RU2776302C2 - Polypeptide linker for production of multi-specific antibodies - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention allows for the production of a mutant polypeptide linker developed from a hinge IgG1 sequence with remote glycosylation sites. The specified linker can be used in the production of different multi-specific antibodies.

EFFECT: compared to known analogues, the use of a linker according to the present invention allows for the production of more uniform preparations of multi-specific antibodies.

20 cl, 5 dwg, 1 ex

Description

The present invention relates to multispecific antibodies with improved properties, useful in the field of medicine.

Prior Art

Therapeutic fusion proteins have become an important tool in drug development. Peptide linkers are often used to construct multidomain proteins from various functional protein modules. The resulting multi-domain proteins are designed to bind to a target recognized by individual modules (or simultaneously to perform the biological function of individual modules) to either enhance the biological effects associated with individual single domains or create new biological activities that are unattainable through individual single domains. There are many examples of molecules that use peptide linkers: single-chain antibody variable domains (scFv), immunocytokines (hybrids of cytokines and antibodies), bispecific antibodies (BsAbs), etc. The choice of linker(s) for a particular fusion protein is dictated by considerations such as: 1) whether the linker(s) require flexibility to allow folding of different domains into a particular tertiary structure (e.g., scFv-based antibodies), 2) whether the linker(s) require ) stiffness to provide the necessary separation between protein domains, or 3) whether the linker(s) need to be cleavable to allow separation of the domains in vivo to obtain the desired activity (Xiaoying Chen, et al., Adv Drug Deliv Rev. 2013. 65(10 ): 1357-1369). The choice of linkers can be critical, as unsuitable linkers can reduce or eliminate the desired activity of the fusion protein (Yumi Maeda, et al., Anal. Biochem. 1997. 249(2): 147-152).

Various linker sequences have been identified for use in constructing fusion proteins (Richard George and Jaap Heringa, Protein Engineering. 2003. 15(11): 871-879; Xiaoying Chen et al, Adv Drug Deliv Rev. 2013. 65(10): 1357 -1369). There are also various databases available that compile linker sequences used to construct fusion proteins: 1) SynLinker compiled by the National University of Singapore (http://synlinker.syncti.org) and 2) International Genetically Engineered Machine Competition ( http://parts.igem.org/Protein_domains/Linker); Center for Integrative Bioinformatics at Vrie University Amsterdam (http://www.ibi.vu.nl/programs/linkerdbwww).

International patent application WO2013/005194 discloses multispecific antibodies constructed with linkers designed from an IgG1 hinge sequence followed by the N-terminus of an IgG1 CH2 domain sequence followed by an 8 amino acid semi-rigid linker sequence from the IgA1 hinge core. This semi-rigid linker, which forms the central part of the IgA1 hinge, is important to ensure sufficient separation of both antibody Fab domains to avoid steric interference from the C-terminus of the outer Fab1, which is exposed to the antigen-binding paratope of the inner Fc-proximal Fab2.

However, the inventors have determined that the presence of a large number of glycoforms makes it problematic to obtain uniform preparations of such multispecific antibodies necessary for the development of therapeutic agents. In addition, the characterization of such preparations will also be quite complex, making it difficult to compare different production batches.

The essence of the invention

For this reason, the inventors have come to the conclusion that an improvement can be made by changing the design of the linker, in particular by removing glycosylation sites from the linker.

Thus, the invention provides a linker polypeptide which comprises or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1),

where X_one, X₂, X_3, X_four, X_5,X₆, X₇, X_eight,X₉, X_ten, the same or different, are any amino acid as defined herein; provided that the polypeptide does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6).

Such a polypeptide is useful as a linker in fusion proteins, in particular multispecific, in particular bispecific antibodies.

Thus, the subject of the invention is a multispecific antigen-binding fragment containing at least two Fab fragments with different CH1 and CL domains, each Fab fragment recognizing a different epitope of interest, and these Fab fragments are located in tandem in any order, so that the C-terminus of the CH1 domain of the first Fab fragment is linked to the N-terminus of the VH domain of the next Fab fragment via a polypeptide linker,

characterized in that the sequence of the polypeptide linker includes or consists of the amino acid sequence EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 1), where X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, the same or different, are any amino acid; provided that the linker sequence does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6).

In addition, a multispecific antibody is provided having two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined herein.

In a preferred embodiment, a multispecific antibody is provided that has an immunoglobulin-like structure, comprising:

- two identical antigen-binding arms, each of which consists of a multispecific antigen-binding fragment, as defined herein;

- dimerized CH2 and CH3 domains of immunoglobulin;

- an IgA, IgG or IgD hinge region linking the C-terminus of the CH1 domains of the antigen-binding arms to the N-terminus of the CH2 domains.

Thus, the present invention more specifically relates to a multispecific, preferably bispecific, antibody containing two heavy chains and four light chains,

where each heavy chain includes

a. an immunoglobulin Fc region containing Hinge-CH2-CH3 domains,

b. where the Fc region is linked to the CH1-VH heavy chain Fab of antibody 1 (Ab1) with the indicated hinge domain,

With. which in turn is linked to the heavy chain CH1-VH of the Fab of an antibody 2 (Ab2) via a polypeptide linker sequence, wherein the polypeptide linker sequence links the N-terminus of said Ab1 Fab heavy chain VH domain to the C-terminus of said Ab2 Fab heavy chain VH domain,

and the four light chains include Ab1 light chains and Ab2 light chains associated with their heavy chain cognate domains;

which is characterized in that the polypeptide linker sequence comprises or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1),

where X_one, X₂, X_3, X_four, X_5,X₆, X₇, X_eight,X₉, X_ten, the same or different, are any amino acids; provided that the linker sequence does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6).

In a particular embodiment, the polypeptide linker sequence comprises or consists of a sequence selected from the group consisting of

EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 2);

EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 3); and

EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 4).

Another object of the invention is a polypeptide that includes, preferably consists of a heavy chain multispecific antigennegative fragment, or multispecific, preferably bispecific antibodies, as defined herein.

The invention further relates to a polynucleotide containing a sequence encoding such a polypeptide.

A host cell transfected with an expression vector containing said polynucleotide is also part of the invention.

