Cysteine ​​engineered antibody constructs, conjugates and methods of use

Abstract

Described are antibody constructs that have been engineered to introduce at least one cysteine ​​insertion mutation ("cysteine ​​engineered antibody constructs"). The inserted cysteine ​​residue(s) can be used as a site for conjugation of one or more active agents to the antibody construct to provide a conjugate, such as an antibody-drug conjugate.

Description

[Technical field]

[0001] The present disclosure relates to the field of antibodies, specifically, antibodies engineered to contain one or more cysteine ​​insertion mutations and conjugates comprising these antibodies and active agents. [Background technology]

[0002] Antibody drug conjugates (ADCs) represent a relatively new and promising class of therapeutics. ADCs are generally composed of a monoclonal antibody linked to a small molecule therapeutic ("drug") via a linker. The drug-antibody ratio (DAR) and the specific site of drug conjugation can affect the stability and exposure of the ADC (Hamblett, et al., 2004, Clin. Cancer Res., 10(20):7063-7070; Shen, et al., 2012, Nature Biotechnology, 30:184-189). Conventional methods for generating ADCs through conjugation via native cysteine ​​or lysine residues typically result in heterogeneous mixtures of conjugates, both in terms of drug loading and conjugation, resulting in potential disadvantages in one or more of the following: antibody stability, specificity, in vivo distribution, pharmacokinetics, and / or efficacy.

[0003] The replacement of natural amino acids in antibodies with cysteine ​​residues for site-specific conjugation was first described by Lyons in the context of antibody labeling (Lyons, et al., 1990, Protein Eng Des Sel., 3(8):703-708). To solve the problem of heterogeneity, site-specific drug conjugation at engineered cysteine ​​residues in antibodies was later described (THIOMAB™) (Junutula, et al., 2008, Nat Biotechnol., 26:925-932; Vollmar, et al., 2017, Bioconjug Chem, 28(10):2538-2548; Ohri, et al., 2018, Bioconjug Chem., 29(2):473-485). Other examples of site-directed mutagenic incorporation of cysteine ​​residues have also been described (e.g., Sussman, et al., 2018, Protein Eng Des Sel., 31(2):47-54).

[0004] Related strategies involving the insertion of cysteine ​​residues rather than the replacement of natural amino acids with cysteine ​​residues have also been reported (Dimasi, et al., 2017, Mol. Pharmaceutics, 14(5):1501-1516, US Patent Application Publication No. US2018 / 0169255). Insertion of a cysteine ​​residue after one of the previously reported THIOMAB™ cysteine ​​substitution positions (S239C), termed C239i, was used in MEDI2228, a clinical stage ADC. Insertion of C239i results in abolition of FcγR binding and antibody-dependent cellular cytotoxicity (ADCC). The structural basis of these changes in function has been described (Gallagher, et al., 2019, Pharmaceutics, 11:546). Other cysteine ​​insertion approaches have also been described (U.S. Patent Application Publication No. US2020 / 0129635 and International Patent Application Publication No. WO2018 / 233572).

[0005] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. Summary of the Invention

[0006] Described herein are cysteine ​​engineered antibody constructs, conjugates, and methods of use.

[0007] In one aspect, the disclosure provides a cysteine ​​engineered antibody construct comprising a VH domain, a VH domain and a VL domain, an Fc region, or a combination thereof, wherein the Fc region comprises a CH2 domain and / or a CH3 domain, and the antibody construct comprises: (a) an insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) an insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) an insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) an insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) an insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) an insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) an insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain, (h) an insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, (i) an insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; wherein the numbering of the amino acids in the VL, CL, VH, and CH1 domains is Kabat numbering and the numbering of the amino acids in the CH2 domain is EU numbering; and wherein the antibody construct is based on immunoglobulin G (IgG).

[0008] In another aspect, the present disclosure relates to a conjugate comprising a cysteine ​​engineered antibody construct as described in any one of the embodiments disclosed herein and one or more active agents conjugated to each of the one or more inserted cysteine ​​residues.

[0009] In another aspect, the present disclosure provides a compound of formula (I): A-(L-(D) q ) p (I)

[0010] wherein A is a cysteine ​​engineered antibody construct, L is a linker, D is an active agent, q is an integer between 1 and 4, and p is an integer between 1 and 8; the cysteine ​​engineered antibody construct comprises a VH domain, a VH domain and a VL domain, an Fc region, or a combination thereof, wherein the Fc region comprises a CH2 domain and / or a CH3 domain; and the cysteine ​​engineered antibody construct comprises (a) an insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain, (b) an insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain, (c) an insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain, (d) an insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain, (e) an insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain. (f) an insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) an insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) an insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) an insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain; and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; wherein the numbering of the amino acids in the VL, CL, VH, and CH1 domains is Kabat numbering and the numbering of the amino acids in the CH2 domain is EU numbering; and the cysteine ​​engineered antibody construct is based on Immunoglobulin G (IgG), and each D is attached via an L to the inserted cysteine ​​residue.

[0011] In another aspect, the present disclosure relates to a composition comprising a conjugate described in any one of the embodiments disclosed herein and a pharma- ceutically acceptable carrier or diluent. In another aspect, the disclosure relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering an effective amount of a conjugate described herein, wherein the active agent comprised by the conjugate is a therapeutic agent.

[0012] In another aspect, the disclosure relates to a conjugate as described herein for use in therapy, wherein the active agent comprised by the conjugate is a therapeutic agent.

[0013] In another aspect, the disclosure relates to the use of a conjugate described herein in the manufacture of a medicament for the treatment of a subject in need thereof, wherein the active agent comprised by the conjugate is a therapeutic agent.

[0014] In another aspect, the present disclosure relates to a method of preparing a conjugate according to any one of the embodiments disclosed herein, comprising subjecting a cysteine ​​engineered antibody construct to reducing conditions such that the thiol group of one or more inserted cysteine ​​residues is reduced, and reacting a thiol-reactive linker-activator with the antibody construct under conditions that allow for the formation of a bond between the linker and the reduced thiol.

[0015] In another aspect, the disclosure provides a method for preparing an antibody-drug conjugate having a predetermined drug-antibody ratio (DAR), comprising: (i) providing a cysteine ​​engineered antibody construct comprising a VH domain, a VH domain and a VL domain, an Fc region, or a combination thereof, wherein the Fc region comprises a CH2 domain and / or a CH3 domain, and the antibody construct comprises: (a) an insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) an insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) an insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) an insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) an insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) an insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) an insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain. (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, (i) insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; and (ii) reacting the cysteine ​​engineered antibody construct with a drug-linker to provide an antibody-drug conjugate, wherein the predetermined DAR is 1, 2, 3, 4, 5, 6, 7, or 8, and the cysteine ​​engineered antibody construct comprises the same number of cysteine ​​insertion mutations as the predetermined DAR,

[0016] In another aspect, the present disclosure relates to a polynucleotide or set of polynucleotides encoding the cysteine ​​engineered antibody construct described in any one of the embodiments disclosed herein.

[0017] In another aspect, the present disclosure relates to a vector comprising one or more polynucleotides encoding the cysteine ​​engineered antibody construct described in any one of the embodiments disclosed herein.

[0018] In another aspect, the present disclosure relates to a host cell comprising a vector comprising one or more polynucleotides encoding a cysteine ​​engineered antibody construct described in any one of the embodiments disclosed herein. [Brief description of the drawings]

[0019] [Figure 1] The structures of the following drug linkers are shown: (A) MCvcPAB-Tulysin M, (B) MCvcPABC-MMAE, and (C) MTvc Compound 1. [Figure 2A]FIG. 1 shows a non-reducing capillary-electrophoresis SDS, (CE-SDS) gel analysis of 30 exemplary antibody-drug conjugates (ADCs) prepared using cysteine ​​insertion variants compared to a non-conjugated control (v17427) and a control ADC.Lane A: unconjugated control (v17427); Lanes B-D. variant v22760 (L_K39.5C) conjugated to MCvcPABC-MMAE (B), MCvcPAB-TulysinM (C), and MTvc Compound 1 (D); Lanes E-G: variant v22761 (L_K126.5C) conjugated to MCvcPABC-MMAE (E), MCvcPAB-TulysinM (F), and MTvc Compound 1 (G); Lanes H-J: MC Lanes K-M: variant v22765 (H_G237.5C) conjugated to vcPABC-MMAE (H), MCvcPAB-TulysinM (I), and MTvc Compound 1 (J); Lanes K-M: variant v22768 (H_Q295.5C) conjugated to MCvcPABC-MMAE (K), MCvcPAB-TulysinM (L), and MTvc Compound 1 (M); Lanes N-P: variant v22768 (H_Q295.5C) conjugated to MCvcPABC-MMAE (N), MCvcPAB-TulysinM (O), and Lanes Q-S: variant v27321 (L_W148.5C) conjugated to MCvcPABC-MMAE (Q), MCvcPAB-TublysinM (R), and MTvc compound 1 (S); Lanes T-V: variant v28983 (L_K149.5C) conjugated to MCvcPABC-MMAE (T), MCvcPAB-TublysinM (U), and MTvc compound 1 (V). _P40.5C); Lanes W-Y: variant v28989 (H_A9.5C) conjugated to MCvcPABC-MMAE (W), MCvcPAB-Tublysin M (X), and MTvc Compound 1 (Y), Lanes Z-BB: variant v28993 (H_G169.5C) conjugated to MCvcPABC-MMAE (Z), MCvcPAB-Tublysin M (AA), and MTvc Compound 1 (BB); Lanes CC-EE: MCvcPABC-MMAE (CC).Lanes FF-HH: variant v22758 (H_A114C, control) conjugated to MCvcPABC-MMAE (FF), MCvcPAB-Tulysin M (GG), and MTvc Compound 1 (HH); Lanes II-KK: MCvcPABC-MMAE (II), MCvcPAB-Tulysin M (JJ), and MTvc Compound 1 (KK). MW markers (top to bottom): 119, 68, 48, 29, 21, 16 kDa. [Figure 2B](B) Reduced CE-SDS gel analysis of 30 exemplary antibody-drug conjugates (ADCs) prepared using cysteine ​​insertion variants compared to an unconjugated control (v17427) and a control ADC. Lane A: unconjugated control (v17427); Lanes B-D. Variant v22760 (L_K39.5C) conjugated to MCvcPABC-MMAE (B), MCvcPAB-TulysinM (C), and MTvc Compound 1 (D); Lanes E-G: Variant v22761 (L_K126.5C) conjugated to MCvcPABC-MMAE (E), MCvcPAB-TulysinM (F), and MTvc Compound 1 (G); Lanes H-J: MC Lanes K-M: variant v22765 (H_G237.5C) conjugated to vcPABC-MMAE (H), MCvcPAB-TulysinM (I), and MTvc Compound 1 (J); Lanes K-M: variant v22768 (H_Q295.5C) conjugated to MCvcPABC-MMAE (K), MCvcPAB-TulysinM (L), and MTvc Compound 1 (M); Lanes N-P: variant v22768 (H_Q295.5C) conjugated to MCvcPABC-MMAE (N), MCvcPAB-TulysinM (O), and Lanes Q-S: variant v27321 (L_W148.5C) conjugated to MCvcPABC-MMAE (Q), MCvcPAB-TublysinM (R), and MTvc compound 1 (S); Lanes T-V: variant v28983 (L_K149.5C) conjugated to MCvcPABC-MMAE (T), MCvcPAB-TublysinM (U), and MTvc compound 1 (V). _P40.5C); Lanes W-Y: variant v28989 (H_A9.5C) conjugated to MCvcPABC-MMAE (W), MCvcPAB-Tublysin M (X), and MTvc Compound 1 (Y), Lanes Z-BB: variant v28993 (H_G169.5C) conjugated to MCvcPABC-MMAE (Z), MCvcPAB-Tublysin M (AA), and MTvc Compound 1 (BB); Lanes CC-EE: MCvcPABC-MMAE (CC).Lanes FF-HH: variant v22758 (H_A114C, control) conjugated to MCvcPABC-MMAE (FF), MCvcPAB-Tulysin M (GG), and MTvc Compound 1 (HH); Lanes II-KK: MCvcPABC-MMAE (II), MCvcPAB-Tulysin M (JJ), and MTvc Compound 1 (KK). MW markers (top to bottom): 119, 68, 48, 29, 21, 16 kDa. [Figure 3A] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22760 (L_K39.5C). [Figure 3B] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22761 (L_K126.5C). [Figure 3C] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22765 (H_G237.5C). [Figure 3D]FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22768 (H_Q295.5C). [Figure 3E] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v27321 (L_W148.5C). [Figure 3F] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v27322 (L_K149.5C). [Figure 3G] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28983 (L_P40.5C). [Figure 3H] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28989 (H_A9.5C). [Figure 3I]FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (filled circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28993 (H_G169.5C). [Figure 3J] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v29001 (H_T299.5C). [Figure 3K] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22758 (H_A114C, control). [Figure 3L] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MTvc compound 1 after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v29013 (H_S239.5C, control). [Figure 4A] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22760 (L_K39.5C). [Figure 4B]Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring-opened (open circles; right axis) are shown. Variant v22761 (L_K126.5C). [Figure 4C] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22765 (H_G237.5C). [Figure 4D] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22768 (H_Q295.5C). [Figure 4E] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring-opened (open circles; right axis) are shown. Variant v27321 (L_W148.5C). [Figure 4F] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v27322 (L_K149.5C). [Figure 4G]Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28983 (L_P40.5C). [Figure 4H] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28989 (H_A9.5C). [Figure 4I] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v28993 (H_G169.5C). [Figure 4J] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring-opened (open circles; right axis) are shown. Variant v29001 (H_T299.5C) [Figure 4K] Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v22758 (H_A114C, control). [Figure 4L]Figure 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPABC-MMAE after incubation with mouse plasma. Both the remaining DAR (closed circles; left axis) and % maleimide ring opening (open circles; right axis) are shown. Variant v29013 (H_S239.5C, control). [Figure 5A] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v22760 (L_K39.5C). [Figure 5B] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v22761 (L_K126.5C). [Figure 5C] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v22765 (H_G237.5C). [Figure 5D]FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v22768 (H_Q295.5C). [Figure 5E] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v27321 (L_W148.5C). [Figure 5F] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v27322 (L_K149.5C). [Figure 5G] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v28983 (L_P40.5C). [Figure 5H]FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v28989 (H_A9.5C). [Figure 5I] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v28993 (H_G169.5C). [Figure 5J] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tublysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tublysin M are shown. Variant v29001 (H_T299.5C). [Figure 5K] FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v22758 (H_A114C, control). [Figure 5L]FIG. 1 shows the results of immunoprecipitation mass spectrometry (IPMS)-mediated DAR analysis of antibody-drug conjugates (ADCs) containing cysteine ​​insertion variants conjugated to the drug-linker MCvcPAB-Tubulysin M after incubation with mouse plasma. The remaining DAR (filled circles, solid line, left axis), % maleimide ring opening (open circles, right axis), and % degradation (acetyl loss) (filled circles, dashed line) of MCvcPAB-Tubulysin M are shown. Variant v29013 (H_S239.5C, control). [Figure 6A] Figure 1 shows the results of in vitro cytotoxicity testing of ADCs containing cysteine ​​insertion variants conjugated to MTvc Compound 1 at DAR 1, 2, or 3 on different c-Met expressing cell lines, (A) EBC-1 cell line (high c-Met expression) compared to a control ADC (v17427-MTvc Compound 1, DAR 4). [Figure 6B] Figure 1 shows the results of in vitro cytotoxicity testing of ADCs containing cysteine ​​insertion variants conjugated to MTvc compound 1 at DAR 1, 2, or 3 on different c-Met expressing cell lines, (A) HT-29 cell line (medium c-Met expression) compared to a control ADC (v17427-MTvc compound 1, DAR 4). [Figure 7A] Shown is the in vivo antitumor activity of ADCs containing cysteine ​​insertion variants conjugated to MTvc compound 1 at DAR1, 2, or 3 in the medium c-Met expressing colon cancer xenograft model HT-29 compared to DAR4 control at toxin-matched doses of 6 mg / kg (DAR1), 3 mg / kg (DAR2), 2 mg / kg (DAR3), and 1.5 mg / kg (DAR4). [Figure 7B] Shown is the in vivo antitumor activity of ADCs containing cysteine ​​insertion variants conjugated to MTvc compound 1 at DAR1, 2, or 3 in the medium c-Met expressing colon cancer xenograft model HT-29 compared to DAR4 control at toxin-matched doses of 12 mg / kg (DAR1), 6 mg / kg (DAR2), 4 mg / kg (DAR3), and 3 mg / kg (DAR4). [Figure 8A]Shown is the in vivo antitumor activity of ADCs containing cysteine ​​insertion variants conjugated to MTvc compound 1 at DAR1, 2, or 3 in the high c-Met expressing non-small cell lung cancer xenograft model H1975 compared to DAR4 control at toxin-matched doses of 4 mg / kg (DAR1), 2 mg / kg (DAR2), 1.3 mg / kg (DAR3), and 1 mg / kg (DAR4). [Figure 8B] Shown is the in vivo antitumor activity of ADCs containing cysteine ​​insertion variants conjugated to MTvc compound 1 at DAR1, 2, or 3 in the high c-Met expressing non-small cell lung cancer xenograft model H1975 compared to DAR4 control at toxin-matched doses of 24 mg / kg (DAR1), 12 mg / kg (DAR2), 8 mg / kg (DAR3), and 6 mg / kg (DAR4). [Figure 9] 1 shows a sequence alignment of the CH1 domains of human IgG1 (allele*01 [SEQ ID NO: 41] and allele*03 [SEQ ID NO: 42]), IgG3 (allele*01 [SEQ ID NO: 43], allele*18 [SEQ ID NO: 44], and allele*17 [SEQ ID NO: 45]), IgG2 (allele*04 [SEQ ID NO: 46]), IgG4 allele*01 [SEQ ID NO: 47], IgG2 allele*01 [SEQ ID NO: 48]), and IgG2 (allele*02 [SEQ ID NO: 49]). [Figure 10] 1 shows a sequence alignment of the CH2 domains of human IgG1 (allele*01 [SEQ ID NO: 3]), IgG3 (allele*01 [SEQ ID NO: 4], IgG3 (allele*16 [SEQ ID NO: 4], allele*09 [SEQ ID NO: 5], allele*09 [SEQ ID NO: 6], allele*11 [SEQ ID NO: 7], allele*14 [SEQ ID NO: 8], and allele*18 [SEQ ID NO: 9]), IgG4 (allele*01 [SEQ ID NO: 10], and allele*02 [SEQ ID NO: 11]), and IgG2 (allele*01 [SEQ ID NO: 12] and allele*02 [SEQ ID NO: 13]). [Figure 11]1 shows a sequence alignment of the CH3 domains of human IgG1 (allele*01 [SEQ ID NO: 14], allele*04 [SEQ ID NO: 15], and allele*03 [SEQ ID NO: 16]), IgG2 (allele*01 [SEQ ID NO: 17] and allele*06 [SEQ ID NO: 18]), IgG3 (allele*15 [SEQ ID NO: 19], allele*17 [SEQ ID NO: 20], human IgG4 (allele*03 [SEQ ID NO: 21]), human IgG3 (allele*14 [SEQ ID NO: 22]), human IgG4 (allele*01 [SEQ ID NO: 23]), human IgG3 (allele*06 [SEQ ID NO: 24]), human IgG3 (allele*08 [SEQ ID NO: 25]), human IgG3 (allele*01 [SEQ ID NO: 26]), human IgG3 (allele*03 [SEQ ID NO: 27]), human IgG3 (allele*13 [SEQ ID NO: 28]). [Figure 12] 3 shows a sequence alignment of the CL domains of human kappa light chains (allele*01 [SEQ ID NO:29], allele*04 [SEQ ID NO:30], allele*05 [SEQ ID NO:31], allele*02 [SEQ ID NO:32], and allele*03 [SEQ ID NO:33]), and human lambda light chains (allele3*02 [SEQ ID NO:34], allele3*03 [SEQ ID NO:35], allele6*01 [SEQ ID NO:36], allele2*01 [SEQ ID NO:37], allele7*01 [SEQ ID NO:38], allele7*03 [SEQ ID NO:39], and allele1*02 [SEQ ID NO:40]). [Figure 13A] FIG. 1 shows the hydrophobic interaction chromatography (HIC) profile of variant v29013 (H_S239.5C, control) conjugated to drug-linker MCvcPABC-MMAE. [Figure 13B] FIG. 1 shows the hydrophobic interaction chromatography (HIC) profile of variant v29001 (H_T299.5C) conjugated to drug-linker MCvcPABC-MMAE. [Figure 14A] FIG. 1 shows the hydrophobic interaction chromatography (HIC) profile of variant v29013 (H_S239.5C, control) conjugated to drug-linker MTvc compound 1. [Figure 14B] FIG. 1 shows the hydrophobic interaction chromatography (HIC) profile of variant v29001 (H_T299.5C) conjugated to drug-linker MTvc compound 1. [Figure 15] A shows the differential scanning calorimetry (DSC) profiles of variant v29013 (H_S239.5C, control) and B shows the differential scanning calorimetry (DSC) profiles of variant v29001 (H_T299.5C) compared to a control variant containing a cysteine ​​substitution mutation (v27320, L_K149C), respectively. [Figure 16A] 1 shows a hydrophobic interaction chromatography (HIC) profile of ADC v35074-MTvc compound 1 (DAR6) in which two distinct peaks were observed: the peak eluted at 7.07 min represents DAR5 and the peak eluted at 7.5 min represents DAR6. [Figure 16B] Size exclusion chromatography (SEC) profile of the same ADC is shown, with the fraction eluting at 3.3 min representing monomer (99%). [Figure 17A] FIG. 13 shows the reduced LC-MS profile of the light chain (LC) of ADC v35074-MTvc compound 1 (DAR6) after EndoS treatment. [Figure 17B] FIG. 13 shows the reduced LC-MS profile of heavy chain 1 of ADC v35074-MTvc compound 1 (DAR6) after EndoS treatment. [Figure 17C] FIG. 13 shows the reduced LC-MS profile of heavy chain 2 of ADC v35074-MTvc compound 1 (DAR6) after EndoS treatment. [Figure 18]Figure 1 shows capillary electrophoresis SDS (CE-SDS) profiles of cysteine ​​insertion variant v35074 as unconjugated antibody and conjugated to drug-linker MTvc compound 1. Lane 1: protein ladder; lane 2: trastuzumab control (non-reduced (NR)); lane 3: unconjugated v35074 (NR); lane 4: v35074-MTvc compound 1 (NR); lane 5: trastuzumab (reduced (R)); lane 6: unconjugated v35074 (R); lane 7: v35074-MTvc compound 1 (R). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure relates to antibody constructs engineered to introduce at least one cysteine ​​insertion mutation ("cysteine ​​engineered antibody constructs"). "Cysteine ​​insertion mutation" in this context refers to a non-native cysteine ​​residue that is introduced between two amino acid residues present in the sequence of a parent antibody construct. The inserted cysteine ​​residue(s) can be used as a site for conjugation of one or more active agents, such as therapeutic agents, diagnostic agents, and labeling agents, to the antibody construct to provide a conjugate.

