阅读说明：本技术 半胱氨酸工程化的抗原结合分子 (Cysteine engineered antigen binding molecules ) 是由 G·沃兹尼亚克-克诺普 F·吕克尔 G·斯塔德迈尔 J·雷布卡 N·拉舍 S·迪克吉塞尔 于 2018-10-03 设计创作，主要内容包括：一种特异性抗原结合成员(ABM),包含特异性抗原结合部分和包含CH2结构域的抗体Fc区域,其经工程化在位置108和/或113处进行半胱氨酸取代,其中根据IMGT进行编号,并且其中抗体Fc区域不包含抗原结合CH3结构域的；以及一种ABM缀合物(ABMC),包含ABM和在CH2结构域的位置108和113处共价缀合至半胱氨酸中的一个或两个的至少一个异源分子。(A specific Antigen Binding Member (ABM) comprising a specific antigen binding portion and an antibody Fc region comprising a CH2 domain, engineered for a cysteine substitution at position 108 and/or 113, wherein numbering is according to IMGT, and wherein the antibody Fc region does not comprise an antigen binding CH3 domain; and an ABM conjugate (ABMC) comprising an ABM and at least one heterologous molecule covalently conjugated to one or both of the cysteines at positions 108 and 113 of the CH2 domain.)

1.A specific Antigen Binding Member (ABM) comprising a specific antigen binding portion and an antibody Fc region comprising a CH2 domain engineered for a cysteine substitution at position 108 and/or 113, wherein numbering is according to IMGT, and wherein the antibody Fc region does not comprise an antigen binding CH3 domain.

2. The ABM of claim 1, wherein an antigen binding moiety is fused to the N-terminus of the antibody CH2 domain.

3. The ABM of claim 1 or 2, wherein said CH2 domain comprises one or two cysteine substitutions, which are N108C and/or L113C, wherein numbering is according to IMGT.

4. The ABM of any one of claims 1-3, wherein said antigen binding portion comprises an antigen binding portion of an antibody, an enzyme, an adhesion protein, a ligand for a receptor, or a ligand binding portion.

5. The ABM of any one of claims 1-4, wherein said antigen binding moiety is selected from the group consisting of Fab, F (ab')₂scFv, Fd, Fv, and one or more antibody domains comprising at least one antibody binding site in a CDR.

6. The ABM of any one of claims 1-5, wherein antigen binding portion is fused to the N-terminus of the CH2 domain by a linker and/or hinge region.

7. The ABM of any one of claims 1-6, wherein the C-terminus of the CH2 domain is fused to the N-terminus of the CH3 domain, preferably wherein the Fc region is comprised in an antibody Fc consisting of a dimer of antibody heavy chains.

8. The ABM of any one of claims 1-7, wherein said Fc region is of the IgG, IgA, IgM or IgE isotype, preferably, of a human antibody.

9. The ABM of any one of claims 1-8, selected from the group consisting of a monoclonal antibody, a bispecific antibody, a multispecific antibody, and an antigen-binding portion of an antibody.

10. The ABM of any one of claims 1-9, which specifically recognizes an antigen of interest expressed on the surface of a cell of interest.

An ABM conjugate (ABMC) comprising the ABM of any one of claims 1 to 10, and at least one heterologous molecule covalently conjugated to one or two cysteines at positions 108 and 113 of the CH2 domain, wherein numbering is according to IMGT.

12. The ABMC of claim 11 wherein the heterologous molecule is a substance suitable for use in the diagnosis, cure, mitigation, treatment or prevention of a disease, preferably selected from the group consisting of pharmaceutical drug substances, toxins, radionuclides, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes, oligonucleotides, DNA, RNA, siRNA, RNAi, microRNA, peptide nucleic acids, photoactive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, fluorescent tags, visualization peptides, biotin, serum half-life modulators, capture tags, chelating agents and solid supports.

13. The ABMC of claim 11 or 12 wherein the heterologous molecule is conjugated to one or both of the cysteines at positions 108 and 113 of the CH2 domain via a conjugated linker, wherein numbering is according to IMGT.

14. The ABMC of claim 13 wherein the conjugate linker comprises a maleimide group.

15. An expression system comprising one or more nucleic acid molecules encoding the ABM of any one of claims 1-10.

16. A host cell comprising the expression system of claim 15.

17. The method of producing an ABM of any one of claims 1-10, wherein the host cell of claim 16 is cultured or maintained under conditions to produce the ABM.

18. A pharmaceutical formulation comprising an ABM according to any one of claims 1 to 10, or an ABMC according to any one of claims 11 to 14, and a pharmaceutically acceptable carrier or excipient in a parenteral formulation.

19. A method of producing an ABMC according to any one of claims 11-14, comprising the steps of:

a) providing an ABM according to any one of claims 1 to 10; and

b) at least one thiol group of one or both cysteines at positions 108 and 113 of the CH2 domain is reacted with the heterologous molecule by a site-specific conjugation method.

20. The method of claim 19, wherein the at least one thiol group reacts with the heterologous molecule by a michael reaction using a conjugated linker comprising a maleimide group.

Technical Field

The present invention relates to Antigen Binding Members (ABMs) comprising a cysteine engineered antibody Fc region, and ABM conjugates in which one or more heterologous molecules, are conjugated to any cysteine.

Background

Monoclonal antibodies have been widely used as therapeutic antigen binding molecules. The basic antibody structure will be explained herein with the example of an intact IgG1 immunoglobulin.

Two identical heavy chains (H) and two identical light chains (L) combine to form a Y-shaped antibody molecule. The heavy chains each have four domains. The amino-terminal variable domain (VH) is located at the tip of Y. Followed by three constant domains: CH1, CH2 and the carboxy terminus CH3, located at the base of the stem of Y. A small stretch, switch, connecting the heavy chain variable and constant regions. The hinge connects CH2 and CH3(Fc fragment) to the remainder of the antibody (Fab fragment). One Fc and two identical Fab fragments can be generated by proteolytic cleavage of the hinge in the intact antibody molecule. The light chain is composed of two domains, Variable (VL) and Constant (CL), separated by a switch.

Disulfide bonds in the hinge region connect the two heavy chains. The light chain is coupled to the heavy chain by an additional disulfide bond. Depending on the kind of immunoglobulin, Asn-linked carbohydrate moieties are linked at different positions of the constant domain. For IgG1, two disulfide bonds in the hinge region between the Cys235 and Cys238 pairs join the two heavy chains together. Between Cys214(EU index and Kabat numbering) in the CL domain, the light chain is coupled to the heavy chain by two additional disulfide bonds, Cys220(EU index numbering) or Cys233 (numbering according to Kabat) in the CH1 domain. The carbohydrate moiety is attached to Asn306 of each CH2, creating a distinct bulge in the stem of Y.

