing the cumulative molar levels of cysteine ​​in the absence of metabolically active cellular consumers over a 14-day period.

[0271] Figures 3A to 3F summarize the titers and proportions of different products purified from cell culture supernatants by protein A affinity chromatography as described in Example 1. Each bar corresponds to a single experiment; for each experimental condition, n = 2. (3A) shows the titers of all products purified from cell culture supernatants by protein A affinity chromatography. (3B) shows the effective product titers obtained from different cultures, calculated as the titers of all products multiplied by the value of the major product (…). Right now The fraction of products (KiH molecules). (3C) shows the fraction of products obtained from different cultures that are the major products ( Right now The fractions of products containing KiH molecules are shown in (3D). (3E) shows the fractions of products as mortar-potassium molecules among the products obtained from different cultures. (3F) shows the fractions of products as mortar-potassium molecules among the products obtained from different cultures. Right now The ratio of KiH molecules is calculated as the fraction (%) of the product as mortar-mortar molecules divided by the fraction of the product as the major product ( Right now The percentage (%) of the product of KiH molecules.

[0272] Figure 4. Histogram summarizing the relative proportions of different molecules detected by non-reducing CE-SDS analysis of cell culture products purified by protein A affinity chromatography. Titers of protein A purified products obtained through culture using different methods are also shown.

[0273] Figure 5. Schematic diagram of six different KiH molecules (products A to F). This diagram was used to test the effects of different cysteine ​​levels in the basal culture medium and feed to analyze the impact on the levels of the major KiH products.

[0274] Figure 6. Example of a Design of Experiments (DoE) model typically used for optimization in a multidimensional parameter space.

[0275] Figures 7A and 7B. Theoretical cumulative cysteine ​​concentration trajectories were calculated and plotted in a 14-day fed-batch bioreactor with representative sampling events but no cell consumers, to represent the overall molar cysteine ​​system “uptake” or “dilution” in different test cases.

[0276] Figure 8. Experimental design (DoE) model for the observation and prediction levels of mortar-mortar and major products and the effects on residues for culture medium A.

[0277] Figure 9. Experimental design (DoE) models for the observation and prediction levels of mortar-mortar and major products and their effects on residues for commercial culture media.

[0278] Figure 10. Global experimental design (DoE) model for the level of observation and prediction of mortar-mortar and major products for combined culture medium types.

[0279] Figures 11A and 11B. Experimental Design (DoE) model prediction analysis tool plotting for mortar-mortar and major product levels in (11A) medium A or (11B) commercial medium.

[0280] Figure 12. δ-modified major product levels of KiH products A to F under low and high cysteine ​​cell culture conditions.

[0281] Figure 13. Bar graph showing the improved osmolar-osmolar (HH) and major product levels of KiH products in cysteine ​​basal medium and cysteine ​​diet by comparing osmolar concentrations of 300 mOsmol / kg and 370 mOsmol / kg.

[0282] Figure 14. Bar graph showing the improved major product and reduced osmolarity (HH) levels of KiH product A in cysteine ​​basal medium and cysteine ​​diet by comparing osmolar concentrations of 300 mOsmol / kg and 370 mOsmol / kg.

[0283] Example

[0284] Example 1

[0285] The inventors investigated the generation of a bispecific molecule comprising a mortar (KiH) Fc moiety, a target antigen-binding Fab moiety, and a 4-1BB binding moiety formed by three copies of the amino acid sequence of the extracellular domain of 4-1BBL.

[0286] The desired product is schematically represented in Figure 1A (“KiH” molecule) and is formed by the following: (i) A polypeptide comprising, from its N-terminus to its C-terminus: (a) The VH region of an antibody that binds to the target antigen; (b) CH1 region; (c) CH1-CH2 hinge region; (d) CH2 region; and (e) CH3 region containing mortar modification; (ii) A polypeptide comprising, from its N-terminus to its C-terminus: (a) Two copies of the extracellular domain of 4-1BBL bound by a linker sequence; (b) CL area; (c) CL-CH2 hinge area; (e) CH2 region; and (f) CH3 regions containing pestle-modified regions; (iii) A polypeptide comprising, from its N-terminus to its C-terminus: (a) (i)(a) of the VL region of the antibody; and (b) CL area; and (iv) A polypeptide comprising, from its N-terminus to its C-terminus: (a) The extracellular domain of 4-1BBL; and (b) CH1 region.

