RU2784673C2 - Poly-specific antibody structures - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to a tri-specific antibody molecule. Polynucleotide encoding the specified antibody; an expression vector, and a host cell are also disclosed. A method for the production of the specified tri-specific antibody is disclosed.

EFFECT: invention has a capability of effective obtaining a tri-specific antibody.

13 cl, 10 dwg, 20 tbl, 5 ex

Description

The present invention relates to certain multispecific antibody constructs, pharmaceutical compositions comprising the construct, DNA encoding the constructs, and vectors comprising them. The present invention also extends to a method for expressing the constructs, for example, in a host cell, and methods for preparing them as a pharmaceutical composition. The present invention also relates to the use of multispecific antibody constructs and compositions in therapy.

There are a number of approaches for generating bispecific antibodies, many of which are based on the format first described by Morrison et al. (Coloma and Morrison 1997, Nat Biotechnol. 15, 159-163), involving the fusion of single-chain variable fragments (scFv) with whole antibodies, such as IgG, or Fab fragments of antibodies, Schoonjans et al., 2000, Journal of Immunology, 165, 7050-7057.

In the case of Fab-scFv, the V _H and V _L scFv domains used in such bispecific and trispecific molecules are usually held together by peptide linkers. However, in the case of some combinations of V _H and V _L variable domains, the peptide linkers are not able to confer sufficient stability to the scFv, resulting in variable domain "breathing" and random intermolecular pairing with the variable domains and thus a tendency to form multimers through their single stranded Fv part. This is shown in FIG. 1 in the context of a bispecific molecule.

The inventors of the present invention have reengineered multispecific antibody molecules in the interest of providing antibody molecules with equivalent functionality while minimizing multimerization during expression and/or after purification. This helps to increase the yield of the resulting "monomeric" material and provides molecules with suitable stability for use in treatment after purification, concentration and drug formulation.

Thus, in one aspect, a polyspecific antibody molecule is provided, comprising:

a) polypeptide(th) chain(s) of formula ( I ): V _H -CH ₁ -X-V ₁ ; and

b) polypeptide(th) chain(s) of formula ( II ): V _L -C _L -YV ₂ ;

or consisting of a) and b), where:

V _H is a heavy chain variable domain;

CH ₁ is a heavy chain constant region domain, eg domain 1;

X is a bond or linker;

Y is a bond or linker;

V ₁ is dsFv, sdAb, scFv or dsscFv;

V _L is a light chain variable domain;

C _L is a domain from a light chain constant region such as Ccappa;

V ₂ is dsFv, sdAb, scFv or dsscFv;

wherein at least one of V ₁ or V ₂ is dsFv or dsscFv.

Advantageously, when the polyspecific antibody molecules of the present invention minimize the amount of aggregation observed after purification and maximize the amount of monomer in formulations of the construct at pharmaceutical concentrations, including maintaining high levels of monomer for long periods of storage, for example, the monomer may be present as 50%, 60%, 70% or 75% or more, such as 80 or 90% or more, such as 91, 92, 93, 94, 95, 96, 97, 98 or 99% or more of total protein.

Detailed description of the invention

"Multispecific antibody", as used here, refers to an antibody molecule described here, which has two or more binding domains, for example, two or three binding domains.

In one embodiment, the antibody construct is a trispecific antibody.

"Trispecific antibody", as used here, refers to an antibody molecule with three antigen-binding sites that can independently bind the same or different antigens. In one example, a trispecific antibody molecule binds to two different antigens, i. two binding sites bind the same antigen, and a third binding site binds a second, different antigen. Preferably, the three binding sites of the trispecific antibody molecule of the present invention independently bind three different antigens.

In one embodiment, the construct is a bispecific antibody.

"Bispecific molecule", as used here, refers to a molecule with two antigen-binding sites that can bind the same or different antigens.

In one embodiment, all domains bind the same antigen, including binding to the same antigen epitope or binding to different antigen epitopes.

In one embodiment, there are three binding domains, and each of the three binding domains binds a different (different) antigen.

In one embodiment, there are three binding domains and two binding domains bind the same antigen, including binding to the same epitope or different epitopes of the same antigen, and the third binding domain binds a different (different) antigen.

"Antigen-binding site", as used here, refers to a part of a molecule that includes a pair of variable regions, in particular a related pair, that interact specifically with a target antigen.

Binding domains as used herein include a single domain antibody, ie. variable region and antigen-binding sites.