These features have profound functional consequences. The variable regions of both the heavy and light chains (VH) and (VL) are located at the N-terminal region, the "tip" of the Y, where they react with antigen. This tip of the molecule is the side at which the N-terminus of the amino acid sequence is located. The stem of Y bulges out in some way to effectively mediate effector functions such as complement activation and interaction with Fc receptors, or ADCC and ADCP. Its CH2 and CH3 domains are raised to facilitate interaction with effector proteins. The C-terminus of the amino acid sequence is located on the opposite side of the tip, which may be referred to as the "bottom" of the Y.

Two types of light chains are found in antibodies, called lambda (λ) and kappa (κ), respectively. A given immunoglobulin has either a kappa chain or a lambda chain, rather than only one of each. No functional differences were found between antibodies with lambda or kappa light chains.

Each domain in an antibody molecule has two beta sheets of similar structure that are tightly packed against each other in a compressed antiparallel beta barrel. This conserved structure is called the immunoglobulin fold. The immunoglobulin fold of the constant domain comprises a 3-strand fold stacked with a 4-strand sheet. The fold is stabilized by hydrogen bonds between the beta strands of each sheet, by hydrophobic bonds between residues of the inner opposing sheets, and by disulfide bonds between the sheets. The 3-chain sheet layer contains chains C, F and G, while the 4-chain sheet layer contains chains A, B, E and D. The letters a to G indicate the sequential positions of the beta chain along the amino acid sequence of the immunoglobulin fold.

The folding of the variable domain has 9 beta strands arranged in two sheets of 4 and 5 strands. The 5-strand layer is structurally homologous to the 3-strand layer of the constant domain, but contains additional strands C' and C ". The remainder of the chain (A, B, C, D, E, F, G) has the same topology and similar structure in constant domain immunoglobulin folds as their counterparts. As in the constant domain, disulfide bonds link chains B and F in opposite sheets.

The variable domains of both the light and heavy immunoglobulin chains comprise three hypervariable loops, or Complementarity Determining Regions (CDRs). The three CDRs (CDR1, CDR2, CDR3) of the V domain are clustered at one end of the β -barrel. CDRs are loops connecting beta strands B-C, C '-C' of the immunoglobulin fold with F-G. The residues in the CDRs vary from one immunoglobulin molecule to the next, giving each antibody antigen specificity.

The VL and VH domains at the tips of the antibody molecules are closely packed such that the 6 CDRs (3 on each domain) cooperate to construct a surface (or cavity) for antigen-specific binding. Thus, the natural antigen-binding site of an antibody consists of loops connecting chains B-C, C '-C "and F-G of the light chain variable domain and chains B-C, C' -C" and F-G of the heavy chain variable domain.

Loops in a native immunoglobulin that are not CDR loops, or that are not part of the antigen binding pocket defined by the CDR loops and optional adjacent loops within the CDR loop regions, have no antigen binding or epitope binding specificity, but contribute to correct folding of the entire immunoglobulin molecule and/or its effectors or other functions, and are therefore referred to as structural loops. Thus, a "structural loop" or a "non-CDR loop" according to the present invention is to be understood in the following manner: immunoglobulins consist of domains with a so-called immunoglobulin fold. In essence, antiparallel beta sheets are connected by loops to form a compressed antiparallel beta barrel. In the variable region, some loops of the domain contribute substantially to the specificity of the antibody, i.e., binding to the antigen. These loops are called CDR loops. All other loops of the antibody domain contribute considerably to the structure and/or effector function of the molecule. These loops are defined herein as structural loops or non-CDR loops.

Antigen binding Fc fragment (also known as Fcab)^TM[ f-star Co; fc fragment with antigen binding site (Wozniak-Knopp et al, 2010)]Comprising, for example, a modified Fc domain of IgG1 which specifically binds to an antigen with high affinity, is described, for example, in WO 2009/132876A1 and WO 2009/000006 a 1.

Various antibody constructs are currently being developed to provide Antibody Drug Conjugates (ADCs).

ADC combines the specificity of the antibody with the cytotoxicity of the drug, thereby improving the therapeutic effect of both. ADCs typically consist of an antibody, a linker, and a cytotoxin. The effect of the antibody is to target the delivery of the drug to the cell. In certain cases, efficient internalization of antigen-antibody complexes is critical to the ADC mechanism of action. After internalization, the linker is cleaved and the toxin is released in its active form. The toxin is inactivated by conjugation prior to release and is therefore stable and harmless in circulation.

In ADCs, the cytotoxins currently used can be divided into two categories: toxins that interact with microtubules by inhibiting microtubule assembly (e.g., maytansinoids and auristatins), and those that bind to the DNA minor groove and cause cell death by inducing DNA strand breaks (e.g., calicheamicin).(Wyeth, gemtuzumab) uses calicheamicin derivatives and is the first ADC approved by the U.S. food and drug administration for the treatment of acute myeloid leukemia. It exited the market in 2010 because safety considerations and benefits to the patient were not satisfactory. Currently, there are several ADCs in various clinical trials.

Generally, linkers that are stable in circulation are used because of the early release of cytotoxins that might otherwise lead to non-specific cell killing. The linker chosen is susceptible to cleavage in lysosomes and release of the drug intracellularly. Currently, there are four different types of joints: an acid labile linker that is stable at neutral pH (e.g., blood) and undergoes hydrolysis in an acidic environment; disulfide bond-based linkers that cleave in the cytosol due to high intracellular concentrations of glutathione; a peptide-based linker that conjugates the drug to the antibody via a peptide bond and is released by lysosomal proteases; thioether-containing, non-cleavable linkers, which are more stable and are believed to release the drug by intracellular proteolytic degradation.

Free thiol (SH) -groups can be introduced by partial reduction of interchain disulfide bonds, or by introduction of new surface cysteines by site-directed mutagenesis to create specific conjugation sites (Junutula, Bhakta, et al, 2008; Voynov et al, 2010). The construct thus having reactive thiol groups is provided as a "pre-ADC". Engineering of surface cysteines at various positions has been described in WO2013/070565A1, WO22014/124316A1, WO2015/157595A1 and WO2017/112624A 1.

There is a need to improve cysteine engineering of antibodies without altering the basic properties of the Fc fragment.

Disclosure of Invention

It is an object of the present invention to provide improved cysteine engineered ABMs which provide reactive thiol groups that are readily conjugated to drugs.

Said object is achieved by the subject of the invention.