[0287] The molecule is a heteropeptide complex in which peptides (i) and (ii) associate with each other via the interaction between their respective CH2-CH3 regions, and in which peptides (i) and (iii) associate with each other via the interaction between the VL-CL region of peptide (iii) and the VH-CH1 region of peptide (i), and in which peptides (ii) and (iv) associate with each other via the interaction between the CH1-4-1BBL region of peptide (iv) and the CL-4-1BBL region of peptide (ii).

[0288] Figure 1B shows an exemplary undesirable product of a cell containing an expression vector for co-expressing peptides (i), (ii), (iii), and (iv).

[0289] The “mortar-mortar” molecule is a heteropeptide complex containing a homodimer of monomeric peptide (i) formed through the interaction between their CH2-CH3 regions. The molecule further contains two monomers of peptide (iii), each of which is associated with the peptide (i) monomer via the interaction between the VL-CL region of peptide (iii) and the VH-CH1 region of peptide (i).

[0290] The “½ pestle” molecule is a heterodimeric polypeptide complex containing polypeptide (ii) monomers, which associate with polypeptide (iv) monomers via the interaction between the CL-4-1BBL region of polypeptide (ii) and the CH1-4-1BBL region of polypeptide (iv).

[0291] The “½ mortar” molecule is a heterodimeric polypeptide complex containing a polypeptide (i) monomer, which associates with the polypeptide (iii) monomer via the interaction between the VL-CL region of polypeptide (iii) and the VH-CH1 region of polypeptide (i).

[0292] The inventors used a fed-batch cell culture process to culture CHO-K1 cells containing expression vectors for co-expressing polypeptides (i), (ii), (iii), and (iv) under different cell culture conditions.

[0293] Pre-culture of the primary culture was carried out in temperature-controlled, CO2-controlled, and humidity-controlled shake flask cultures and incubators from Mytron.

[0294] Fed-batch cell cultures were conducted in a 2 L fully controlled bioreactor connected to a control unit to regulate temperature, pH, dissolved oxygen (pO2), aeration, and agitation. Online processing data (such as pO2, temperature, pH, agitator speed, and aeration) were recorded using the fermentation control system. External pH and pCO2 measurements were monitored using a blood gas analyzer system (pHOx, IUL instrument). Viable and total cell density and viability were assessed using a Cedex HiRes system (Roche). Concentrations of products, substrates, and metabolites in the cell culture supernatant were determined using a Cedex BioHT (Roche) and RRLC 1200 system (Agilent). Osmolality was determined using an OSMOMAT® auto osmometer system (Gonotec).

[0295] Cell culture was performed using chemically defined basal media A or B. Cells seeded in basal media A were seeded at a density of 10E5 cells / mL, and cells seeded in basal media B were seeded at a density of 3.5E5 cells / mL. Chemically defined basal media A and B consist of a suitable carbon source ( For example It consists of various mixtures of glucose, amino acids and amino acid derivatives, vitamins and vitamin-like substances, polyamines, lipids and bulk salts.

[0296] During the 14- to 16-day fed-batch cell culture process, the temperature was maintained at 36.5°C, 30% pO2, and pH 7.0.

[0297] For the supplemental feeding process, two different nutritional feeds (1 and 2) were provided, as summarized below.

[0298]

[0299] Control the concentration of cysteine ​​in the feed to achieve the final cysteine ​​concentration during the breeding period.

[0300] Cysteine ​​levels accumulate in the absence of metabolically active cellular consumers, as illustrated in Figure 2. If cellular consumers are present, the actual measurable cysteine ​​level will be lower. When cellular consumers are present, the difference between the theoretically accumulated cysteine ​​and the actually measured cysteine ​​concentration, δ, represents the amount of cysteine ​​consumed by the active biomass. Considering both biomass and consumption time, a specific cysteine ​​consumption rate qS can be calculated. Cys .

