TW201639595A - Chemically-locked bispecific antibodies - Google Patents

Abstract

There is disclosed a process for forming chemically-locked bispecific or heterodimer antibodies, preferably in the IgG class, in high specificity and with high homogeneity. More specifically, there is disclosed a chemically-locked bispecific IgG class antibody having a linkage region joined together with bio-orthogonal click chemistry.

Description

Chemically locked bispecific antibody

The present disclosure provides methods for forming chemically-locked bispecific or heterodimeric antibodies, preferably IgG species, with high specificity and high homogeneity. More specifically, the present disclosure provides a chemically-locked bispecific IgG species antibody having a linkage region joined together by bioorthogonal click chemistry.

Cross-reference to related applications

This patent application claims priority from U.S. Provisional Patent Application Serial No. 61/991,508, filed on May 10, 2014.

Human immunoglobulin G or IgG antibodies exist in 4 seed classes, each having different structural and functional properties. The IgG line consists of two heavy chain-light chain pairs (half antibodies) which are directly linked to the Cys residues in the hinge region (EU index number: cysteine residues 226 and 229) Kabat number: heavy chain interchain disulfide linkage of cysteine residues 239 and 242). Human IgG4 molecules differ in the absence or presence of various molecular forms of inter-heavy chain disulfide bonds.

Various recombinant antibody formats have been developed, for example, by fusion of IgG antibody formats and single-stranded domains to obtain tetravalent bispecific antibodies (Coloman et al, Nature Biotech 15 (1997) 159-163; WO 2001/077342; And Morrison, Nature Biotech 25 (2007) 1233-1234). Another format no longer retains the antibody core structure (IgA, IgD, IgE, IgG or IgM), such as bispecific antibodies, trispecific antibodies or tetraspecific antibodies, minibodies, several single-stranded formats (scFv, Bis-scFv) ). However, such formats are capable of binding two or more antigens (Holliger et al, Nature Biotech 23 (2005) 1126-1136; Fischer and Leger, Pathobiology 74 (2007) 3-14; Shen et al, J. Immunological Methods 318 (2007) 65-74; and Wu et al, Nature Biotech. 25 (2007) 1290-1297).

Separating or preferentially synthesizing the former dimerization from a mixture comprising the two types of polypeptide dimers via a dimer linked via at least one interchain disulfide linkage and a dimer not linked via at least one interchain disulfide linkage The method of the body is reported in US 2005/0163782.

Bispecific antibodies are difficult to produce in sufficient amounts and qualities of materials using conventional hybrid-hybridoma and chemical coupling methods. In addition, WO2005/062916 and US Patent Application No. 2010/0105874 describe how to separate a disulfide bond into a single heavy chain-light chain unit (A or B) having a single binding region by reducing antibody "AA" and antibody "BB". ) (wherein A and B bind to different targets) to form a bispecific antibody. These antibodies then allow for the isomerization of the disulfide bonds such that the antibodies AB, BA, AA and BB are each reformed with a probability of about 25%. However, AB and BA are the same bispecific antibodies and thus represent a yield of up to about 50%. Therefore, this requires an additional step to isolate the desired bispecific antibody formed from the original reconstituted antibody. However, U.S. Patent Application No. 2010/0105874 relates to a hinge region having a CPSC sequence in IgG4 and states: "The CPSC sequence results in a more flexible core hinge and can form an intrachain disulfide bond. It is believed to have an IgG4-like core hinge. The antibody of the sequence may have the inherent activity of rearranging disulfide bonds, as simulated by the conditions used in the methods of the invention ("paragraph 0013). In addition, other forms of bispecific antibodies were prepared using "bulge and pore" structures made by altering the heavy chain sequences of antibodies A and B.

Accordingly, the present disclosure provides a process for the production of chemically-locked bispecific IgG antibodies that address the industry's extremely high bispecific antibodies for bulging and pore methods that alter the amino acid sequence in the immobilized antibody region. The need for yield and better stability.

The present disclosure provides a chemically-locked bispecific antibody "AB" or "BA" from an IgG 1, IgG2 or IgG4 antibody or its Fab2 fragment "A" and an IgG1, IgG2 or IgG4 antibody or its Fab2 fragment "B" Process. The process comprises: (a) contacting the first antibody "A" with a reducing agent under substantially all disulfide linkages between the heavy chains in the hinge region to obtain a pair of first antibody fragments A ' , Each of which comprises a single light chain attached to a single heavy chain, the heavy chain having one or more reactive thiol groups formed by the reduction of the disulfide linkages; (b) the first heterobifunctional linker attached Linked to the first antibody fragment A ' , wherein the first heterobifunctional linker comprises (i) a first thiol reactive functional group for reactivity of the heavy chain covalently attached to the first antibody fragment A ' a thiol group, and (ii) an azide to form an azide functionalized first antibody fragment; (c) a second antibody "B" and a reducing agent between the heavy chains sufficient to cleave the hinge region Substantially contacting under conditions of all disulfide linkages to obtain a pair of second antibody fragments B ' , each comprising a single light chain attached to a single heavy chain, the heavy chain having one or more reductions from the disulfide linkages Forming a reactive thiol group; (d) attaching a second heterobifunctional linker to the second antibody fragment B ' The second heterobifunctional linker comprises: (i) a second thiol reactive functional group for covalent attachment to a reactive thiol group of the heavy chain of the second antibody fragment; Ii) an alkyne; thereby forming an alkyne-functionalized second antibody fragment; (e) reacting the azide-functionalized first antibody fragment with the alkyne-functionalized second antibody fragment to pass the azide to the alkyne The 3-dipolar cycloaddition covalently attaches the first antibody fragment to the second antibody fragment to form a chemically-locked bispecific antibody "AB" or "BA".

The step of reducing the disulfide linkage in the hinge region is preferably carried out without substantially reducing the disulfide linkage between the heavy chain and the light chain, which means, in some embodiments, at least about 90% or at least about 95% or at least about 99% of the disulfide linkages between the heavy and light chains are The disulfide bond in the hinge region remains intact after cleavage.

Preferably, the first heterobifunctional linker has the form QLN ₃ wherein Q is a thiol reactive functional group, an L-based hydrocarbon linker, and an N ₃ -based azide. Preferably, the thiol reactive functional group Q is an alkyl halide (e.g., alkyl chloride, bromide, or iodide), benzyl halide, maleimide, halo-maleimide (bromomaleimide) or dihalo-maleimide (dibromomaleimide). Preferably, L system having 3 to 60 atoms (e.g., more generally 6 to 50) in said hydrocarbon linker between the direct link and N is ₃ Q. More preferably, an L-based polyalkylene oxide (PEF) group or an L-based polymer, wherein each polymer unit is -(CH ₂ CH ₂ -O) _n - or -(O-CH ₂ CH ₂ ) _n - (where "n" is independently an integer from 1-20, more usually 1-8).

Preferably, the first heterobifunctional linker (attached to the first antibody fragment) is

Wherein Q can be any group suitable for linking the linker to the antibody fragment, but is preferably capable of covalently bonding to a thiol from a cysteine residue in the hinge region of the heavy chain. An example group Q system:

Wherein Z is independently selected from the group consisting of H, Br, I and SPh. Preferably, Z is not H at least once, but in the case of maleimide, Z can be hydrogen at each occurrence. M is independently CR* or N.

X ₁ , X ₂ , X ₃ , X ₂ , X ₄ and X _{5 are} independently selected from the group consisting of: a bond (ie, it is absent), -O-, -NR ^N -, -N=C-, -C=N-, -N=N-, -CR*=CR*-(cis or trans), -C≡C-, -(C=O)-, -(C=O)-O- , -(C=O)-NR ^N -, -(C=O)-(CH ₂ ) _n -, -(C=O)-O-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ ) _n - and -(C=O)-NR ^N - (CH ₂ CH ₂ -O) _n -, wherein " n " is zero or an integer from 1 to 10; R ^a , R ^b , R ^c and R ^{d are} independently selected from the group consisting of -O-, -NR ^N -, -CH ₂ -, -(CH ₂ ) _n -, -(CR* ₂ ) _n -, -(CH ₂ CH ₂ -O) _n -, -(CR* ₂ CR* ₂ -O) _n -, -(O-CH ₂ CH ₂ ) _n -, -(O-CR* ₂ CR* ₂ ) _n -, -CR*= CR*-(cis or trans), -N=C-, -C=N-, -N=N-, -C≡C-, -(C=O)-, -(CH ₂ ) _n - (C=O)-, -(C=O)-(CH ₂ ) _n -, -(CH ₂ ) _n -(C=O)-(CH ₂ ) _n -, -O-(C=O)- , -(C=O)-O-, -O-(C=O)-O-, -(CH ₂ ) _n -(C=O)-O-, -O-(C=O)-(CH ₂ ) _n , -(C=O)-O-(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C=O)-, -(CH ₂ ) _n -(C=O)-O -(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C=O)-(CH ₂ ) _n -, -NR ^N -(C=O)-, -(C=O)-NR ^N -, -NR ^N -(C=O)-O-, -O-(C=O)-NR ^N -, -NR ^N -(C=O )-NR ^N -, -(CH ₂ ) _n -(C=O)-NR ^N -, -NR ^N -(C=O)-(CH ₂ ) _n , -(C=O)-NR ^N -( CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N -(C=O)-, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH _{2 n} -NR ^N -(C=O)-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -( C=O)-NR ^N -(CH ₂ ) _n - and a 2-8 membered cyclic hydrocarbon, heterocyclic ring, aryl or heteroaryl ring; wherein " n " is independently zero or an integer from 1 to 10; Wherein " l ", " p ", " q " and " r " are independently zero or an integer from 1 to 10; an Ω key (ie, not present) or a _C3-26 hydrocarbon ring or fused ring system It optionally includes up to 4 fused rings, each ring having 3-8 members and optionally 1-4 heteroatoms selected from O, S and N in each ring. Preferably, the Ω is fused to a cyclooctane ring or a 1,2,3-triazole ring fused to an 8-membered heterocyclic ring or ring system; R* and R ^N are independently H in each occurrence or a C _1-12 hydrocarbon, optionally substituted with from 1 to 6 heteroatoms selected from the group consisting of halogen, O, S and N; and any two of the groups R* and/or R ^N may together form a 3-8 membered ring .