According to the present invention there is provided a specific Antigen Binding Member (ABM) comprising a specific antigen binding portion and an antibody Fc region comprising a CH2 domain, the antibody Fc region being engineered (with mutation or otherwise modified as compared to the wild-type CH2 domain) to carry out a cysteine substitution at position 108 and/or 113, wherein numbering is according to IMGT. Specifically, the ABM comprises a specific antigen binding portion and an antibody Fc region comprising a CH2 domain, wherein the CH2 domain comprises a cysteine substitution at position 108 and/or 113, wherein numbering is according to IMGT.

Specifically, one or both cysteines are engineered into the F-G loop of the CH2 domain at a predetermined position by point mutation by substituting cysteine for the naturally occurring amino acid. Thus, one or more free thiol groups are engineered into the ABM.

Free thiol groups are herein understood to be mercapto (-SH) functional groups of cysteine residues, which are unpaired with (not cross-linked to) other cysteine residues of the ABM, and which may be unblocked or blocked by chemical entities (other than the ABM), for example by cysteine or glutathione, which may be present in the cell culture medium when the ABM is expressed in cell culture. Specifically, free (unpaired) cysteine residues are introduced into the ABM for site-specific labeling and/or drug conjugation.

The indicated positions are surprisingly well suited when it is determined that the exposure of the amino acid residue interacts with the solvent, despite being "hidden" or "buried". In the prior art, solvent exposure of a site indicates a favorable accessibility for drug conjugation.

In particular, the antigen binding portion includes the antigen binding portion of an antibody, or the binding site of any of an enzyme, an adhesion protein, a ligand of a receptor, or a ligand binding portion of a ligand, which is capable of binding to the cognate structure of a binding partner. In particular, the antigen binding portion consists of the binding site of a naturally occurring receptor.

In particular, the antigen-binding portion comprises one or more antibody variable domains, in particular VH and VL domains, which associate to form a VH/VL binding site comprising or consisting of three VH-CDR regions and three VL-CDR regions.

A specific ABM as described herein comprises a CH3 domain comprising an antigen binding site, e.g., wherein one or more amino acid sequences in at least one structural loop region are modified, thereby obtaining a modified structural loop region that specifically binds to an epitope of an antigen, e.g., a surface antigen to which the unmodified CH3 domain does not significantly bind. The antigen-binding CH3 domain comprising the antigen-binding site in the structural loop has been shown to have advantageous properties in antigen-binding Fc or in the antigen-binding Fc portion of antibodies, or in the antigen-binding Fc portion of any other ABM comprising such Fc.

Specific ABMs as described herein consist of or comprise an antigen binding Fc comprising an antigen binding site, e.g. wherein one or more amino acid sequences in at least one structural loop region are modified, thereby obtaining a modified structural loop region specifically binding to an antigenic epitope, e.g. a surface antigen, such as Her2, to which the unmodified Fc does not significantly bind. Antigen binding Fc comprising an antigen binding site in a structural loop has been shown to have advantageous properties as the antigen binding Fc part of Fcab or as the antigen binding Fc part of an antibody or any other ABM comprising such Fc.

Specific ABMs described herein include antibodies that include an antigen-binding CH3 domain or Fc, e.g., full-length antibodies, such as those having an IgG structure, that comprise one or more (e.g., only 2) antigen-binding CH3 domains, or that comprise an antigen-binding Fc in place of a wild-type CH3 domain and Fc, respectively. Exemplary binding members are full-length bispecific antibodies, referred to as mabs^2TM(f-star Co.).

According to a preferred embodiment, the antigen binding moiety is selected from the group consisting of Fab, F (ab')₂scFv, Fd, Fv, antigen binding CH3, Fcab, and one or more antibody domains comprising at least one antibody binding site in a CDR or non-CDR (or structural) loop.

In particular, the amount of the solvent to be used,

a) an antigen-binding portion fused to the N-terminus of the antibody CH2 domain; and/or

b) The antigen binding portion is comprised in the CH3 domain and/or the Fc region.

Specifically, the antigen binding portion is contained in a structural loop of the Fc region, particularly in the C-terminal structural loop of one or both of the CH3 domains in the Fc region.

According to a specific embodiment, the antigen binding portion is comprised in an antigen binding Fc, or in a full length multivalent or bispecific antibody comprising an antigen binding Fc.

In particular, the antigen binding portion is fused to the N-terminus of the CH2 domain via a linker and/or hinge region. In particular, the hinge region is any peptide hinge region consisting of an amino acid sequence, which is the hinge region of a naturally occurring immunoglobulin. In particular, the hinge region is a human immunoglobulin, e.g., comprising or consisting of the amino acid sequence identified as SEQ ID NO. 7.

In the ABMs described herein, the linking of the antibody domains is specifically by recombinant fusion or chemical ligation. Specific ligation may be performed by ligating the C-terminus of one domain to the N-terminus of the other domain. For example, wherein one or more amino acid residues in the terminal region are deleted to shorten the domain size, or extended to increase the flexibility of the domain.

In particular, shortened domain sequences may be used which include deletions of the C-terminal and/or N-terminal region, for example deletions of at least 1, 2, 3, 4 or 5, up to 6, 7, 8, 9 or 10 amino acids.

In particular, a linker sequence may be used which is a linker or hinge region of an immunoglobulin or at least a part of a hinge region, such as a peptide linker consisting of an amino acid sequence, e.g. comprising at least 1, 2, 3, 4 or 5 amino acids, up to 10, 15 or 20 amino acids. The joining sequence is also referred to herein as a "joining point". The domains may be extended by linkers, for example by amino acid sequences derived from the N-or C-terminal regions of antibody domains which are naturally located adjacent to the domains, for example including the natural point of attachment between these domains. Alternatively, the linker may comprise an amino acid sequence derived from the hinge region. However, the linker may also be an artificial sequence, e.g. consisting of consecutive Gly and/or Ser amino acids, preferably of a length of 5 to 20 amino acids, preferably of 8 to 15 amino acids.

In particular, the C-terminus of the CH2 domain is fused to the N-terminus of the CH3 domain, preferably wherein the Fc region is comprised in an antibody Fc consisting of a dimer of antibody heavy chains.

Specifically, the Fc region is comprised in the Fc portion of an antibody (referred to herein as "antibody Fc" or "Fc") which consists of two CH2 domains and two CH3 domains, wherein a first chain of the CH2 domain is fused to the CH3 domain, thereby forming a dimer with a second chain of the CH2 domain fused to the CH3 domain.