[0021] Certain embodiments of the present disclosure relate to cysteine ​​engineered antibody constructs and conjugates comprising active agents covalently attached to inserted cysteine ​​residues in the antibody constructs, which may be used for a variety of therapeutic and diagnostic applications.

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0023] As used herein, the term "about" refers to approximately a ±10% variation from a given value. It is to be understood that such a variation is always included in any given value presented herein, whether or not that variation is specifically referred to.

[0024] The use of the words "a" or "an" when used herein in conjunction with the term "comprising" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0025] As used herein, the terms "comprising," "having," "including," and "containing," as well as grammatical variations thereof, are inclusive, i.e., open ended, and do not exclude additional unrecited elements and / or method steps. The term "consisting essentially of," when used herein in connection with a composition, use, or method, means that additional elements and / or method steps may be present, but these additions do not substantially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used herein in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. When a composition, use, or method described herein is said to include certain elements and / or steps, in certain embodiments it may consist essentially of those elements and / or steps, and in other embodiments it may consist of those elements and / or steps, whether or not those embodiments are specifically mentioned.

[0026] As used herein, the term "antibody construct" encompasses full-length antibodies and functional fragments of full-length antibodies. Functional antibody fragments include antigen-binding fragments (such as Fab' fragments, F(ab')2 fragments, Fab fragments, single chain variable regions (scFv), and single domain antibodies (sdAbs)), as well as Fc fragments that contain an Fc region capable of binding to one or more Fc receptors (FcRs). The term "antibody construct" also encompasses Fc fusion proteins that contain an Fc region and one or more heterologous polypeptides.

[0027] A full-length antibody comprises heavy and light chains assembled as a heterotetramer comprising two heavy chains and two light chains. The heavy chain typically comprises the domains (N-terminus to C-terminus): VH-CH1-hinge-CH2-CH3, and the light chain typically comprises the domains (N-terminus to C-terminus): VL-CL. Unless otherwise specified, the numbering of amino acids in the VH, CH1, VL, and CL domains used herein is the Kabat numbering, and the numbering of amino acid residues in the CH2 and CH3 domains and hinge region used herein is the EU numbering, also referred to as the EU index (both numbering systems are described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0028] The terms "Fc region" and "Fc" are used interchangeably herein and refer to the C-terminal region of an immunoglobulin heavy chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region sequence is usually defined as extending from position 239 (EU numbering) to the C-terminus of the heavy chain. The "Fc polypeptide" of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., the polypeptide that comprises the C-terminal constant region of an immunoglobulin heavy chain capable of stable self-association. The Fc region typically comprises a CH2 domain and a CH3 domain, but in some embodiments may comprise only the CH2 domain or only the CH3 domain. The Fc region may also be considered to include the hinge region in certain embodiments.

[0029] The "CH2 domain" of the human IgG Fc region is typically defined as extending from position 239 to position 340. The "CH3 domain" is usually defined as including the amino acid residues C-terminal to the Fc region, i.e., the CH2 domain from positions 341 to 447. The "hinge region" of human IgG1 is generally defined as extending from positions 216 to 238 (Burton, 1985, Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by matching the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond.

[0030] An "Fc fusion protein," in the context of this disclosure, is a protein that comprises all or part of an Fc region (eg, a CH2 or CH3 domain) fused to a heterologous protein or polypeptide.

[0031] The terms "derived from" and "based on," when used herein to describe an amino acid sequence, mean that the subject amino acid sequence is substantially identical to the recited reference amino acid sequence.

[0032] The term "substantially identical" as used herein in relation to amino acid sequences means that, when optimally aligned (e.g., using the methods described below), an amino acid sequence shares at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with its reference amino acid sequence. The percent identity between two amino acid sequences can be determined by a variety of methods known in the art, including, for example, commonly available computer software, such as Smith Waterman Alignment (Smith & Waterman, 1981, J Mol Biol 147:195-7), "BestFit" (Smith & Waterman, 1981, Advances in Applied Mathematics, 482-489), BLAST (Basic Local Alignment Search Tool; (Altschul, et al., 1990, J Mol Biol, 215: 403-10), and its variations and updates, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software can be used to determine alignment. In addition, a person skilled in the art can determine the appropriate parameters for measuring alignment, including the algorithm required to achieve maximum alignment over the length of the sequences being compared. Generally, for peptides, the length of the comparison sequence is at least 10 amino acids, but a person skilled in the art will understand that the actual length depends on the full length of the sequences being compared. In certain embodiments, the length of the comparison sequence can be the full length of the protein or peptide sequence.

[0033] The term "isolated" as used herein with respect to a substance means that the substance has been removed from its original environment (e.g., its natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in an animal organism is not isolated, but the same polynucleotide or polypeptide separated from some or all of the coexisting substances in the natural system is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, but such a vector or composition is isolated in that it is not part of its natural environment.

[0034] It should be understood that the positive recitation of a feature in an embodiment described herein serves as a basis for excluding features in alternative embodiments. In particular, when a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form alternative embodiments, whether or not such alternative embodiments are specifically mentioned.

[0035] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0036] Cysteine ​​Engineered Antibody Constructs A cysteine ​​engineered antibody construct of the present disclosure is an antibody construct that comprises one or more cysteine ​​insertion mutations. The cysteine ​​engineered antibody construct can be, for example, a full-length antibody, a functional fragment of a full-length antibody, or an Fc fusion protein. Functional antibody fragments include, for example, antigen-binding fragments and Fc fragments. Examples of antigen-binding fragments include, but are not limited to, antibody light and / or heavy chain variable regions (VL, VH), variable fragments (Fv), Fab' fragments, F(ab')2 fragments, Fab fragments, single chain variable regions (scFv), complementarity determining regions (CDRs), and single domain antibodies (sdAbs). Fc fragments typically comprise the CH2 and CH3 domains of an antibody and are capable of binding to one or more Fc receptors (FcRs). Fc fragments may optionally comprise a hinge region.

[0037] An Fc fusion protein comprises an Fc region fused or covalently linked to one or more heterologous polypeptides. In certain embodiments, an Fc fusion protein comprises an Fc region fused or covalently linked to one or more target binding domains. Examples of target binding domains that may be included in an Fc fusion protein in certain embodiments include, but are not limited to, receptors, receptor fragments (such as extracellular portions), ligands, cytokines, and heterologous antigen-binding antibody fragments (such as antigen-binding fragments from different antibody classes or subclasses). One or more heterologous polypeptides may be fused or covalently linked to the Fc region directly or via a linker, e.g., an amino acid-based linker.

[0038] Certain embodiments of the present disclosure relate to cysteine ​​engineered antibody constructs comprising an antigen binding domain, an Fc region, or both an antigen binding domain and an Fc region. In some embodiments, the cysteine ​​engineered antibody construct comprises an antigen binding domain comprising a VH domain or a VH domain and a VL domain. In some embodiments, the cysteine ​​engineered antibody construct comprises an Fc region comprising a CH2 domain and / or a CH3 domain. In some embodiments, the cysteine ​​engineered antibody construct comprises an Fc region comprising a CH2 domain and a CH3 domain.

[0039] Certain embodiments of the present disclosure relate to cysteine ​​engineered antibody constructs that are full-length antibodies. In such embodiments, the cysteine ​​engineered antibody can be, for example, a monoclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody. In this context, a full-length antibody can include one or more Fab regions. For example, a full-length antibody can be a one-arm (monovalent) antibody (OAA), a bivalent antibody, or a multivalent antibody.

[0040] Certain embodiments relate to cysteine ​​engineered antibody constructs that are functional antibody fragments. In some embodiments, the cysteine ​​engineered antibody constructs are functional fragments that include at least one antigen binding domain, such as a Fab, scFv, or sdAb. In some embodiments, the cysteine ​​engineered antibody constructs include two or more antigen binding domains, which can be, for example, a Fab, scFv, or a combination thereof. In some embodiments, the cysteine ​​engineered antibody constructs include two or more antigen binding domains linked with a linker, such as in a tandem scFv format or a scFv-Fab format.

[0041] In some embodiments, cysteine ​​engineered antibodies may be bispecific or multispecific antibodies that contain two or more antigen binding domains, each binding a different antigen epitope.

[0042] Certain embodiments relate to cysteine ​​engineered antibody constructs that are Fc fusion proteins.

[0043] When a cysteine ​​engineered antibody construct comprises one or more antigen binding domains, each antigen binding domain binds to a target antigen. A target antigen is typically a cell surface molecule, such as a protein, lipid, or polysaccharide, that is found on the surface of a target cell, such as a tumor cell, a virus-infected cell, a bacteria-infected cell, an injured red blood cell, an arterial plaque cell, an inflamed tissue cell, or a fibrotic tissue cell. Examples of target antigens include, but are not limited to, tumor-associated antigens (TAA), cell surface receptor proteins, transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis, and molecules associated with angiogenesis. Certain embodiments relate to cysteine ​​engineered antibody constructs comprising at least one antigen binding domain that binds to a tumor-associated antigen (TAA).

[0044] The cysteine ​​engineered antibody constructs of the present disclosure are derived from immunoglobulin G (IgG). In certain embodiments, the cysteine ​​engineered antibody constructs are derived from human IgG. In some embodiments, the cysteine ​​engineered antibody constructs are derived from human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the cysteine ​​engineered antibody constructs are derived from IgG1. In some embodiments, the cysteine ​​engineered antibody constructs are derived from human IgG1.

[0045] In certain embodiments where the cysteine ​​engineered antibody construct comprises a cysteine ​​insertion in the light chain, the antibody construct may comprise a kappa light chain or a lambda light chain. In some embodiments where the cysteine ​​engineered antibody construct comprises a cysteine ​​insertion in the light chain, the antibody construct comprises a kappa light chain.

[0046] The amino acid sequences of the CH1, CH2, and CH3 domains of human IgG1, IgG2, IgG3, and IgG4, as well as the amino acid sequences of the kappa and lambda light chains, are known in the art (see, for example, the sequences provided on the International ImMunoGeneTics information system (IMGT®) website). Representative amino acid sequences of the CH1, CH2, and CH3 domains for various alleles of human IgG1, IgG2, IgG3, and IgG4 are also provided in Figures 9-11, respectively, and representative amino acid sequences for alleles of the kappa and lambda CL domains are provided in Figure 12.