A further aspect of the invention is a method for producing a multispecific antibody, preferably a bispecific antibody, as described herein, said method comprising the steps of: a) culturing in a suitable medium and culture conditions a host cell expressing an antibody heavy chain as defined herein, and expressing the light chain of an antibody, as defined herein; and b) recovering said produced antibodies from the culture medium or from said cultured cells.

Drawing captions

Figure 1 is a schematic representation of a full length BiXAb bispecific antibody of the invention.

Figure 2 is a schematic representation of a bispecific construct that includes only two Fab domains connected by a linker without an Fc domain (Fab-Fab).

Figure 3A shows the analysis of BiXAb2b by size exclusion chromatography.

Figure 3B shows the analysis of BiXAb3b size exclusion chromatography.

Figure 3C shows the analysis of Fab-Fab3b by size exclusion chromatography.

Figure 4A shows the MS spectrum of the LC-MS analysis of Fab-Fab3a.

Figure 4B shows the MS spectrum of the LC-MS analysis of Fab-Fab3b.

Figure 5A shows the MS spectrum of the LC-MS analysis of BiXAb3a.

Figure 5B shows the MS spectrum of the LC-MS analysis of BiXAb3b.

Detailed description of the invention

Definitions

The basic structure of a naturally occurring antibody molecule is a Y-shaped tetrameric quaternary structure consisting of two identical heavy chains and two identical light chains held together by non-covalent interactions and interchain disulfide bonds.

In mammalian species, there are five types of heavy chains: α, δ, ε, γ and μ, which define the class (isotype) of immunoglobulin: IgA, IgD, IgE, IgG and IgM, respectively. The N-terminal heavy chain variable domain (VH) is followed by a constant region containing three domains (numbered CHI, CH2, and CH3 from N-terminus to C-terminus) in the γ, α, and δ heavy chains, while the heavy chain constant regions the μ and ε chains consist of four domains (numbered CHI, CH2, CH3, and CH4 from the N-terminus to the C-terminus). The CH1 and CH2 domains of IgA, IgG and IgD are separated by a flexible hinge, the length of which varies between different classes, and in the case of IgA and IgG, between different subtypes: lgGl, lgG2, IgG3 and IgG4 have, respectively, hinges of 15, 12, 62 (or 77) and 12 amino acids, and IgAl and IgA2 have hinges of 20 and 7 amino acids, respectively.

There are two types of light chains, λ and κ, which can be associated with any heavy chain isotype, but both light chains are of the same type in a given antibody molecule. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a single domain constant region called CL.

The heavy and light chains are paired by a protein/protein interaction between the CH1 and CL domains and between the VH and VL domains, and the two heavy chains are linked by a protein/protein interaction between their CH3 domains.

Antigen-binding regions correspond to the arms of a Y-shaped structure, each of which consists of a complete light chain paired with heavy chain VH and CH1 domains and are called Fab fragments (Fragment antigen binding - an antigen binding fragment). Fab fragments were first generated from native immunoglobulin molecules by cleavage with papain, which cleaves the antibody molecule at the hinge region on the amino-terminal side of the interchain disulfide bonds, thus freeing two identical antigen-binding arms. Other proteases, such as pepsin, also cleave the antibody molecule at the hinge region, but at the carboxy-terminal side of the interchain disulfide bonds, releasing fragments consisting of two identical Fab fragments and remaining linked via disulfide bonds; reduction of disulfide bonds in F(ab')2 fragments results in the formation of Fab' fragments.

The part of the antigen-binding region corresponding to the VH and VL domains is called the Fv fragment (Fragment variable - variable fragment); it contains CDRs (complementarity determining regions) that form an antigen-binding site (also called a paratope).

The effector region of an antibody, which is responsible for its binding to effector molecules on immune cells, corresponds to the Y-shaped stem and contains the paired heavy chain CH2 and CH3 domains (or CH2, CH3 and CH4 domains, depending on the antibody class), and is called Fc -area (Fragment crystallisable - crystallizable fragment).

Due to the identity of the two heavy chains and two light chains, natural antibody molecules have two identical antigen-binding sites and thus bind simultaneously to two identical epitopes.

In the context of the present invention, a "multispecific antigen binding fragment" is defined herein as a molecule having two or more antigen binding regions, each of which recognizes a different epitope. Different epitopes may be carried by the same antigenic molecule or by different antigenic molecules. The term "recognizing" or "recognizing" means that the fragment specifically binds the target antigen.

An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more easily and/or for a longer duration than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, a binding reference means a preferential binding.

The terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal that is being evaluated for treatment and/or subjected to treatment. The subjects may be humans, but may also be other mammals, in particular those mammals that can be used as laboratory models of human disease, such as mice, rats, rabbits, dogs, etc.

The term "treatment" or "treat" refers to an act, application, or therapy in which an object, including a human being, is subjected to medical care for the purpose of improving the condition of the object, directly or indirectly. In particular, this term refers to the reduction of morbidity or alleviation of symptoms, the elimination of relapses, the prevention of relapses, the prevention of morbidity, the improvement of symptoms, the improvement of prognosis, or a combination of both in some embodiments. One skilled in the art will appreciate that treatment does not necessarily result in complete absence or elimination of symptoms. For example, in relation to cancer, "treatment" or "treat" may refer to slowing down the growth, proliferation or metastasis of tumor or malignant cells, preventing or delaying the development of growth, proliferation or metastasis of tumor or malignant cells, or some combinations thereof.

Construction of multispecific antibodies

Provided herein are constructs of multispecific antigen binding fragment(s) and multispecific antibodies comprising said fragments, wherein each multispecific antigen binding fragment consists essentially of tandem Fab fragments separated by a linker of the invention.

Such fragments and constructs preferably contain chains from human immunoglobulins, preferably from IgG, and even more preferably from IgG1.

In the case of a multispecific antigen-binding fragment containing more than two different Fab fragments, the polypeptide linkers separating the Fab fragments may be the same or different.

In accordance with a preferred embodiment of the multispecific antibody of the invention, it has two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined above. The antigen binding arms can be linked to each other in various ways, depending on the intended use of the antibody.