The Fc region is specifically characterized by a dimer of Fc chains, each Fc chain characterized by comprising a CH2-CH3 antibody domain, which dimer may be a homodimer or a heterodimer, e.g., wherein a first Fc chain differs from a second Fc chain by at least one point mutation in the CH2 and/or CH3 domains.

In particular, one or two cysteines of the CH2 domain of Fc are engineered to comprise one or two cysteine substitutions at positions 108 and/or 113, wherein numbering is according to IMGT. Specifically, Fc contains one or two cysteine substitutions in each CH2 domain, such that Fc contains only 1, 2, 3, or4 free thiol groups.

Specifically, the antigen binding portion is fused to the Fc or Fc region, particularly to the N-terminus of the CH2 domain, or to the hinge region connecting the antigen binding portion to the CH2 domain.

According to a particular embodiment, the antigen binding moiety is incorporated within the Fc region, e.g., in the C-terminal loop region of the CH3 domain, which is understood to be the "structural loop region".

According to a specific embodiment, the antigen binding moiety is the antigen binding site of Fcab. Specifically, Fcab comprises one or two antigen binding moieties. Specifically, Fcab comprises two antigen binding moieties, wherein the first antigen binding moiety is incorporated into the C-terminal structural loop region of the first CH3 domain and the second antigen binding moiety is incorporated into the C-terminal structural loop region of the second CH3 domain.

Furthermore, Fcab may be part of a construct comprising one or more antigen binding moieties, e.g. two antigen binding moieties, wherein the first is fused to the N-terminus of the first CH2-CH3 chain, e.g. via a linker or hinge region, and the second is fused to the N-terminus of the second CH2-CH3 chain, e.g. via a linker or hinge region.

In particular, the ABM is an antigen-binding Fc (particularly Fcab), or comprises an antigen-binding Fc (particularly a mAb)²) The full-length multivalent or bispecific antibody of (a).

According to a specific embodiment, the ABM is a full length immunoglobulin having the structure of any one of IgG, IgA, IgM, or IgE, wherein Fc is exchanged for Fcab. Thus, the ABM comprises three, four or at least three or four antigen binding moieties, and optionally two, three or more different antigen binding specificities.

In a particular embodiment, the ABM is a full-length multivalent or bispecific antibody comprising

i) Two antigen binding portions, each having one antigen binding site (e.g., each being a Fab arm of an antibody), wherein each antigen binding site consists of a CDR loop, an

ii) an antigen binding portion comprising one or two antigen binding sites (e.g., an antigen binding Fc comprising one or two antigen binding sites in the CH3 domain), wherein each antigen binding site consists of a non-CDR loop.

Specifically, the ABM is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a multispecific antibody, an antigen-binding portion of an antibody, an Fcab molecule, and an antibody comprising an Fcab molecule. In particular, the ABM is a human, humanized or chimeric antibody. Specifically, ABMs are human antibodies, particularly human IgG antibodies, modified for introduction of a point mutation in the CH2 domain as described herein, and optionally further modified to introduce one or more additional antigen binding sites.

Specifically, ABMs are bispecific or multispecific, specifically recognizing two or more different antigens, wherein a specific antigen is recognized by one, two or more antigen binding moieties. Specifically, the ABM is bivalent or multivalent, wherein the antigen is specifically recognized by two or more antigen binding moieties, respectively.

Specifically, the ABM; are cross-reactive, wherein two or more antigens are specifically recognized by one cross-specific binding site of the antigen-binding portion.

Specifically, ABMs are monoclonal antibodies. Specifically, preparations of monoclonal antibodies are provided, which are obtained by culturing a cell line of host cells that has been engineered by recombinant techniques to express the monoclonal antibodies.

According to a particular embodiment, the CH2 domain includes one or two cysteine substitutions, which are N108C and/or L113C, wherein numbering is according to IMGT.

In particular, the CH2 domain is of a mammalian species, such as human, murine, rabbit, goat, camelid, llama, bovine or equine, or an avian species, such as a hen.

In particular, the CH2 domain is a wild-type CH2 domain consisting of an amino acid sequence that is naturally occurring except for one or two cysteines engineered to predetermined positions, thereby obtaining an artifact.

In particular, the CH2 domain is an immunoglobulin of any of the IgG, IgA, IgM or IgE isotypes, in particular any of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM antibodies, preferably human antibodies.

In particular, the CH2 domain is the CH2 domain of human IgG, in particular IgG1, and comprises one or two cysteine substitutions, which are N108C and/or L113C, wherein numbering is according to IMGT.

In particular, the CH2 domain is the CH2 domain of human IgG, in particular IgG1, and comprises one or two cysteine substitutions, which are N325C and/or L328C, wherein numbering is according to the EU index of Kabat.

Specifically, the CH2 domain includes or consists of an amino acid sequence that is identified as any one of SEQ ID NOs 1, 2 or 3, or an amino acid sequence that has at least 90% sequence identity to any one of SEQ ID NOs 1, 2 or 3.

In particular functional variants of the CH2 domain are characterized by a certain degree of sequence identity, e.g. especially at least 90% or at least 95% of the naturally occurring sequence is characterized by a β -barrel structure of the antibody domain, which is similar to the structure of the individual domains in the human IgG, IgA, IgM or IgE structure, especially the human IgG1 structure.

In particular, functionally active variants of the CH2 domain comprising one or more point mutations, preferably up to 10 point mutations, in particular any of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 point mutations in the naturally occurring sequence may be used.

In particular, the Fc or Fc region is of a mammalian species, such as human, murine, rabbit, goat, camelid, llama, bovine or equine, or an avian species, such as a hen.

In particular, the Fc region comprises the wild-type CH2-CH3 domain sequence consisting of an amino acid sequence that is naturally occurring except for one or two cysteines in CH2 engineered at predetermined positions, thereby obtaining an artifact.

In particular, the Fc or Fc region is an immunoglobulin of any of the IgG, IgA, IgM or IgE isotypes, in particular any of the IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE or IgM antibodies, preferably a human antibody.

Specifically, the Fc region consisting of one CH2 and one CH3 domain is human IgG, in particular IgG1, and comprises one or two cysteine substitutions in the CH2 domain, which are N108C and/or L113C, wherein numbering is according to IMGT.

In particular, the Fc region consisting of one CH2 and one CH3 domain is human IgG, in particular IgG1, and comprises one or two cysteine substitutions in the CH2 domain, which are N325C and/or L328C, wherein the numbering is according to the EU index of Kabat.

Specifically, the Fc domain comprises or consists of an amino acid sequence that is identified as any one of SEQ ID NOs 4, 5, or 6, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs 4, 5, or 6.