The second heterobifunctional linker has the form QLG wherein Q is a thiol reactive functional group, an L series hydrocarbon linker, and the G system contains an alkyne group. The thiol-reactive functional group Q is selected from the group consisting of alkyl halides (e.g., alkyl chlorides, bromides, or iodides), benzyl halides, maleimides, halo-malays. An imine (for example, bromide) and a dihalo-maleimide (for example, dibromomaleimide). L is a hydrocarbon linker having from 3 to 60 atoms (e.g., preferably from 6 to 50) in the direct chain between Q and G. Preferably, the L-based polyalkylene oxide group (PEG), preferably the polymer of the L-based unit -(CH ₂ CH ₂ -O) _n - or -(O-CH ₂ CH ₂ ) _n - Wherein "n" is independently 1-20, more usually an integer from 1-8).

G is an alkyne-containing group capable of undergoing cycloaddition with an azide. In some embodiments, G includes a terminal alkyne, such as -C≡CH. In other embodiments, G includes a ring or ring system having a -C≡C- bond in the ring. In an embodiment, G comprises a C8 ring having a -C≡C- bond. In one embodiment, the -C≡C-ring is strained.

The second heterobifunctional linker has the following form:

Q is the same as or different from the first heterobifunctional linker. Q is usually a thiol reactive group of the form:

Wherein Z is independently selected from H, Br, I and SPh at each occurrence. In some embodiments, Z is not H at least once, but in the case of maleinide, Z can be hydrogen at each occurrence. M is independently CR* or N at each occurrence.

G is usually a C _8-20 hydrocarbon group including a -C≡C- bond capable of undergoing a 1,3 dipolar cycloaddition reaction with an azide. In some embodiments, G has the form -C≡CH. In other embodiments, G comprises a ring having a -C≡C- bond. In particular, G can include an 8-membered ring having a triple bond (eg, cyclooctyne). The ring may optionally be fused to one, two or more other rings, typically a _C3-6 cyclic hydrocarbon ring comprising an aryl group, a heteroaryl group and a heterocyclic ring. An 8-membered ring containing a triple bond may contain one or more (e.g., 1-4) heteroatoms, such as nitrogen and oxygen, in the ring. In one embodiment, the 8-membered ring contains a nitrogen atom in the ring that provides an attachment point to L, as in the exemplary structure shown below:

Preferably, G has the following form:

X ₁ , X ₂ , X ₃ , X ₂ , X ₄ and X ₅ are each independently selected from the group consisting of: a bond (ie, it is absent), -O-, -NR ^N -, -N=C-, -C=N-, -N=N-, -CR*=CR*-(cis or trans), -C≡C-, -(C=O)-, -(C =O)-O-, -(C=O)-NR ^N -, -NR ^N -(C=O)-, -NR ^N -(C=O)-O-, -(C=O)-( CH ₂ ) _n -, -(C=O)-O-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ ) _n - and -(C=O)-NR ^N -( CH ₂ CH ₂ -O) _n -, wherein " n " is zero or an integer from 1 to 10; R ^a , R ^b , R ^c and R ^d are independently selected from -O-, -NR ^N at each occurrence -, -CH ₂ -, -(CH ₂ ) _n -, -(CR* ₂ ) _n -, -(CH ₂ CH ₂ -O) _n -, -(CR* ₂ CR* ₂ -O) _n -, -(O-CH ₂ CH ₂ ) _n -, -(O-CR* ₂ CR* ₂ ) _n -, -CR*=CR*-(cis or trans), -N=C-, -C= N-, -N=N-, -C≡C-, -(C=O)-, -(CH ₂ ) _n -(C=O)-, -(C=O)-(CH ₂ ) _n - , -(CH ₂ ) _n -(C=O)-(CH ₂ ) _n -, -O-(C=O)-, -(C=O)-O-, -O-(C=O)- O-, -(CH ₂ ) n -(C=O)-O-, -O-(C=O)-(CH ₂ ) _n , -(C=O)-O-(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C=O)-, -(CH ₂ ) _n -(C=O)-O-(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C= O)-(CH ₂ ) _n -, -NR ^N -(C=O)-, -(C=O)-NR ^N -, -NR ^N -(C=O)-O-, -O-(C=O)-NR ^N -, -NR ^N -(C=O)-NR ^N -, -(CH ₂ ) _n -(C= O)-NR ^N -, -NR ^N -(C=O)-(CH ₂ ) _n , -(C=O)- NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N - (C=O)-, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N -(C=O)-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -(CH ₂ ) _n - or 2 -8 membered cyclic hydrocarbon, heterocyclic, aryl or heteroaryl ring; wherein " n " is independently zero or an integer from 1 to 10 at each occurrence; and wherein " l ", " p ", " q " And " r " are independently zero or an integer from 1 to 10; an Ω linkage (ie, absent) or a _C3-26 hydrocarbon or fused ring system, optionally including up to 4 fused rings. Each ring has 3-8 members and optionally includes 1-4 heteroatoms selected from O, S and N in each ring. In one embodiment, Ω comprises a 1,2,3-triazole ring fused to a cyclooctane ring or fused to an 8-membered heterocyclic ring or ring system.

^N R and R * are independently H or C _1-12 hydrocarbon-based, at each occurrence, which is optionally substituted with 1-6 groups selected from halogen, O, N, and S substituted with the hetero atom; and wherein the two groups R * and / or R ^N can form a 3-8 member ring together.

The cycloaddition reaction between the azide and the alkyne can be carried out via a 1,3 dipolar cycloaddition reaction. This reaction can be catalyzed by copper ions. In some embodiments, the cycloaddition reaction occurs at a neutral or physiological pH.

The disclosure further provides for the reduction of antibody "A" having a hinge residue sequence (EU index number: residue 226-229; Kabat number: residue 239-242) CPPC or CPSC or SPPC or SPSC and having a hinge residue sequence ( Residue 226-229) CPPC or CPSC or SPPC or SPSC second antibody "B" to form half antibody A and half antibody B, comprising: (a) reducing each antibody A and antibody B, wherein reducing conditions Rupture of any interchain or intrachain disulfide bond in the hinge region of each antibody with a hinge residue sequence (EU index number: residue 226-229; Kabat number: residue 239-242) CPPC or CPSC or SPPC or SPSC (b) linking a compound selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N; to one of the hinge core sequences of half antibody A or two Cys residues (EU index number: residues 226 and 229; Kabat number: residues 239 and 242) to form Attaching half antibody A; (c) linking a compound selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

To one of the hinge core sequences of antibody B or two Cys residues 226 and 229 (EU index number: residues 226 and 229; Kabat number: residues 239 and 242) to form linked antibody B; and (d) Approximately one molar amount of linked antibody A and linked antibody B are incubated together under neutral conditions to form a linked bispecific antibody AB.

Preferably, the antibody A is reduced in a reducing agent to form a half antibody A and the antibody B is reduced to form a half antibody B, wherein the reducing agent is selected from the group consisting of L-cysteine, dithiothreitol,巯-mercaptoethanol, cysteamine, TCEP (叁(2-carboxyethyl)phosphine), 2-MEA (2-mercaptoethylamine), and combinations thereof. Preferably, the hinge region having one or two Cys residues in antibody A is linked to a portion A having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N. Preferably, the hinge region having one or two Cys residues in Antibody B is linked to a portion B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

To form a linked half antibody B.

The disclosure further provides a chemically-locked bispecific antibody AB, wherein the linked half antibody A

Wherein N ₃ is -N=N=N; linked to linked antibody B

To form the bispecific antibody AB having the structure shown in Figure 10.

The present disclosure provides a chemically-locked bispecific antibody "AB" or "BA" derived from an IgG type antibody "A" or a fragment thereof and an IgG type antibody "B", which comprises a structure having a group selected from the group consisting of Half antibody A:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N, and wherein the Z is a leaving group; and a half antibody B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

The disclosure further provides a bispecific antibody comprising: (a) a first antibody fragment A ' comprising a single heavy chain and a light chain from antibody A, the heavy chain having one or more reactive thiol groups (b) a second antibody fragment B ' comprising a single heavy chain and a light chain, the heavy chain having one or more reactive thiol groups; wherein the first and second antibody fragments are passed through 1, 2, 3 - a triazole covalently linked, the 1,2,3-triazole being attached via an azide (a reactive thiol attached to the first antibody fragment via a linker) and an alkyne (via a linker) A cycloaddition reaction of a reactive thiol on the second antibody fragment is formed. Linkers are set forth above. Antibody fragments A ' and B ' are IgGl, IgG2 or IgG4 immunoglobulins derived from their Fab2 fragments.