[0047] In certain embodiments, the cysteine ​​insertion mutation does not affect or only minimally affects the stability of the cysteine ​​engineered antibody construct as determined by melting temperature (Tm). By "does not affect or only minimally affects" is meant that the Tm of the domain of the cysteine ​​engineered antibody construct into which the cysteine ​​residue is inserted is within (i.e. ±) 0°C to 8°C of the Tm of the same domain in the corresponding parent antibody construct (lacking the cysteine ​​insertion mutation). For example, for a cysteine ​​engineered antibody construct comprising a cysteine ​​residue inserted into the CH2 domain, the CH2 domain Tm of the cysteine ​​engineered antibody construct is within 0°C to 8°C of the CH2 domain Tm of the corresponding parent antibody construct. In some embodiments, the Tm of the domain of the cysteine ​​engineered antibody construct into which the cysteine ​​residue is inserted is within 0°C to 7°C of the Tm of the same domain in the corresponding parent antibody construct. In some embodiments, the Tm of a domain of a cysteine ​​engineered antibody construct into which a cysteine ​​residue is inserted is within 0° C. to 6° C., or within 0° C. to 5° C., of the Tm of the same domain in the corresponding parent antibody construct.

[0048] The Tm of an antibody construct can be determined by various techniques known in the art, such as circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In certain embodiments, the Tm difference between a cysteine ​​engineered antibody construct and the corresponding parent antibody construct is determined by DSC.

[0049] In certain embodiments, the cysteine ​​engineered antibody constructs of the present disclosure contain the same cysteine ​​insertion mutation in each chain of the antibody construct, e.g., both heavy chains or both light chains, resulting in an antibody construct with an average drug-to-antibody ratio (DAR) of 2 when conjugated to an active agent.

[0050] Certain embodiments of the present disclosure relate to "DAR-tuned" cysteine ​​engineered antibody constructs. A "DAR-tuned" antibody construct in this context is a cysteine ​​engineered antibody construct that contains a cysteine ​​insertion mutation in only one chain of the construct (allowing for a DAR1 conjugate) or a combination of cysteine ​​insertion mutations (allowing for a conjugate with two or more DARs, e.g., DAR3, DAR4, or DAR6).

[0051] Cysteine ​​insertion mutation By combining structure-based computational approaches and experimental studies, suitable sites for cysteine ​​insertion mutations were identified in IgG structures, as described in the Examples herein. In certain embodiments, the cysteine ​​engineered antibody constructs of the present disclosure comprise: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0052] It is understood that the cysteine ​​insertion mutation(s) that may be included in a given antibody construct will depend on the format of the antibody construct. A full-length antibody construct may, for example, include the above-mentioned cysteine ​​insertion mutation(s) in any of the VH, VL, CL, CH1, and / or CH2 domains, while an antibody construct that includes only an Fc region, such as an Fc fusion protein, may include the above-mentioned cysteine ​​insertion mutation(s) in the CH2 domain. Similarly, an antibody construct that includes an antigen-binding domain, such as an scFv or Fab, but lacks an Fc region, may include the cysteine ​​insertion mutation(s) in the VH, VL, CL, and / or CH1 domains.

[0053] In some embodiments, the cysteine ​​engineered antibody construct comprises in the Fc region: (a) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (b) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (c) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0054] In some embodiments, the cysteine ​​engineered antibody construct comprises in the Fab region: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; and (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain.

[0055] In some embodiments, the cysteine ​​engineered antibody construct comprises in the CL domain or the CH1 domain: (a) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (b) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (c) the insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain, and (d) an insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain.

[0056] In some embodiments, the cysteine ​​engineered antibody construct comprises in the variable region: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) the insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain, and (c) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain.

[0057] In some embodiments, the cysteine ​​engineered antibody construct comprises a cysteine ​​insertion mutation as described above in the CH2 domain or variable region. In some embodiments, the cysteine ​​engineered antibody construct comprises a cysteine ​​insertion mutation as described above in the CH2 domain or variable region, the cysteine ​​insertion mutation being (a) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (b) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (c) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, and (d) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0058] The cysteine ​​insertion mutations described herein may be introduced into an antibody construct symmetrically (i.e., the same cysteine ​​insertion mutation may be introduced into each respective heavy or light chain) or they may be introduced asymmetrically (i.e., one cysteine ​​insertion mutation is introduced into one heavy or light chain and a different cysteine ​​insertion mutation, or no cysteine ​​insertion mutation is introduced into the other heavy or light chain). In certain embodiments, a cysteine ​​engineered antibody construct comprises symmetric cysteine ​​insertion mutations. In some embodiments, a cysteine ​​engineered antibody construct comprises one or more asymmetric cysteine ​​insertion mutations. In some embodiments, a cysteine ​​engineered antibody construct comprises a combination of symmetric and asymmetric cysteine ​​insertion mutations.

[0059] Introducing two cysteine ​​insertion mutations symmetrically into an antibody construct, e.g., into both heavy chains or both light chains, results in a cysteine ​​engineered antibody construct that has an average drug-antibody ratio (DAR) of 2 when conjugated to an active agent. Introducing asymmetric cysteine ​​insertion mutations and / or combinations of cysteine ​​insertion mutations into an antibody construct allows for "tuning" the DAR of the final conjugate. For example, an antibody construct containing cysteine ​​insertion mutations in only one chain of the construct allows for a DAR1 conjugate, while an antibody construct containing a combination of cysteine ​​insertion mutations allows for a conjugate with a DAR of 2 or more. In those embodiments in which the cysteine ​​engineered antibody construct comprises a combination of cysteine ​​insertion mutations, the mutations may be introduced symmetrically (i.e., the same cysteine ​​insertion mutation is included in both chains of the antibody construct), asymmetrically (i.e., the cysteine ​​insertion mutation(s) in one chain of the antibody construct are different or absent from the other chain of the antibody construct), or a combination thereof (i.e., at least one cysteine ​​insertion mutation in one chain of the antibody construct is the same as the cysteine ​​insertion mutation in the other chain of the antibody construct and at least one cysteine ​​insertion mutation is different or absent from the other chain). Typically, when the antibody construct comprises a single cysteine ​​insertion mutation or an asymmetric cysteine ​​insertion mutation, the cysteine ​​insertion mutation(s) are introduced in the heavy chain of the antibody construct. However, asymmetric light chain cysteine ​​insertion mutations are contemplated in certain embodiments.

[0060] Certain embodiments of the present disclosure relate to cysteine ​​engineered antibody constructs that contain two cysteine ​​insertion mutations that are symmetric (i.e., each inserted cysteine ​​residue is at the same position on each respective heavy or light chain).

[0061] Certain embodiments of the present disclosure relate to "DAR tuned" cysteine ​​engineered antibody constructs comprising one or a combination of the cysteine ​​insertion mutations described herein. In some embodiments, the cysteine ​​engineered antibody construct comprises between 1 and 8 cysteine ​​insertion mutations. In some embodiments, the cysteine ​​engineered antibody construct comprises between 1 and 6 cysteine ​​insertion mutations. In some embodiments, the cysteine ​​engineered antibody construct comprises between 1 and 4 cysteine ​​insertion mutations.

[0062] Certain embodiments of the present disclosure relate to DAR-tuned cysteine ​​engineered antibody constructs that contain an odd number of cysteine ​​insertion mutations, for example, 1, 3, 5, or 7 cysteine ​​insertion mutations. Some embodiments relate to DAR-tuned cysteine ​​engineered antibody constructs that contain 1, 3, or 5 cysteine ​​insertion mutations. Some embodiments relate to DAR-tuned cysteine ​​engineered antibody constructs that contain 1 or 3 cysteine ​​insertion mutations.

[0063] Certain embodiments relate to cysteine ​​engineered antibody constructs comprising a single (1) cysteine ​​insertion mutation. In some embodiments, the cysteine ​​engineered antibody construct comprises a single cysteine ​​insertion mutation in the heavy chain of the antibody construct. In some embodiments, the cysteine ​​engineered antibody construct comprises: (a) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (b) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (c) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (d) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (e) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0064] In some embodiments, the cysteine ​​engineered antibody construct comprises: (a) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (b) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, and (c) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0065] Certain embodiments of the present disclosure relate to cysteine ​​engineered antibody constructs comprising three cysteine ​​insertion mutations as described herein. In such embodiments, the cysteine ​​engineered antibody construct may comprise three different (asymmetric) cysteine ​​insertion mutations, or may comprise two symmetric cysteine ​​insertion mutations (i.e., at the same position on each respective heavy or light chain) and one asymmetric cysteine ​​insertion (one inserted cysteine ​​residue on one light or heavy chain). In some embodiments, the cysteine ​​engineered antibody construct comprises three cysteine ​​insertion mutations, two of which are the same (symmetric) and one of which is different (asymmetric).

[0066] In certain embodiments, the cysteine ​​engineered antibody construct comprises three cysteine ​​insertion mutations, two of which are identical (symmetric) and one of which is different (asymmetric), the symmetric cysteine ​​insertion mutation being: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain;

[0067] Asymmetric cysteine ​​insertion mutations: (i) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (ii) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (iii) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (iv) insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain; and (v) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0068] In certain embodiments, the cysteine ​​engineered antibody construct comprises three cysteine ​​insertion mutations, two of which are identical (symmetric) and one of which is different (asymmetric), the symmetric cysteine ​​insertion mutation being: (a) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (b) the insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain, and (c) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain;

[0069] Asymmetric cysteine ​​insertion mutations: (i) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (ii) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, and (iii) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0070] Certain embodiments of the present disclosure relate to DAR-tuned cysteine ​​engineered antibody constructs that contain an even number of cysteine ​​insertion mutations, for example, 4, 6, or 8 cysteine ​​insertion mutations. Some embodiments relate to DAR-tuned cysteine ​​engineered antibody constructs that contain 4 or 6 cysteine ​​insertion mutations. Typically, in such embodiments, the cysteine ​​insertion mutations are symmetric cysteine ​​insertion mutations. However, in some embodiments, asymmetric cysteine ​​insertion mutations are also contemplated.

[0071] In certain embodiments, the cysteine ​​engineered antibody construct comprises 4, 6, or 8 cysteine ​​insertion mutations, the cysteine ​​insertion mutations being (i) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (ii) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (iii) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (iv) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (v) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0072] In certain embodiments, the cysteine ​​engineered antibody construct comprises four or six cysteine ​​insertion mutations, the cysteine ​​insertion mutations being (i) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (ii) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (iii) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (iv) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (v) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0073] Additional mutations In certain embodiments of the present disclosure, the cysteine ​​engineered antibody construct may include additional mutations known in the art to provide the antibody construct with desired functional changes. For example, in some embodiments, mutations may be introduced into the CH2 domain of the cysteine ​​engineered antibody construct to alter binding to one or more Fc receptors and / or mutations may be introduced into the CH3 domain of the cysteine ​​engineered antibody construct to improve heterodimer formation when the antibody construct includes a heterodimeric Fc region. In some embodiments where the antibody construct is bispecific or multispecific, mutations may be introduced into the Fab region to promote correct pairing between each heavy and light chain. Examples of such Fab region mutations include those described in International Patent Application Publication Nos. WO2014 / 082179, WO2015 / 181805, and WO2017 / 059551.

[0074] CH2 domain mutations In certain embodiments, a cysteine ​​engineered antibody construct may comprise one or more additional mutations within the CH2 domain, for example, a cysteine ​​engineered antibody construct may comprise a modified CH2 domain with altered binding to one or more Fc receptors, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0075] Various amino acid mutations in the CH2 domain that selectively change the affinity to different Fcγ receptors are known in the art. Amino acid mutations that result in increased binding and amino acid modifications that result in decreased binding can both be useful in certain indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) improves antibody-dependent cell-mediated cytotoxicity (ADCC), thus increasing the lysis of target cells. Similarly, decreasing the binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations. Increasing the binding to FcγRIIb, or decreasing or eliminating the binding of the Fc region to all of the Fcγ receptors ("knockout" variants), can be useful when decreasing or eliminating ADCC and complement-mediated cytotoxicity (CDC) is desired.

[0076] Examples of amino acid mutations that alter binding by Fcγ receptors include, but are not limited to, S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom, et al., 2011, Breast Cancer Res, 67(18):8882-90), and F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom, et al., 2011, Breast Cancer Res, 67(18):8882-90). Res,13(6):R123), F243L (increased affinity for FcγRIIIa) (Stewart,et al.,2011,Protein Eng Des Sel.,24(9):671-8), S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields,et al.,2001,J Biol Chem,276(9):6591-604), S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar,et al.,2006,Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) Chu, et al., 2008, Mol Immunol, 45(15):3926-33).

[0077] Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No11, ISBN1 907568 37 9, Oct 2012, page 283).

[0078] Various publications describe strategies that have been used to engineer antibodies to produce "knockout" variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691; Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication Nos. 2011 / 0212087, 2012 / 0225058, and 2012 / 0251531, International Publication No. WO 2006 / 105338, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0079] Specific non-limiting examples of known amino acid mutations to reduce FcγR and / or complement binding to Fc include, but are not limited to, N297A, L234A / L235A, C220S / C226S / C229S / P238S, C226S / C229S / E3233P / L235V / L235A, L234F / L235E / P331S, IgG2 V234A / G237A, IgG2 H268Q / V309L / A330S / A331S, IgG4 L235A / G237A / E318A, and IgG4 S228P / L236E. Additional examples include Fc regions engineered to contain the amino acid modifications L235A / L236A / D265S, and the asymmetric amino acid modifications described in International Patent Application Publication No. WO2014 / 190441.

[0080] CH3 domain mutations In certain embodiments, the cysteine ​​engineered antibody constructs described herein may comprise one or more additional mutations in the CH3 domain, for example, the cysteine ​​engineered antibody constructs may comprise a modified CH3 domain comprising one or more amino acid mutations that favor the formation of a heterodimeric Fc over the formation of a homodimeric Fc. Heterodimeric Fc regions may be useful, for example, in bispecific antibody constructs and in those cysteine ​​engineered antibody constructs that comprise a single cysteine ​​insertion mutation or an asymmetric combination of cysteine ​​insertion mutations.

[0081] Various amino acid mutations that can be made in the CH3 domain of an Fc to promote the formation of a heterodimeric Fc are known in the art, such as those described in International Patent Application Publication No. WO 96 / 027011 ("knobs-into-holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include asymmetrically modified Fc regions, as described in International Patent Application Publication Nos. WO2012 / 058768 and WO2013 / 063702.

[0082] In certain embodiments, the cysteine ​​engineered antibody construct comprises a modified CH3 domain, where one Fc polypeptide comprises an amino acid mutation at position F405 selected from F405A, F405S, F405T, and F405V, and an amino acid mutation at position Y407 selected from Y407I and Y407V, and the other Fc polypeptide comprises an amino acid mutation at position T366 selected from T366I, T366L, or T366M, and the amino acid mutation T394W. In some embodiments, the amino acid mutation at position T366 is T366I or T366L.

[0083] In some embodiments, one Fc polypeptide comprises amino acid mutations at positions F405 and Y407 as described above, and further comprises the amino acid mutation L351Y.

[0084] In some embodiments, one Fc polypeptide comprises amino acid mutations at positions T366 and T394 as described above, and further comprises an amino acid mutation at position K392 selected from K392F, K392L, or K392M. In some embodiments, the amino acid mutation at position K392 is K392L or K392M.

[0085] In some embodiments, the cysteine ​​engineered antibody construct comprises a modified CH3 domain, as described above, in which one Fc polypeptide comprises amino acid mutations at positions F405 and Y407, and optionally further comprises an amino acid mutation at position L351, and the other Fc polypeptide comprises amino acid mutations at positions T366 and T394, and optionally further comprises an amino acid mutation at position K392, and one or both of the Fc polypeptides further comprises the amino acid mutation T350V.

[0086] In certain embodiments, the cysteine ​​engineered antibody construct comprises a modified CH3 domain, where one Fc polypeptide comprises the amino acid mutation F405A, F405S, F405T, or F405V together with the amino acid mutation Y407I or Y407V, and optionally further comprises the amino acid mutation L351Y, and the other Fc polypeptide comprises the amino acid mutation T366I or T366L together with the amino acid mutation T394W, and optionally further comprises the amino acid mutation K392L or K392M. In some embodiments, one or both of the Fc polypeptides further comprises the amino acid mutation T350V. In some embodiments, both Fc polypeptides further comprise the amino acid mutation T350V.