Functional variants of the Fc region in particular are characterized by a certain degree of sequence identity, for example at least 90% or at least 95% of the naturally occurring sequences in particular are characterized by a β -barrel structure of the CH2 and CH3 antibody domains, which is similar to the structure of the individual domains in the human IgG, IgA, IgM or IgE structure, in particular the human IgG1 structure.

In particular, functionally active variants of the Fc region may be used which comprise one or two point mutations in the naturally occurring sequence comprised in one or both of the CH2 and CH3 domains of the Fc region, preferably up to 10 point mutations, in particular any of the 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 point mutations in one or both of the antibody domains CH2 and CH 3.

According to certain embodiments, the ABM specifically recognizes an antigen of interest expressed on the surface of a cell of interest, in particular via one or more antigen binding moieties. Such surface antigens are specific on the surface of a target cell, which is any one of the mammals, in particular a human cell, which upon binding to the antigen is targeted to react with the ABM or any heterologous moiety linked to said ABM.

In particular, the antigen of interest is selected from cell surface antigens including receptors, in particular from the group consisting of erbB receptor tyrosine kinases (e.g. EGFR, Her2 including Her2neu, Her3 and Her 4). In addition, other antigens may also be targeted, such as molecules of the TNF receptor superfamily, e.g. Apo-1 receptor, TNFR1, TNFR2, nerve growth factor receptor NGFR, CD40, CD 40-ligand, OX40, TACI, BCMA, BAFF receptor, T cell surface molecule, T cell receptor, T cell antigen, Apo-3, DR4, DR5, DR6, decoy receptors, e.g. DcR1, DcR2, CAR1, HVEM, GITR, ZTNFR-5, NTR-1, TNFL1, IGFR-1, c-Met, but not limited to these molecules, B cell surface antigens, e.g. CD10, CD19, CD20, CD21, CD22, DC-SIGN, antigens or markers of solid or blood cancer cells, lymphoma or leukemia cells, other blood cells including platelets, but not limited to these molecules.

According to a specific embodiment, the surface antigen is selected from the group consisting of receptor tyrosine kinases (ErbB family).

Specifically, ABMs internalize upon binding to target cells. According to a specific embodiment, the internalized ABM specifically recognizes an antigen selected from the group consisting of receptor tyrosine kinases (ErbB family). Internalization of ABMs upon binding to target cells is determined by standard techniques including, for example, flow cytometry, radiolabeled antibody studies, image analysis, or cytotoxicity assays using antibody drug conjugates.

In particular, ABMs comprise a functional antigen-binding site consisting of a VH/VL domain pair, which is capable of binding to a target with high affinity and has a KD of less than 10^-6M、10^-7M、10^-8M、10^-9M or 10^-10Any of M. In particular, ABMs are bispecific or heterodimeric antibodies targeting two different antigens, each of which is recognized by the antibody with a KD of less than 10^-6M、10^-7M、10^-8M、10^-9Or 10^-10Any of M.

Specifically, ABMs are monospecific or bispecific antibodies targeting at least EGFR. According to a specific embodiment, the antibody is cetuximab (Imclone Systems, Bristol-Myers Squibb, Merck KGaA).

Specifically, the ABM is a bispecific or multispecific antibody, wherein the first target is any one of CD3, CD16, or Her2neu, and the second target is EGFR.

According to a specific embodiment, the ABM comprises two different Fab arms, thereby providing two different Fv structures, each with specific binding characteristics. Specifically, ABMs are heterodimers or bispecific antibodies targeting two different antigens or two different epitopes.

In particular, ABMs are heterodimers or bispecific antibodies comprising a first and a second Fab arm recognizing different antigens or epitopes, e.g. bispecific full length immunoglobulins.

For example, the antigen binding portion used in the ABMs described herein is a Fab arm, which is a dimer of a Heavy Chain (HC) consisting of a VH-CH1 domain sequence and a Light Chain (LC) consisting of a VL-CL (kappa or lambda) domain sequence, with or without any disulfide bonds, hinge domains, and/or linker sequences that link antibody domains. When cleaved from an antibody, a Fab arm is generally understood to be a Fab fragment (or Fab portion). The Fab arms are specifically characterized by the formation of only one antigen binding site by pairing of the VH and VL domains and are only capable of monospecific and monovalent binding to the target.

According to a particular aspect, the ABMs described herein are heterodimeric antibodies comprising two different HCs, each HC comprising a CH2 and a CH3 domain, and optionally a CH4 domain, the HCs of which dimerize to an Fc region.

Specifically, the ABM comprises an Fc or Fc region of a heterodimer, wherein a first Fc chain differs from a second Fc chain by at least one point mutation in the CH2 and/or CH3 domains.

In particular, the heterodimeric Fc region comprises two CH3 domains, the CH3 domain being engineered to be introduced, and/or characterized by one or more of:

a) chain exchange engineered domain (SEED) CH3 heterodimer, consisting of alternating fragments of human IgA and IgG CH3 sequences;

b) one or more knob (knob) or hole (hole) mutations, preferably, any of T366Y/Y407'T, F405A/T394' W, T366Y: F405A/T394'W: Y407' T, T366W/Y407'A and S354C: T366W/Y349' C: T366 'S: L368' A: Y407V;

c) the cysteine residue in the first CH3 domain is covalently linked to the cysteine residue in the second CH3 domain, thereby introducing an interdomain disulfide bond, preferably linking the C-termini of the two CH3 domains;

d) one or more mutations that repel charge inhibit heterodimer formation, preferably any of K409D/D399' K, K409D/D399' R, K409E/D399' K, K409E/D399' R, K409D: K392D/D399' K: E356' K or K409D: K392D: K370D/D399' K: E356' K: E357' K; and/or

e) The one or more mutations selected for heterodimer formation and/or thermostability are preferably any of:

T350V:L351Y:F405A:Y407V/T350V:T366L:K392L:T394W，

T350V:L351Y:F405A:Y407V/T350V:T366L:K392M:T394W，

L351Y:F405A:Y407V/T366L:K392M:T394W，

F405A Y407V/T366L K392M T394W or

F405A:Y407V/T366L:T394W，

Wherein numbering is according to the EU index of Kabat.

Such CH3 mutations are engineered to yield two different Fc chains and HCs, respectively (at least differing by sequence differences of the CH3 domain), which preferably pair with each other, to obtain heterodimers of Fc chains or HCs, thereby significantly reducing the tendency to produce HC homodimers, i.e., dimers of two HCs of the same sequence.