The present disclosure further provides an antibody covalently bonded to the linker fragment, wherein the connecting member comprises a ring having a C ₈ -C≡C- bond capable of cycloaddition reaction of the azide. The disclosure further provides an antibody fragment covalently bonded to a linker, wherein the linker comprises an azide capable of undergoing a cycloaddition reaction with a -C≡C- bond.

Preferably, the antibody A is reduced in a reducing agent to form a half antibody A and the antibody B is reduced to form a half antibody B, wherein the reducing agent is selected from the group consisting of L-cysteine, dithiothreitol,巯-mercaptoethanol, cysteamine, TCEP (叁(2-carboxyethyl)phosphine), 2-MEA (2-mercaptoethylamine), and combinations thereof. Preferably, the hinge region having one or two Cys residues in antibody A is linked to a portion A having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N; to one of the hinge core sequences of half antibody A or two Cys residues (EU index number: residues 226 and 229; Kabat number: residues 239 and 242) to form Attaching half antibody A; (c) linking a compound selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

One of the hinge core sequences to antibody B or two Cys residues 226 and 229 (EU index) Number: residues 226 and 229; Kabat number: residues 239 and 242) to form linked antibody B; (d) Approximately equimolar amount of ligated antibody A and ligated antibody B are incubated together under neutral conditions to form a linked bispecific antibody AB.

Preferably, the antibody A is reduced in a reducing agent to form a half antibody A and the antibody B is reduced to form a half antibody B, wherein the reducing agent is selected from the group consisting of L-cysteine, dithiothreitol,巯-mercaptoethanol, cysteamine, TCEP (叁(2-carboxyethyl)phosphine), 2-MEA (2-mercaptoethylamine), and combinations thereof. Preferably, the hinge region having one or two Cys residues in antibody A is linked to a portion A having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N. Preferably, the hinge region having one or two Cys residues in Antibody B is linked to a portion B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

To form a linked half antibody B.

The disclosure further provides a chemically-locked bispecific antibody AB, wherein the linked half antibody A

Wherein N ₃ is -N=N=N; linked to linked antibody B

To form the bispecific antibody AB having the structure shown in Figure 10.

The present disclosure provides a chemically immobilized bispecific antibody "AB" or "BA" derived from an IgG type antibody "A" and an IgG type antibody "B", which comprises a half antibody A having a structure selected from the group consisting of :

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N, and a leaving group in which the Z system is bonded; and a half antibody B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Figure 1 shows a schematic representation of the creation of a bispecific mAb by chemical coupling to a single Cys residue in the hinge region of an IgG class of antibodies.

2 shows a schematic representation of interchain cross-linking via a single Cys residue chemically coupled to the hinge region of an IgG class antibody, according to the disclosure herein.

Figure 3 shows a schematic representation of intracellular cross-linking to two Cys residues within the hinge region of an IgG class of antibodies.

Figure 4 shows (top and bottom panels) a schematic representation of the generation of bispecific mAbs by cross-linking between two Cys residues in the hinge region of an IgG class of antibodies.

Figure 5 shows SDS PAGE analysis of a chemically locked half mAb fragment.

Figure 6 shows MS analysis of HC Fab from naked mAb (top panel), azide coupled mAb fragment (middle panel) and alkyne coupled mAb fragment (bottom panel).

Figure 7 shows SDS PAGE of crosslinked products from azide attached half mAb and alkyne attached half mAb fragments.

Figure 8 shows MS analysis of (Fab) ₂ from the starting mAb (upper panel) and the crosslinked product (bottom panel).

Figure 9 shows non-reducing SDS PAGE of the chemically modified half antibody fragments (lanes 2 and 3) produced in Example 4 herein.

Figure 10 shows the generation of bispecific antibodies via click coupling between two half antibody fragments.

Figure 11 shows non-reducing SDS PAGE of half antibody fragment (a) and click product (b). The half antibody-azide is located in lane 2 of gel (a) and the half antibody-DBCO is located in lane 3 of gel (a). Click on the product located in lane 2 of gel (b).

Figure 12 shows a mass spectrum of the IdeS digestion click product showing the mass of the bispecific antibody CBA-0710.

Figure 13 shows SEC size exclusion chromatography of the bispecific antibody CBA-0710.

Figure 14 shows the binding of the bispecific antibody CBA-0710 on BIAcore. More specifically, Figure 14 shows the simultaneous binding of the bispecific antibody CBA-0710 on BIAcore to both antigens.

Figure 15 shows the bispecific antibody CBA-0710 bound to triple negative breast cancer cell line MDA-MB-231. These cells express antigen-1 and antigen-2. STI-A0607 is an anti-antigen-1 monoclonal antibody and STI-A1010 is an anti-antigen-2 monoclonal antibody.

Figure 16 shows the antagonistic activity of the bispecific antibody CBA-0710 in triple negative breast cancer cells. Sex. STI-A0607 is an anti-antigen-1 monoclonal antibody and STI-A1010 is an anti-antigen 2 monoclonal antibody. HGF is a natural ligand for antigen-1.

Figure 17 shows IFN-[gamma] release in response to an increase in the bispecific antibody CBA-0710. STI-A1010 is an anti-antigen-2 (immunoassay) monoclonal antibody. The competitor mAb is a humanized anti-antigen-2 (immunoassay) monoclonal antibody.

Figure 18 shows IL-2 release in response to an increase in the bispecific antibody CBA-0710. STI-A1010 is an anti-antigen-2 (immunoassay) monoclonal antibody. The competitor mAb is a humanized anti-antigen-2 (immunoassay) monoclonal antibody.

Figure 19 shows the improved efficacy of a chemically immobilized bispecific antibody composed of an A anti-c-Met antibody and a B anti-PD-L1 antibody compared to a regular anti-c-Met IgG1 and anti-PD-L1 IgG1 antibody.

(Ab) a schematic diagram 'of FIG. ₂ shows bispecific F 20 via chemical coupling.

Figure 21 shows an SDS_PAGE gel analysis of F(ab)'2 and F(ab)'. Column 1 was an antibody A digested with IdeS. Column 2 is an antibody B digested with IdeS. Column 3 was digested and antibody A of FC was removed. Column 4 is digested and FC antibody B is removed. Column 5 was subjected to digestion and removal of FC, antibody A reduced with 2MEA and TCEP and coupled with DBCO (dibenzylcyclooctyl)-maleimide. Column 6 was subjected to digestion and removal of FC, antibody 2 reduced with 2MEA and TCEP, coupled with azide-maleimide.

Figure 22 shows a schematic representation of the generation of F(ab)' ₂ chemically locked bispecific antibodies via click coupling between two F(ab)' fragments.

Figure 23 shows the SEC from the click F(ab)' ₂ of Example 7 herein.

Figure 24 shows the click-specific bispecific F(ab)' ₂ fragment analysis SDS PAGE. Column 1 was antibody A which was digested and removed with FC and reduced with DBA (dibenzylcyclooctyl)-maleimide using 2MEA and TCEP reduction. Column 2 was digested and FC was removed and antibody B coupled to azide-maleimide was reduced using 2MEA and TCEP. Column 3 is clicked on _bispecific F(ab)' ₂ .

Figure 25 shows a mass spectrum of a click product showing the mass of the bispecific F(ab)' ₂ .

Figure 26 shows the SEC clicking F(ab)'2.

Figure 27 shows the simultaneous binding of bispecific F(ab)' ₂ to two antigens on Octet Red.

Figure 28 shows a schematic representation of the production of bispecific IgG2 via chemical coupling.

Figure 29 shows IgG2 fragment analysis SDS PAGE. Column 1 is antibody A. Column 2 is an antibody A reduced with TCEP and coupled to azide-maleimide. Column 3 is antibody B. Column 4 is an antibody B reduced with TCEP and coupled to azide-maleimide.

Figures 30A-C show mass spectra showing (Figure 30A) IgG2_A coupled to the linker, (Figure 30B) IgG2_B coupled to the linker and (Figure 30C) bispecific-IgG2 formed between IgG2_A and B the quality of.

Figure 31 shows SEC of IgG2_A, IgG2_B and click products.

The bispecific antibody (BsAb) is composed of two semi-antibody fragments chemically linked at the hinge region (Fig. 1). The anti-system IgGl or IgG4 isotype is initiated. The starting antibody may contain a modified hinge region in which the Cys residue is first mutated to Ser, leaving only one disulfide at the hinge. The production of bispecific antibodies involves three major steps. The first step is the selective reduction of both antibodies A and B to form a half antibody fragment. The second step is to introduce functional moiety X or Y into the hinge region of each antibody half fragment via cysteine-based coupling to obtain chemically modified antibody half fragments A' and B', respectively. In the final step, two antibody half fragments are ligated together via a chemical linkage between the X and Y moieties to form a bispecific antibody.

The present disclosure provides a process for producing a chemically-locked bispecific antibody "AB" or "BA" from an IgG type antibody "A" and an IgG type antibody "B", which comprises: (a) reduction of a hinge residue sequence ( EU index number: residue 226-229; Kabat number: residue 239-242) CPPC or CPSC or SPPC or SPSC first antibody "A" and hinge residue sequence (EU index number: residues 226-229; Kabat number: residues 239-242) CPPC or CPSC or SPPC or SPSC second antibody "B" to form half antibody A and half antibody-B, wherein antibody A binds to the first target and antibody B binds to the second target Wherein the reducing condition ruptures any interchain or intrachain disulfide bond in the hinge region of the antibody sequence of the hinge residue sequence (residues 226-229) CPPC or CPSC or SPPC or SPSC; (b) the compound of formula I and half One of the hinge core sequences of antibody A or two Cys residues (EU index number: residues 226 and 229; Kabat number: residues 239 and 242) are joined to form a linked half having a structure selected from the group consisting of Antibody A:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N; (c) one or two Cys residues of the hinge core sequence of the compound of formula II and antibody B (EU index number: residues 226 and 229; Kabat number: residue 239) And 242) linked to form a linked antibody B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

And (d) cultivating approximately one molar amount of linked antibody A and linked antibody B under neutral conditions to form a linked bispecific antibody AB.