"Specific" as used herein is intended to refer to a binding site or binding domain that only recognizes the antigen for which it is specific, or a binding site or binding domain that has a significantly higher affinity for the antigen, relative to to which it is specific, as compared to the affinity for antigens for which it is non-specific, for example, 5, 6, 7, 8, 9, 10 times higher affinity.

Affinity can be determined using a standard assay such as surface plasmon resonance such as BIAcore.

In one embodiment, a multispecific antibody molecule according to the present invention is provided as a heavy and light chain dimer: formulas (I) and (II), respectively, where V _H -CH ₁ is part together with V _L -C _L is part form a functional Fab or Fab' fragment.

V _H is a variable domain, for example, a heavy chain variable domain. In one embodiment, V _H is a heavy chain variable domain. In one embodiment, V _H is a chimeric variable domain, that is, it contains components derived from at least two species, eg, a human framework and non-human CDRs. In one embodiment, V _H is humanized. In one embodiment, V _H is human.

V _L is a variable domain, for example, a light chain variable domain. In one embodiment, V _L is a light chain variable domain. In one embodiment, V _L is a chimeric variable domain, that is, it contains components from at least two species, eg, a human framework and non-human CDRs. In one embodiment, V _L is humanized. In one embodiment, V _L is human.

Typically, V _H and V _L together form an antigen-binding domain. In one embodiment, V _H and V _L form a cognate recognition pair.

A "cognate recognition pair" as used herein refers to a pair of variable domains from a single antibody that has been generated in vivo, i.e. naturally occurring pairing of variable domains isolated from the host. Therefore, the pair of cognate recognition is the pair V _H and V _L . In one example, a cognate recognition pair binds an antigen in cooperation.

"Variable region", as used here, refers to the region in the chain of the antibody, including the CDR and the frame region, in particular the appropriate framework region.

Variable regions for use in the present invention will generally be derived from an antibody, which can be generated by any method known in the art.

"Derived from (from)" refers to the fact that the sequence used, or a sequence very similar to the sequence used, was derived from the original genetic material, such as the light or heavy chain of an antibody.

The term "very similar" as used herein is intended to refer to an amino acid sequence that is 95% or more similar throughout its length, such as 96%, 97%, 98%, or 99% similar.

Variable regions for use in the present invention, as described above for V _H and V _L , may be from any suitable source and may be, for example, fully human or humanized.

In one embodiment, the binding domain formed by V _H and V _L are specific for the first antigen.

In one embodiment, the binding domain formed by V ₁ is specific for the second antigen.

In one embodiment, the binding domain formed by V ₂ is specific for the second or third antigen.

In one embodiment, the CH ₁ domain is a naturally occurring domain 1 from the heavy chain of an antibody, or a derivative thereof.

In one embodiment, the C _L fragment, in the light chain, is a kappa constant region sequence or a lambda constant region sequence, or a derivative thereof.

The term "derived from a naturally occurring domain, as used herein, is intended to refer to when one, two, three, four, or five amino acids in a naturally occurring sequence have been changed or removed, e.g., to optimize domain properties, e.g., as a result of eliminating undesirable properties, while retaining the distinguishing feature(s) of the domain.

In one embodiment, one or more naturally occurring or engineered interchain (ie, between the light and heavy chains) disulfide bonds are present in a functional Fab or Fab' fragment.

In one embodiment, a "natural" disulfide bond is present between CH ₁ and _CL in the polypeptide chains of formula (I) and (II).

When the _CL domain is derived from either kappa or lambda, the natural position for the bonding cysteine is 214 in human cKappa and cLambda (Kabat numbering ^4th edition 1987).

The exact location of the disulfide-forming cysteine in CH ₁ depends on the particular domain actually used. For example, in human gamma-1, the natural position of the disulfide bond is at position 233 (Kabat numbering ^4th edition 1987). The position of the bonding cysteine for other human isotypes such as gamma 2, 3, 4, IgM and IgD is known, for example, position 127 for human IgM, IgE, IgG2, IgG3, IgG4 and 128 in the heavy chain of human IgD and IgA2B.

Optionally, the disulfide bond may be between V _H and V _L of the polypeptides of formulas I and II.

In one embodiment, a polyspecific antibody of the present invention has a disulfide bond at a position equivalent to or corresponding to that naturally occurring between CH ₁ and C _L .

In one embodiment, a constant region comprising CH ₁ and a constant region such as _CL has a disulfide bond that is in a non-naturally occurring position. It can be created in the molecule by introducing cysteine(s) into the amino acid chain at the desired position or positions. This non-natural disulfide bond is present in addition to or as an alternative to the natural disulfide bond present between CH ₁ and _CL . The cysteine(s) at natural positions can be replaced by an amino acid such as serine which is incapable of forming a disulfide bridge.