[0087] In certain embodiments, the cysteine ​​engineered antibody construct comprises a modified CH3 domain comprising an amino acid mutation as set forth in any one of variant 1, variant 2, variant 3, variant 4, or variant 5 of Table 1. [Table 1]

[0088] Preparation of cysteine ​​engineered antibody constructs The cysteine ​​engineered antibody constructs described herein can be prepared using standard recombinant methods. Recombinant production generally involves synthesizing one or more polynucleotides encoding the cysteine ​​engineered antibody construct, cloning the one or more polynucleotides into an appropriate vector(s), and introducing the vector(s) into a suitable host cell for expression of the cysteine ​​engineered antibody construct. Recombinant production of proteins is well known in the art and can be accomplished using standard techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, (1987 & updates), John Wiley & Sons, New York, NY, and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990).

[0089] Accordingly, certain embodiments of the present disclosure relate to an isolated polynucleotide or set of polynucleotides that encode the cysteine ​​engineered antibody constructs described herein. Thus, a polynucleotide in this context may encode all or a portion of a cysteine ​​engineered antibody construct.

[0090] The terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide that "encodes" a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0091] For expression of cysteine ​​engineered antibody constructs, one or more polynucleotides encoding the cysteine ​​engineered antibody constructs can be inserted into a suitable expression vector using standard ligation techniques, either directly or after one or more subcloning steps. Examples of suitable vectors include, but are not limited to, plasmids, phagemids, cosmids, bacteriophages, baculoviruses, retroviruses or DNA viruses. A vector is typically selected to be functional in the particular host cell to be used, i.e., the vector is compatible with the host cell machinery and allows for the amplification and / or expression of the polynucleotide(s). In this regard, the selection of an appropriate vector and host cell combination is within the ordinary skill of the art.

[0092] Accordingly, certain embodiments of the present disclosure relate to vectors (such as expression vectors) that comprise one or more polynucleotides encoding the cysteine ​​engineered antibody constructs described herein. The polynucleotide(s) may be contained in a single vector or in two or more vectors. In some embodiments, the polynucleotides are contained in a multicistronic vector.

[0093] Typically, an expression vector contains one or more regulatory elements for maintaining the plasmid and cloning and expressing an exogenous polynucleotide sequence. Examples of such regulatory elements include a promoter, an enhancer sequence, an origin of replication, a transcription termination sequence, donor and acceptor splice sites, a leader sequence for secreting a polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a polynucleotide encoding a polypeptide to be expressed, and a selection marker.

[0094] Regulatory elements can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of regulatory sequences from two or more sources), or synthetic. Thus, the source of the regulatory sequences can be any prokaryotic or eukaryotic organism, provided that the regulatory sequences are functional in and can be activated by the machinery of the host cell in which they are used.

[0095] Optionally, the vector may contain a "tag" coding sequence, which is a nucleic acid sequence located at the 5' or 3' end of the coding sequence that encodes a heterologous peptide sequence, such as polyHis (e.g., 6xHis), FLAG®, HA (influenza virus hemagglutinin), myc, metal affinity, avidin / streptavidin, glutathione-S-transferase (GST) or biotin tag. This tag typically remains fused to the expressed protein and can serve as a means for affinity purification or detection of the protein. Optionally, the tag can subsequently be removed from the purified protein by various means, for example, by using specific peptidases for cleavage.

[0096] A variety of expression vectors are readily available from commercial sources. Alternatively, if a commercially available vector that contains all the desired regulatory elements is not available, a commercially available vector can be used as a starting vector to construct an expression vector. If one or more of the desired regulatory elements are not already present in the vector, they can be obtained separately and ligated into the vector. Methods for obtaining various regulatory elements and for constructing expression vectors are well known to those skilled in the art.

[0097] Once an expression vector containing the polynucleotide(s) encoding a cysteine ​​engineered antibody construct has been constructed, the vector may be inserted into a suitable host cell for amplification and / or protein expression. Transformation of the expression vector into a selected host cell may be accomplished by well-known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, and other known techniques. The method selected will depend, in part, on the type of host cell used. These and other suitable methods are well known to those of skill in the art (see, e.g., Sambrook, et al., ibid.).

[0098] A host cell transformed with an expression vector, when cultured under appropriate conditions, will express the protein encoded by the vector, and the protein can then be collected from the culture medium (if the host cell secretes the protein) or directly from the host cell that produces it (if the protein is not secreted). Host cells can be prokaryotic (e.g., bacterial cells) or eukaryotic (e.g., yeast, fungal, plant or mammalian cells). Selection of an appropriate host cell can be readily made by one of ordinary skill in the art, taking into consideration a variety of factors, such as the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) desired or necessary for activity, and ease of folding into a biologically active molecule.

[0099] Accordingly, certain embodiments of the present disclosure relate to host cells comprising a polynucleotide(s) encoding a cysteine ​​engineered antibody construct, or one or more vectors comprising a polynucleotide(s) encoding a cysteine ​​engineered antibody construct, In certain embodiments, the host cell is a eukaryotic cell.

[0100] For example, eukaryotic microbes such as filamentous fungi or yeast can be used as host cells, including fungal and yeast strains in which the glycosylation pathway has been "humanized" (see, e.g., Gerngross, 2004, Nat. Biotech., 22:1409-1414 and Li et al., 2006, Nat. Biotech., 24:210-215). Plant cells can also be utilized as host cells (see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429, which describe the PLANTIBODIES™ technology).

[0101] In some embodiments, the host cell is a mammalian cell. Various mammalian cell lines can also be used as host cells. Examples of useful mammalian host cell lines include, but are not limited to, SV40 transformed monkey kidney line CV1 (COS-7), human embryonic kidney line 293 (e.g., HEK293 cells as described in Graham, et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, 1980, Biol. Reprod., 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT060562), TRI cells (e.g., Mather, et al., 1982, Annals of NYAcad. Sci., 383:44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR as described in Urlaub, et al., 1980, Proc. Natl. Acad. Sci. USA, 77:4216). - including CHO cells, and myelomas (such as Y0, NS0 and Sp2 / 0). See also Yazaki and Wu, 2003, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed., Humana Press, Totowa, NJ).

[0102] Certain embodiments of the present disclosure relate to methods of preparing a cysteine ​​engineered antibody construct described herein, comprising transfecting a host cell with one or more polynucleotides encoding the cysteine ​​engineered antibody construct, e.g., as one or more vectors comprising the polynucleotide(s), and culturing the host cell under conditions suitable for expression of the encoded cysteine ​​engineered antibody construct.

[0103] Typically, cysteine ​​engineered antibody constructs can be isolated from host cells after expression and optionally purified. Methods for isolating and purifying expressed proteins are well known in the art. Standard purification methods include chromatographic techniques such as ion exchange, hydrophobic interaction, affinity, sizing, gel filtration, or reversed phase, which can be performed at atmospheric pressure or at medium or high pressure using systems such as FPLC, MPLC, and HPLC. Other purification methods include electrophoretic methods, immunological techniques, precipitation methods, dialysis methods, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques in combination with protein concentration can also be useful.

[0104] A variety of natural proteins that bind to the Fc region or other regions of antibodies are known in the art and can therefore be used to purify Fc-containing proteins. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can often be enabled by specific fusion partners or affinities as described above. For example, antibodies can be bound to glutathione resins when GST fusions are used, Ni when His tags are used, etc. +2 Purification may be performed using affinity chromatography or, if a Flag tag is used, immobilized anti-Flag antibodies. Examples of useful purification techniques are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), and Protein Purification: Principles and Practice, 3 rd Ed., Scopes, Springer-Verlag, NY (1994).

[0105] Conjugates Certain embodiments of the present disclosure relate to conjugates comprising a cysteine ​​engineered antibody construct as described herein and an active agent conjugated to the antibody construct via the inserted cysteine ​​residue. The active agent can be, for example, a therapeutic agent, a diagnostic agent, or a labeling agent.

[0106] Conjugation of the selected active agent to the cysteine ​​engineered antibody construct can be accomplished in a variety of ways known in the art, and can be direct or via a linker. The linker for conjugation of the active agent is a bifunctional or multifunctional moiety that can bind one or more active agents to the antibody construct. A bifunctional (or monovalent) linker binds a single active agent to a single site on the antibody construct, while a multifunctional (or multivalent) linker binds two or more active agents to a single site on the antibody construct. A linker that can bind one active agent to two or more sites on the antibody construct can also be considered multifunctional.

[0107] When a linker is used to conjugate an active agent to a cysteine ​​engineered antibody construct, the linker contains a thiol-reactive functional group and is reacted with an inserted cysteine ​​residue in the antibody construct. Examples of thiol-reactive functional groups include, but are not limited to, maleimides, α-haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isothiocyanates, and isocyanates.

[0108] The linker also comprises a functional group that can react with the targeting group on the active agent. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (GTHermanson, 2013, Academic Press). Groups on the active agent that can serve as targeting groups for linker attachment include, but are not limited to, thiol, hydroxyl, carboxyl, amine, aldehyde, and ketone groups.

[0109] Non-limiting examples of functional groups for reacting with thiols are described above. Non-limiting examples of functional groups for reacting with amines include activated esters (such as N-hydroxysuccinamide (NHS) esters and sulfo-NHS esters), imidoesters, (such as Traut's reagent), isothiocyanates, aldehydes, and anhydrides (such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include succinimide-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).

[0110] Non-limiting examples of functional groups that can react with electrophilic groups (such as aldehyde or ketone carbonyl groups) on an activator include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0111] Linker may be cleavable or non-cleavable.Cleaving linker is typically susceptible to cleavage under intracellular conditions, for example, via lysosomal process.Examples include protease-sensitive, acid-sensitive, or reduction-sensitive linkers.In contrast, non-cleavable linker relies on the degradation of antibody in cells, which typically results in the release of amino acid-linker active agent moiety.

[0112] Suitable cleavable linkers include, for example, peptide-containing linkers that are cleavable by an intracellular protease, such as a lysosomal or endosomal protease. For example, the linker may include a dipeptide such as valine-citrulline (Val-Cit) or phenylalanine-lysine (Phe-Lys). Other examples of dipeptides suitable for inclusion in the linker include Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, and Met-(D)Lys. Linkers may also include longer peptide sequences, such as the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, or (D)Ala-Phe-Lys, or the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, or Ala-Leu-Ala-Leu.

[0113] Additional examples of cleavable linkers include disulfide-containing linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimidyl-4-(2-pyridyldithio)butanoic acid (SPBD) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoic acid (sulfo-SPBD). Disulfide-containing linkers can optionally include inclusion of additional groups, such as geminal dimethyl groups, to provide steric hindrance adjacent to the disulfide bond to improve the extracellular stability of the linker. Other suitable linkers include hydrolyzable linkers at a specific pH or within a pH range, such as hydrazone linkers.

[0114] A further example of a cleavable linker is a linker that contains a β-glucuronide that is cleavable by the enzyme β-glucuronidase, which is present in lysosomes and the tumor stroma (see, e.g., Graaf, et al., 2002, Curr. Pharm. Des. 8:1391-1403).

[0115] Cleavable linkers may optionally further comprise one or more additional functionalities, such as self-immolative and self-leaving groups, stretchers, or hydrophilic moieties.

[0116] Self-immolative and self-eliminating groups used in linkers include, for example, p-aminobenzyloxycarbonyl (PAB or PABC) and p-aminobenzyl ether (PABE) groups, as well as methylated ethylenediamine (MED). Other examples of self-immolative groups include, but are not limited to, heterocyclic derivatives, such as aromatic compounds that are electronically similar to PABC or PABE groups, such as 2-aminoimidazole-5-methanol derivatives as described in U.S. Pat. No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues, et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-immolative / self-eliminating groups, alone or in combination, are often included in peptide-based linkers and may be included in other types of linkers as well.

[0117] Stretchers used in linkers of ADCs include alkylene groups, and aliphatic acid, diacid, amine, or diamine based stretchers, such as diglycolate, malonic acid, caproic acid, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers.

[0118] Various non-cleavable linkers for attaching active agents to antibodies are also known in the art. Examples include, but are not limited to, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) ("long chain" SMCC or LC-SMCC), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), gamma-maleimidobutyric acid N-succinimidyl ester (GMBS), epsilon-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N- These include linkers based on hydroxysuccinimide esters (MBS), N-(α-maleimidoacetoxy)-succinimide esters (AMAS), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl-4-iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0119] The number of active agent molecules that can be conjugated to a given cysteine ​​engineered antibody construct (drug-to-antibody ratio or DAR) depends on the number of cysteine ​​insertion mutations contained by the antibody construct and the type of linker used (monovalent or multivalent).

[0120] Certain embodiments of the present disclosure include compounds of formula (I): A-(L-(D) q ) p (I) A conjugate having the formula: A is a cysteine ​​engineered antibody construct described herein, L is a linker (e.g., a linker described in any one of the embodiments above); D is an activator; q is an integer from 1 to 4; p is an integer from 1 to 8; Conjugates in which D is attached via L to an inserted cysteine ​​residue in the cysteine ​​engineered antibody construct.

[0121] In some embodiments, in formula (I), q is 1, 2, or 3. In some embodiments, in formula (I), q is 1 or 2. In some embodiments, in formula (I), p is an integer from 1 to 6. In some embodiments, in formula (I), p is 1, 2, 3, or 4. In some embodiments, in formula (I), p is 6.

[0122] In some embodiments, in formula (I), q is 1, 2, or 3, and p is 1, 2, 3, or 4. In some embodiments, in formula (I), q is 1 or 2, and p is an integer from 1 to 8. In some embodiments, in formula (I), q is 1 or 2, and p is 1, 2, 3, or 4. In some embodiments, in formula (I), q is 1 or 2, and p is 6.

[0123] In certain embodiments, the conjugate has formula (II): A-(LD) p (II) wherein A is a cysteine ​​engineered antibody construct described herein, L is a linker (e.g., a linker described in any one of the embodiments above); D is an activator; p is an integer from 1 to 8; D is attached via L to an inserted cysteine ​​residue in the cysteine ​​engineered antibody construct.

[0124] In some embodiments, in formula (II), p is an integer of 1 to 6. In some embodiments, in formula (II), p is 1, 2, 3, or 4. In some embodiments, in formula (II), p is 1, 2, or 3. In some embodiments, in formula (II), p is 2. In some embodiments, in formula (II), p is 1 or 3. In some embodiments, in formula (II), p is 4 or 6.

[0125] Methods for conjugating various agents to free thiol groups on proteins, including antibodies, are known in the art (see, e.g., Bioconjugate Techniques (GT Hermanson, 2013, Academic Press) and exemplary methods are also described in the Examples herein.

[0126] Certain embodiments of the present disclosure relate to methods of preparing conjugates comprising the cysteine ​​engineered antibody constructs of the present disclosure. In some embodiments, the methods comprise subjecting a cysteine ​​engineered antibody construct comprising at least one inserted cysteine ​​residue as described herein to reducing conditions such that the thiol group of the inserted cysteine ​​residue is reduced, and reacting a thiol-reactive linker-activator with the antibody construct under conditions that allow for the formation of a bond between the linker and the reduced thiol.

[0127] Certain embodiments of the present disclosure relate to a method of preparing an antibody-drug conjugate having a predetermined drug-antibody ratio (DAR), the method comprising reacting a cysteine ​​engineered antibody construct comprising one or more cysteine ​​insertion mutations described herein with a drug-linker to provide an antibody-drug conjugate, the predetermined DAR being 1, 2, 3, 4, 5, 6, 7, or 8, and the cysteine ​​engineered antibody construct comprising the same number of cysteine ​​insertion mutations as the predetermined DAR. In certain embodiments of this method, the predetermined DAR is 2. In some embodiments, the predetermined DAR is 1 or 3. In some embodiments, the predetermined DAR is 4 or 6.

[0128] In some embodiments, the predetermined DAR is 1 or 3 and the cysteine ​​insertion mutation comprised by the cysteine ​​engineered antibody construct is (a) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain, and (d) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0129] In some embodiments, the predetermined DAR is 1 or 3 and the cysteine ​​insertion mutation comprised by the cysteine ​​engineered antibody construct is (i) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (ii) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (iii) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (iv) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (v) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

[0130] Activator Active agents that may be conjugated to cysteine ​​engineered antibody constructs include therapeutic agents, diagnostic agents, and labeling agents.

[0131] Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, toxins, apoptotic agents, thrombotic agents, antiangiogenic agents, biological response modifiers, growth factors, radioactive materials and macrocyclic chelates useful for conjugating radioactive metal ions. Examples of diagnostic agents include, but are not limited to, various imaging agents such as fluorescent materials, luminescent materials, and radioactive materials. Examples of labeling agents include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, and radioactive materials.

[0132] Certain embodiments of the present disclosure relate to conjugates comprising the cysteine ​​engineered antibody constructs described herein and a therapeutic agent. Some embodiments relate to conjugates comprising the cysteine ​​engineered antibody constructs described herein and an anti-cancer agent. Exemplary anti-cancer agents include, but are not limited to, maytansinoids, auristatins, hemiasterins, tubulysins, dolastatins, trichothecenes, duocarmycins, camptothecins, calicheamicins, and other enediyne antibiotics, taxanes, anthracyclines, Pseudomonas exotoxins (PEs), pyrrolobenzodiazapenes (PBDs), and analogs and derivatives thereof.