Preferably, in a reducing agent (for example, L-cysteine, dithiothreitol, β-mercaptoethanol, cysteamine, TCEP (叁-(2-carboxyethyl)phosphine), 2-MEA (2- Antibody A is reduced in decylethylamine and combinations thereof to form half antibody A and reduce antibody B to form half antibody B. Preferably, the hinge region having two Cys residues in Antibody A is linked to a portion A having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N. Preferably, the hinge region having two Cys residues in Antibody B is linked to a portion B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

The disclosure further provides a chemically-locked bispecific antibody AB, wherein the linked half antibody A

Wherein N ₃ is -N=N=N; is linked to the linked antibody B

To form the bispecific antibody AB having the structure shown in Figure 10.

The present disclosure provides a chemically immobilized bispecific antibody "AB" or "BA" derived from an IgG type antibody "A" and an IgG type antibody "B", which comprises a half antibody A having a structure selected from the group consisting of :

M=N, C

M’=N, C

Z=I, Br, SPh

Wherein N ₃ is -N=N=N; and half antibody B having a structure selected from the group consisting of:

M=N, C

M’=N, C

Z=I, Br, SPh

Preferably, in a reducing agent (for example, L-cysteine, dithiothreitol, β-mercaptoethanol, cysteamine, TCEP (叁-(2-carboxyethyl)phosphine), 2-MEA (2- Antibody A is reduced in decylethylamine and combinations thereof to form half antibody A and reduce antibody B to form half antibody B.

Preferably, antibodies A and B are monoclonal antibodies. Monoclonal antibodies can be produced by hybridoma methods or by recombinant DNA and protein expression methods. Further, antibodies A and B are full-length antibodies or antibody fragments.

Antibodies A and B have a CPPC core hinge region sequence or a CPSC core hinge region sequence or a SPPC core hinge region sequence or a SPSC core hinge region sequence (EU index number: residue 226- 229; Kabat number: residues 239-242). Further, the step (d) cultivating further comprises the step of adding a reducing agent, wherein the reducing agent is selected from the group consisting of L-cysteine, dithiothreitol, β-mercaptoethanol, cysteamine, TCEP ( Bis(2-carboxyethyl)phosphine), 2-MEA (2-mercaptoethylamine), and combinations thereof.

The quality and purity of the resulting bispecific antibodies can be analyzed using conventional biochemical techniques (eg, absorbance spectroscopy, HP-SEC, SDS-PAGE, original PAGE, and RP-HPLC). It should be noted that the disclosed methods generally avoid any purification step due to the affinity specificity of the linker of formula I for the linker of formula II. However, there are various purification steps that are provided in US 2010/0105874, the disclosure of which is incorporated herein by reference.

The disclosed process further includes the step of formulating a bispecific antibody for therapeutic use. This is achieved by formulating an effective amount of a bispecific antibody in an aqueous solution suitable for human use, particularly for parenteral or intravenous administration.

Figure 2 shows a reaction diagram for the generation of bispecific monoclonal antibodies (mAbs) via chemical coupling. The bispecific mAbs described herein are composed of two semi-antibody fragments chemically linked at the hinge region. The process of bispecific mAb generation involves three main steps (Figure 2). The first step is to selectively reduce the hinge disulfide in two different mAbs A and B, respectively. The second step introduces an intrachain linkage between the two cysteines on the same heavy chain in each mAb via linker X or Y. The in-chain linkage process produces two chemically-locked mAb fragments A' and B'. In the final step, two mAb fragments are ligated together via a chemical linkage between X and Y to form a bispecific antibody AB.

IgG1, wt IgG4 with hinge mutation (CPSC) and IgG4 with hinge mutation (SPSC) were used in this study.

The first step is the reduction of each of antibody A and antibody B. In one embodiment, the antibody (10 mg) is administered at 10 ° C in 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) using 10 molar equivalents of 2 -mercaptoethyl-amine (2-MEA). In the process of 2h. Excess 2-MEA was purified using a 50 kDa filter centrifuge tube using a centrifugation from partial reduction of mAb at 3,000 RPM for 20 minutes. A total of three washes were performed using 0.1 M PBS. The protein concentration was quantified using an absorbance value of 1.58 for a 1.0 mg/mL solution at 280 nm, and the molar concentration was determined using a molecular weight of 150,000 g/mol.

In another embodiment of the reduction step, the antibody (10 mg) is subjected to 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) at 24 ° C using 3.0 molar equivalents of dithiothreitol (DTT). In the process of 2h. Excess DTT was purified using a 50 kDa filter centrifuge tube using a 20 min centrifugation from the partially reduced mAb at 3,000 RPM. A total of 3 washes were performed using 0.1 M PBS.

In another embodiment of the reduction step, mAb (10 mg) is used at 0.1 ° C pH 8.0 PBS, 1.0 mM diethylene three at 24 ° C using 2.0 molar equivalents of ruthenium (2-carboxyethyl)-phosphine (TCEP). Treatment in amine pentaacetic acid (DTPA) for 2 h. The mAb concentration was 8.0 mM. The partially reduced mAb was used directly in the coupling step without purification.

The second step is the coupling step. The partially reduced mAb "antibody A" from the reduction step in 0.1 M PBS was added to 2.5 molar equivalents of crosslinker Z-X-Z (Figures 2 and 3). The crosslinker was obtained from a pre-prepared stock solution (1 mg/mL) stored in DMSO. The partially reduced antibody was at a concentration of 8.0 mg/mL and a DMSO content of 5% (v/v) in the reaction mixture. Coupling was carried out at 24 ° C for 2 hr. Any unreacted, excess crosslinker was quenched using cysteine (1 mM final). The coupled mAbs were purified using a PD-10 column equilibrated with phosphate buffered saline. The coupled mAb structure is illustrated in Figure 4. Under the same conditions, the second mAb (antibody B) was coupled to the cross-linking agent Z-Y-Z (Fig. 5 and Fig. 6) and purified. The structure of the coupled mAb is illustrated in Figures 7 and 8.

The third step is an interchain coupling step. A click coupling diagram for interchain crosslinking is illustrated in Figure 9. Briefly, 3.0 mg of acetylene decorative antibody in 0.5 mL PBS (0.1 M, pH 7.4) was added to the azide decorative antibody fragment (3.0 mg) in 0.5 mL PBS (0.1 M, pH 7.4). Fragment. To this mixture was added 50 μL of acetonitrile and the final content of acetonitrile was 5% (v/v). After reacting for 3 hr at room temperature, use a 100 kDa filter centrifuge tube for use in The mixture was purified by centrifugation at 3,000 RPM for 20 minutes. The mixture was washed 3 times with PBS and the resulting product was subjected to in vitro characterization.

Example 1

This example demonstrates the synthesis of bispecific antibodies according to the disclosed processes. Figure 4 shows a reaction diagram for the production of bispecific monoclonal antibodies (mAbs) by chemical coupling to two Cys residues in the hinge region of an IgG class of antibodies. The disclosed bispecific mAbs are composed of two half-antibody fragments chemically linked at their respective hinge regions. The process of synthesizing bispecific mAbs involves the three main steps shown in Figure 5. The first step is to selectively reduce the hinge disulfide in two different mAbs A and B, respectively. The second step introduces an intrachain linkage between the two cysteines on the same heavy chain in each mAb via linker X or Y. The in-chain linkage process produces two chemically-locked mAb fragments A' and B'. In the final step, two mAb fragments are ligated together via a chemical linkage between X and Y to form a bispecific antibody AB.

More specifically, antibody "A" IgG1 and antibody "B" wild type IgG4 having a hinge mutation (CP S C) were obtained. The first step is antibody reduction. Condition 1: Antibody (10 mg) was separately used in 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) at 10 ° C using 10 molar equivalents of 2- mercaptoethyl-amine (2-MEA). Processed for 2h. Excess 2-MEA was purified using a 50 kDa filter centrifuge tube using a centrifugation from partial reduction of mAb at 3,000 RPM for 20 minutes. A total of three washes were performed using 0.1 M PBS. The protein concentration was quantified using an absorbance value of 1.58 for a 1.0 mg/mL solution at 280 nm, and the molar concentration was determined using a molecular weight of 150,000 g/mol.

Condition 2: The antibody (10 mg) was treated with 3.0 molar equivalents of dithiothreitol (DTT) in 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) for 2 h at 24 °C. Excess DTT was purified using a 50 kDa filter centrifuge tube using a 20 min centrifugation from the partially reduced mAb at 3,000 RPM. A total of 3 washes were performed using 0.1 M PBS.

Condition 3: mAb (10 mg) in 2.0 M pH 8.0 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) at 24 ° C using 2.0 molar equivalents of hydrazine (2-carboxyethyl)-phosphine (TCEP) Processed for 2h. The mAb concentration was 8.0 mM. The partially reduced mAb was used directly in the coupling without purification.

Example 2

This example demonstrates that the bispecific antibody produced in Example 1 retains two of its original semi-Mab binding properties.