If it is desired to produce an antibody without Fc-mediated effects, the antibody will not contain Fc regions. In this case, the two antigen-binding branches can be linked together, for example:

- homodimerization of antigen-binding arms through interchain disulfide bonds provided by the polypeptide(s) linker(s) separating(s) Fab fragments; and/or

- by adding at the C-terminus of each antigen-binding arm a polypeptide extension containing cysteine residues, allowing the formation of interchain disulfide bonds, and homodimerizing said polypeptide extension, resulting in a hinge structure; as non-limiting examples, said polypeptide extension may be, for example, an IgG1, IgG2, or IgG3 hinge sequence;

- through a semi-rigid linker connecting the C-terminals of the heavy chains of two antigen-binding arms to form a single polypeptide chain and maintaining these antigen-binding arms at a sufficient distance from each other.

Alternatively, if effector functions such as CDC, ADCC or ADP are desired, the multispecific antibody of the invention may further comprise an Fc domain providing these effector functions. The choice of Fc domain will depend on the type of effector functions desired.

In this case, the multispecific antibody of the invention has an immunoglobulin-like structure comprising:

two identical multispecific antigen-binding arms as defined above;

- dimerized CH2 and CH3 domains of immunoglobulin;

- either an IgA, IgG or IgD hinge region linking the C-terminus of the CH1 domains of the antigen-binding arms to the N-terminus of the CH2 domains, or alternatively when the CH4 domains following the CH3 domains are derived from IgM or IgE, the C-terminus of the domains The CH1 antigen-binding arms in this case can be linked directly to the N-termini of the CH2 domains.

Preferably, the CH2 and CH3 domains, the hinge region and/or the CH4 domains are derived from the same immunoglobulin or immunoglobulins of the same isotype and subclass as the CH1 domains of the antigen binding arm.

CH2, CH3 and optionally CH4 domains and hinge regions from native immunoglobulins can be used. If desired, they can also be mutated, for example, to modulate the effector function of the antibody. In some cases, all or part of the CH2 or CH3 domain may be discarded.

More specifically, the invention relates to bispecific tetravalent antibodies containing two binding sites for each of their targets and a functional Fc domain allowing activation of effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis.

Such preferred antibodies of the invention are full length antibodies. They preferably contain heavy chains and light chains from human immunoglobulins, preferably IgG, even more preferably IgG1.

The light chains may be lambda or kappa light chains; preferably they are kappa light chains.

In a preferred embodiment, the linker of the invention binds IgG Fab domains in the format of a tetra-Fab bispecific antibody, the amino acid sequence of which contains the heavy chain sequences of at least two Fab linked by said polypeptide linker, followed by a native hinge sequence, followed by the sequence Fc from IgG co-expressed with the corresponding IgG light chain sequences.

An example of the antibodies of the invention, named BiXAb antibodies, which have an IgG-like structure, is shown in Figure 1. The antibodies designated BiXAb2a, BiXAb2b, BiXAb2c and BiXAb3b described below are specific examples.

The bispecific antibodies of the invention typically include

- continuous heavy chain constructed from Fc (hinge-CH2-CH3)

- this is followed by the heavy chain Fab (CH1-VH) of antibody 1 and the subsequent heavy chain of the Fab (CH1-VH) of antibody 2, the latter is attached by a polypeptide linker sequence according to the invention,

- and during protein expression, the resulting heavy chain assembles into dimers, while the co-expressed light chains of antibody 1 and antibody 2 (VL-CL) bind to cognate heavy chains to form the final tandem F(ab)'2-Fc molecule,

while antibody 1 (Ab1) and antibody 2 (Ab2) are different.

In a preferred embodiment, bispecific antibodies are described which include

• two Fab fragments with different CH1 and CL domains, consisting of

a) a Fab fragment having CH1 and C-Kappa domains derived from human IgG1/Kappa and VH and VL domains from Ab1,

b) a Fab fragment having CH1 and C-kappa domains derived from human IgG1/Kappa and VH and VL domains from Ab2,

c) a mutated light chain CL constant domain derived from a human kappa constant domain,

d) mutated heavy chain CH1 constant domain

while the Fab fragments are arranged in tandem in the following order

- The C-terminus of the CH1 domain of the Fab fragment from Ab1 is linked to the N-terminus of the VH domain of the Fab fragment from Ab2 via a polypeptide linker,

- hinge region of human IgG1, connecting the C-terminus of the CH1 domain of the Ab2 fragment with the N-terminus of the CH2 domain,

- dimerized CH2 and CH3 domains of human IgG1.

Ab1 and Ab2 may be any antibody of interest, especially any antibody of therapeutic interest.

In a specific embodiment, Ab1 and Ab2, while different, are independently selected from the group consisting of an anti-EGFR antibody and an anti-HER2/neu antibody. In a preferred embodiment, Ab1 and Ab2, being different, are independently selected from the group consisting of cetuximab or a mutated derivative thereof on the one hand and trastuzumab or a mutated derivative thereof on the other hand.

In another specific embodiment, Ab1 and Ab2 different from each other are independently selected from the group consisting of an anti-CD38 antibody and an anti-PD-L1 antibody.

Such antibodies are useful as a drug, in particular in the treatment of oncological diseases.

Throughout this description, amino acid sequences are defined according to Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

Another example of the constructs of the invention, which is a Fab-Fab multispecific antigen-binding fragment that does not contain an Fc domain, is illustrated in Figure 2. A specific example, designated Fab-Fab3a, is described below.

Such Fab-Fab constructs typically contain two different Fab domains. Such antibodies have only one Fab domain, each of which binds to antigen 1 and antigen 2. They have the same light chains as in the corresponding BiXAbs; however, the Fab-Fab heavy chain is shortened such that their last C-terminal residue is cysteine-220 (in EU numbering).

Linker design

The polypeptide sequence of the linker according to the invention comprises or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1),

where X_one, X₂, X_3, X_four, X_5,X₆, X₇, X_eight,X₉, X_ten, the same or different, are any amino acid; provided that the linker sequence does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6).

The polypeptide linker sequence is less than 80 amino acids, preferably less than 60 amino acids, even more preferably less than 40 amino acids.

In a specific embodiment, X ₁ , X ₂ and X ₃ , identical or different, are threonine (T) or serine (S).