[0133] Pharmaceutical Compositions Certain embodiments of the present disclosure relate to pharmaceutical compositions for therapeutic or diagnostic use, comprising the conjugates described herein and pharma- ceutical acceptable carriers or diluents.The compositions can be prepared by known procedures using well-known and readily available ingredients, and can be formulated for administration to subjects, for example, orally (including buccal or sublingual), topically, parenterally, rectally or vaginally, or by inhalation or spray.The term "parenteral" as used herein includes injection or infusion by subcutaneous, intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal or intrathecal routes.

[0134] The compositions are typically formulated in a form suitable for administration to a subject by a selected route, such as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable, or liquid. The compositions may be provided as unit dose formulations.

[0135] Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or or gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0136] In certain embodiments, the composition may be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions may contain the conjugate in a non-toxic parentally acceptable diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution. In addition, sterile fixed oils may be employed as a solvent or suspending medium. For this purpose, various brands of fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are also used in the preparation of injectables. Auxiliary agents such as local anesthetics, preservatives and / or buffers known in the art may also be included in the injectable solution or suspension.

[0137] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art, see, e.g., "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0138] How to use Conjugates comprising the cysteine ​​engineered antibody constructs of the present disclosure conjugated to an active agent may be used in therapeutic, diagnostic and screening methods, the exact nature of which will depend on the nature of the conjugate, including the type of active agent conjugated to the cysteine ​​engineered antibody construct.

[0139] For example, certain embodiments of the present disclosure relate to methods of treating a disease or disorder by administering to a subject having the disease or disorder a conjugate comprising a cysteine ​​engineered antibody construct described herein conjugated to a therapeutic agent. In some embodiments where the therapeutic agent is an anti-cancer agent, the conjugate may be used in methods of treating cancer.

[0140] Certain embodiments of the present disclosure relate to methods of diagnosing a disease or disorder comprising administering to a subject suspected or known to have the disease or disorder a conjugate comprising a cysteine ​​engineered antibody construct described herein conjugated to a diagnostic agent. Some embodiments relate to methods of diagnosing a disease or disorder comprising contacting a biological sample obtained from a subject suspected or known to have the disease or disorder with a conjugate comprising a cysteine ​​engineered antibody construct described herein conjugated to a diagnostic agent.

[0141] Certain embodiments of the present disclosure relate to a method of screening a biological sample, such as a sample taken from a subject, for the presence of a target moiety, comprising contacting the sample with a conjugate comprising a cysteine ​​engineered antibody construct described herein conjugated to a labeling agent, wherein the cysteine ​​engineered antibody construct specifically binds to the target moiety.

[0142] The following examples are offered for illustrative purposes and are not intended to limit the scope of the claimed invention in any way. EXAMPLES

[0143] General Procedure 1. Cloning, Expression, and Purification of Cysteine ​​Engineered Variants An IgG1 antibody targeting c-Met or FRα and with a heterodimeric Fc region (HetFc) was used to construct the cysteine ​​engineered constructs and controls described in the Examples below. HetFc contains the following mutations in the CH3 domain:

[0144] Chain A (HC-A): T350V_L351Y_F405A_Y407V

[0145] Chain B (HC-B): T350V_T366L_K392L_T394W

[0146] Cysteine ​​engineered constructs and controls were cloned and expressed as follows: Genes encoding the antibody heavy and light chains were constructed via gene synthesis using codons optimized for human / mammalian expression. The signal peptide MAVMAPRTLVLLLSGALALTQTWAG [SEQ ID NO: 1] was included at the N-terminus of each polypeptide sequence. In some cases, the light chain contained the peptide ESSCDVKLV [SEQ ID NO: 2] fused directly to the C-terminal residue.

[0147] The final gene products were subcloned into the mammalian expression vector PTT5 (NRC-BRI, Canada) and expressed in CHO cells (Durocher, et al., 2002, Nucl Acids Res., 30(2):E9). Briefly, CHO-3E7 cells were grown in suspension in FreeStyle™ F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 0.1% w / v Pluronic and 4 mM glutamine to a cell density of 1.5-2 million cells / ml with viability >97%. Transfections were performed using a mixture of plasmid DNA: 5% pTTo-GFP plasmid (green fluorescent protein to determine transfection efficiency), 15% pTT22-AKT plasmid, 21% antibody construct DNA (at a ratio of 1:1:3 HC-A, HC-B, LC), 68.37% salmon sperm DNA as described by Durocher and coworkers (Delafosse, et al., 2016, J Biotechnol, 227:103-111; Raymond, et al., 2015, MAbs, 7(3):571-83). After transfection, shake flasks containing cells were placed on an orbital shaker set at 120 rpm in a humidified incubator at 37°C and 5% CO2. 24 hours after transfection, 1% w / v tryptone N1 (TN1) and 0.5 mM valproic acid were added to the cultures. Cultures were then transferred to an orbital shaker (120 rpm) placed in a humidified incubator at 32°C and 5% CO2. At 24-48 hours, GFP positive cells should be 30-60% as determined by flow cytometry. Cells were harvested 7-10 days after transfection, spun at 4,000 rpm, then filter sterilized (clarified) using a 0.45 μm filter (Millipore Sigma, Burlington, MA) and frozen at -80°C.

[0148] Thawed clarified medium was loaded onto a MabSelect™ SuRe™ Protein-A column (GE Healthcare, Chicago, IL) and washed with 10 column volumes of PBS buffer (pH 7.2). Antibody was eluted with 10 column volumes of citrate buffer (pH 3.6) and the pooled antibody-containing fractions were neutralized with TRIS (pH 11).

[0149] The antibody-containing Protein-A eluate was further purified by size-exclusion chromatography (SEC). For SEC, the sample was loaded onto a Sephadex200 HiLoad® 16 / 60 200prep grade column (GE Healthcare, Chicago, IL) using an AKTA™ purification system (GE Healthcare, Chicago, IL; Express, FPLC or Purifier system) at a flow rate of 1 mL / min. PBS buffer (PH 7.4) was used at a flow rate of 1 mL / min. Fractions corresponding to purified antibodies were pooled based on SDS-PAGE or capillary electrophoresis analysis (LabChip GX®; PerkinElmer, Inc., Waltham, MA) and concentrated to 5-10 mg / mL as needed. In addition, if low endotoxin was a requirement for downstream analysis, the system, column, and resin (if applicable) were depyrogenated using NaOH solution with standard protocols prior to protein purification.

[0150] 2. Drug Conjugation Cysteine ​​engineered variants and controls were expressed in cysteine-capped forms: L-cysteine ​​capped, glutathione capped, or a combination of both. To reduce sample heterogeneity and increase conjugation efficiency, all antibodies were subjected to a reduction-oxidation step prior to conjugation with maleimide-activated drug-linkers. A representative procedure is provided below.

[0151] 3 mg of a 5 mg / mL solution of variant v28983 (see Table 2.2; MW146301Da) was reduced in PBS (pH 7.4) with a molar excess of 25 equivalents of tris(2-carboxyethyl)phosphine (TCEP) in the presence of 1 mM diethylenetriaminepentaacetic acid (DTPA) for 3 hours at 37°C (water bath) based on the following calculations (Table A): [Table 2]

[0152] After reduction was complete, excess TCEP was removed using a 5 mL 40 kD Zeba™ Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA) equilibrated with PBS (pH 7.4). The reduced antibody was oxidized overnight (18 hours) at 4° C. with a 25 molar excess of dehydroascorbic acid (DHAA) (assuming 100% recovery from Zeba™ column purification) to reform interchain disulfide bonds while keeping the inserted cysteines in the reduced (free thiol) form. The addition of DHAA was based on the following calculations (Table B): [Table 3]

[0153] The oxidized antibody was divided into three aliquots of 1 mg for conjugation to three different drug-linkers: MTvc compound 1, MCvcPABC-MMAE and MCvcPAB-Tulysin M. The structures of the three drug-linkers are shown in Figure 1. Conjugation was achieved by incubation with a 5 molar excess of drug-linker at room temperature. Drug-linkers were prepared as 10 or 20 mM DMSO stocks and added to the reactions based on the following calculations (Table C). [Table 4]

[0154] 3. Differential Scanning Calorimetry (DSC) The thermal stability of cysteine ​​engineered antibodies was measured using DSC as follows: 400 μL of purified sample at a concentration of either 0.2 mg / ml or 0.4 mg / mL in PBS was used for DSC analysis using a MicroCal VP-Capillary DSC™ (GE Healthcare, Chicago, IL). At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline and a buffer injection was set up before each sample injection for reference. Each sample was scanned from 20 to 100°C at a rate of 60°C / hr with low feedback, 8 sec filter, 5 min preTstat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin7 software (OriginLab Corporation, Northampton, MA).

[0155] 4. Hydrophobic Interaction Chromatography (HIC) For HIC runs, a TSKgel Butyl-NPR (2.5 μm, 4.6 × 35 mm) column (TOSOH Bioscience GmbH, Griesheim, Germany) was loaded with 5 column volumes of buffer A (1.5 M (NH4)2SO4, 25 mM PO4) at room temperature. 3- Typically, 20-30 ug of sample at a concentration of 2-3 mg / mL was equilibrated with 95% buffer A and 5% buffer B (75% of 25 mM PO4 3- The column was loaded with 100% ethanol (pH 6.95 + 25% isopropanol) and run at 0.5 mL / min for 15 min using the following gradient (Table D): [Table 5]

[0156] For each sample, the HIC chromatogram was integrated using appropriate parameters that provided a full baseline-to-baseline integral of each peak followed by an integral of each peak showing reasonable separation. Peaks corresponding to different DAR species within the sample were identified. The DAR0 peak showed a retention time consistent with the naked (reduced and oxidized) antibody. For ADCs containing single cysteine ​​insertion variants, each subsequent peak represents DAR1 and DAR2.

[0157] DAR by HIC was calculated based on the AUC for each DAR species (0, 1, and 2). Calculated DAR=(%AUCx0+%AUCx1+%AUCx2) / 100

[0158] The HIC retention time (HIC-RRT) of each ADC was calculated as follows: HIC-RRT = RT of target DAR / RT of DAR0

[0159] To minimize the effect of cysteine ​​capping of antibodies on HIC-RRT, the RT of DAR0 refers to the retention time of reduced and oxidized antibody without conjugation to a payload. Each variant has its own DAR0 RT for HIC-RRT calculations.

[0160] 5. Analytical Size Exclusion Chromatography (SEC) For analytical SEC runs, an Agilent Advance Bio SEC column (300 Å, 2.7 μm, 7.8×150 mm) (Agilent Technologies, Inc., Santa Clara, CA; serial #6377910-24) was loaded with 5 column volumes of buffer A (150 mM NaCl) at room temperature. xThe column was equilibrated with 500 mL of PO4, pH 6.95. Typically, 20-30 ug of sample at a concentration of 2-3 mg / mL was loaded onto the column and run isocratically at 1 mL / min for 7 min, with absorbance at 280 nm reported. For each sample, the chromatogram was integrated to obtain a full baseline-to-baseline integral for each peak, with reasonable placement of separation between partially resolved peaks. The peak corresponding to the major component of IgG (approximate retention time 3.3 min) was reported as monomeric based on the SEC profile of a control IgG1 antibody, trastuzumab. Any peak occurring before 3.3 min was designated as HMWS, and any peak occurring after 3.3 min was designated as LMWS, excluding the solvent peak (greater than 5.2 min).

[0161] 6. Liquid Chromatography-Mass Spectrometry (LC-MS) For DAR by LC-MS, ADCs were diluted to 1 mg / mL in PBS (pH 7.4) and then deglycosylated. For deglycosylation, typically 1 ug of EndoS was used for every 10 ug of ADC, and the reaction mix was incubated at room temperature for 1 hour. The samples were reduced by adding 3 uL of 500 mM TCEP to each 10 uL sample, followed by incubation at 70° C. for 1 hour. Finally, the samples were run on an LC-MS quadrupole time-of-flight (QTOF) system (Agilent 1290 HPLC connected to an Agilent 6545 QTOF; Agilent Technologies, Inc., Santa Clara, CA) with an injection of 1 uL each. The detailed procedure is described below. Column: PLRP-S1000Å, 8uM, 50x2.1mm (Agilent Technologies, Inc., Santa Clara, CA) Mobile phase C: 0.1% formic acid, 0.025% trifluoroacetic acid, and 10% isopropyl alcohol in H2O. Mobile phase D: 0.1% formic acid and 10% isopropyl alcohol in acetonitrile Detection: Signal A (280 nm, 4.0 bandwidth), Signal B (220 nm, 4.0 bandwidth) Gradient: [Table 6] ● Time after execution: 2 minutes

[0162] 7. Capillary Electrophoresis-SDS (CE-SDS) All samples were first diluted to 1 mg / mL before preparing samples in 96-well PCR plates according to the manufacturer's protocol (Protein Express Assay LabChip™; PerkinElmer, Inc., Waltham, MA). Briefly, 2 uL of ADC was mixed with 7 uL of Protein Express buffer in the presence or absence of 400 mM dithiothreitol (DTT) as a reducing agent, followed by heat denaturation at 95° C. for 5 minutes. Samples were then diluted in a 1:2 ratio in dH2O prior to data acquisition. After each CE-SDS run, the gels and corresponding electropherograms were analyzed using LabChip™ Reviewer (PerkinElmer, Inc., Waltham, MA).

[0163] Example 1: Identification of potential cysteine ​​insertion sites by in silico engineering Identification of putative cysteine ​​insertion sites in IgG1 was performed on implicit molecular dynamics (MD) trajectories of a model Fab (from PDB ID1JPT, D3H44) and a model Fc molecule (from PDB ID1E4K) according to the following guiding criteria. Elimination of CDR Avoidance of secondary structures Relative Solvent Accessible Surface Area (SASA): rSASA>30% ●Avoid interference with Protein A and FcRn binding

[0164] The locations were then labeled according to rSASA and root mean square variation (RMSF) as follows: Type 1: Desirable rSASA (30-60%) and mobile (RMSF above the mean + 1 standard deviation (SD) threshold) Type 2: Desirable rSASA (30–60%), but ordered (RMSF below the mean + 1 SD threshold) Type 3: exposed (rSASA>60%) and mobile (RMSF above the mean + 1 SD threshold) ● Type 4: exposed (rSASA>60%) but ordered (RMSF below the mean + 1SD threshold)

[0165] Cysteine ​​insertion sites ("designs") in Phase I were proposed based on a structure-guided, semi-rational approach. Putative insertion sites were ranked based on their risk (interference with other known ligands and disulfide scrambling), environmental (types 1-4 labels as described above), as well as estimated potential for structural impact and conjugation stability. A total of 13 designs were proposed in Phase I, sampling different structural regions of IgG1.

[0166] The second phase involved performing modeling experiments for each individual putative cysteine ​​insertion and selecting variants based on parameters calculated from implicit solvent molecular dynamics (MD) trajectories for the modeled insertion. Briefly, each loop of interest (n residues) was removed and n+1 loops from the deposited structure in the RCSB PDB (Research Collaboratory for Structural Bioinformatics-Protein Data Bank) were grafted in its place based on lowest root mean square deviation (RMSD) with the anchor boundary residues. Each loop was then mutated to match the original sequence with the cysteine ​​residues inserted at each relevant position. For each grafted loop model, implicit solvent MD trajectories were calculated and designs were ranked according to the parameters and criteria listed in Table 1.1 below.

[0167] In the few cases where the loop grafting algorithm failed to provide a solution, designs from these loops were selected using the methodology described for Phase 1 design. A total of 32 variants were generated by this process, with 13 variants generated in Phase 1 and 19 variants generated in Phase 2.

Table 7

[0168] Example 2: In Vitro Characterization of Initial Variants Thirty-two variants from Example 1 were cloned together with the controls shown in Table 2.1 and expressed as described in General Procedure 1. Two additional constructs, v27321 and v27322, were also included in this initial characterization. These two variants each contain a cysteine insertion around position K149. Cysteine substitutions at position K149 have been described previously (Vollmar, et al., 2017, Bioconjug Chem, 28(10):2538-2548).

[0169] Throughout the examples, the following nomenclature is used for cysteine insertion mutations. The position numbers are Kabat for the Fab regions (VH, VL, CH1, and CL) and EU for the Fc regions (CH2 and CH3). All cysteine insertions are numbered based on the residue prior to insertion, with reference to the unmodified heavy chain (H) or light chain (L) followed by ".5". For example, L_K149.5C indicates a cysteine inserted after residue Lys149 in the light chain.