[0301] For the "osmolality shift" experiment, using NaCl solution, the osmolality of cell cultures was shifted from 300 mOsmol / kg to 400 mOsmol / kg by either a bolus injection on day 7 (osmolality shift on day 7) or by a ramping profile from day 3 to day 7 (osmolality shift from day 3 to day 7). The shift strategy was designed to be effective after the growth phase and before the production phase, which typically occurs from day 8 of culture.

[0302] The table below summarizes the different cell culture conditions evaluated:

[0303] 14 to 16 days after the start of fermentation, the cell culture supernatant was harvested by centrifugation (10 min, 1000 rpm, followed by 10 min, 4000 rpm) and clarified by 0.22 µm filtration.

[0304] To determine the yield and proportion of cell culture products, the product concentration in the cell-free supernatant was measured using a Cedex BioHT (Roche) instrument. The cell-free supernatant was micropurified using protein A. In short, protein A affinity chromatography is used to purify antibody-derived target proteins because the Fc moiety contained in the target molecule is known to have a high affinity for protein A. Small volumes of cell-free supernatant (150 µl to 3 ml) were purified using PureSpeed ​​tips (Mettler Toledo, Columbus, OH) or OPUS RoboColumns (Repligen, Waltham, MA).

[0305] The protein A-purified molecules were then analyzed by non-reducing CE-SDS for validation of the major and byproducts (as abundance percentages). Small δ values ​​at 100% indicate a portion of unknown peaks that could not be annotated.

[0306] CE-SDS was performed under non-reducing conditions using the LabChipGXII (Perkin Elmer) system, according to the manufacturer's instructions. The expected KiH molecule product had a size of approximately 189 kDa, the mortar-mortar molecule approximately 165 kDa, the half-mortar molecule approximately 143 kDa, and the half-mortar molecule approximately 95 kDa. Unknown byproducts with sizes of approximately 130 kDa and 125 kDa were also detected.

[0307] The titers of the products purified from cell culture supernatant by protein A affinity chromatography and the relative proportions of the different molecules detected are shown in Figures 3A to 3F and summarized in Figure 4.

[0308] Example 2

[0309] To elucidate the effects of cysteine ​​concentration in cell culture basal medium and feed, basal medium osmolality, and cell culture pH set point on the level of KiH major product in the harvested cell culture fluid, a comprehensive experiment based on Design of Experiments (DoE) was designed. Furthermore, the transferability of cysteine ​​correlations for KiH content formation was tested using commercially available mammalian cell culture media. Additionally, we combined six different KiH molecules (Figure 5) with different high- and low-cysteine ​​cell culture nutrient strategies and analyzed their corresponding effects on KiH major product levels. In the first experimental series (Experiments 1–24), different cysteine ​​levels in cell culture basal medium and nutrient feed were tested on product A under both process C and commercial medium backgrounds. In subsequent experimental series (Experiments 25–48), six different KiH molecules (Figure 5) were used to test the effects of different cysteine ​​levels in basal medium and feed, and to assess cell culture pH at inoculation and the initial osmolality of the basal medium. In summary, 48 individual experiments were conducted using the ambr15 fermentation robot device (Sartorius Stedim, Göttingen, Germany).

[0310]

[0311] DoE Experiment

[0312] Statistical experimental planning using Design of Experiments (DoE) is a powerful and well-known technique, particularly for optimizing biological and chemical reactions where many factors interdependently influence the results. For example, DoE has been successfully used to optimize cell culture media (Zhang et al., 363-78), fermentation processes (Fu et al., 1095-105), protein purification processes (Pezzini et al., 8197-208), and cell culture conditions (Chen et al., 1211-21).

[0313] Depending on the desired outcome, different DoE methods can be applied to the current process (Figure 6). For example, a fully factorial design is well-suited for situations where 1) only a few factors are considered, 2) the main interest is in linear effects, and 3) a large number of experiments are feasible.