In another specific embodiment, X ₁ , X ₂ and X ₃ , the same or different, are any amino acids other than threonine (T) or serine (S), preferably where X ₁ , X ₂ and X ₃ , the same or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I), still preferably X ₁ , X ₂ and X ₃ , identical or different, may be Ala (A) or Gly (G).

Alternatively, X ₁ , X ₂ and X ₃ , the same or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F ), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E) or Gln (Q).

In a specific embodiment, X ₄ and X _5, identical or different, are any amino acid selected from the group consisting of serine (S), cysteine (C), alanine (A) and glycine (G).

In a preferred embodiment, X ₄ is serine (S) or cysteine (C).

In a preferred aspect, X ₅ is alanine (A) or cysteine (C).

In a specific embodiment, X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, are any amino acid other than threonine (T) or serine (S). Preferably X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I).

Alternatively, X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , the same or different, may be Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg ( R), Phe (F), Tyr (T), His (H) Trp (W), preferably Leu (L), Glu (E) or Gln (Q).

In a preferred embodiment, X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, are selected from the group consisting of Ala (A) and Gly (G).

In another preferred embodiment, X ₆ and X ₇ are the same and are preferably selected from the group consisting of Ala (A) and Gly (G).

In a preferred embodiment, the polypeptide linker sequence includes or consists of the sequence of SEQ ID NO: 1, where

X ₁ , X ₂ and X ₃ , identical or different, are threonine (T), serine (S);

X ₄ is serine (S) or cysteine (C);

X ₅ is alanine (A) or cysteine (C);

X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, are selected from the group consisting of Ala (A) and Gly (G).

In a particular embodiment, the polypeptide linker sequence comprises or consists of a sequence selected from the group consisting of

EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 2);

EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 3); and

EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 4).

In another preferred embodiment, the polypeptide linker sequence comprises or consists of the sequence of SEQ ID NO: 1, where

X ₁ , X ₂ and X ₃ , the same or different, are Ala (A) or Gly (G);

X ₄ is serine (S) or cysteine (C);

X ₅ is alanine (A) or cysteine (C);

X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , identical or different, are selected from the group consisting of Ala (A) and Gly (G).

Obtaining antibodies

Nucleic acids encoding the heavy and light chains of the antibodies of the invention were inserted into expression vectors. The light and heavy chains may be cloned into the same or different expression vectors. The DNA segments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector(s) that allow expression of the immunoglobulin polypeptides. Such control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence. Expression vectors typically replicate in host organisms either as episomes or as part of the host's chromosomal DNA. Typically, expression vectors contain selection markers, such as tetracycline or neomycin, to allow detection of those cells that have been transformed with the desired DNA sequences.

In one example, both heavy and light chain coding sequences (eg, sequences encoding VH and VL, VH-CH1 or VL-CL) are included in a single expression vector. In another example, each of the heavy and light chains of an antibody is cloned into a separate vector. In the latter case, expression vectors encoding heavy and light chains can be co-transfected into a single host cell to express both chains, which can be assembled to form intact antibodies either in vivo or in vitro.

In a specific embodiment, the host cell is co-transfected with three independent expression vectors, such as plasmids, resulting in the co-production of all three chains (namely the heavy chain HC and the two light chains LC1 and LC2, respectively) and the secretion of a multispecific antibody.

More specifically, three vectors can be advantageously used in the following molecular ratio of 3:2:2 (HC:LC1:LC2).

The recombinant vectors for expressing the antibodies described herein typically contain nucleic acid encoding antibody amino acid sequences operably linked to a promoter, either constitutive or inducible. Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or both. Exemplary vectors contain transcription and translation terminators, initiation sequences, and promoters useful in regulating the expression of an antibody-encoding nucleic acid. The vectors optionally contain common expression cassettes containing at least one independent terminator sequence, sequences allowing the cassette to be replicated in both eukaryotes and prokaryotes, i.e. shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

Multispecific antibodies as described herein can be produced in prokaryotic or eukaryotic expression systems such as bacteria, yeast, filamentous fungi, insect and mammalian cells. It is not necessary that the recombinant antibodies of the invention be glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are human embryonic kidney line (293 cells), neonatal hamster kidney cells (BHK cells), Chinese hamster ovary cells/- or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in cells). CHO), African green monkey kidney cells (VERO cells), and human liver cells (Hep G2 cells).

Mammalian tissue cell culture is preferred for polypeptide expression and production because several suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art and include CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines. or transformed B cells or hybridomas.

In a most preferred embodiment, the multispecific, preferably bispecific, antibodies of the invention are generated using a CHO cell line, most preferably a CHO-S cell line.

Expression vectors for these cells may include expression control sequences such as an origin of replication, a promoter and an enhancer, and necessary information processing sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, and the like.

Vectors comprising polynucleotide sequences of interest (eg, heavy and light chain coding sequences and expression control sequences) can be transferred into the host cell by well known methods, which vary depending on the type of cellular host. For example, calcium phosphate treatment or electroporation can be used for other cell hosts (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). When heavy and light chains are cloned into separate expression vectors, the vectors are co-transfected to obtain the expression and assembly of intact immunoglobulins.

Host cells are transformed or transfected with vectors (eg, by chemical transfection or electroporation methods) and cultured in conventional nutrient media (or modified as appropriate) to induce promoters, select transformants, or amplify genes encoding the desired sequences.

Once expressed, whole antibodies, their dimers, individual light and heavy chains, or other forms of the immunoglobulins of the present invention can be further isolated or purified to provide preparations that are substantially homogeneous for further assays and applications. Standard protein purification methods known in the art may be used. For example, suitable purification procedures may include immunoaffinity or ion exchange column fractionation, ethanol precipitation, high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, and gel filtration (see generally Scopes , Protein Purification (Springer-Verlag, N.Y., 1982) Substantially pure immunoglobulins with homogeneity of at least about 90 to 95% are preferred, and homogeneity of 98 to 99% or more is most preferred for pharmaceutical use.

In vitro production allows scale up to produce large quantities of the desired multispecific, preferably bispecific, antibodies of the invention. Such methods may use a homogeneous suspension culture, eg in an airlift or continuously stirred reactor, or a culture of immobilized or trapped cells, eg in hollow fibers, microcapsules, on agarose microbeads, or ceramic cartridges.