Synthesis of 1-(2-(2-azidoethoxy)ethyl)-3,4-dibromo-1H-pyrrole-2,5-dione :

To 2.5 g of 3,4-dibromo-1H-pyrrole-2,5-dione (10 mmol) and 1 g of NMM in 60 mL of THF, MeOCOCl (10 mmol, 940 mg, stored in 10 ml of DCM) was added dropwise and stirred. After 20 min, the reaction solution was diluted with 60 mL of DCM, washed with water three times, and the organic phase was stirred with anhydrous sodium sulfate and concentrated to give 2.65 g of 3,4-dibromo-2,5-di- oxy-2H-pyrrole. -1 (5H)-methyl formate. To 311 mg, 1 mmol of this compound was added 2-(2-azidoethoxy)ethylamine (130 mg, 1 mmol) and 5 mL of DCM, and TLC showed that the reaction was completed in 20 min, then extracted with DCM and brine, with NH ₄ Cl solution was washed, dried over anhydrous sodium sulfate, and then concentrated for column purification, and subjected to flash analysis by 2:1 hexane and ethyl acetate to obtain 230 mg of 1-(2-(2-azido) Ethoxy)ethyl)-3,4-dibromo-1H-pyrrole-2,5-dione. ¹ H NMR: 3.32 ppm (t, J = 5.0 Hz, 1H), 3.40 ppm (t, J = 5.0 Hz, 1H), 3.50 ppm (q, J = 5.0 Hz, 1H), 3.62 ppm (t, J = 5.0) Hz, 1H), 3.63 - 3.69 ppm (m, 3H), 3.84 ppm (t, J = 5hz, 1H). Fw: 365.9, C ₈ H ₈ Br ₂ N ₄ O ₃ ; mass peak (1:2:1): 366.9, 368.9, 370.9.

Example 3

This example illustrates the chemical production of bispecific antibodies using a single Cys residue located in the hinge region of an IgG class of antibodies. The starting mAbs described herein contain engineered hinge regions in which one Cys at the same position on each chain is mutated to Ser, thereby obtaining a hinge that leaves only a single disulfide. The process of bispecific mAb generation involves three main steps (Figure 1). The first step is to selectively reduce the hinge disulfide in two different mAbs A and B, respectively. The second step introduces functional moiety X or Y via cysteine-based coupling. The Cys-ligation step produces two chemically locked mAb fragments A' and B'. In the final step, two mAb fragments are ligated together via a chemical linkage between X and Y to form a bispecific antibody AB. And IgG1 monoclonal antibody having a hinge region mutation (S PPC) of this study.

Condition 1: Antibody (10 mg) was treated with 10 molar equivalents of 2- mercaptoethyl-amine (2-MEA) in 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) at 37 °C. 2h. Excess 2-MEA was purified using a 50 kDa filter centrifuge tube using a centrifugation from partial reduction of mAb at 3,000 RPM for 20 minutes. A total of three washes were performed using 0.1 M PBS. The protein concentration was quantified using an absorbance value of 1.58 for a 1.0 mg/mL solution at 280 nm, and the molar concentration was determined using a molecular weight of 150,000 g/mol.

Condition 2: The antibody (10 mg) was treated with 3.0 molar equivalents of dithiothreitol (DTT) in 0.1 M pH 7.4 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) for 2 h at 24 °C. Excess DTT was purified using a 50 kDa filter centrifuge tube using a 20 min centrifugation from the partially reduced mAb at 3,000 RPM. A total of 3 washes were performed using 0.1 M PBS.

Condition 3: mAb (10 mg) in 2.0 M pH 8.0 PBS, 1.0 mM diethylenetriaminepentaacetic acid (DTPA) at 24 ° C using 2.0 molar equivalents of hydrazine (2-carboxyethyl)-phosphine (TCEP) Processed for 2h. The mAb concentration was 8.0 mM. The partially reduced mAb was used directly in the coupling without purification.

Example 4

This example demonstrates a method of preparing a bispecific antibody having the disclosed chemical linkage structure linking the hinge regions of each half antibody fragment to each other according to the processes disclosed herein. The antibody scaffold is: IgG4 with a hinge mutation ( S PSC).

Each Ig antibody is first modified by chemical modification to generate a half antibody. in particular, Buffer exchange reaction Add the antibody (0.5-3 mg) to a 15 mL filter centrifuge tube (Millipore, UFC903024) and add an appropriate volume of pH 8.0 PBS, 1 mM DTPA (diethylenetriaminepentaacetic acid) buffer to the 50 mL mark on the tube. At the office. The tubes were centrifuged at 3,000 RPM and 5 °C for 20 minutes. The antibody was transferred to a 1.5 mL plastic vial and the concentration was checked using a Nanodrop (Fisher, ND-2000 UV-Vis spectrophotometer). The final antibody concentration is between 5-8 mg/mL.

A stock solution of 1 mg/mL TCEP ((叁(2-carboxyethyl)phosphine)) (Sigma-Aldrich, C4706) in pH 8.0 PBS (1.0 mM DTPA) buffer was prepared. The volume of TCEP solution that needs to be added to the antibody is calculated using the table below, depending on the number of equivalents of antibody recovered after buffer exchange and the resulting mass.

An appropriate volume of TCEP solution (calculated by the above table) was added to the antibody solution, vortexed slightly and the vial was placed on a rotating disk. The reduction reaction was carried out at room temperature for 90 minutes.

A stock solution of DBCO-maleimide (Click Chemistry Tools, A108-100) in DMSO (Sigma-Aldrich, 472301) was prepared based on calculations from the above table. DBCO-maleimide in DMSO was added to the antibody sample (unpurified TCEP). The final volume of DMSO in the antibody sample was about 5% (v/v). The coupling reaction was carried out by rotating the disk for 1 hour at room temperature and under mixing. Each sample was placed in a separate 15 mL filter centrifuge tube (Millipore, UFC903024) and an appropriate volume of 1X DPBS (Corning, 21-031-CM, no calcium or magnesium) buffer was added to the 50 mL mark on the tube. The samples were centrifuged at 3,000 RPM and 5 ° C for 20 minutes. Repeat the washing step again After washing, the sample was transferred to a separate 1.5 mL plastic vial and placed in a refrigerator (5 ° C).

For IgG4 antibodies, 4.0 equivalents of TCEP have provided a large number of half antibodies. For IgG1 antibodies, 3.5 equivalents of TCEP have provided a large number of half antibodies. 5.0 equivalents of DBCO were used for both types of antibodies.

Add azide-PEG4-azide (2.5 mg, in DMSO (0.6 mL) to a solution of DBCO-maleimide (1.0 mg, 1.0 eq.) in DMSO (0.12 mL). 5.0 equivalents). The mixture was stirred at room temperature for 2 hr. The reaction has been completed as indicated by LC/MS. The molecular weight of the obtained azide-maleimide was 627.65 g/mol.

Azide-maleimide synthesis (reaction Figure 1)

Reaction Figure 1. Synthesis of azide-maleimide

The azide-maleimide (5.0 equivalents) in DMSO was added to the antibody sample. The final volume of DMSO in the antibody sample was about 5% (v/v). The coupling reaction was carried out by rotating the disk for 1 hour at room temperature and under mixing. The sample was washed as previously described.

Each half antibody fragment was purified by a hydrophobic interaction column (HIC). HIC analysis was performed using a TOSOH butyl-NPR column at a column temperature of 40 ° C and a flow rate of 0.6 mL/min. The elution was achieved using a 30 min gradient in 50 mM sodium phosphate buffer (pH 7.0) with decreasing salt concentration (from 1.5 M to 0 M ammonium sulfate) and an increase in organic modifier (from 0% to 25% isopropanol) ). Semi-antibody fragments were analyzed by SDS PAGE. Specifically, 20 μL and a concentration of 0.6 mg/mL were required for each sample to be analyzed. Follow established protocols for running SDS-PAGE gels (RTP AD001-01 and AD002-01). Figure 9 shows non-reducing SDS PAGE of chemically modified half antibody fragments (lanes 2 and 3).

Example 5

This example illustrates the generation of bispecific antibodies via a click reaction. Figure 10 shows the generation of bispecific antibodies via click coupling between two half antibody fragments. The click reaction between the two half antibody fragments is shown in Figure 10. The half antibody-DBCO fragment (500 μg) in PBS (5.0 mg/mL) was added to the half antibody-azide fragment (500 μg) in PBS (5.0 mg/mL). The reaction was carried out by rotating a disk for 2 hours at room temperature and under mixing. The mixture was subjected to SDS PAGE analysis (Fig. 11) and purified by ion exchange chromatography.

The bispecific antibody was purified on a Agilent 1200 HPLC using a Thermo WCX-10 column at a flow rate of 0.6 mL/min. Elution was achieved using a 30 min gradient of increasing salt concentration (0 mM to 100 mM NaCl) at pH 5.7 in 10 mM MES buffer. The bispecific antibody STI CBA-0710 was digested by IdeS protease and analyzed by Water Xevo G-2 QTOF mass spectrometry (Figure 12).

The biophysical properties of the bispecific antibody STI CBA-0710 were determined by size exclusion chromatography (SEC) of bispecific antibodies (Fig. 13). Specifically, bispecific antibodies were analyzed on a Agilent 1200 HPLC using a TSK gel SuperSW3000 column (4.6 mm ID x 30 cm, 4 [mu]m). The buffer was 0.2 M potassium phosphate, 0.25 M KCl (pH 6.2).