In the description of CH3 point mutations described herein, a "diagonal line" distinguishes a point mutation on one strand or domain of a respective pair from a point mutation on the other strand or other domain of the corresponding pair; an "indentation" in the amino acid position numbering represents the second strand of the heterodimer, or dimer. "colon" respectively identify the chain or domain on one of the point mutations in the combination.

Any mutation selected for heterodimer formation as described above, or other mutations according to the disclosure of Von Kreudenstein et al (Landes Bioscience, vol.5, No.5,2013, pp 646-654) may be used.

Preferably, (i) a carina; or (ii) a hole mutation, or (iii) a protuberance and hole mutation engineered on one strand or domain, and the corresponding (i) hole, or (ii) protuberance mutation, or (iii) a hole and protuberance mutation engineered on the other strand of the heterodimer.

In particular, a pair of CH3 domains comprising one or two engineered CH3 domains, the CH3 domain may comprise more than one (additional) interdomain disulfide bridge, e.g. 2 or 3, connecting the pair of two CH3 domains.

In particular, in the two CH3 domains of the respective CH3 domain pair, different mutations (according to a above) are engineered to produce homologous (matched) pairs, wherein one domain comprises a steric modification of the contact surface in the β -sheet region, preferably linked to the respective contact surface of the other domain by a complementary steric modification. Such steric modifications are mainly produced by different amino acid residues and side chains, e.g. to produce "protuberance" or "hole" structures, which complement each other to form "protuberance into hole" dimers.

According to a particular aspect, each CH3 domain in the Fc region is of the IgG type, is identified as SEQ ID NO:8 (CH 3 of human wild-type IgG 1), or is a functional variant of SEQ ID NO:8, is engineered by combining at least one beta-chain IgA fragment of at least 2 amino acids in length to obtain a chain exchange, and preferably the Fc region comprises a cognate pair of CH3 domains by pairing an IgA fragment of the first CH3 domain with an IgA fragment of the second CH3 domain. According to a specific embodiment of strand-exchanged CH3, the first CH3 domain comprising the AG chain is characterized by the amino acid sequence identified as SEQ ID NO. 9; the matching second CH3 domain comprising the GA strand is characterized by the amino acid sequence identified as SEQ ID NO 10.

Such a chain-swapped CH3 domain may specifically comprise alternating fragments of IgA and IgG amino acid sequences, e.g., comprising at least 1, 2, 3, 4, or 5 different IgA fragments, each at a different position, and separated from each other by a non-IgA fragment (e.g., an IgG fragment).

According to a particular aspect, the ABM is an effector function competent antibody comprising an Fc γ receptor binding site, and/or a C1q binding site, optionally in the Fc region.

Specifically, the antibody is characterized by any of ADCC and/or CDC activity.

However, according to a particularly preferred aspect, the ABM is an Effector Negative (EN) antibody comprising an Fc region that is deficient in binding to fey receptors and/or C1 q.

In particular, the antibodies are effector deficient (also referred to herein as effector negative), with significantly reduced or no binding by the Fc region to Fc γ receptor or CD16 a.

In particular, the effector negative antibody is characterized by the CH2 sequence of human IgG2 or an engineered variant thereof, comprising the CH2 domain of modified human IgG2 (F296A, N297Q), described in US8562986, fused to the N-terminus of the C-terminal CH3 domain (numbering according to the EU index of Kabat).

In particular, the EN antibodies have a significant reduction, or lack, ADCC and/or CDC.

Specifically, the ABM comprises positions CH2 and/or CH, if any3 domain, pH-dependent FcRn binding site. Specifically, ABMs comprise the Fc portion of an antibody, which comprises an FcRn binding site at the intersection of CH2 and CH3 domains. In particular, the FcRn binding site has affinity for binding FcRn with a KD of less than 10 in a pH dependent manner^-4M, or less than 10^-5M、10^-6M、10^-7M or 10^-8M。

In particular, binding affinity for FcRn is at least 1-log in a pH dependent manner, preferably at least 2-log or 3-log increase at pH5-6 compared to the same binding affinity at physiological pH (pH 7.4).

According to another aspect, ABM is engineered to alter pH-dependent FcRn binding. For example, the CH3 domain of at least one human IgG1 is engineered to comprise at least one mutation at the FcRn binding site to reduce pH-dependent FcRn binding, in particular at least one of the H433A or H435A mutations, or at least one of the H433A and H435A mutations, wherein the numbering is according to the EU index of Kabat. Reducing the binding of pH-dependent FcRn can result in binding FcRn with a binding affinity of less than 1-log in a pH-dependent manner, preferably about the same or lower than at pH5-6, compared to the same binding affinity at physiological pH (pH 7.4).

Drawings

FIG. 1:mass Spectrometry of H561-4 Asn325 Cys: treatment with the cysteine modifier resulted in a maximum peak shift of about 829.5Da, corresponding to a modified cysteine residue. Mass Spectrometry of H561-4 Leu328 Cys: treatment with the cysteine modifier resulted in a maximum peak shift of approximately 1659Da, corresponding to two modified cysteine residues.

FIG. 2:HIC analysis of the double cysteine substitution mutant CX _ N325CL 328C-mal-val-cit-MMAE.

FIG. 3: mass spectrometry analysis of mono-and di-cysteine substitution mutants.

FIG. 4: HIC analysis of B10v5x225M SEED-mal-val-cit-MMAE conjugates.

FIG. 5: CX _ Alexafluor488 and the single cysteine substituted mutants CX _ N325C (Asn325Cys) and CX _ L328C (Leu328Cys) mutants, coupled with maleimide-Alexafluor 488, internalize into EGFR strongly positive A431 and MB-MDA468 cells.

FIG. 6: FcRn binding to CX and CX _ Asn325CysLeu328Cys _ CysP6 at pH 5.8, then pH adjusted to 7.4 for dissociation.

FIG. 7: sequence of

1, SEQ ID NO: an amino acid sequence of human CH2 comprising a N325C substitution, wherein numbering is according to the EU index of Kabat;

2, SEQ ID NO: an amino acid sequence of human CH2 comprising a substitution of L328C, wherein numbering is according to the EU index of Kabat;

3, SEQ ID NO: an amino acid sequence of human CH2 comprising the substitutions N325C and L328C, wherein numbering is according to the EU index of Kabat;

4, SEQ ID NO: an amino acid sequence comprising a human Fc substituted with N325C, wherein numbering is according to the EU index of Kabat;

5, SEQ ID NO: an amino acid sequence comprising a human Fc substituted with L328C, wherein numbering is according to the EU index of Kabat;

6 of SEQ ID NO: an amino acid sequence of human CH2 comprising the substitutions N325C and L328C, wherein numbering is according to the EU index of Kabat;

7, SEQ ID NO: amino acid sequence of human IgG1 hinge region

8, SEQ ID NO: amino acid sequence of CH3 of human IgG1

9 of SEQ ID NO: the amino acid sequence of human IgG1, engineered to include an AG chain according to SEED technology;

10, SEQ ID NO: the amino acid sequence of human IgG1 engineered to include a GA chain according to SEED technology

FIG. 8: cetuximab-based ADCs were evaluated for cytotoxicity in vitro using Mal-Val-Cit-MMAE.