The introduction of engineered cysteines can be carried out using any method known in the art. These methods include, but are not limited to, PCR mutagenesis with overlapping primers, site-directed mutagenesis, or cassette mutagenesis (see, in general, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be generated by general gene synthesis via annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

In one embodiment, the disulfide bond between CH ₁ and _CL is completely absent, for example, interchain-forming cysteines can be replaced with another amino acid, such as serine. Thus, in one embodiment, there are no interchain disulfide bonds in a functional Fab fragment of the molecule. Communications such as WO2005/003170, incorporated herein by reference, describe how to provide Fab fragments without an interchain disulfide bond.

V ₁ is dsFv, sdAb, scFv or dsscFv, for example dsFv, scFv or dsscFv.

V ₂ is dsFv, sdAb, scFv or dsscFv, such as dsFv, scFv or dsscFv.

In one embodiment, both V ₁ and V ₂ are dsscFv.

In one embodiment, both V ₁ and V ₂ are dsFv. When both V ₁ and V ₂ are dsFv or V _H or V _L the variable domain is the same for V ₁ and V ₂ . In one embodiment, V ₁ and V ₂ have the same V _H variable domain. In another embodiment, V ₁ and V ₂ have the same V _L variable domain. In one embodiment, the V _H and V _L variable domains are the same for V ₁ and V ₂ . The latter enables crosslinking, which may be desirable for some purposes.

In one embodiment, V ₁ is dsFv and V ₂ is scFv. In one embodiment, V ₁ is scFv and V ₂ is dsFv. In one embodiment, V ₁ is dsscFv and V ₂ is dsFv. In one embodiment, V ₁ is dsFv and V ₂ is dsscFv. In one embodiment, V ₁ is dsscFv and V ₂ is scFv. In one embodiment, V ₁ is scFv and V ₂ is dsscFv. In one embodiment, V ₁ is not scFv. In one embodiment, V ₂ is not scFv. In one embodiment, neither V ₁ nor V ₂ is scFv.

Thus, in one aspect, a polyspecific antibody molecule is provided, comprising:

a) polypeptide(th) chain(s) of formula ( I ): V _H -CH ₁ -X-V ₁ ; and

b) polypeptide(th) chain(s) of formula ( II ): V _L -C _L -YV ₂ ;

or consisting of a) and b), where:

V _H is a heavy chain variable domain;

CH ₁ is a heavy chain constant region domain, eg domain 1;

X is a bond or linker;

Y is a bond or linker;

V ₁ is dsFv or dsscFv;

V _L is a light chain variable domain;

C _L is a domain from a constant region, for example a domain from a light chain constant region such as Ckappa;

V ₂ is dsFv or dsscFv.

"Single chain variable fragment" or "scFv" as used herein refers to a single chain variable fragment consisting of or including a heavy chain variable domain (V _H ) and a light chain variable domain (V _L ) that is stabilized with a peptide linker between V _H and V _L variable domains. The variable domains of V _H and V _L may be in any suitable orientation, for example, the C-terminus of V _H may be linked to the N-terminus of V _L , or the C-terminus of V _L may be linked to the N-terminus of V _H .

"Disulfide-stabilized single chain variable fragment" or "dsscFv" as used herein refers to a single chain variable fragment that is stabilized with a peptide linker between the V _H and V _L variable domains and also includes an interdomain disulfide bond between V _H and V _L.

"Disulfide-stabilized variable fragment" or "dsFv" as used herein refers to a single-chain variable fragment that does not include a peptide linker between the V _H and V _L variable domains, but is instead stabilized via an interdomain disulfide bond between V _H and V _L.

"Single domain antibody" or "sdAb", as used here, refers to an antibody fragment consisting of a single monomeric variable domain of an antibody, such as V _H or V _L or VHH.

In one embodiment, when V ₁ and/or V ₂ are dsFv or dsscFv, the disulfide bond between the V _H and V _L variable domains of V ₁ and/or V ₂ is between two of the residues listed below (unless in context otherwise, Kabat numbering is used in the list below). Wherever reference is made to Kabat numbering, the corresponding reference is Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

In one embodiment, the disulfide bond is in a position selected from the group consisting of:

• V _H 37+V _L 95C, see eg Protein Science 6, 781-788 Zhu et al. (1997);

• V _H 44+V _L 100, see eg Biochemistry 33 5451-5459 Reiter et al (1994); or Journal of Biological Chemistry Vol. 269 no. 28 pp.18327-18331 Reiter et al. (1994); or Protein Engineering, vol.10 no.12 pp.1453-1459 Rajagopal et al. (1997);