Therapeutic Applications

Another aspect of the invention is a pharmaceutical composition containing an antibody of the invention. Another aspect of the invention is the use of an antibody of the invention for the manufacture of a pharmaceutical composition. Another aspect of the invention is a method for manufacturing a pharmaceutical composition comprising an antibody of the invention.

In another aspect, the present invention relates to a composition, for example, a pharmaceutical composition containing an antibody as defined herein, which is included in the composition together with a pharmaceutical carrier.

The composition of the present invention can be administered in a variety of ways known in the art.

The present invention, thus generally described above, will be better understood with reference to the following examples, which are presented by way of illustration and are not intended to limit the present invention.

EXAMPLES

Design

The first bispecific antibody of the invention is an antibody designated BiXAb2a having the following structure:

i) a continuous heavy chain that includes

- heavy chain variable region (VH) of trastuzumab corresponding to SEQ ID NO: 7

- Wild type CH1 constant domain (residue at position Kabat 192 is threonine) from human IgG1 corresponding to SEQ ID NO: 8

Polypeptide linker connecting 2 Fab heavy chains, consisting of EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 2);

- Variable region of the heavy chain of cetuximab (VH), corresponding to SEQ ID NO: 9

- Mutated CH1 constant domain (residue at position Kabat 192 was mutated from threonine to glutamic acid) from human IgG1 corresponding to SEQ ID NO: 10

- Wild-type human IgG1 hinge corresponding to SEQ ID NO: 11

- Wild type CH2 domain of human IgG1 corresponding to SEQ ID NO: 12

- Wild type CH3 domain of human IgG1 corresponding to SEQ ID NO: 13

Thus, the bispecific antibody of the invention has a continuous heavy chain (701 residues) SEQ ID NO: 14

ii) wild-type trastuzumab light chain consisting of SEQ ID NO: 15

iii) constant domain mutated cetuximab light chain (residues at positions Kabat Ser 114 and Asn 137 were mutated to Ala and Lys, respectively) from human kappa corresponding to SEQ ID NO: 16.

The second bispecific antibody of the invention is the antibody designated BiXAb2b, which consists of the same sequences except for the linker, which is

EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 3).

The third bispecific antibody of the invention is the antibody designated BiXAb2c, which consists of the same sequences except for the linker, which is

EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 4).

The fourth bispecific antibody of the invention is an antibody designated BiXAb3b having the following structure:

i) a continuous heavy chain that includes

- Heavy chain variable region (VH) of atezolizumab corresponding to SEQ ID NO: 23

(EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISP YGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQ GTLVTVSS)

- Mutated constant domain CH1 (residue at position 192 Kabat was mutated from threonine to glutamic acid) from human IgG1 corresponding to SEQ ID NO: 10

The polypeptide linker connecting 2 Fab heavy chains consists of SEQ ID NO: 3;

Daratumumab heavy chain variable region (VH) corresponding to SEQ ID NO: 17

(EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGS GGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYW GQGTLVTVSS)

- Wild type CH1 constant domain (residue at position Kabat 192 is threonine) from human IgG1 corresponding to SEQ ID NO: 8

- Wild-type human IgG1 hinge corresponding to SEQ ID NO: 11

- Wild type CH2 domain of human IgG1 corresponding to SEQ ID NO: 12

- Wild type CH3 domain of human IgG1 corresponding to SEQ ID NO: 13

[Thus, the heavy chain of the invention has a continuous heavy chain of SEQ ID NO: 18

ii) constant domain mutated atezolizumab light chain (residues at positions Kabat Ser 114 and Asn 137 were mutated to Ala and Lys, respectively) from human kappa corresponding to SEQ ID NO: 19

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSYVACEVTHQ

iii) wild-type daratumumab light chain consisting of SEQ ID NO: 20

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

For comparison, an antibody designated BiXAb3a was also prepared, which differs from the BiXAb3b antibody by a linker that consists of

EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6).

Another construct of the invention is designated Fab-Fab3b; it consists of the same sequence as BiXAb3b except that the heavy chain lacks the hinge, CH2 and CH3 domains. Thus, Fab-Fab3b has a continuous heavy chain of SEQ ID NO: 21

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVEVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGGEVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

For comparison purposes, a construct, designated Fab-Fab3a, was also generated, which consists of the same sequence as BiXAb3a, except that the heavy chain lacks the loop, CH2 and CH3 domains.

SEQ ID NO: 7-16 are shown below.

- SEQ ID NO: 7

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

- SEQ ID NO: 8

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV

- SEQ ID NO: 9

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

- SEQ ID NO: 10

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVEVPSSSLGTQTYICNVNHKPSNTKVDKKV

- SEQ ID NO: 11

EPKSCDKTHTCPPCP

- SEQ ID NO: 12

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

- SEQ ID NO: 13

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- SEQ ID NO: 14

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVEVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

- SEQ ID NO: 15

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKPVNSFTHQ

- SEQ ID NO: 16

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPAVFIFPPSDEQLKSGTASVVCLLKNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQECGLSSPV

Gene synthesis

Anti-HER2 (trastuzumab, clone humAb4D5-8) and anti-EGFR (cetuximab) amino acid sequences were used to construct DNA sequences after codon optimization for mammalian expression using the GeneScript program. For the heavy chain, the DNAs encoding signal peptides, Fab1 variable region and CH1 constant domain were followed by a pseudo-hinge linker, and Fab2 variable region and CH1 constant domain with flanking sequences for restriction enzyme digestion were synthesized using GeneScript. For the light chain, GeneScript synthesized DNA encoding signal peptides and variable and constant kappa regions.

PCR reactions using PfuTurbo Hot Start were performed to amplify the inserts, which were then cleaved with NotI + ApaI and NotI + HindIII for the heavy and light chains, respectively. The double-cleaved heavy chain fragments were ligated into the NotI + ApaI treated expression vector pcDNA3.1 (Invitrogen) into which the CH1 + hinge + CH2 + CH3 domains from human IgG1 had already been inserted. The double cleaved light chain fragments were ligated into the NotI + HindIII treated expression vector pcDNA3.1 (Invitrogen). Plasmid DNA was verified by double-stranded DNA sequencing.