FIG. 9: HIC chromatograms of unconjugated and toxin-conjugated HER2 binding antibodies with cysteine mutations at positions N325 and L328C.

Specific embodiments refer to any ABM exemplified herein, or include any heavy and light chain, or any pair of heavy and light chains described in the examples section. Specifically, the ABMs described herein may comprise or consist of the heavy and light chains described in the examples section.

The invention further provides ABM conjugates (ABMC) comprising an ABM as described herein, and at least one heterologous molecule covalently conjugated to one or two cysteines at positions 108 and 113 of the CH2 domain, wherein numbering is according to IMGT. Specifically, ABMC is a cysteine-linked ADC.

Specifically, the ABM to heterologous molecule (drug) stoichiometric ratio ranges between 1:2 and 1: 4.

In particular, conjugation chemistry is used, which is commonly used to bioconjugate drugs to macromolecules by reaction with free cysteine. Cysteine residues are specifically alkylated by reacting them with alpha-haloketones or Michael (Michael) acceptors (e.g. maleimide derivatives). Specifically, any or each free thiol of the ABM reacts to covalently link a heterologous molecule by a reaction known as michael addition. Specifically, a thiol may be reacted with a maleimide group to give a thiol-maleimide adduct (michael adduct).

Suitable methods for conjugating an antibody to one or more drug moieties by reaction with non-crosslinked highly reactive cysteine amino acids are well known in the art, for example as described in US7521541B 2.

Specifically, the free cysteine of an ABM is unpaired with another cysteine of the same ABM molecule, and thus is not cross-linked within the ABM, or has no part of the intra-molecular disulfide bond of the ABM.

Specifically, the free cysteine of ABM binds to other thiol-bearing molecules (other than the same ABM molecule), such as unbound cysteine or glutathione, which may be present after recombinant expression of ABM in cell culture. Such thiol binding is understood to be "thiol-terminated" which will prevent reaction with the thiol-reactive reagent and is preferably removed by reduction of the antibody with a reducing agent such as TCEP (tris- (2-carboxyethyl) -phosphine).

As used herein, the term "reducing agent" refers to a chemical that provides electrons to another chemical. Exemplary reducing agents include Dithiothreitol (DTT), 2-mercaptoethanol (2-ME), and tris (2-carboxyethyl) phosphine (TCEP), as well as their related salts (e.g., TCEP-hydrochloride).

Treatment with a reducing agent will generally reduce the interchain disulfide bonds of the antibody. Therefore, it is preferable that the reduction step is followed by a re-oxidation step using an oxidizing agent (e.g., dehydroascorbic acid). Between the reduction and oxidation steps, a purification step may be included. Reoxidized antibodies typically contain free cysteines of the highly reactive cysteine amino acids.

As herein describedThe term "oxidizing agent" is used to refer to a compound that causes the conversion of a pair of free thiols to disulfide bonds. Examples of oxidizing agents include, for example, 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB), dehydroascorbic acid (DHAA), and copper sulfate (CuSO)₄). The "reoxidation step" is a defined step which serves to convert a pair of free thiols to disulfide bonds. The step of determining includes introducing an exogenous oxidizing agent and/or intentionally holding for a period of time to allow for autoxidation.

As an alternative to thiol-reactive maleimides, disulfide bonds can be obtained by oxidizing the thiol group of cysteine with a thiol-bearing linker.

Alpha, beta-unsaturated carbonyl compounds, as well as 1, 4-addition reactions of alpha, beta-unsaturated nitriles with resonance-stable carbon nucleophiles (e.g., enolate ions and enamines), such as enolate ions and enamines, are known Michael additions. The α, β -unsaturated compounds that undergo michael addition are referred to as michael acceptors, nucleophilic michael donors, and product michael adducts.

Thus, the present invention provides site-specific conjugation of heterologous molecules through site-directed mutagenesis of the ABM, through site-specific chemical conjugation, or through engineered sites in the ABM.

According to a particular embodiment, the heterologous molecule is a substance suitable for use in the diagnosis, cure, mitigation, treatment or prevention of a disease, preferably selected from the group consisting of pharmaceutical substances, toxins, radionuclides, immunomodulators, cytokines, lymphokines, chemokines, growth factors, tumor necrosis factors, hormones, hormone antagonists, enzymes (e.g., L-asparaginase), oligonucleotides, DNA, RNA, siRNA, RNAi, microRNA, peptide nucleic acids, photoactive therapeutic agents, anti-angiogenic agents, pro-apoptotic agents, peptides, lipids, carbohydrates, fluorescent tags, visualization peptides, biotin, serum half-life modulators, capture tags, chelating agents and solid supports.

In particular, the heterologous molecule is any of a dye, a radioisotope, or a cytotoxin. Particular examples include fluorescent proteins, dyes, or tethered conjugation with functional molecules (e.g., PEG, porphyrins, peptides, peptide nucleic acids, and drugs).