[0314] The DoE method in this study was planned and analyzed using the statistical software tool JMP (SAS Institute GmbH, Böblingen, Germany). The "Actual vs. Predicted" plot feature of JMP software illustrates the importance of the DoE results and test parameters. For this DoE study, "good model quality" was defined by the following criteria: p-value < 0.05, RMSE value < 10% of the median of the observed / analyzed parameters, and r² > 0.8.

[0315] Table 1 lists the ranges of test parameters and variables in 48 individual experiments embedding the DoE method. For the fed-batch process, two different media were used: medium A and commercially available DMEM (Thermo Fisher Scientific, Waltham, MA USA; catalog number 21013024; in Table 1, it is referred to as "Com" = commercial medium). Feeding was achieved using two different nutrient feeds (feed 1 and feed 2), as summarized below.

[0316] Control the concentration of cysteine ​​in the feed to achieve the final cysteine ​​concentration during cultivation. No "consumers" (such as "cells," chemical reactions, or sediment) are present. wait However, the theoretical cysteine ​​concentration trajectory in the fed-batch experiments of representative sampling events was calculated and plotted in Figures 7A and 7B to represent the "intake" or "dilution" of the overall molar cysteine ​​system in different test cases (increasing / decreasing global cysteine ​​concentration by mass balance of basal medium and nutrient feed).

[0317] Table 1: Conditions for fermentation runs used for DoE and parameter testing

[0318] (Abbreviation for basal culture medium: Com = Commercial)

[0319] As described above, the harvested and cell-separated cell culture supernatants were processed for CE-SDS analysis to assess the levels of KiH major products and the abundance of HH byproducts (where applicable).

[0320] The experimentally obtained data can be described by statistically significant linear multiple regression models for product A, which demonstrate good model quality: Process A KiH main product: p-value <0.0001, RMSE: 1.5566 [%], r2: 0.89; HH product: p-value 0.0004, RMSE: 1.2749 [%], r2: 0.82 (Figures 8A and 8D); Commercial: KiH main product: p-value <0.0001, RMSE: 1.5766 [%], r2: 0.96; HH product: p-value <0.0001, RMSE: 2.0924 [%], r2: 0.88 (Figures 9A and 9D).

[0321] Both cysteine ​​levels in the cell culture basal medium and the nutrient diet showed highly significant contributions to the observed effects. When cysteine ​​was present alone in either the cell culture basal medium or the nutrient diet, or when it was stronger in both, the cysteine ​​showed a dose-dependent negative effect on the abundance of the KiH major product level and a dose-dependent positive effect on the HH byproduct level for product A in a standalone linear regression model (as shown by the observations vs. predictions plot and statistics). Effects were analyzed by considering all data for product A to construct a global model (Figure 10) and separate models, specific to medium A (Figure 8) and specific to commercial media (Figure 9). Both data analysis methods yielded the same conclusion: high levels of cysteine ​​(alone in basal medium or in combination with nutrient diets) produce high levels of product AHH byproducts; while low levels of cysteine ​​(alone in basal medium or in combination with nutrient diets) produce high levels of KiH target proteins (as illustrated in the predictive profiling tool plots, Figures 11A and 11B for medium A and commercial medium, respectively).

[0322] In subsequent experimental series, as described in Table 1, experiments 25–48, along with two additional 2+1 extracellular domain fusion mAbs (products B and C), a 3+1 x mAb (product D), a 2+1 TCB (T cell bispecific) mAb (product E), and a cytokine fusion mAb (product F) (Figure 5), were used and tested in cell culture experiments to demonstrate the transferability of the effects on KiH products other than product A. A comparison between low-homocysteine ​​cell culture conditions (3 mM cysteine ​​in basal medium / 0 mM cysteine ​​in nutrient diet) and high-homocysteine ​​cell culture conditions (6 mM cysteine ​​in basal medium / 15 mM cysteine ​​in nutrient diet) showed higher levels of the major KiH product for all molecules (products A–F) under low-homocysteine ​​cell culture conditions, although the improvement in δ varied depending on the starting product used (0.2% for product C and 18.7% for product A) (Table 3, Figure 12). The differences in KiH levels [%] between “low” and “high” cysteine ​​basal media and feeding strategies were calculated as follows: Difference in KiH levels [%] = 100 x (KiH major product level) 低 Cys [%] - KiH major product level 高 Cys [%]) / KiH Major product level 低 Cys [%]

[0323] Table 2: Increasing KiH target protein levels through cysteine-based culture medium and feed strategies.