Table 8

[0170] Each antibody was then conjugated to MTvc compound 1 or MCvcPABC-MMAE as described in General Procedure 2. Preliminary in vitro characterization of each antibody and ADC was performed as described in General Procedures 3-6. The variants were ranked based on the following criteria. ● DSC: Tm difference from the parental antibody defined as follows ○ No change (0 °C ≤ Tm difference ≤ 3 °C) ○ Small (3 °C < Tm difference ≤ 8 °C) ○ High (Tm difference > 8 °C) High Antibody yield from a single 500 mL transfection in CHO cells DAR(MS) drug-to-antibody ratio determined by LC-MS DAR(HIC) drug-antibody ratio determined by HIC Relative retention time (RRT D2 / D0) calculated by dividing the HIC retention time of DAR2 by the HIC retention time of DAR0

[0171] Ten variants that displayed the most favorable characteristics were selected for further characterization. The properties determined from the preliminary characterization of these ten variants and five controls are shown in Table 2.2. [Table 9]

[0172] Examples of HIC profiles of one variant (v29001(H_T299.5C)) and a control variant (v29013(H_S239.5C)) conjugated to MCvcPABC-MMAE or MTvc compound 1 are shown in Figures 13 and 14, respectively. DSC profiles of the same two variants (unconjugated) are shown in Figure 15.

[0173] The HIC profile of the control variant v29013 (H_S239.5C) can be seen to consist of multiple peaks (Figures 13A and 14A) indicating the presence of multiple species, whereas the HIC profile of variant v29001 (H_T299.5C) shows a single monomeric peak (Figures 13B and 14B). Figure 14 also shows that the MTvc compound 1 conjugate made with variant v29001 appeared to be less hydrophobic (lower HIC-RRT) than the MTvc compound 1 conjugate made with the control variant v29013. The DSC profile in Figure 15 shows that a more pronounced destabilization was observed for the control variant v29013 (Tm of the CH2 domain is 62°C) than for variant v29001 (Tm of the CH2 domain is 65°C). The Tm of the CH3 domain for both of these variants was very similar, confirming that the cysteine ​​insertion does not affect the stability of this domain.

[0174] Example 3: Preparation of antibody-drug conjugates containing cysteine ​​insertion variants Each of the 10 variants shown in Table 2.2, along with two of the controls, v22758 (H_A114C) and v29013 (H_S239.5C), were conjugated to three different drug-linkers (MTvc compound 1, MCvcPABC-MMAE, and MCvcPAB-Tublysin M; see Figure 1) following general procedure 2.

[0175] The resulting 36 ADCs were characterized by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis SDA (CE-SDS), and on-cell binding assays as described in Examples 4-8.

[0176] Example 4: In vitro characterization - Hydrophobic interaction chromatography The ADCs from Example 3 were characterized by hydrophobic interaction chromatography (HIC) as described in General Procedure 4. HIC allows the separation of different proteins based on their inherent hydrophobicity, and because HIC is a non-denaturing method, it allows the separation of different DAR species contained by a given ADC. HIC is also a useful technique for ranking ADCs based on their relative retention time (RRT). Because hydrophobic ADCs may be cleared more quickly from the in vivo circulation, the HIC-RRT value is a potentially useful biophysical parameter for identifying the most useful cysteine ​​insertion sites.

[0177] HIC was used to determine the DAR of all ADCs conjugated to MTvc compound 1, MCvcPABC-MMAE, and MCvcPAB-Tulysin M, and the HIC-RRT of all ADCs conjugated to MTvc compound 1 and MCvcPABC-MMAE. The results are shown in Tables 4.1 and 4.2. [Table 10] [Table 11]

[0178] Overall, all ADCs exhibited a DAR range of 1.4-2.0. When conjugated to each of the three drug-linkers, one variant, v29001, showed a single peak in HIC, indicating full loading with a DAR of 2.0 in each case. Variant v22768 showed the lowest drug loading, only 1.4-1.5 with each drug-linker. Conjugation efficiency was expected to be site-dependent due to the influence of thiol pKa, SASA, and the local environment of the inserted cysteine, which was reflected in the DAR values ​​(Table 4.1).

[0179] The drug-linker MTvc compound 1 is relatively hydrophilic. Conjugation of this drug-linker to any of the site-specific cysteine ​​insertion variants had minimal effect on the HIC retention time. For most of the variants conjugated to MTvc compound 1, the HIC-RRT values ​​were less than 1.15, which was also the HIC-RRT value observed for the control v22758 ADC conjugated to the same drug-linker. Two variants conjugated to MTvc compound 1, v22768 and v28993, showed slightly higher HIC-RRT values ​​than the control v22758 conjugated to the same drug-linker: 1.18 and 1.19, respectively. The variant v29001 conjugated to MTvc compound 1 appeared to be less hydrophobic than both of the controls (v22758 and v29013) conjugated to the same drug-linker.

[0180] The drug-linker MCvcPABC-MMAE is more hydrophobic than MTvc compound 1, and therefore the HIC-RRT values ​​of all ADCs containing MCvcPABC-MMAE were higher than their respective MTvc compound 1 conjugates. Overall, for the MCvcPABC-MMAE conjugates, a similar trend was observed as that observed for the MCvcC compound 1 conjugates: v28993 conjugated to MCvcPABC-MMAE showed the highest HIC-RRT value, and v29001 conjugated to MCvcPABC-MMAE showed the lowest HIC-RRT value (1.05). For one variant, v27321, conjugated to MCvcPABC-MMAE, and the control v29013 conjugated to the same drug-linker, the HIC-RRT values ​​could not be determined due to poor resolution of the HIC profiles of each of these two ADCs.

[0181] Example 5: In vitro characterization - Size exclusion chromatography The ADC from Example 3 was further characterized by size exclusion chromatography (SEC), a useful technique for estimating protein size in protein preparations and determining the presence of aggregates / high molecular weight species (HMWS) and fragmentation / low molecular weight species (LMWS).

[0182] During preparation of the ADCs, any inappropriate oxidation due to the formation of disulfide bonds through the inserted cysteine ​​residues may lead to the formation of concatemers and other HMWS. To determine the molecular size and relative abundance of the different species, each of the ADCs was analyzed by SEC as described in General Procedure 5.

[0183] The results are shown in Table 5.1. [Table 12]

[0184] As can be seen from Table 5.1, all ADC preparations contained greater than 90% monomer by HPLC-SEC. In general, ADCs containing the drug-linker MTvc compound 1 showed the highest monomer content, except for variant v22768, which showed the highest HMWS content (6%) and lowest monomer content (94%) for the MTvc compound 1 conjugate. The control ADC, v22758(A114C), conjugated to MTvc compound 1 showed a monomer content of 95%.

[0185] ADCs containing the MCvcPABC-MMAE drug-linker showed similar trends to those containing MTvc compound 1, with monomeric content ranging from 97% to 99% and lower amounts of HMWS and LMWS.

[0186] In contrast, ADCs containing the MCvcPAB-Tublysin M drug-linker exhibited lower monomer content than ADCs containing either of the other two drug-linkers. The ADC, v29001-MCvcPAB-Tublysin M, exhibited the lowest monomer content (92%) and highest LMWS content (7%) of all ADCs tested.

[0187] Example 6: In vitro characterization - Liquid chromatography-mass spectrometry (LC-MS) The ADC from Example 3 was further characterized by liquid chromatography-mass spectrometry (LC-MS) as described in General Procedure 6.

[0188] LC-MS is the standard analytical method for measuring the drug-antibody ratio (DAR) and drug loading distribution of ADCs. The general procedure for LC-MS-based DAR measurement at the intact ADC level involves deconvoluting the mass spectrum into a series of "zero charge" masses, then integrating and weighting the spectral peak areas or peak intensities to obtain the DAR distribution or calculate the average DAR.

[0189] For analysis of the ADCs from Example 3, the ADCs were deglycosylated prior to LC-MS analysis by treatment with EndoS, which removes all linked carbohydrate moieties except for reduced terminal N-acetylglucosamine (GlcNAc) and fucose (Fuc). Some of the ADCs were deglycosylated prior to LC-MS analysis by treatment with EndoS, which removes all linked carbohydrate moieties except for reduced terminal N-acetylglucosamine (GlcNAc) and fucose (Fuc). 236 GG 237 The ADCs were also treated with IdeS, a protease that cleaves just after the hinge cysteine ​​at position 1. Reduction of the IdeS-treated samples yielded three distinct species: Fc / 2, Fd, and LC. DAR determination by MS on the IdeS-treated samples further improved the accuracy of the DAR measurements and provided additional structural information of the ADCs.

[0190] The average DARs determined by LC-MS for each of the ADCs are shown in Table 6.1. [Table 13]

[0191] As shown in Table 6.1, the DARs of the ADCs determined by LC-MS ranged from 1.7 to 2.0, indicating that all three drug-linkers had similar conjugation efficiencies. No detectable conjugation was observed for the hinge or interchain disulfide cysteine ​​residues.

[0192] Overall, the DARs determined by LC-MS correlated well with those obtained by HIC (see Table 4.1). For example, when conjugated to each of the three drug-linkers, variant v29001 showed a DAR2 by both LC-MS and HIC measurements. For variants v27321 and v29013 conjugated to MCvcPABC-MMAE, calculation of the DAR by HIC was unsuccessful due to poor separation of the relevant peaks. However, the DARs of these two ADCs were successfully determined by LC-MS (DAR1.8 and 2.0, respectively).

[0193] Example 7: In vitro characterization - Capillary electrophoresis-SDS (CE-SDS) To assess sample purity, each of the ADCs from Example 3 was assessed by capillary electrophoresis SDS (CE-SDS) under reducing and non-reducing conditions as described in General Procedure 7.

[0194] Under reducing conditions, the interchain disulfide bonds in the ADC antibody are reduced to obtain the corresponding heavy chain (HC) and light chain (LC), which can be separated according to their molecular weight difference. In contrast, under non-reducing conditions, the antibody remains intact and can be separated from any partial antibodies or antibody fragments, as well as any concatemers. Intact full-length IgG1 (2H-2L) has the highest molecular weight of about 150 kDa, followed by 2H-L, HH, HL, H, L fragments with molecular weights of about 125, 100, 75, 50, and 25 kDa, respectively. Incomplete or partial oxidation of the antibody during the preparation of the ADC leads to the presence of some or all of these LMWS in the sample, while overoxidation leads to cysteine-cysteine ​​linked oligomeric HMWS.

[0195] The results are shown in Figure 2. Note that the intact non-reduced ADCs, including the unconjugated control (v17427), showed a MW of approximately 160 kDa (as shown in Figure 2(A)) rather than the calculated 150 kDa. This discrepancy may be due to either the effect of the conjugation dye used in the LabChip™ protocol on peptide binding or the inherent precision limitations of the instrument (±20% size precision based on the manufacturer's protocol). Similarly, all reduced samples, including the unconjugated control (v17427), showed a MW of approximately 28 kDa for LC and approximately 64 kDa for HC, both slightly higher than the calculated values ​​of approximately 23 kDa and 50 kDa, respectively (see Figure 2(B)).

[0196] Comparison of all 36 ADCs shown in Figure 2 with the unconjugated control (v17427) indicates that the reduction-oxidation step in the conjugation protocol was successful and each antibody refolded into its original full-length conformation prior to conjugation to the drug-linker.

[0197] Example 8: Antigen binding assay on cells by flow cytometry The apparent binding affinity of the ADC from Example 3 for the target c-Met, both as naked antibodies and as conjugates, was assessed by flow cytometry as described below and compared to the binding affinity of a control unconjugated parent antibody (v17427).

[0198] Cells from the high c-Met expressing cell line EBC-1 (4 million receptors / cell) (XenoTech, LLC, Lenexa, KS) were seeded at 25,000 cells / well with a minimum seeding volume of 100 μL. Briefly, adherent EBC-1 cells were detached from the culture vessel using cell dissociation buffer and seeded into 96-well plates at 25,000 cells / well. Cells were kept on ice for 10 minutes, pelleted by centrifugation at 400g× for 3 minutes, and the supernatant was removed by tapping the inverted plate. The cell pellet was kept on ice. Test articles were titrated in cold FACS buffer and cells were treated with 50 uL per well of the designated treatment. Assay plates were parafilmed and incubated overnight at 4°C. The cells were then washed and incubated with secondary A647-goat anti-human Fc (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 2ug / mL and washed again. The cells were resuspended in 25uL of FACS buffer and analyzed using a BD LSRFortessa™ (X-20) High-Throughput Sampler (HTS) (BD Biosciences, San Jose, CA). The resulting GeoMean values ​​were then used to plot specific binding using Prism8 software (GraphPad Prism Software) and the K D and B. max was used to calculate the values.

[0199] In this example, all ten cysteine ​​insertion variants from Example 3 and both controls were evaluated as naked antibodies and as corresponding ADCs with the MTvc compound 1 drug-linker. Both the control and two selected cysteine ​​insertion variants containing either light or heavy chain cysteine ​​insertions (variants v27321 and v22765, respectively) were also tested as ADCs with the MCvcPABC-MMAE drug-linker. In addition, the control v22758 (A114C) and the two selected cysteine ​​insertion variants v27321 and v22765 were tested as ADCs with the MCvcPAB-Tublysin M drug-linker.

[0200] The results are shown in Table 8.1. [Table 14-1] [Table 14-2] [Table 14-3]

[0201] Overall, the cysteine ​​insertion variants showed comparable binding to the parent (v17427) naked antibody and the v17427-MTvc Compound 1 ADC, however variant v29001 and its MTvc Compound 1 conjugate showed lower binding (greater Kd, 5-fold) compared to control v17427.

[0202] All ADCs generally showed very similar binding to their naked antibody counterparts. Cysteine ​​insertion variant v29001 and control v22758 MTvc Compound 1 ADCs showed slightly lower B than their respective naked antibody counterparts. max showed.

[0203] The control v29013 (S239.5), both as a naked antibody and as an ADC with either drug-linker, showed lower B max The values ​​were shown.

[0204] Overall, this example also shows that, with the exception of variant v29001, the ADCs containing cysteine ​​insertion variants did not exhibit differential binding compared to their naked antibody counterparts and exhibited comparable binding to the parent antibodies. However, the binding exhibited by the ADCs containing variant v29001 was still within a similar range as the corresponding ADCs containing the controls v22758 and v29013.

[0205] Example 9: In vitro cytotoxicity The cytotoxicity of the ADCs from Example 3 containing either MCvcPABC-MMAE or MTvc Compound 1 was tested in vitro against various tumor cell lines expressing the target surface antigen (c-Met), as described below. The following cell lines were used (Table 9.1). [Table 15]

[0206] Each of the cell lines shown in Table 9.1 was grown in their respective complete growth medium until the day of assay. After removal from the culture vessel using trypsin-EDTA, cells were counted using a Scepter™ Cell Counter (Sigma-Aldrich Canada, Oakville, ON). Cells were diluted to 20,000 cells / mL in complete growth medium such that 50 uL / well in a 384-well plate equaled 1,000 cells / well unless otherwise stated. All ADCs were diluted to a starting concentration of 15 nM in complete growth medium (RPMI1640) and subsequently diluted 1:3 in a sterile 96-well dilution plate (final volume 200 uL / well). Samples (20 uL / well) were transferred in duplicate to the 384-well plate. Plates were then seeded with a 20,000 cell / mL suspension (50 uL / well) of the test cell line unless otherwise stated. Cells were allowed to settle to the bottom of the wells by leaving them at room temperature for 5-10 min followed by incubation at 37°C / 5% CO2 for 4 days. After incubation, cell viability was quantified by adding 1x CellTiter-Glo® reagent (Promega Corporation, Madison, WI) at 10 uL / well. After 30 min of incubation in the dark, luminescence was measured using a Synergy™ H1 Hybrid Multi-Mode Reader (BioTek Instruments Inc., Winooski, VT). Data were analyzed with Prism7 software (GraphPad Prism Software).

[0207] The results are shown in Table 9.2. [Table 16-1] [Table 16-2]

[0208] As expected, ADCs containing MTvc compound 1 showed the greatest potency in the high c-Met expressing cell line EBC-1 compared to activity in lower c-Met expressing cell lines. All ADCs containing this drug-linker, including the HIC-purified DAR2 wild-type control (v17427-MTvc compound 1), had ECs ranging from 0.02 to 0.04 nM in the EBC-1 cell line. 50 In the low c-Met expressing BT-20 cell line, wild-type control (v17427-MTvc compound 1 DAR2 fraction) (EC 50 0.15 nM) than the cysteine ​​insertion variant v28993 (EC 50 0.11nM), v29001(EC 50 0.12nM), v28983(EC 50 0.12nM), v28989(EC 50 0.13nM), v22765(EC 50 0.14 nM), and v27321 (EC 50 High potency was observed for EC50 / CpG (0.14 nM), indicating the importance of site-specific conjugation over stochastic conjugation. All ADCs containing the MTvc Compound 1 drug-linker had an EC50 / CpG of >15 nM in the moderately c-MET-expressing H292 cell line. 50 For the moderately c-Met expressing HCC827 cell line, the cysteine ​​insertion variants conjugated to MTvc compound 1 showed EC values ​​ranging from 0.24 to 0.71 nM. 50 The control ADC, v29013 (S239.5), conjugated to MTvc compound 1, showed the highest in vitro potency (EC 50 0.16 nM).