Figure 14 shows the binding of the bispecific antibody CBA-0710 on BIAcore. The first antigen was immobilized on a CM5 sensor wafer using standard NHS/EDC coupling methods up to approximately 1500 RU. Run the buffer for the baseline. The bispecific antibody is loaded and then the buffer is loaded and then the binding to the second antigen is run.

Example 6

This example shows the results of various analyses of the bispecific antibodies produced herein. Bispecific antibodies have cell-based binding and function. The bispecific antibody CBA-0710 binds to MDA-MB-231 (human breast cancer) cells (Fig. 15) as analyzed by flow cytometry. Determination of EC ₅₀ values of the antibodies. MDA-MB-231 triple negative breast cancer (TNBC) cells expressing antigen-1 and antigen-2 were harvested using enzyme-free cell dissociation buffer (GIBCO) and transferred to V-bottom 96-well plates (50,000 cells/well). Serial dilutions of cells with bispecific antibody CBA-0710 or parental monospecific anti-antigen-1 or anti-antigen-2 antibody in FACS buffer (PBS + 2% FBS) + NaN ₃ on ice Cultivate for 45 minutes. After washing twice in FACS buffer, a 1:1000 dilution of phycoerythrin-conjugated anti-human IgG (gamma-chain specific) was added and incubated for 30 min. After the final wash, the fluorescence intensity was measured on an Intellicyt high throughput flow cytometer (HTFC). Data were analyzed using Graphpad Prism software and nonlinear regression fitting. Data points are shown as positive marker cell median fluorescence intensity (MFI) +/- standard error. EC ₅₀ values are reported as the antibody concentration reaching 50% of the maximum binding of the cells.

The results are graphically illustrated in Table 3 and Figure 15, and demonstrate that the binding of the bispecific antibody to MDA-MB-231 cells is improved compared to the parental type antibody, wherein the sub-millimeter EC50 value is similar to the anti-antigen -2 antibody, and the binding strength is generally higher than that of the anti-antigen-1 antibody.

The bispecific antibody CBA-0710 displays antagonistic activity. Specifically, inhibition of c-MET phosphorylation by the bispecific antibody CBA-0710 was performed following the PathScan ^® Phospho-Met (panTyr) Sandwich ELISA kit No. 7333 protocol. Briefly, cell lysates are added to the reconstituted detection antibody and incubated, followed by the addition of a reconstituted HRP-linker secondary antibody. After washing, TMB was added and incubated. After the termination solution was added, the results were read. Figure 16 shows the antagonistic activity of the bispecific antibody CBA-0710 in triple negative breast cancer cells. STI-A0607 is an anti-antigen-1 monoclonal antibody and STI-A1010 is an anti-antigen 2 monoclonal antibody. HGF is a natural ligand for antigen-1.

The bispecific antibody CBA-0710 has immunomodulatory activity. To measure the ability of the bispecific antibody CBA-0710 to modulate T cell reactivity, purified CD4+ cells and allogeneic dendritic cells (by culturing mononuclear spheres in GM-CSF and IL-4 for 7 days) Get) cultivate together. Parallel plates were set up to allow collection of supernatants on days 3 and 5 to measure IL-2 and IFNy, respectively, using commercial ELISA kits. A humanized anti-antigen-2 (immunoassay) mAb of the competitor was generated and used as a positive control IgG1 and a non-related STI human mAb was used as a negative control IgG antibody. Figure 17 shows IFN-[gamma] release in response to an increase in the bispecific antibody CBA-0710. STI-A1010 is an anti-antigen-2 (immunoassay) monoclonal antibody. The competitor mAb is a humanized anti-antigen-2 (immunoassay) monoclonal antibody.

Example 7

This example illustrates the protocol for the synthesis of the disclosed bispecific antibodies using F(ab)' ₂ antibodies A' and B'. The bispecific F(ab)' ₂ set forth herein is composed of two F(ab)' fragments chemically linked at the hinge region (Figure 20). The anti-system IgGl or IgG4 isotype is initiated. The starting antibody contains a modified hinge region in which the Cys residue is mutated to the Ser residue, leaving only one disulfide at the hinge region. The production of bispecific F(ab)' ₂ chemically locked bispecific antibodies involves four major steps. The first step is to remove the Fc fragment. The second step is the selective reduction of antibodies A and B, respectively. The third step is to introduce functional moiety X or Y into the hinge region via cysteine-based coupling to obtain chemically modified antibody fragments A' and B', respectively. In the final step, the two antibody fragments are ligated together via a chemical linkage between X and Y to form a bispecific F(ab)' ₂ . The reaction scheme used for this synthesis is shown in Figure 21.

Specifically, a bispecific antibody for synthesizing F(ab)' ₂ chemically locked antibody containing an IgG1 A antibody (having a hinge mutation ( S PPC)) and an IgG4 B antibody (having a hinge mutation ( S PSC)) was carried out. Process. The antibody (1.5 mg) was added to each tube of IdeS (A0-FR1-008) at 37 ° C using the enzyme IdeS, which cleaves the IgG digestive enzyme at only one specific site below the hinge region. Incubate overnight in a head to head rotator. The Fc fragment was then removed using Protein A purification.

The antibody (1-10 mg) was added to a 15 mL filter centrifuge tube (Millipore, UFC903024) and an appropriate volume of 50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA pH 7.7 buffer was added to the 50 mL mark on the tube. The tubes were centrifuged at 3,000 RPM and 22 °C for 20 minutes. The antibody was transferred to a 1.5 mL plastic vial and the concentration was checked using a Nanodrop (Fisher, ND-2000 UV-Vis spectrophotometer). The final antibody concentration is up to 10 mg/mL.

1 mL of 50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA in pH 7.7 buffer was added to a vial containing 6 mg of 2-mercaptoethylamine HCl (to give 50 mM 2-MEA). 50 mM 2-MEA was added to F(ab)' ₂ and the final concentration was 15 mM, and mixed well. Incubate for 15 min at 37 °C. The 2-MEA was separated from the reduced F(ab)' ₂ using a NAP-5 (GE17-0853-02) demineralization column.

A stock solution of 1 mg/mL TCEP ((叁(2-carboxyethyl)phosphine)) (Sigma-Aldrich, C4706) in pH 8.0 PBS (1.0 mM DTPA) buffer was prepared. The number of equivalents of F(ab)' ₂ recovered after protein A purification and the resulting mass were evaluated, and 5 equivalents of TCEP were added to desalted F(ab)', shaken well and incubated for 5 min at room temperature.

For DBCO (dibenzylcyclooctyl)-maleimide and azide-maleimide coupling, DBCO-Malayia was prepared in DMSO (Sigma-Aldrich, 472301) A stock solution of amine (Click Chemistry Tools, A108-100) and 20 equivalents of DBCO-maleimide in DMSO were added to the F(ab)'(A) sample (unpurified TCEP). The final volume of DMSO in the antibody sample was about 5% (v/v). The coupling reaction was carried out by rotating the disk for 2 hours at room temperature with mixing. The azide-maleimide (20 equivalents) in DMSO was added to the F(ab)' (B) sample. The final volume of DMSO in the antibody sample was about 5% (v/v). The coupling reaction was carried out by rotating the disk for 2 hours at room temperature with mixing.

For the washing step, each sample was placed in a separate 15 mL filter centrifuge tube (Millipore, UFC903024) and an appropriate volume of 1X DPBS was added (Corning, 21- 031-CM, no calcium or magnesium) buffer to the 50 mL mark on the tube. The samples were centrifuged at 3,000 RPM and 22 ° C for 20 minutes. Repeat the washing step again. After washing, the samples were transferred to a separate 1.5 mL plastic vial and placed in a refrigerator (5 ° C) or used in a click step. For the F(ab)' fragment analysis, the SDS PAGE program was used. For each sample to be analyzed, 20 μL was required and the concentration was 0.6 mg/mL. Follow established protocols for running SDS-PAGE gels (RTP AD001-01 and AD002-01) (Figure 21).

A click reaction reaction diagram between two F(ab)' fragments is shown in Figure 23. To the F(ab)'-azide fragment (500 μg) in PBS (5.0 mg/mL), F(ab)'-DBCO fragment (500 μg) in PBS (5.0 mg/mL) was added. The reaction was carried out overnight by rotating the disk at room temperature with mixing. The mixture was subjected to SEC analysis (Fig. 24).

The biophysical properties of the bispecific antibodies produced in this example were analyzed using size exclusion chromatography (SEC). Analysis of F(ab)'_A, F(ab)'_B and bispecific click_F using size exclusion chromatography (SEC) Agilent 1200 HPLC (using TSK gel SuperSW3000 column (4.6 mm ID x 30 cm, 4 μm)) (ab)' ₂ (Figure 24). The buffer was 0.2 M potassium phosphate, 0.25 M KCl and the pH was 6.2.

The bispecific F(ab)' _{2 was} confirmed by mass spectrometry. Bispecific F(ab)' ₂ was analyzed on a Water Xevo G-2 QTOF9 (Figure 25). The bispecific F(ab)' _{2 was} purified using size exclusion chromatography (SEC). The bispecific click _F(ab)' ₂ (Figure 26) was purified using size exclusion chromatography (SEC) Agilent 1200 HPLC (using a TSK gel SuperSW3000 column (4.6 mm ID x 30 cm, 4 [mu]m)). The buffer was 0.2 M potassium phosphate, 0.25 M KCl and the pH was 6.2.