[0324]

[0325] Example 3

[0326] As chemophysical parameters, cell culture pH and initial osmolality of the culture medium were tested as putative regulators for KiH and HH levels, as described in Table 1. As described in Example 1, applying higher initial osmolality of the basal medium (370 mOsmol / kg vs. 300 mOsmol / kg) had a moderate but clear effect on the KiH and HH levels of product A; higher KiH levels and lower HH levels were detected (Figure 13). Under low-cysteine ​​cell culture conditions (3 mM cysteine ​​in the basal medium / 0 mM cysteine ​​in the nutrient diet), the beneficial effect of the basal medium osmolality was stronger, showing an improvement of up to 6% in the major KiH product and a reduction of up to 3% in the HH byproduct (Figure 14).

[0327] Interestingly, no effect of cell culture pH on the levels of KiH and HH byproducts was observed.

[0328] In summary, a clear and unexpected regulation of KiH target protein levels and the abundance of unintended HH byproducts by cysteine ​​was observed in cell cultures. This effect was synergistic with the osmolar concentration of the cell culture basal medium and independent of the KiH molecules tested.

Claims

1. A method for generating a molecule comprising a polypeptide complex formed by an interaction between a first polypeptide comprising a CH3 region containing a gluten-modified region and a second polypeptide comprising a CH3 region containing a gluten-modified region, wherein the method comprises culturing cells comprising nucleic acids encoding the first polypeptide and the second polypeptide in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM for a substantial portion of the culture period.

2. The method according to claim 1, wherein the cell culture medium has an osmolar concentration greater than 300 mOsmol / kg.

3. The method according to claim 1 or claim 2, wherein the method comprises seeding the cells in a cell culture medium containing cysteine ​​at a concentration of less than 6 mM, optionally wherein the method comprises seeding the cells in a cell culture medium containing cysteine ​​at a concentration of 1 mM to 5 mM.

4. The method according to any one of claims 1 to 3, wherein the method comprises seeding the cells in a cell culture medium containing cysteine ​​at a concentration of 2.5 mM to 4.75 mM.

5. The method according to any one of claims 1 to 4, wherein the method comprises seeding the cells in a cell culture medium having an osmolar concentration greater than 300 mOsmol / kg, optionally wherein the method comprises seeding the cells in a cell culture medium having an osmolar concentration of 320 mOsmol / kg to 420 mOsmol / kg.

6. The method according to any one of claims 1 to 5, wherein the method comprises seeding the cells in a cell culture medium having an osmolar concentration of about 370 mOsmol / kg.

7. The method according to any one of claims 1 to 6, wherein the culture period is at least 3 days, and optionally wherein the culture period is at least 7 days or at least 14 days.

8. The method according to any one of claims 1 to 7, wherein the cell culture medium has a pH of 6.8 to 7.4, optionally wherein the cell culture medium has a pH of about 7.

2.

9. The method according to any one of claims 1 to 8, wherein the first polypeptide or the second polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the ligand for the co-stimulatory molecule.

10. The method according to any one of claims 1 to 9, wherein: (i) The first polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the ligand used for co-stimulatory molecule, and the second polypeptide comprises all or part of an amino acid sequence forming an antigen-binding moiety that binds to the target antigen; or (ii) The first polypeptide comprises all or part of an amino acid sequence forming an antigen-binding portion that binds to the target antigen, and the second polypeptide comprises an amino acid sequence having at least 70% amino acid sequence identity with the amino acid sequence of the ligand used for co-stimulatory molecules.

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