• V _H 44+V _L 105, see eg J Biochem. 118, 825-831 Luo et al. (1995);

• V _H 45+V _L 87, see eg Protein Science 6, 781-788 Zhu et al. (1997);

• V _H 55+V _L 101, see eg FEBS Letters 377 135-139 Young et al. (1995);

• V _H 100+V _L 50, see eg Biochemistry 29 1362-1367 Glockshuber et al. (1990);

• V _H 100b+V _L 49;

• V _H 98+V _L 46, see eg Protein Science 6, 781-788 Zhu et al. (1997);

• V _H 101+V _L 46;

• V _H 105+V _L 43, see eg Proc. Natl. Acad. sci. USA Vol. 90 pp.7538-7542 Brinkmann et al. (1993); or Proteins 19, 35-47 Jung et al. (1994)

• V _H 106+V _L 57, see eg FEBS Letters 377 135-139 Young et al. (1995)

and its corresponding position in a pair of variable regions located in the molecule.

In one embodiment, a disulfide bond is formed between the V _H 44 and V _L 100 positions.

The amino acid pairs listed above are in positions conducive to cysteine substitution so that disulfide bonds can be formed. Cysteines can be engineered into these desired positions using known methods. In one embodiment, therefore, an engineered cysteine according to the present invention refers to a site where a naturally occurring residue at a given amino acid position is replaced by a cysteine residue.

The introduction of engineered cysteines can be carried out using any method known in the art. These methods include, but are not limited to, PCR mutagenesis with overlapping primers, site-directed mutagenesis, or cassette mutagenesis (see, in general, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, 1989; Ausbel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Site-directed mutagenesis kits are commercially available, such as the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be generated by general gene synthesis via annealing, ligation and PCR amplification and cloning of overlapping oligonucleotides.

Accordingly, in one embodiment, when V ₁ and/or V ₂ are dsFv or dsscFv, the V _H and V _L variable domains of V ₁ and/or the V _H and V _L variable domains of V ₂ may be disulfide-linked between two cysteine residues, and the position of the pair of cysteine residues is selected from the group consisting of: V _H 37 and V _L 95, V _H 44 and V _L 100, V _H 44 and V _L 105, V _H 45 and V _L 87, V _H 100 and V _L 50, V _H 100b and V _L 49, V _H 98 and V _L 46, V _H 101 and V _L 46, V H 105 and V _L 43 and _{V H 106} and V _L 57.

In one embodiment, when V ₁ and/or V ₂ are dsFv or dsscFv, the V _H and V _L variable domains of V ₁ and/or the V _H and V _L variable domains of V ₂ may be linked by a disulfide bond between two residues cysteine, one in V _H and one in V _L that are outside the CDR, and the position of the pair of cysteine residues is selected from the group consisting of V _H 37 and V _L 95, V _H 44 and V _L 100, V _H 44 and V _L 105, V _H 45 and V _L 87, V _H 100 and V _L 50, V _H 98 and V _L 46, V _H 105 and V _L 43 and V _H 106 and V _L 57.

In one embodiment, when V ₁ is dsFv or dsscFv, the V _H and V _L variable domains of V ₁ are linked by a disulfide bond between two engineered cysteine residues, one at position V _H 44 and the other at V _L 100.

In one embodiment, when V ₂ is dsFv or dsscFv, the V _H and V _L variable domains of V ₂ are linked by a disulfide bond between two engineered cysteine residues, one at position V _H 44 and the other at V _L 100.

In one embodiment, when V ₁ is dsFv, dsscFv, or scFv, the V _H domain of V ₁ is attached to X.

In one embodiment, when V ₁ is dsFv, dsscFv, or scFv, the V _L domain of V ₁ is attached to X.

In one embodiment, when V ₂ is dsFv, dsscFv, or scFv, the V _H domain of V ₂ is attached to Y.

In one embodiment, when V ₂ is dsFv, dsscFv, or scFv, the V _L domain of V ₂ is attached to Y.

The skilled artisan will appreciate that when V ₁ and/or V ₂ is a dsFv, the multispecific antibody will include a third polypeptide encoding the corresponding free V _H or V _L domain that is not attached to X or Y. When and V ₁ , and V ₂ are dsFv, then the "free variable domain" (ie, the domain linked via a disulfide bond to the remainder of the polypeptide) will be common to both chains. Thus, while the actual variable domain fused or X or Y linked to a polypeptide may be different in each polypeptide chain, the free variable domains paired with it will generally be identical to each other.