Expression and purification

The bispecific antibodies of the invention were generated by transient gene expression by co-transfection of 3 genes encoded by separate vectors in a molar ratio of 2:3:3=HC:LC1:LC2 (1 continuous heavy chain (HC) and 2 light chains (LC)) in CHO- S adapted to serum-free medium in suspension (CHO SFM-II medium from Life Technologies™). Typically, to test expression in an average volume of 50 ml, 50 mg of plasmid DNA (25 μg of heavy chain 1, 12.5 μg of tratuzumab light chain and 12.5 μg of cetuximab light chain) were mixed in a 1.5 ml Eppendorf tube, 1 ml of CHO SFM medium containing 25 μl of 3 mg/ml PEI transfection reagent (Polyplus) pH 7.0 was incubated at room temperature for 20 minutes. The DNA-PEI mixture was loaded into 49 ml Invitrogen FreeStyle™ CHO-S cells from Life Technologies at 1~2×10 ⁶ /ml in a 125 ml shake flask. Cells were shaken for another 6 days. The supernatant was collected by centrifuging the cells at 3000 rpm for 15 minutes. BiXAb expression titer in the supernatant was determined using FortéBio protein A biosensors (Octet® Systems). The bispecific monoclonal antibody (BiXAb) was then purified in protein A affinity medium using MabSelect SuRe (GE Healthcare Life Sciences). The antibody was eluted from protein A, using 0.1 M glycine, pH 3.5, neutralized in 1 M TRIS. The purified antibody in Dulbecco's PBS (Lonza BE17-512Q) was sterile filtered (0.2 μM sterile filters from Techno Plastic Products AG) and the final concentration determined by reading the OD at 280 nm using an Eppendorf BioSpectrometer®.

SDS-PAGE analysis

Electrophoresis was performed under reducing and non-reducing conditions using Biorad Stain-Free 4-15% gels and the appropriate working buffer. Samples were prepared by combining purified BiXAb or Fab-Fab antibodies with 2X SDS sample buffer and heating for 5 minutes at 95°C. The preparation of the recovered samples included the addition of the NuPAGE reducing agent prior to heating. Apparent MW was determined using Ladder Precision Plus unstained protein standards (Biorad).

Analysis by size exclusion chromatography

Protein aggregation is often observed in engineered protein molecules. We performed analytical size exclusion chromatography (SEC) to analyze the content of high molecular weight particles in our antibodies. We used a SEC-s3000 (300x7.8 mm) column (BioSep) and an Aktapurifier 10 system (GE Healthcare); analysis was performed at a flow rate of 1 ml/min using PBS buffer pH 7.4.

SEC chromatograms of BiXAb2b (Figure 3A), BiXAb3a, BiXAb3b (Figure 3B), Fab-Fab3a and Fab-Fab3b (Figure 3C) showed that the main peak corresponds to the expected sizes of BiXAb and Fab-Fab monomeric antibodies; These peaks accounted for 99.9-100.0% of the total sample. Thus, we concluded that antibodies containing the new linker do not have high molecular weight varieties. The narrow and symmetrical shape of the monomeric peaks suggests that all BiXAbs and Fab-Fabs were correctly assembled and represented by a single species.

Characterization of BiXAb by Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was used to test the thermal stability of BiXAb2b. A Microcal™ VP-Capillary DSC system (Malvern Instruments) was used to perform differential scanning calorimetry experiments.

Samples were centrifuged (20,000 x g, 5 min, 4°C) and their protein content was quantified prior to DSC analysis using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) using an IgG assay program. For analysis, samples were diluted in PBS to a final concentration of 1 mg/mL.

The pre-equilibration time was 3 min, and the obtained thermograms were obtained at temperatures from 20 to 110°C at a scanning rate of 60°C/h, a filtration period of 25 s, and feedback from the medium. Prior to sample analysis, 5 buffer/buffer scans were performed to stabilize the instrument, and a buffer/buffer scan was performed between each protein/buffer scan. The data was matched against a non-two-state deployment model with pre- and post-transition adjusted by subtracting baseline.

The DSC results demonstrated that the DSC profile of BiXAb2b showed two transitions. The smaller peak had a Cp max of 96 kcal/mol/°C and a Tm1 of 71.5°C, which corresponds to the unfolding of both CH2 and Fab domains, and a larger peak had a Cp max of 190 kcal/mol/°C and a Tm2 of 80.5°C , which corresponds to the unfolding of the CH3 domain.

Analysis by Liquid Chromatography/Mass Spectroscopy (LC-MS)

LC-MS/MS data were obtained using a Dionex Ultimate 3000 system coupled to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) and a Proswift RP-4H reverse phase column (250 mm × 1 mm; Thermo Fisher ). The temperature in the column oven was set to 65°C. Ten microliters were injected for LC separation. A gradient of mobile phases consisting of LC-MS grade water with 0.1% formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B) was applied at a flow rate of 0.2 ml/min (total run time - 20 minutes). The eluted antibody species were injected into a Q-Exactive instrument using electrospray ionization (ESI) running in positive ion mode using full scan at 15,000 resolution. Xcalibur 2.2 software (Thermo Fisher Scientific, Bremen, Germany) was used for instrumental control and processing. data files.

Figures 4A and 4B show LC-MS analysis of Fab-Fab3a (containing the linker of SEQ ID NO: 6) and Fab-Fab3a (containing the linker of SEQ ID NO: 3), respectively. Figure 4A demonstrates that the LC-MS spectrum of Fab-Fab3a with the linker corresponding to SEQ ID NO: 6 is significantly more heterogeneous than that of the related Fab-Fab3b antibody, which differed only in linker composition (SEQ ID NO: 3). The linker sequence in the Fab-Fab3a antibody contains the sequence PSTPPTPSPS (SEQ ID NO: 22) which is found in the human IgA1 loop and is known to undergo O-linked glycosylation at 2 threonine and 2 serine residues. Since the glycosylation of these sites is heterogeneous, the product usually has multiple glycoforms whose populations are highly dependent on the conditions for expression of the recombinant proteins. The total number of N-linked and O-linked glycosylation sites in Fab-Fab3a is at least 4, which explains the complex spectrum of MS seen in Figure 4A. The linker sequence corresponding to SEQ ID NO: 3 was designed to reduce heterogeneity due to inhomogeneous O-linked glycosylation. As such, several serine and threonine residues in the linker portion whose sequence matches that of the human IgA1 loop known to undergo O-linked glycosylation were replaced by glycine residues; In addition, some other serine and threonine residues were replaced by glycine in the linker. Figure 4B shows that the MS spectrum for Fab-Fab3b containing the linker of SEQ ID NO: 3 is greatly simplified; this is because the Fab-Fab3b analyte is less complex than the Fab-Fab3a analyte whose spectrum is shown in Figure 4A. Thus, we concluded that removing the 4 O-linked glycosylation sites from the Fab-Fab3b antibody by replacing the SEQ ID NO: 6 linker with the SEQ ID NO: 3 linker resulted in a significantly more homogeneous BiXAb preparation.