[0209] For ADCs containing MCvcPABC-MMAE, the control v29013 (S239.5) was administered to the high c-Met expressing cell line EBC-1 (EC 50 All ten cysteine ​​insertion variants conjugated to MCvcPABC-MMAE showed the highest potency at EC 50 The potency ranged from 0.05 to 0.09 nM. MCvcPABC-MMAE (EC 50 1.27 nM) than the control v29003 (S239.5) conjugated to MCvcPABC-MMAE. 50 0.59nM), v29001(EC 50 0.64nM), v28983(EC 50 0.64nM), and v27322 (EC 50 High potency was observed for all ADCs containing the MCvcPABC-MMAE drug-linker with an EC value of >15 nM in the moderate c-Met expressing cell lines H292 and HCC827. 50 The values ​​were shown.

[0210] Overall, this example shows that the potency of ADCs containing cysteine ​​insertion variants does not appear to be affected by the location of the individual cysteine ​​insertion sites.

[0211] Example 10: Stability of antibody drug conjugates in mouse plasma For multiple reasons, including enzymatic metabolism and retro-Michael reactions, ADCs can lose their payload while circulating in vivo, or the payload can be modified in a way that renders the ADC ineffective. The ADCs from Example 3 were evaluated in a mouse plasma stability assay, as described below, to determine loss of payload (drug-linker).

[0212] ADCs were diluted in mouse plasma at 0.5 mg / mL and incubated in a 37°C water bath for 0, 1, 3, and 7 days before evaluating drug loss for each. Samples were removed from the water bath at the indicated time points and immediately frozen at -80°C. Time points within 24 hours were prepared separately for ADCs containing MCvcPAB-Tublysin M drug-linker. ADCs and antibodies were recovered by immunoprecipitation. Samples were first deglycosylated with 250 ng EndoS enzyme (2 ug ADC in 50 uL PBS) for 1 hour at room temperature (RT). The deglycosylated ADC was then captured on streptavidin magnetic beads (GE Healthcare Life Sciences, Chicago, IL) pre-bound to biotinylated goat anti-human IgG F(ab')2 capture antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) (15ug capture antibody per 100uL of magnetic bead slurry per sample) for 1.5 hours at room temperature. After ADC capture, samples were reduced with 25mM DTT (ThermoFisher Scientific, Waltham, MA) per 100uL of sample for 1 hour at room temperature and then eluted in 20uL of elution buffer (20% acetonitrile, 1% formic acid in dH2O) for 1 hour at room temperature. A control ADC (v22758) spiked into mouse plasma at 2.0ug was included as a control to validate the immunoprecipitation procedure. To determine the amount of drug-linker loss, the DAR for each sample was assessed by LC-MS, as described in General Procedure 6.

[0213] In addition to the potential loss of the drug-linker, the maleimide ring in the linker could potentially undergo water-mediated ring-opening, stabilizing the ADC. Maleimide ring-opening would result in an increase of 18 Da in the ADC mass. For all samples, the amount of maleimide ring-opening in addition to drug-linker loss was calculated.

[0214] Tubulysin M is susceptible to metabolism by loss of acetyl groups during circulation. Understanding whether this type of degradation occurs in cysteine ​​insertion variant ADCs will provide additional information regarding the stability / exposure / accessibility of each cysteine ​​insertion site. To evaluate whether any of the cysteine ​​insertion sites serve to protect the tubulysin M payload from acetyl loss, plasma stability was monitored and compared to ADCs containing controls v22758 (Thiomab HC-A114C) and v29013 (S239.5). In general, % degradation of tubulysin M ADCs was calculated as the percentage of all drug-loaded species that lost acetyl group mass (ring-open and non-ring-open) divided by the sum of all drug-loaded species.

[0215] The results of the stability study are shown in Figures 3, 4, and 5.

[0216] As can be seen from Figure 3, for ADCs containing the drug-linker MTvc compound 1, DAR loss was similar across most variants, with the greatest decrease occurring within the first 24 hours, reaching a final DAR of approximately 1.6 by day 7. For ADCs containing variants v27322, v29001 and control v29013, DAR loss was nearly negligible throughout the incubation period. Maleimide ring opening for most variants began at 0-20% and progressed to complete ring opening over 7 days. ADCs containing variant v22765 and control v29013 only reached approximately 70% ring opening by day 7.

[0217] Figure 4 shows that for ADCs containing the drug-linker MCvcPABC-MMAE, the DAR loss was about 10% for most variants over the incubation period. The least stable ADCs were those containing variants v22760 and v22768, which lost 50% DAR over 7 days. For MCvcPABC-MMAE ADCs, ring opening and DAR loss did not correlate perfectly. ADCs containing variants v22760 and v22768 showed about 70% ring opening, but also the highest DAR loss. The most stable ADCs were those containing the controls v29013 and v22758, as well as those containing cysteine ​​insertion variants v22761, v22765, v27321, and v27322, all of which showed less than 10% DAR loss.

[0218] Figure 5 shows that among the ADCs containing the drug-linker MCvcPAB-Tulysin M, ADCs containing variants v22761, v27321, v27322, and control v22758 exhibited rapid Tubulysin M degradation, with over 70% degradation at 24 hours and 100% degradation by day 7. This degradation was responsible for the majority of the DAR loss in the ADCs containing variants v22761 and v27321. The most stable MCvcPAB-Tulysin M ADC contained variant v29001, which showed only about 20% degradation over 7 days, moderate ring opening, and very little DAR loss. Degradation in this ADC was 5% less than the amount of degradation exhibited by the control v29013-MCvcPAB-Tulysin M ADC.

[0219] Example 11: Preparation of DAR-tuned antibody drug conjugates The ADCs described in the previous examples, with the same cysteine ​​insertion in either both heavy chains (1xcys HC) or both light chains (1xcys LC), had an average DAR of about 2. In this example, cysteine ​​insertions were evaluated for potential combinations to generate constructs with more than two or fewer than two cysteine ​​residue insertions per antibody, allowing for the generation of ADCs with average DARs of 1, 2, or 3, as described below.

[0220] Constructs containing one insertion per antibody molecule (1xcys Ab) were generated by heterodimeric assembly of heavy chains. These constructs included one heavy chain without any insertion and one heavy chain with a single inserted cysteine ​​residue (1xcys HC).

[0221] Constructs containing three insertions per antibody molecule (3xcys Ab) were generated by heterodimeric assembly of heavy chains, either by combining two light chains each with a single cysteine ​​insertion (1xcys LC) with one heavy chain with a single cysteine ​​insertion (1xcys HC) and one heavy chain without any insertion, or by combining one heavy chain with a single cysteine ​​insertion (1xcys HC) with one heavy chain with two cysteine ​​insertions (2xcys HC).

[0222] Details are provided in Table 11.1.

[0223] Additionally, constructs with four inserted cysteines per antibody molecule (4xCys Ab) may be generated by combining two different sets of insertion designs, either by combining two light chains each with a single cysteine ​​insertion (1xCys LC) with two heavy chains each with a single cysteine ​​insertion (1xCys HC) or by combining two heavy chains each with two cysteine ​​insertions (2xCys HC) or two light chains each with two cysteine ​​insertions (2xCys LC). [Table 17]

[0224] The antibodies were prepared as described in General Procedure 1 and each antibody was conjugated to MTvc compound 1 as described in General Procedure 2, except for v17427, which was stochastically conjugated to NHS-ester activated compound 1 at a lysine residue in DAR2 to produce ADC v34293.

[0225] In vitro characterization of each resulting "DAR-tuned" ADC by HIC, UPLC-SEC, LC-MS, and CE-SDS was performed as described in General Procedures 4-7. The results are shown in Table 11.2. [Table 18]

[0226] As can be seen from Table 11.2, all variants were successfully conjugated to MTvc compound 1 at their respective target DARs as determined by LC-MS. The HIC-RRT values ​​of each ADC correlated well with the estimated values ​​of the MTvc compound 1 ADC at DAR2 (see Examples 4 and 6). The UPLC-SEC profiles showed greater than 90% monomer for each of the DAR-adjusted ADCs.

[0227] CE-SDS showed that all DAR-tuned ADCs appeared as full-sized antibodies with only minor amounts of non-specific conjugation.

[0228] Example 12: Binding assay on cells - DAR tuned ADCs In vitro characterization of the ADCs from Example 11 by binding on cells was evaluated as described in Example 8 on the cMet expressing cell lines EBC-1, H292, and BT-20. ADC v34281 (DAR3) was tested only on EBC-1 and H292 cell lines. Parent antibody (v17427) conjugated to drug-linker ADvc compound 1 (stochastic, lysine conjugation) at DAR2 or MTvc compound 1 (stochastic, cysteine ​​conjugation) at DAR4 was included as an additional control.

[0229] The results are shown in Table 12.1. As expected, all DAR-tuned ADCs demonstrated similar target binding to the unconjugated parent antibody (v17427), regardless of DAR. [Table 19]

[0230] Example 13: In vitro cytotoxicity - DAR tuned ADCs In vitro cytotoxicity of the ADCs from Example 11 was evaluated as described in Example 9. A parent antibody (v17427) conjugated to drug-linker MTvc compound 1 (stochastic, cysteine ​​conjugation) at DAR4 was included as an additional control.

[0231] The results are shown in Figure 6 and Table 13.1. In general, the cytotoxicity of ADCs with DAR3 was greater than that of ADCs with DAR2 and DAR1. No significant differences in cytotoxicity were observed between ADCs with DAR1. [Table 20]

[0232] Overall, the results show that all DAR-tuned ADCs were active against the c-Met-expressing cell lines EBC-1 and HT-29. The in vitro potency of these ADCs correlated well with the DAR value (i.e., the number of toxins conjugated).

[0233] Example 14: In vivo antitumor activity A selection of ADCs from Example 11 were evaluated for in vivo antitumor activity in the high c-Met expressing non-small cell lung cancer xenograft model H1975 and the medium c-Met expressing colon cancer xenograft model HT-29. For comparison, the activity of the control ADCs v17427-MCvcPABC-MMAE (DAR4), v17427-MTvc-compound 1 (DAR4), and v17427-ADvc-compound 1 (DAR2) was evaluated.

[0234] For the HT-29 model, tumor cell suspension (3 × 10 in 0.1 ml of PBS) was 6 The cells were subcutaneously implanted into BALB / c nude mice. The average tumor volume was approximately 160 mm. 3 Upon reaching 14.1 days, animals were randomly assigned to groups (n=8 per group) and treated with a single IV dose of test article as shown in Table 14.1. Dose levels of ADC at different DARs were molar matched to the toxin. Tumor volumes and body weights were measured twice weekly for the 32 day study period.

[0235] For the H1975 model, tumor cell suspension (5 × 10 in 0.1 mL of PBS) 6 The cells were subcutaneously implanted into BALB / c nude mice. The average tumor volume was approximately 150 mm. 3 Upon reaching IV dose, animals were randomly assigned to groups (n=8 per group) and treated with a single IV dose of test article as shown in Table 14.1. Tumor volumes and body weights were measured twice weekly for the 38 day study period. [Table 21]

[0236] The results are shown in Figures 7 and 8.

[0237] In the HT-29 model, site-specific and stochastic conjugated DAR2, DAR3, and DAR4 Compound 1 ADCs all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 6, 4, and 3 mg / kg, respectively (p<0.05, mixed effects model for tumor growth rate) (Figure 7B). DAR1 Compound 1 ADCs did not inhibit tumor growth at a toxin-matched dose of 12 mg / kg (Figure 7B). Site-specific and stochastic conjugated DAR2, DAR3, and DAR4 Compound 1 ADCs, except for v28983-MTvc-Compound 1, also all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 3, 2, and 1.5 mg / kg, respectively (Figure 7A). In comparison, v17427-MCvcPABC-MMAE did not significantly inhibit tumor growth at the 1.5 mg / kg dose tested (Figure 7A). When antibody dose was toxin-matched, there was a trend for a positive correlation between DAR and antitumor activity. The activity of the site-specific v29001-MTvc-Compound 1 DAR2 ADC was comparable to that of the v17427-ADvc Compound 1 stochastic DAR2 control (Figure 7A).

[0238] In the H1975 model, Compound 1 ADCs of DAR1, DAR2, DAR3, and DAR4 all significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 24, 12, 8, and 6 mg / kg, respectively (p<0.05, mixed effects model for tumor growth rate) (Figure 8B). Compound 1 ADCs of DAR2, DAR3, and DAR4 all also significantly inhibited tumor growth compared to vehicle at toxin-matched antibody doses of 2, 1.3, and 1 mg / kg, respectively, whereas the ADC of DAR1 did not significantly inhibit tumor growth at a toxin-matched antibody dose of 4 mg / kg (Figure 8A).

[0239] No significant weight loss was observed in any treatment group in either study.

[0240] Example 15: FcγR and FcRn binding of cysteine ​​insertion variants The ten cysteine ​​insertion variants shown in Table 2.2 were evaluated for their ability to bind to the neonatal Fc receptor (FcRn), and the Fcγ receptors (FcγR) CD64a (FcγRI), CD32a (FcγRIIA; allele types His131 and Arg131), CD32b (FcγRIIB), and CD16a (FcγRIIIA; allele types V158 and F158), as described below.

[0241] Binding to FcγR: The affinity of FcyR for the tested variants was measured by surface plasmon resonance (SPR) using a Biacore™ T200 System (Cytiva, Marlborough, MA) with PBS buffer (pH 7.4) containing 0.05% Tween 20 and 3.4 mM EDTA. Protein A (Genscript Biotech Corporation, Piscataway, NJ; Cat. Z02201) was covalently immobilized on a CM5 sensor chip through standard amine coupling to 2000 RU (response units) at 15 ug / mL in 10 mM sodium acetate (pH 4.5). 2.5 ug / mL of each tested variant was injected at a flow rate of 10 uL / min for 30 seconds for Protein A capture. FcγR was injected at 25 uL / min over the antibody immobilized surface using single cycle kinetics. For CD32aH, CD32aR, and CD32bY, which have weak affinity and fast on and off interactions, increasing concentrations of 0.15 to 12 uM for 15 seconds were used. For CD16aF and CD16aV, 40 second injections of increasing concentrations of 0.06 to 5 uM were used. For CD64a, 100 second injections of increasing concentrations of 0.41 to 300 uM were used. For all FcγRs, a 120 second dissociation was used, and the Protein A surface was regenerated with a 30 second pulse of 10 mM glycine (pH 1.5) between injection cycles. Sensorgrams were double referenced and fitted to a steady state model for affinity determination, or a 1:1 binding model for kinetics and affinity when the dissociation phase is very slow for CD64a. The reported K D Values ​​are the average of two independent runs. All experiments were performed at 25°C.

[0242] Binding to FcRn: The affinity of FcRn for the tested variants was measured by surface plasmon resonance (SPR) using a Biacore™ T200 System (Cytiva, Marlborough, MA) with PBS buffer containing 0.05% Tween 20 and 3.4 mM EDTA adjusted to pH 5.9. Neutravidin (ThermoFisher Scientific, Waltham, MA; Cat. 31000) was covalently immobilized on a CM5 sensor chip through standard amine coupling to 2000 RU (response units) at 10 ug / mL in 10 mM sodium acetate, pH 4.5. Human FcRn with C-terminal biotin on the large subunit at 5 ug / mL was injected for 20 seconds at a flow rate of 20 uL / min to reach an FcRn capture level of 70 RU. Each test variant was injected over the FcRn immobilized surface using single cycle kinetics. Antibody variants were injected for 45 s at increasing concentrations from 5 to 1200 nM at 50 uL / min with a 180 s dissociation using single cycle methodology in pH 5.9 running buffer. The FcRn surface was regenerated with a 30 s pulse of PBST (pH 7.4) between injection cycles. Sensorgrams were double referenced and fitted to a steady-state binding model to generate affinity values. Reported K D Values ​​are the average of two independent runs. All experiments were performed at 25°C.

