Screening of fixed-point coupling sites of cysteine-modified antibody-toxin conjugate (TDC)

1 . A cysteine-modified antibody-toxin conjugate, comprising an antibody and a cytotoxin, wherein the antibody comprises an inserted cysteine at one or more cysteine insertion sites at positions 110, 111, or 142 within the light chain constant region, or at positions 254, 255, 258, 259, 354, 355, 357, 378, 379, 386, 387 or 410 within the heavy chain constant region.

2 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein the amino acid sequence of said cysteine insertion site comprises one or more of:

LC-110ins:

(SEQ ID NO: 45)

EIKRTCVAAPS;

LC-111ins:

(SEQ ID NO: 46)

IKRTVCAAPSV;

LC-142ins:

(SEQ ID NO: 47)

NNFYPCREAKV;

HC-254ins:

(SEQ ID NO: 48)

ISRTPCEVTCV;

HC-255ins:

(SEQ ID NO: 49)

SRTPECVTCVV;

HC-258ins:

(SEQ ID NO: 50)

PEVTCCVVVDV;

HC-259ins:

(SEQ ID NO: 51)

EVTCVCVVDVS;

HC-354ins:

(SEQ ID NO: 52)

PSRDECLTKNQ;

HC-355ins:

(SEQ ID NO: 53)

SRDELCTKNQV;

HC-357ins:

(SEQ ID NO: 54)

DELTKCNQVSL;

HC-378ins:

(SEQ ID NO: 55)

IAVEWCESNGQ;

HC-379ins:

(SEQ ID NO: 56)

AVEWECSNGQP;

HC-386ins:

(SEQ ID NO: 57)

GQPENCNYKTT;

HC-387ins:

(SEQ ID NO: 58)

QPENNCYKTTP;

HC-410ins:

(SEQ ID NO: 59)

KLTVDCKSRWQ.

3 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein the cytotoxin is conjugated to the sulfhydryl group of the inserted cysteine via a linker, wherein the antibody light chain have an amino acid sequence corresponding to: EIKRTCVAAPS (SEQ ID NO:45), IKRTVCAAPSV (SEQ ID NO:46), or NNFYPCREAKV (SEQ ID NO:47), wherein the antibody heavy chain have an amino acid sequence corresponding to: ISRTPCEVTCV (SEQ ID NO:48), SRTPECVTCVV (SEQ ID NO:49), PEVTCCVVVDV (SEQ ID NO: 50), EVTCVCVVDVS (SEQ ID NO:51), PSRDECLTKNQ (SEQ ID NO:52), SRDELCTKNQV (SEQ ID NO:53), DELTKCNQVSL (SEQ ID NO:54), IAVEWCESNGQ (SEQ ID NO:55), AVEWECSNGQP (SEQ ID NO:56), GQPENCNYKTT (SEQ ID NO:57), QPENNCYKTTP (SEQ ID NO:58), or KLTVDCKSRWQ (SEQ ID NO:59), and wherein C corresponds to the inserted cysteine in the light or heavy chain of the cysteine-modified antibody.

4 . The cysteine-modified antibody-toxin conjugate of claim 3 , wherein said cytotoxin is selected from a group consisting of MMAE, MMAF, PBD, SN-38, Dox, Amanitin having the following molecular formulas:

5 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein said antibody light chain comprises kappa isotype.

6 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein said antibody heavy chain comprises IgG1, IgG2, IgG3 or IgG4 isotypes.

7 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein said inserted cysteine comprises a sulfhydryl group before being coupled to the cytotoxin.

8 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein said antibody light chain comprises lambda isotype.

9 . The cysteine-modified antibody-toxin conjugate of claim 1 , wherein said cytotoxin to the antibody ratio ranges from 1.6 to 2.0 or from 3.2 to 4.0.

10 . The cysteine-modified antibody-toxin conjugate of claim 1 , prepared by,

reducing a cysteine-modified antibody comprising a protected cysteine having a protected sulfhydryl group and a protecting group,

using a reducing agent to remove the protecting group from the protected cysteine to provide a removed protecting group and a reduced antibody comprising a de-protected cysteine having a sulfhydryl group,

removing the removed protecting group and any excess reducing agent using a method comprising cation exchange chromatography or ultrafiltration,

oxidizing the reduced antibody using an oxidizing agent to reattach the interchain disulfide bonds of the antibody,

coupling the cytotoxin with the sulfhydryl group on the inserted cysteine to provide the cysteine-modified antibody-toxin conjugate, and

removing any uncoupled cytotoxin using a method comprising cation exchange chromatography or ultrafiltration.