The difference in homogeneity between the bispecific antibodies containing the linkers of SEQ ID NO: 6 and SEQ ID NO: 3 was particularly evident from the analysis of full-length BiXAbs, which are symmetrical molecules that have two linker sequences and thus have an increased number of glycoforms per preparation BiXAb. In addition, BiXAbs have two N-linked glycosylation sites, one for each heavy chain Fc domain. LC-MS spectra of BiXAb3a (generated with linker SEQ ID NO: 6) and BiXAb3b (generated with linker SEQ ID NO: 3) are presented in Figures 5A and 5B, respectively. BiXAb3a is expected to contain 8 additional O-linked glycosylation sites relative to BiXAb3b based on linker sequence differences. The large number of O-linked glycosylation sites in BiXAb3a explains the very complex spectrum of MS in Figure 5A. Both BiXAbs have 2 N-glycosylation sites in the Fc domain resulting in additional glycoforms contributing to the heterogeneity of both BiXAbs, which explains the residual amount of heterogeneity in BiXAb3b observed in Figure 5B.

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SEQUENCE LIST

<110> BIOMUNEX pharmaceuticals

<120> A polypeptide linker for preparing multispecific antibodies

<130> B2412PC

<160> 23

<170>PatentIn version 3.5

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<211> 34

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<213> Artificial Sequence

<220>

<223> linker

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<221> misc_feature

<222> (4)..(4)

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<220>

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<222>(8)..(8)

<223> Xaa can be any naturally occurring amino acids

<220>

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<222> (10)..(11)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (14)..(14)

<223> Xaa can be any naturally occurring amino acids

<220>

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<222> (24)..(25)

<223> Xaa can be any naturally occurring amino acids

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<222> (28)..(28)

<223> Xaa can be any naturally occurring amino acids

<220>

<221> misc_feature

<222> (30)..(30)

<223> Xaa can be any naturally occurring amino acids

<220>

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<222> (32)..(32)

<223> Xaa can be any naturally occurring amino acids

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Glu Pro Lys Xaa Cys Asp Lys Xaa His Xaa Xaa Pro Pro Xaa Pro Ala

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Pro Glu Leu Leu Gly Gly Pro Xaa Xaa Pro Pro Xaa Pro Xaa Pro Xaa

20 25 30

Gly Gly

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala

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Gly Gly

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala

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Pro Glu Leu Leu Gly Gly Pro Ala Ala Pro Pro Ala Pro Ala Pro Ala

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ala Pro Ala

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Gly Gly

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

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Pro Glu Leu Leu Gly Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

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Gly Gly

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala

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Pro Glu Leu Leu Gly Gly Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

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Gly Gly

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

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Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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Lys Val

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Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

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Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

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Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

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Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

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Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

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Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 10

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<212> PRT

<213> Homo sapiens

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Glu Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

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Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

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Lys Val

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<213> Homo sapiens

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Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

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<210> 12

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<212> PRT

<213> Homo sapiens

<400> 12

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

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<210> 13

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<212> PRT

<213> Homo sapiens

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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

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Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

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Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

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Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 14

<211> 701

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<213> Homo sapiens

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

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Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

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Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Ser Pro Pro Ala Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Gly Gly Pro Pro Gly Pro Gly Pro Gly Gly Gly Gln Val Gln Leu

245 250 255

Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile

260 265 270

Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp

275 280 285

Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp

290 295 300

Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser

305 310 315 320

Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser

325 330 335

Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr

340 345 350

Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr

355 360 365

Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

370 375 380

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

385 390 395 400

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

405 410 415

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

420 425 430

Leu Tyr Ser Leu Ser Ser Val Val Glu Val Pro Ser Ser Ser Leu Gly

435 440 445

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

450 455 460

Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

465 470 475 480

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

485 490 495

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

500 505 510

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

515 520 525

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

530 535 540

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

545 550 555 560

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

565 570 575

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

580 585 590

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

595 600 605

Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

610 615 620

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

625 630 635 640

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

645 650 655

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

660 665 670

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

675 680 685

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

690 695 700

<210> 15

<211> 214

<212> PRT

<213> Homo sapiens

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 214

<212> PRT

<213> Homo sapiens

<400> 16

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ala Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Lys Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 17

<211> 122

<212> PRT

<213> Homo sapiens

<400> 17

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asn Ser Phe

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Lys Asp Lys Ile Leu Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 702

<212> PRT

<213> Homo sapiens

<400> 18

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Glu Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Ser Pro Pro Ala Pro Ala Pro Glu Leu Leu Gly Gly Pro Ala

225 230 235 240

Ala Pro Pro Ala Pro Ala Pro Ala Gly Gly Glu Val Gln Leu Leu Glu

245 250 255

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

260 265 270

Ala Val Ser Gly Phe Thr Phe Asn Ser Phe Ala Met Ser Trp Val Arg

275 280 285

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

290 295 300

Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

305 310 315 320

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

325 330 335

Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Lys Asp Lys Ile Leu

340 345 350

Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

355 360 365

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

370 375 380

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

385 390 395 400

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

405 410 415

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

420 425 430

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

435 440 445

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

450 455 460

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

465 470 475 480

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

485 490 495

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

500 505 510

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

515 520 525

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

530 535 540

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

545 550 555 560

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

565 570 575

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

580 585 590

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

595 600 605

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

610 615 620

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

625 630 635 640

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

645 650 655

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

660 665 670

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

675 680 685

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

690 695 700

<210> 19

<211> 214

<212> PRT

<213> Homo sapiens

<400> 19

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ala Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Lys Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 20