Specific examples are those heterologous molecules, which are artificial or biochemical compounds or molecules that interfere with the physiological function of a cell, such as a cancer or cancer cell. Drugs that may be linked to the ABM may include cytostatic or cytotoxic agents. For example, cytostatics, including alkylating agents, antimetabolites, antibiotics, mitotic inhibitors, hormones, or hormone antagonists, can be used to covalently couple to the ABM. Alkylating agents may, for example, include busulfan (malilan), carboplatin (parapidin), chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine (DTIC-Dome), estramustine phosphate, ifosfamide, methoxyethylamine (nitrogen mustard), melphalan (sinapine), procarbazine, thiotepa, uracil mustard, antimetabolites, and may, for example, include cladribine, cytarabine (cytarabine arabine), floxuridine (FUDR, 5-fluorodeoxyuridine), fludarabine, 5-fluorouracil (5FU), gemcitabine, hydroxyurea, 6-mercaptopurine (6MP), methotrexate (methotrexate), 6-mercaptoguanine, pentostatin, Pibobrooman, tegafur, troxate, glucuronic acid, antibiotics, can be, for example, comprising aclacinomycin, bleomycin, actinomycin (actinomycin D), daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, mitomycin C, mitoxantrone, plicamycin (mithramycin), or mitotic inhibitors, can for example comprise etoposide (VP-16, Van Pesid), teniposide (VM-26, Vernamon (Vumon)), vinblastine, vincristine, vindesine, hormones, or hormone antagonists, which can for example be used, comprising buserelin, conjugated equine estrogens (proimalin), cortisone, clothene (Tace), dexamethasone (Decadron), Diethylstilbestrol (DES), ethinyl estradiol (ethinyl estradiol), fluorohydroxyketon (methyltestosterone), flutamide, goserelin acetate (gorerel), hydroxyprogesterone caproate (caproic acid), hydroxyprogesterone (caproic acid), and, Leuprorelin, medroxyprogesterone acetate (provitamin), medroxyprogesterone acetate (megestrol), prednisone, tamoxifen (novladex), testolactone (dehydrotestosterone), testosterone. Cytostatic or antineoplastic compounds, such as those disclosed above, are known in The art and can be found, for example, in D.S. Fischer & T.M. Knobf (1989), The cancer chemother handbook (3 rd edition), Chicago, Yeast Book Medical and Association of Community cancer centers (Spring,1992), Compendia-based drug bulletin, Rockville, Md.

In particular, the heterologous molecule is conjugated to one or two cysteines at positions 108 and 113 of the CH2 domain via a conjugation linker, wherein numbering is according to IMGT. Such a conjugate linker is also understood as a spacer, which is coupled to a heterologous molecule. Typically, the linker is covalently attached to the heterologous molecule prior to reaction with the ABM.

Specifically, the conjugate linker comprises a maleimide group.

In particular, the conjugate linker is a cleavable or non-cleavable linker. In particular, the linker is a synthetic or artificial amino acid sequence that links or links the ABM to a heterologous molecule or drug.

In particular, cleavable linkers are used that cleave in response to a physiological stimulus (e.g., low pH, high glutathione concentration, and/or proteolytic cleavage). Specific cleavable linkers are cleaved by proteases, acids, or by reduction of disulfide bonds (e.g., glutathione-mediated or glutathione-sensitive). For example, the cleavable linker may comprise a valine-citrulline linker, a hydrazone linker, or a disulfide linker.

In particular, a non-cleavable linker is used in combination with an internalizing ABM. In this case, ABMC rely on degradation in the lysosome after internalization. Specific non-cleavable linkers include maleimidocaproyl linkers linked to MMAF (mc-MMAF), N-maleimidomethylcyclohexane-1-carboxylate (MCC), or thiol-acetamidoacryloyl linkers.

The invention further provides expression systems comprising one or more nucleic acid molecules, particularly isolated nucleic acid molecules, encoding the ABMs described herein. Depending on the number of different strands, each consisting of an amino acid sequence, one or more encoding nucleic acid molecules may be used in an expression system comprising one or more expression cassettes comprised in one or more expression vectors.

In particular, the expression cassette is incorporated into a plasmid that comprises or incorporates a nucleic acid as described herein, which optionally comprises other sequences, to express the ABM encoded by the nucleic acid sequence, e.g., regulatory sequences.

The invention further provides host cells comprising the expression systems described herein. In particular, the host cell is a production host cell comprising at least one expression cassette or plasmid incorporating one or more nucleic acid molecules encoding the ABMs described herein.

In particular, the host cell transiently or stably expresses ABM. According to a specific embodiment, the host cell is a eukaryotic host cell, preferably any yeast or mammalian cell.

The invention further provides methods of producing the ABMs described herein, wherein the host cells described herein are cultured or maintained under conditions that produce the ABMs.

Specifically, ABMs may be isolated and/or purified from cell culture supernatants. According to a specific embodiment, the ABM is a bispecific full length antibody which is a heterodimer comprising two different HCs and two different LCs, and the ABM comprises a correctly paired homologous HC/LC pair and homologous CL and CH1 domains, respectively, and the ABM is produced by a host cell, wherein less than 10% of the produced antibodies are mis-paired, preferably less than 5%, as measured by mass spectrometry (LC-ESI-MS) comparing the maximum peak intensities.

In particular, the ABMs or ABMCs described herein are provided for medical, diagnostic or analytical use.

In particular, the ABMs or ABMCs described herein are provided for the treatment of cancer, autoimmune diseases or allergy, targeting at least one antigen associated with said disease. Thus, the invention further relates to a method of treating a subject having cancer, an autoimmune disease, or an allergy by administering an effective amount of an ABM or ABMC as described herein, wherein the ABM or ABMC targets at least one antigen associated with the disease.

In particular, the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, metastatic colorectal cancer (mCRC), unresectable liver metastases, head and neck squamous cell carcinoma, non-small cell lung cancer (NSCLC) and Head and Neck Squamous Cell Carcinoma (HNSCC).

The invention further provides a pharmaceutical formulation comprising an ABM or ABMC as described herein, preferably in the form of a parenteral or mucosal formulation, optionally comprising a pharmaceutically acceptable carrier or excipient.

Specifically, the ABM or ABMC described herein are provided in a pharmaceutical formulation comprising a pharmaceutically acceptable carrier or excipient in a parenteral formulation.

The present invention further provides a method of producing the ABMC described herein, comprising the steps of:

a) providing an ABM as described herein; and

b) at least one thiol group of one or both cysteines at positions 108 and 113 of the CH2 domain is reacted with the heterologous molecule by a site-specific conjugation method, in particular a chemical conjugation method.

In particular, the at least one thiol group is reacted with the heterologous molecule by a michael reaction using a conjugated linker comprising a maleimide group.

In particular, the production process does not include measures and/or reaction steps to cleave the intra-molecular sulfur or disulfide bonds that would otherwise generate free thiol groups, e.g., under reducing conditions. Thus, it is preferred to prepare ABM and/or ABMC under non-reducing conditions.

However, according to a specific aspect, the ABM is pretreated with a reducing agent to carry out reduction and reoxidized with an oxidizing agent to prepare the free reactive cysteine of the ABM in preparation for conjugation, which may improve conjugation efficiency.

Unless otherwise noted, locations herein are according to IMGT system numbering (Lefranc et al, 1999, Nucleic Acids Res.27: 209-212). However, in the examples section, the numbering according to the EU index of Kabat was used. An explanation of the Kabat numbering scheme can be found in Kabat, EA, et al, Sequences of proteins of immunological interest (NIH publication No. 91-3242, 5 th edition (1991)). Table 23 shows the correspondence of the name and number of the mutein, referring to the EU index and IMGT numbering according to Kabat at the positions indicated herein.