The in vitro affinity force measurement system used Octet Red (Figure 27). The bispecific F(ab)' ₂ antigen interaction was measured on Octet Red (ForteBio) using a sensor AR2G. Briefly, the measurement protocol was as follows: 300 second baseline, 300 seconds loading 10 μg/ml bispecific F(ab)' ₂ , 120 second baseline, 300 second antigen A, 300 second dissociation, 300 second antigen B and 300 seconds Dissociation (Figure 27). Sensor hydration and baseline and dissociation measurements were performed in PBS.

Example 8

This example illustrates the protocol for the synthesis of the disclosed bispecific antibodies using IgG2 antibodies A' and B'. The bispecific IgG2 line set forth herein consists of two IgG2 fragments chemically linked at the hinge region (Figure 29). The initial anti-system IgG2 isotype is initiated. The generation of bispecific IgG2 involves three major steps. The first step is to reduce one or two (total of 4) disulfide bindings in the hinge region of the IgG2 antibody while still maintaining the homodimeric structure. The second step introduces the functional moiety X or Y into the hinge via cysteine-based coupling to obtain chemically modified antibody fragments A' and B', respectively. In the final step, two antibodies are joined together via a chemical linkage between X and Y to form bispecific IgG2.

Specifically, a process for synthesizing an IgG2 chemically immobilized bispecific antibody is carried out. The antibody (1-10 mg) was added to a 15 mL filter centrifuge tube (Millipore, UFC903024) and an appropriate volume of 50 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA pH 7.7 buffer was added to the 50 mL mark on the tube. The tubes were centrifuged at 3,000 RPM and 22 °C for 20 minutes. The antibody was transferred to a 1.5 mL plastic vial and the concentration was checked using a Nanodrop (Fisher, ND-2000 UV-Vis spectrophotometer). The final antibody concentration is up to 10 mg/mL.

A stock solution of 1 mg/mL TCEP ((叁(2-carboxyethyl)phosphine)) (Sigma-Aldrich, C4706) in pH 8.0 PBS (2.0 mM DTPA) buffer was prepared. The equivalents of IgG2 and the resulting mass were added. Two equivalents of TCEP were added to the IgG2 solution, shaken well and incubated for 90 min at room temperature.

DBCO (dibenzylcyclooctyl)-maleimide and azide-maleimide

For coupling, a stock solution of DBCO-maleimine (Click Chemistry Tools, A108-100) in DMSO (Sigma-Aldrich, 472301) was prepared and 5 equivalents of DBCO-ma in DMSO were prepared. The imine was added to the IgG2_A sample (unpurified TCEP). The final volume of DMSO in the antibody sample was about 5% (v/v). The coupling reaction was carried out by rotating the disk for 1 hour at room temperature and under mixing. The azide-maleimide (5 equivalents) in DMSO was added to the IgG2 (B) sample. The final body of DMSO in the antibody sample The product is about 5% (v/v). The coupling reaction was carried out by rotating the disk for 1 hour at room temperature and under mixing.

For the washing step, each sample was placed in a separate 15 mL filter centrifuge tube (Millipore, UFC903024) and an appropriate volume of 1X DPBS (Corning, 21-031-CM, no calcium or magnesium) buffer was added to the tube. 50mL mark. The samples were centrifuged at 3,000 RPM and 22 ° C for 20 minutes. Repeat the washing step again. After washing, the samples were transferred to a separate 1.5 mL plastic vial and placed in a refrigerator (5 ° C) or used in a click step.

For each sample to be analyzed, 20 μL was required and the concentration was 0.6 mg/mL. Follow established protocols for running SDS-PAGE gels (RTP AD001-01 and AD002-01) (Figure 29).

For mass spectrometry, antibody A was reduced using TCEP and coupled to DBCO for analysis on a Water Xevo G-2 QTOF. This data indicates that 2 DBCO (reducing only one disulfide bond) or 4 DBCO (reducing two disulfide bonds) is coupled to IgG2 (Figures 30A-C).

The biophysical properties of the bispecific antibodies produced in this example were analyzed using size exclusion chromatography (SEC). Bispecific IgG2 was analyzed using size exclusion chromatography (SEC) Agilent 1200 HPLC (using a TSK gel SuperSW3000 column (4.6 mm ID x 30 cm, 4 [mu]m)) (Figure 31). The buffer was 0.2 M potassium phosphate, 0.25 M KCl and the pH was 6.2.

Claims (26)