<211> 214

<212> PRT

<213> Homo sapiens

<400> 20

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 21

<211> 475

<212> PRT

<213> Homo sapiens

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Glu Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Ser Pro Pro Ala Pro Ala Pro Glu Leu Leu Gly Gly Pro Ala

225 230 235 240

Ala Pro Pro Ala Pro Ala Pro Ala Gly Gly Glu Val Gln Leu Leu Glu

245 250 255

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

260 265 270

Ala Val Ser Gly Phe Thr Phe Asn Ser Phe Ala Met Ser Trp Val Arg

275 280 285

Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser

290 295 300

Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

305 310 315 320

Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

325 330 335

Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys Ala Lys Asp Lys Ile Leu

340 345 350

Trp Phe Gly Glu Pro Val Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

355 360 365

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

370 375 380

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

385 390 395 400

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

405 410 415

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

420 425 430

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

435 440 445

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

450 455 460

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

465 470 475

<210> 22

<211> 10

<212> PRT

<213> Homo sapiens

<400> 22

Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

1 5 10

<210> 23

<211> 118

<212> PRT

<213> Homo sapiens

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<---

Claims (45)

1. A multispecific antigen-binding fragment containing at least two Fab fragments, where each Fab fragment recognizes a different epitope of interest, and these Fab fragments are arranged in tandem in any order, where the C-terminus of the CH1 domain of the first Fab fragment is linked to N- the end of the VH domain of the next Fab fragment through a polypeptide linker, characterized in that the sequence of the polypeptide linker includes or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1), where X_one, X₂, X_3, X_four, X_5,X₆, X₇, X_eight,X₉, X_ten, the same or different, are any amino acid; provided that the linker sequence does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6). 2. The multispecific antigen binding fragment of claim 1, wherein said multispecific antigen binding fragment contains at least two Fab fragments with different CH1 and CL domains. 3. A multispecific antibody having two identical antigen-binding arms, each of which consists of a multispecific antigen-binding fragment according to claim 1 or 2. 4. A multispecific antibody according to claim 3, which has an immunoglobulin-like structure, including: - two identical antigen-binding arms, each of which consists of a multispecific antigen-binding fragment, as defined in paragraph 1; - dimerized CH2 and CH3 domains of immunoglobulin; - an IgA, IgG or IgD hinge region linking the C-terminus of the CH1 domains of the antigen-binding arms to the N-terminus of the CH2 domains. 5. A multispecific antibody, preferably a bispecific antibody, comprising two heavy chains and four light chains, characterized in that each heavy chain includes - an immunoglobulin Fc region containing Hinge-CH2-CH3 domains, - where the Fc region is linked to CH1-VH from the heavy chain Fab of antibody 1 (Ab1) by the specified hinge domain, - which, in turn, is linked to CH1-VH from the heavy chain of Fab antibody 2 (Ab2) via a polypeptide linker sequence, where the polypeptide linker sequence links the N-terminus of said VH domain from the heavy chain of Fab Ab1 to the C-terminus of said CH1 domain from Ab2, and the four light chains include Ab1 light chains and Ab2 light chains associated with their heavy chain cognate domains; where the sequence of the polypeptide linker includes or consists of the amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1), where X_one, X₂, X_3, X_four, X_5,X₆, X₇, X_eight,X₉, X_ten, the same or different, are any amino acid; provided that the linker sequence does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6). 6. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-5, where X ₁ , X ₂ and X ₃ , the same or different, are threonine (T) or serine (S). 7. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-5, characterized in that X ₁ , X ₂ and X ₃ , the same or different, represent any amino acid other than threonine (T) or serine (S), preferably, where X ₁ , X ₂ and X ₃ , identical or different, selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D) and Ile (I). 8. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-7, characterized in that X ₄ and X ₅ , the same or different, represent any amino acid selected from the group consisting of serine (S), cysteine (C), alanine (A) and glycine (G). 9. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-8, characterized in that X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , the same or different, are any amino acid other than threonine (T) or serine (S). 10. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-9, characterized in that X ₄ is serine (S) or cysteine (C). 11. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-10, characterized in that X ₅ is alanine (A) or cysteine (C). 12. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-9, characterized in that X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ , the same or different, are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn ( N), Asp (D) and Ile (I). 13. The multispecific antigen-binding fragment or multispecific antibody according to claim 12, characterized in that X ₆ , X ₇ , X _{8 ,} X ₉ , X ₁₀ are the same or different, are selected from the group consisting of Ala (A) and Gly (G ). 14. The multispecific antigen binding fragment or multispecific antibody of claim 13, wherein X ₆ and X ₇ are identical and are preferably selected from the group consisting of Ala (A) and Gly (G). 15. A multispecific antigen binding fragment or a multispecific antibody according to claim 13, characterized in that the polypeptide linker sequence includes or consists of a sequence selected from the group consisting of EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 3); EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 2); and EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 4). 16. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-15, which recognizes both EGFR and HER2/neu. 17. Multispecific antibody according to claim 5, characterized in that Ab1 and Ab2, which differ from each other, are independently selected from the group consisting of cetuximab or its mutated derivative, on the one hand, and trastuzumab or its mutated derivative, on the other hand. 18. Multispecific antigennegative fragment or multispecific antibody according to any one of paragraphs. 1-15 which recognizes both CD38 and PD-L1. 19. A linker polypeptide that includes or consists of an amino acid sequence EPKX ₁ CDKX ₂ HX ₃ X ₄ PPX ₅ PAPELLGGPX ₆ X ₇ PPX ₈ PX ₉ PX ₁₀ GG (SEQ ID NO: 1), where X ₁ , X ₂ , X _3, X ₄ , X _5, X ₆ , X ₇ , X _8, X ₉ , X ₁₀ , identical or different, represent any amino acid as defined in any of paragraphs. 1-11; provided that the polypeptide does not include or consist of the sequences EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 5) or EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 6). 20. The linker polypeptide according to claim 19, which includes or consists of a sequence selected from the group including EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 3); EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 2); and EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 4).

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