[0243] Results: The results are shown in Table 15.1. All cysteine ​​insertion variants had a K D Most of the cysteine ​​insertion variants bound to FcRn with similar affinity, with K values ​​within 2-fold of that of the wild-type control (v17427). D The variants v29001 (H_T299.5C) and v22765 (H_G237.5C) had significantly reduced binding to FcγRs, but also bound to all FcγRs with similar affinity, within 2-fold of the K of the wild-type control v17427, and the variant v22768 (H_Q295.5C) had reduced binding to CD16aF, CD16aV, CD32aH, and CD32aR. DThe control cysteine ​​insertion variant v29013 (H_S239.5C) also showed significantly reduced binding to FcγR, as expected. [Table 22]

[0244] Example 16: Transferability of cysteine ​​insertion mutations To demonstrate that the cysteine ​​insertion mutations are transferable to other antibodies, four of the insertion sites were selected and introduced into four different antibodies as detailed in Table 16.1, for a total of seven new variants. [Table 23]

[0245] Cysteine ​​insertion variants were prepared in various antibody backgrounds following the same protocol as described in General Protocol 1. Trastuzumab, CR8071, H3, and SGNCD19a based variants (v34014, v34012, v34010, v33996, v34004, and v34015) were conjugated to the drug-linker MTvc compound 1 as described in General Procedure 2 with the following exceptions: For variants v34012 and v34010, once reduction was complete, the reduced variants were buffer exchanged into PBS (pH 6.5) for either oxidation at room temperature or oxidation prior to conjugation with MTvc compound 1. Samples prepared with oxidation at 4° C. showed better biophysical properties.

[0246] Variant v34217 was conjugated to three different camptothecin-based drug-linkers (MC-GGFG-Camptothecin 1, MC-GGFG-Camptothecin 2, and MC-GGFG-*Camptothecin 2), as described below. Drug-linker MC-GGFG-*Camptothecin 2 contains the same camptothecin analog as drug-linker MC-GGFG-Camptothecin 2, but the linker is attached at a different position within the drug molecule.

[0247] A solution (647.2 μL) of variant v34217 (6 mg) was diluted to 6.4 mg / mL with a 5 mM solution of DTPA (diethylenetriaminepentaacetic acid, final concentration 1 mM, volume: 188 μL) in PBS (pH 7.4), and to this solution was added 25 mM tris(2-carboxyethyl)phosphine (TCEP) (25 equivalents, 104 μL). After 3 hours of incubation in a 37° C. water bath, the reduced antibody was purified using a 40 kDa 5 mL Zeba™ Spin Desalting Column (Thermo Fisher Scientific, Waltham, MA) pretreated with 10 mM sodium acetate (pH 5.5). The reduced antibody was oxidized overnight (18 hours) at 4°C with a 25 molar excess of dehydroascorbic acid (DHAA) (assuming 100% recovery from Zeba™ column purification) to reform the interchain disulfide bonds while keeping the inserted cysteines in the reduced (free thiol) form. The reoxidized antibody was split into three uniform aliquots and conjugated to maleimide-functionalized drug-linkers MC-GGFG-Camptothecin1, MC-GGFG-Camptothecin2, or MC-GGFG-*Camptothecin2 in the presence of 10% (v / v) DMSO by incubating with a 4 molar excess of 10 mM DMSO stock at room temperature for 60-75 minutes after thorough mixing by pipetting. The formed conjugates were then purified by a 40k Zeba™ column pre-equilibrated with 10 mM sodium acetate (pH 4.5).

[0248] Upon completion of conjugation, all ADCs were analyzed by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-SDS (CE-SDS) as described in general protocols 4, 5, 6, and 7, respectively.

[0249] The results are shown in Table 16.2 and overall show that cysteine ​​insertion mutations can be successfully used in different antibodies and to conjugate different drug-linkers. For most ADCs, the DAR measured by either HIC or LC-MS was as expected (approximately DAR2). For v33996, even for the naked antibody, the elution in HIC was relatively broad, and therefore no clear peak was observed after conjugation, and neither the DAR nor the HIC-RRT could be measured. For v34012, some degree of non-specific conjugation was observed, resulting in a DAR of 2.1. The ADCs showed at least 95% monomer by SEC, except for two ADCs conjugated to MTvc compound 1: v34012 and v34010. For these two ADCs, approximately 15% LMWS was observed after oxidation (before the addition of the drug-linker) and did not change after conjugation. Further optimization of the oxidation step improves the amount of LMWS observed for these two variants. [Table 24]

[0250] Example 17: Preparation of additional DAR-tuned antibody drug conjugates In this example, multiple cysteine ​​insertion sites were combined to generate antibodies capable of site-specific conjugation to provide ADCs with DARs of 4 and 6. The cysteine ​​insertion site combinations used are shown in Table 17.1. [Table 25]

[0251] Cysteine ​​insertion variants were prepared according to the protocol described in General Protocol 1. Anti-cMet variants of DAR4 and DAR6 were expressed in cysteine-capped form, either L-cysteine ​​capped, glutathione capped, or a combination of both. Reduction, oxidation, and conjugation to drug-linker MTvc compound 1 were performed as described in General Procedure 2 with the following modifications. To allow for additional cysteine ​​capping, reduction was performed with a molar excess of 30 and 40 equivalents of tris(2-carboxyethyl)phosphine (TCEP) for DAR4 and DAR6 variants under similar conditions, respectively. Oxidation was performed with a molar excess of 30 and 40 equivalents of dehydroascorbic acid (DHAA) for DAR4 and DAR6 variants, respectively.

[0252] Conjugation of the anti-FRα variants of DAR4 to the drug-linkers MC-GGFG-Camptothecin 1, MC-GGFG-Camptothecin 2, and MC-GGFG-*Camptothecin 2 was performed as described in Example 16.

[0253] Upon completion of conjugation, all ADCs were analyzed by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-SDS (CE-SDS) as described in general protocols 4, 5, 6, and 7, respectively.

[0254] The results are shown in Table 17.2 (anti-cMet antibody) and Table 17.3 (anti-FRα antibody).

[0255] Overall, site-specific conjugation of DAR4 and DAR6 on the anti-cMet antibody scaffold was successful, with up to three different cysteine ​​insertions introduced into the antibody and conjugated to drug-linkers. Representative HIC, SEC, LC-MS, and CE-SDS profiles of the ADC of DAR6, v35074-MTvc compound 1, are shown in Figures 16-18.

[0256] For ADCs v33943-MTvc compound 1 and v35074-MTvc compound 1, the DAR values ​​calculated by HIC were lower than expected due to the presence of co-eluting peaks. However, for the other anti-cMet ADCs, the DARs calculated by HIC were close to the target DARs. DARs measured by LC-MS are a more direct approach and should reflect values ​​closest to the absolute values. Except for v33952-MTvc compound 1, all other anti-cMet ADCs showed at least 99% monomer by UPLC-SEC. Further optimization of the oxidation step of v33952 should reduce the amount of LMWS, as this antibody showed more than 10% LMWS formation before the addition of the drug-linker. CE-SDS performed under non-denaturing conditions showed a higher percentage of half-antibody for this variant compared to the full-sized antibody.

[0257] For the conjugation of site-specific DAR4 on the anti-FRα antibody scaffold, all ADCs appeared as >99% monomers by UPLC-SEC. Only one peak was observed for these ADCs by HIC. Both the conjugated and unconjugated antibodies had very similar elution times, and the DAR estimation by HIC was less accurate than the LC-MS DAR calculation. Four of the six ADCs showed DAR4 by LC-MS. MC-GGFG-camptothecin 1 or MC-GGFG- * Variant v34456 conjugated to either camptothecin 2 showed approximately 25% non-specific conjugation, which could be reduced by further optimization. [Table 26] [Table 27]

[0258] Example 18: In vitro cytotoxicity of DAR-tuned anti-FRα ADCs The cell growth inhibition (cytotoxicity) capabilities of the anti-FRα ADCs (DAR2 and DAR4) generated in Examples 16 and 17 were compared to the stochastic DAR4 ADC in a 3D cytotoxicity assay described below using the FRα-expressing cancer cell lines JEG-3 (placental choriocarcinoma) and T-47D (breast cancer).

[0259] Briefly, 3,000 cells / well were seeded into 384-well Ultra-Low Attachment (ULA) plates, centrifuged at 200xg for 2 min, and incubated under standard culture conditions for 3 days to allow spheroid formation (1 spheroid / well). After 3 days, spheroids were treated with titrations of test articles prepared in complete growth medium and incubated under standard culture conditions for 6 days. After incubation, CellTiter-Glo® 3D Reagent (Promega Corporation, Madison, WI) was added to all wells. Plates were incubated at room temperature in the dark for 1 hour, and luminescence was quantified using a BioTek Cytation5 Cell Imaging Multi-Mode Reader (Agilent Technologies, Inc., Santa Clara, CA). Percent cytotoxicity values ​​were calculated and plotted against test article concentrations based on blank wells (no test article added) using GraphPad Prism9 software (GraphPad Software, San Diego, CA).

[0260] The results are shown in Table 18.1. The site-specific DAR4 ADCs demonstrated comparable 3D in vitro cytotoxicity as the payload-matched stochastic DAR4 ADCs against both JEG-3 and T-47D spheroids. The site-specific DAR2 ADCs demonstrated log-fold lower potency than the payload-matched DAR4 ADCs against JEG-3 and T-47D spheroids, as expected due to the lower drug loading on the DAR2 ADCs.

[0261] Site-specific and stochastic ADCs of DAR4 containing camptothecin 2 showed similar efficacy to payload-matched stochastic ADCs of DAR8 against both JEG-3 and T-47D spheroids.

[0262] Site-specific and stochastic ADCs of DAR4 containing camptothecin 1 showed similar potency as payload-matched DAR8 stochastic ADCs on high FRα-expressing JEG-3 spheroids. In lower FRα-expressing T-47D spheroids, DAR4 ADCs showed a drug load-dependent dose response and payload-matched DAR8 stochastic ADCs showed 3- to 9-fold higher potency. [Table 28]

[0263] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are specifically incorporated by reference in their entireties into this specification to the same extent as if each such individual patent, patent document, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0264] Modifications of the specific embodiments described herein that will be obvious to those of skill in the art are intended to be included within the scope of the following claims.

Claims

1. 1. A cysteine ​​engineered antibody construct comprising: The antibody construct comprises: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; the numbering of amino acids in the VL, CL, VH, and CH1 domains is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering; The cysteine ​​engineered antibody construct, wherein the antibody construct is based on Immunoglobulin G (IgG).

2. 2. The cysteine ​​engineered antibody construct of claim 1, wherein the melting temperature (Tm) of said domain of said cysteine ​​engineered antibody construct comprising said cysteine ​​insertion mutation is within 0° C. to 8° C. of the Tm of the same domain in a corresponding parent antibody construct lacking said cysteine ​​insertion mutation.

3. 2. The cysteine ​​engineered antibody construct of claim 1 , wherein the antibody construct comprises one, two, or three cysteine ​​insertion mutations.

4. the antibody construct comprises two cysteine ​​insertion mutations; 4. The cysteine ​​engineered antibody construct of claim 3, wherein the two cysteine ​​insertion mutations are symmetric mutations.

5. the antibody construct comprises a three cysteine ​​insertion mutation; 4. The cysteine ​​engineered antibody construct of claim 3, wherein two of the three cysteine ​​insertion mutations are symmetric mutations.

6. the antibody construct comprises one or three cysteine ​​insertion mutations; Each cysteine ​​insertion mutation independently (a) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (d) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

7. 3. The cysteine ​​engineered antibody construct of claim 1 or 2, wherein the antibody construct comprises four or six cysteine ​​insertion mutations.

8. The cysteine ​​insertion mutation is (a) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (b) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (c) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (d) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (e) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain.

9. 9. The cysteine ​​engineered antibody construct of any one of claims 1 to 8, wherein the antibody construct comprises one or more antigen binding domains, at least one of the antigen binding domains comprising a VH domain or a VH domain and a VL domain.

10. 10. The cysteine ​​engineered antibody construct of claim 9, wherein the antibody construct comprises two antigen binding domains.

11. The cysteine ​​engineered antibody construct of claim 10, wherein the antibody construct is bispecific.

12. 12. The cysteine ​​engineered antibody construct of any one of claims 9 to 11, wherein at least one of the antigen binding domains binds to a tumor associated antigen.

13. 13. The cysteine ​​engineered antibody construct of any one of claims 1 to 12, wherein the antibody construct comprises an Fc region comprising a CH2 domain and a CH3 domain.

14. 14. The cysteine ​​engineered antibody construct of any one of claims 1 to 13, wherein the antibody construct is IgG1 based.

15. The cysteine ​​engineered antibody construct of any one of claims 1 to 14, wherein the IgG is human IgG.

16. 16. The cysteine ​​engineered antibody construct of any one of claims 1 to 15, wherein the antibody construct comprises a heterodimeric Fc region comprising a CH2 domain and a CH3 domain.

17. 17. The cysteine ​​engineered antibody construct of Claim 16, wherein the heterodimeric Fc region comprises a modified CH3 domain comprising amino acid mutations that favor formation of the heterodimeric Fc over a homodimeric Fc.

18. A conjugate comprising the cysteine ​​engineered antibody construct of any one of claims 1 to 17 and one or more active agents conjugated to each of the one or more inserted cysteine ​​residues.

19. (a) Formula (I): A-(L-(D) ｑ ) ｐ (I) A conjugate having the formula: A is a cysteine ​​engineered antibody construct, L is a linker, D is an activator; q is an integer from 1 to 4, p is an integer from 1 to 8; or (b) Formula (II): (a) ｐ (II) A conjugate having the formula: A is a cysteine ​​engineered antibody construct, L is a linker, D is an activator; p is an integer from 1 to 8; 4. The cysteine ​​engineered antibody construct, (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; the numbering of amino acids in the VL, CL, VH, and CH1 domains is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering; the cysteine ​​engineered antibody construct is based on immunoglobulin G (IgG), The conjugate, wherein each D is linked via L to an inserted cysteine ​​residue.

20. 20. The conjugate of claim 19, wherein p is 1, 2, 3, 4, or 6.

21. The conjugate of any one of claims 18 to 20, wherein the active agent is a diagnostic agent, a labeling agent, or a therapeutic agent.

22. A composition comprising the conjugate of any one of claims 18 to 21 and a pharma- ceutically acceptable carrier or diluent.

23. 22. Use of a conjugate according to claim 21 in the manufacture of a medicament for the treatment of a subject in need thereof, comprising: The use, wherein the active agent is a therapeutic agent.

24. 23. A method for preparing a conjugate according to any one of claims 18 to 22, comprising subjecting the cysteine ​​engineered antibody construct to reducing conditions such that the thiol group of the one or more inserted cysteine ​​residues is reduced, and reacting a thiol-reactive linker-activator with the antibody construct under conditions allowing the formation of a bond between the linker and the reduced thiol.

25. 1. A method for preparing an antibody-drug conjugate having a predetermined drug-antibody ratio (DAR), comprising: (i) providing a cysteine ​​engineered antibody construct, The antibody construct comprises: (a) insertion of a cysteine ​​residue between positions 39 and 40 in the VL domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (d) insertion of a cysteine ​​residue between positions 148 and 149 in the CL domain; (e) insertion of a cysteine ​​residue between positions 149 and 150 in the CL domain; (f) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (g) insertion of a cysteine ​​residue between positions 169 and 170 in the CH1 domain; (h) insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; (i) the insertion of a cysteine ​​residue between positions 295 and 296 in the CH2 domain, and (j) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; (ii) reacting the cysteine ​​engineered antibody construct with a drug-linker to provide the antibody-drug conjugate; the predetermined DAR is 1, 2, 3, 4, 5, 6, 7 or 8, and the cysteine ​​engineered antibody construct comprises the same number of cysteine ​​insertion mutations as the predetermined DAR; the numbering of amino acids in the VL, CL, VH, and CH1 domains is Kabat numbering, and the numbering of amino acids in the CH2 domain is EU numbering; The method, wherein the cysteine ​​engineered antibody construct is based on immunoglobulin G (IgG).

26. 26. The method of claim 25, wherein the predetermined DAR is 2.

27. i) the predetermined DAR is 1 or 3; and The cysteine ​​insertion mutation is (a) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (b) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (c) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (d) an insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; or ii) the predetermined DAR is 4 or 6; and The cysteine ​​insertion mutation is (a) insertion of a cysteine ​​residue between positions 40 and 41 in the VL domain; (b) insertion of a cysteine ​​residue between positions 126 and 127 in the CL domain; (c) insertion of a cysteine ​​residue between positions 9 and 10 in the VH domain; (d) the insertion of a cysteine ​​residue between positions 237 and 238 in the CH2 domain; and (e) insertion of a cysteine ​​residue between positions 299 and 300 in the CH2 domain; 26. The method of claim 25.

28. A polynucleotide or set of polynucleotides encoding the cysteine ​​engineered antibody construct of any one of claims 1 to 17.

29. A vector comprising one or more polynucleotides encoding the cysteine ​​engineered antibody construct of any one of claims 1 to 17.

30. A host cell comprising the vector of claim 29.

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