A method for preparing a bispecific antibody "AB" or "BA" from a first antibody "A" and a second antibody "B", comprising: (a) allowing the first antibody A and the reducing agent to be sufficient to cleave the hinge region Contacting substantially all disulfide linkages between the heavy chains to obtain a pair of first antibody fragments A ' , each comprising a single light chain attached to a single heavy chain, wherein the heavy chain has one or more a reactive thiol group formed by the reduction of the disulfide linkages; (b) attaching a first heterobifunctional linker to the first antibody fragment A ' , the first heterobifunctional linker comprising i) a first thiol reactive functional group for covalent attachment to a reactive thiol group of the heavy chain of the first antibody fragment, and (ii) an azide, thereby forming an azide Functionalizing the first antibody fragment; (c) contacting the second antibody B with a reducing agent under conditions sufficient to cleave substantially all disulfide linkages between the heavy chains in the hinge region to obtain a pair of second antibody fragments B ', each comprising a single light chain attached to a single heavy chain, wherein the heavy chain has one or more from the Reactive thiol groups formed in the reduction of disulfide links; (d) the second heterobifunctional linkers attached to the second antibody fragment B ', the second heterobifunctional linker comprises: (i) first a dithiol reactive functional group for a reactive thiol group covalently attached to the heavy chain of the second antibody fragment; and (ii) an alkyne; thereby forming an acetylenically functionalized second antibody fragment; And (e) reacting the azide-functionalized first antibody fragment with the alkyne-functionalized second antibody fragment to covalently attach the first antibody fragment to the cycloaddition of the azide to the alkyne to The second antibody fragment forms a chemically-locked bispecific antibody "AB" or "BA". A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the first heterobifunctional linker has the form QLN ₃ , wherein Q a thiol reactive functional group comprising an alkyl halide, a benzyl halide, a maleimide, a halo-maleimide or a dihalo-maleimide; and the L system has 3 Up to 60 atomic hydrocarbon connectors, and N ₃ is an azide group. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the first heterobifunctional linker has the form QLN ₃ , wherein Q a thiol reactive functional group comprising a maleimide, a halo-maleimide or a dihalo-maleimine group; and the L system has 3 to 60 atoms and has a unit a hydrocarbon coupling of a polymer configuration of -(CH ₂ CH ₂ -O) _n - and/or -(O-CH ₂ CH ₂ ) _n - wherein "n" is independently an integer from 1 to 20; ₃ series azide group. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 2, wherein the first heterobifunctional linker has the following form: Wherein, the Q group is in the form of a thiol reactive group: Wherein Z is independently selected from the group consisting of H, Br, I, and SPh, provided that Z is not H at least once; and M is independently CR* or N; wherein X ₁ , X ₂ , X ₃ , X ₂ , X ₄ and X _{5 are} independently selected from the group consisting of: bond, -O-, -NR ^N -, -N=C-, -C=N-, -N=N-, -CR*=CR *-(cis or trans), -C≡C-, -(C=O)-, -(C=O)-O-, -(C=O)-NR ^N -, -(C=O )-(CH ₂ ) _n -, -(C=O)-O-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ ) _n - and -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, wherein " n " is zero or an integer from 1 to 10; wherein R ^a , R ^b , R ^c and R ^{d are} independently selected from the group consisting of: -O- , -NR ^N -, -CH ₂ -, -(CH ₂ ) _n -, -(CR* ₂ ) _n -, -(CH ₂ CH ₂ -O) _n -, -(CR* ₂ CR* ₂ -O _n -, -(O-CH ₂ CH ₂ ) _n -, -(O-CR* ₂ CR* ₂ ) _n -, -CR*=CR*-(cis or trans), -N=C- , -C=N-, -N=N-, -C≡C-, -(C=O)-, -(CH ₂ ) _n -(C=O)-, -(C=O)-(CH ₂ ) _n -, -(CH ₂ ) _n -(C=O)-(CH ₂ ) _n -, -O-(C=O)-, -(C=O)-O-, -O-(C =O)-O-, -(CH ₂ ) _n -(C=O)-O-, -O-(C=O)-(CH ₂ ) _n , -(C=O)-O-(CH _{2 n} -, -(CH ₂ ) _n -O-(C=O)-, -(CH ₂ ) _n -(C=O)-O-(CH _{2 ) n} -, -(CH ₂ ) _n -O-(C=O)-(CH ₂ ) _n -, -NR ^N -(C=O)-, -(C=O)-NR ^N -, -NR ^N -(C=O)-O-, -O-(C=O)-NR ^N -, -NR ^N -(C=O)-NR ^N -, -(CH ₂ ) _n -(C=O) -NR ^N -, -NR ^N -(C=O)-(CH ₂ ) _n , -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N -(C =O)-, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N -(C=O)-(CH ₂ ) _n - , -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -, -(CH ₂ ) _n - (C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -(CH ₂ ) _n - or 2 to 8 a cyclic hydrocarbon, heterocyclic, aryl or heteroaryl ring; wherein " n " is independently zero or an integer from 1 to 10; and wherein " l ", " p ", " q " and " r " are independently Zero or an integer from 1 to 10; an Ω linkage or a C _3-26 hydrocarbon or fused ring system, optionally including up to 4 fused rings, each having 3 to 8 members and optionally a ring comprising from 1 to 4 heteroatoms selected from O, S and N; wherein R* and R ^{N are} independently H or C _1-12 hydrocarbons, optionally 1 to 6 selected from halogen, O, S And a hetero atom substitution of the group consisting of N; and wherein R* and/or R ^N can form a 3 to 8 member ring together. A method for preparing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 4, wherein the Q system is maleimide, bromine-maleimide Or dibromomaleimide. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the second heterobifunctional linker has the form QLG, wherein the Q system Thiol reactive functional group, including alkyl halide, benzyl halide, maleimide, halo-maleimide or dihalo-maleimide; and L system has 3 to 60 A hydrocarbon chain of atoms, and the G system contains an alkyne group. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 1, wherein the G system is -C≡CH. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" of claim 1, wherein G includes a C8 loop having a -C≡C- bond. A method for preparing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 8, wherein G has the following form: A method for preparing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 8, wherein G has the following form: A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the second heterobifunctional linker has the form QLG, wherein the Q system a thiol reactive functional group comprising a maleimide, a halo-maleimide or a dihalo-maleimine group; and the L system has 3 to 60 atoms and includes a unit-( a hydrocarbon chain of a polymer of CH ₂ CH ₂ —O) _n — or —(O—CH ₂ CH ₂ ) _n — wherein “n” is independently an integer from 1 to 20; and the G series includes capable of The nitride undergoes a _C8-20 hydrocarbon of the C8 ring of the -C≡C-bond of the 1,3 dipolar cycloaddition reaction. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 10, wherein the second heterobifunctional linker has the following form: Wherein, the Q group is in the form of a thiol reactive group: Wherein Z is independently selected from the group consisting of H, Br, I, and SPh, provided that Z is not H at least once; and M is independently CR* or N; the G system includes having the ability to react with the azide. 1,8 dipolar cycloaddition reaction - C _≡ C-bonded C ₈ ring C _8-20 hydrocarbon group; X ₁ , X ₂ , X ₃ , X ₂ , X ₄ and X ₅ at each occurrence Independently selected from the group consisting of: bond, -O-, -NR ^N -, -N=C-, -C=N-, -N=N-, -CR*=CR*- (cis or anti Formula, -C≡C-, -(C=O)-, -(C=O)-O-, -(C=O)-NR ^N -, -NR ^N -(C=O)-,- NR ^N -(C=O)-O-, -(C=O)-(CH ₂ ) _n -, -(C=O)-O-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ ) _n - and -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, wherein " n " is zero or an integer from 1 to 10; R ^a , R ^b , R ^c and R ^{d are} independently selected from the group consisting of -O-, -NR ^N -, -CH ₂ -, -(CH ₂ ) _n -, -(CR* ₂ ) _n -, -(CH ₂ CH ₂ -O) _n -, -(CR* ₂ CR* ₂ -O) _n -, -(O-CH ₂ CH ₂ ) _n -, -(O-CR* ₂ CR* ₂ ) _n -, -CR*= CR*-(cis or trans), -N=C-, -C=N-, -N=N-, -C≡C-, -(C=O)-, -(CH ₂ ) _n - (C=O)-, -(C=O)-(CH ₂ ) _n -, -(CH ₂ ) _n -(C=O)-(CH ₂ ) _n -, - O-(C=O)-, -(C=O)-O-, -O-(C=O)-O-, -(CH ₂ ) _n -(C=O)-O-, -O- (C=O)-(CH ₂ ) _n , -(C=O)-O-(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C=O)-, -(CH ₂ ) _n -(C=O)-O-(CH ₂ ) _n -, -(CH ₂ ) _n -O-(C=O)-(CH ₂ ) _n -, -NR ^N -(C=O)-,- (C=O)-NR ^N -, -NR ^N -(C=O)-O-, -O-(C=O)-NR ^N -, -NR ^N -(C=O)-NR ^N -, -(CH ₂ ) _n -(C=O)-NR ^N -, -NR ^N -(C=O)-(CH ₂ ) _n , -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N -(C=O)-, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ ) _n -, -(CH ₂ ) _n -NR ^N - (C=O)-(CH ₂ ) _n -, -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O) -NR ^N -, -(CH ₂ ) _n -(C=O)-NR ^N -(CH ₂ CH ₂ -O) _n -, -(CH ₂ CH ₂ -O) _n -(C=O)-NR ^N -(CH ₂ ) _n - or 2 to 8 membered cyclic hydrocarbon, heterocyclic, aryl or heteroaryl ring; wherein " n " is independently zero or an integer from 1 to 10; and wherein " l ", " p ′′, “ q ” and “ r ” are independently zero or an integer from 1 to 10; Ω is independently a bond or a C _3-26 hydrocarbon ring or a fused ring system, optionally including up to 4 fused rings, as appropriate Each ring has 3 to 8 members and optionally includes 1 to 4 independently selected from each ring in each ring. a hetero atom of S and N; wherein R* and R ^N are independently H or C _1-12 hydrocarbons at each occurrence, and optionally 1 to 6 heteroatoms selected from halogen, O, S and N Substituting; and wherein two of the groups R* and/or ^RN may together form a 3 to 8 membered ring. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the cycloaddition reaction occurs in the presence of copper ions. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the cycloaddition reaction occurs at a neutral pH. A method for preparing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 1, wherein at least 90% of the heavy chain and the light chain are such The disulfide linkage remains substantially intact after cleavage of the disulfide bonds in the hinge region. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" in claim 1, wherein the antibody A or the antibody B is an IgG1 immunoglobulin. A method for producing a bispecific antibody "AB" or "BA" from the first antibody "A" and the second antibody "B" according to claim 1, wherein the antibody A or the antibody B is an IgG4 immunoglobulin. A method for producing a chemically immobilized bispecific antibody "AB" or "BA" from an IgG1, IgG2 or IgG4 antibody or a Fab2 fragment "A" thereof, and an IgG1, IgG2 or IgG4 antibody or a fragment "B" thereof The method comprises the following steps: (a) reducing the first antibody "A" having a hinge residue sequence (EU index number: residues 226 to 229) selected from the group consisting of CPPC, CPSC, SPPC and SPSC and having a hinge residue sequence (EU index) ID: residues 226 to 229) are selected from the group consisting of antibodies "B" consisting of CPPC, CPSC, SPPC, and SPSC to form half antibody A and half antibody-B, wherein antibody A binds to the first target and antibody B binds to a two target, wherein the reducing condition ruptures any of the interchain or intrachain disulfide bonds in the hinge region of antibody A and antibody B; (b) one of the antibody hinge core sequences or two Cys residues that link the first compound to half antibody A Base 226 or/and 229 (EU index number: residue 226 or/and 229) to form a linked half antibody A, wherein the first compound has a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh wherein N ₃ is -N=N=N; (c) linking the second compound to a CPPC having a hinge residue sequence (residues 226 to 229) Or one of the hinge core sequences of antibody B of CPSC or SPPC or SPSC or two Cys residues 226 and 229 to form a linked antibody B, wherein the second compound has a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh; and (d) cultivating approximately one molar amount of linked antibody A and linked antibody B under neutral conditions to form the chemical lock Bispecific antibody AB. A method of producing a chemically immobilized bispecific antibody according to claim 18, wherein the reduction of the antibody A to form the half antibody A and the reduction of the antibody B to form the half antibody B are carried out in a reducing agent, wherein the reducing agent Is selected from the group consisting of L-cysteine, dithiothreitol, β-mercaptoethanol, cysteamine, TCEP (叁(2-carboxyethyl)phosphine), 2-MEA (2-mercapto) Ethylamine) and combinations thereof. A method of producing a chemically-locked bispecific antibody according to claim 18, wherein the hinge region having two Cys residues (EU index number: residue 226 or/and 229) in antibody A has a composition selected from the group consisting of Part A of the structure of the group: M = N, C M ' = N, CZ = I, Br, SPh wherein N ₃ is -N = N = N. A method of producing a chemically-locked bispecific antibody according to claim 18, wherein the hinge region having one or two Cys residues (EU index number: residue 226 or/and 229) in antibody B is selected from Part B of the structure of the following group is connected: M=N, C M'=N, CZ=I, Br, SPh to form a linked half-antibody A having a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh wherein N ₃ is -N=N=N; and the linked antibody B having a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh. A chemically-locked bispecific antibody AB comprising a linked half-antibody A linked to: Wherein N ₃ is -N=N=N; the linked half-antibody A is linked to a linked antibody B linked to: A chemically-locked bispecific antibody "AB" or "BA" derived from an IgG type antibody "A" and an IgG type antibody "B", which comprises a half antibody A linked to a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh wherein N ₃ is -N=N=N; the half antibody A is linked to a half antibody B linked to a structure selected from the group consisting of: M=N, C M'=N, CZ=I, Br, SPh. A bispecific antibody comprising: (a) a first antibody fragment A ' comprising a single heavy chain and a light chain from antibody A, wherein the single heavy chain has one or more reactive thiol groups; b) a second antibody fragment B ' comprising a single heavy chain and a light chain from antibody B, wherein the single heavy chain has one or more reactive thiol groups; wherein the first and second antibody fragments are via a 1,2,3-triazole covalently linked by a cycloaddition reaction of an azide and an alkyne, the azide being attached to the reactive thiol on the first antibody fragment via a linker, the alkyne A reactive thiol attached to the second antibody fragment via a linker. The bispecific antibody of claim 25, wherein the fragments A ' and B ' are derived from an IgGl or IgG4 immunoglobulin. Type of covalent bonding of the linker to the antibody fragment, having the connecting member ₈ comprises a ring capable of undergoing a cycloaddition reaction of C -C≡C- bond with the azide.