In one embodiment, X is a bond.

In one embodiment, Y is a bond.

In one embodiment, both X and Y are bonds.

In one embodiment, X is a linker, preferably a peptidic linker, eg a suitable peptide for joining the CH ₁ and V ₁ moieties.

In one embodiment, Y is a linker, preferably a peptidic linker, eg a suitable peptide for joining the C _L and V ₂ moieties.

In one embodiment, both X and Y are linkers. In one embodiment, X and Y are peptide linkers.

As used herein, the term "peptide linker" refers to a peptide composed of amino acids. A number of suitable peptide linkers will be known to those skilled in the art.

In one embodiment, the length of the peptide linker is 50 amino acids or less, such as 20 amino acids or less, such as about 15 amino acids, or less, such as 9, 10, 11, 12, 13, or 14 amino acids.

In one embodiment, the linker is selected from the sequence shown in sequence 1-67.

In one embodiment, the linker is selected from the sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

In one embodiment, X has the sequence SGGGGTGGGGS (SEQ ID NO:1).

In one embodiment, Y has the sequence SGGGGTGGGGS (SEQ ID NO:1).

In one embodiment, X has the sequence SGGGGSGGGGS (SEQ ID NO:2). In one embodiment, Y has the sequence SGGGGSGGGGS (SEQ ID NO:2).

In one embodiment, X has the sequence shown in SEQ ID NO:1 and Y has the sequence shown in SEQ ID NO:2.

In one embodiment, X has the sequence shown in SEQ ID NO:69 or 70. In one embodiment, Y has the sequence shown in SEQ ID NO:69 or 70. In one embodiment, X has the sequence shown in SEQ ID NO: 69 and Y has the sequence shown in SEQ ID NO:70.

Table 1. Hinge linker sequences

SEQID NO: Subsequence 3 DKTHTCAA four DKTHTCPPCPA 5 DKTHTCPPCPATCPPCPA 6 DKTHTCPPCPATCPPCPATCPPCPA 7 DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY eight DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 9 DKTHTCCVECPPCPA ten DKTHTCPRCPEPKSCDTPPPCPRCPA eleven DKTHTCPSCPA

Table 2 Flexible linker sequences

SEQID NO: Subsequence 12 SGGGGSE 13 DKTHTS fourteen (S)GGGGS fifteen (S)GGGGSGGGGS 16 (S)GGGGSGGGGSGGGGS 17 (S)GGGGSGGGGSGGGGSGGGGS eighteen (S)GGGGSGGGGSGGGGSGGGGSGGGGS 19 AAAGSG-GASAS twenty AAAGSG-XGGGS-GASAS 21 AAAGSG-XGGGSXGGGS-GASAS 22 AAAGSG-XGGGSXGGGSXGGGS-GASAS 23 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 24 AAAGSG-XS-GASAS 25 PGGNRGTTTTRRPATTTGSSPGPTQSHY 26 ATTTGSSPGPT 27 ATTTGS - GS 28 EPSGPISTINSPPSKESHKSP 29 GTVAAPSVFIFPPSD thirty GGGGIAPSMVGGGGS 31 GGGGKVEGAGGGGGS 32 GGGGSMKSHDGGGGS 33 GGGGNLITIVGGGGS 34 GGGGVVPSLPGGGGS 35 GGEKSIPGGGGS 36 RPLSYRPPFPFGFPSVRP 37 YPRSIYIRRRHPSPSLTTT 38 TPSHLSHILPSFGLPTFN 39 RPVSPFTFPRLSNSWLPA 40 SPAAHFPRSIPRPPGPIRT 41 APGPSAPSHRSLPSRAFG 42 PRNSIHFLHPLLVAPLGA 43 MPSLSGVLQVRYLSPPDL 44 SPQYPSPLTLTLPPPHPSL 45 NPSLNPPSYLHRAPSRIS 46 LPWRTSLLPSLPLRRRP 47 PPLFAKGPVGLLSRSFFPP 48 VPPAPVVSLRSAHARPPY 49 LRPTPPRVRSYTCCPTP- fifty PNVAHVLPLLTVPWDNLR 51 CNPLLPLCARSPAVRTFP

(S) is optional in sequences 14-18.

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQ ID NO:52), PPPP (SEQ ID NO:53) and PPP.

In one embodiment, the peptide linker is an albumin binding peptide.

Examples of albumin binding peptides are provided in WO2007/106120 and include:

Table 3

SEQID NO: Subsequence 54 DLCLRDWGCLW 55 DICLPRWGCLW 56 MEDICLPRWGCLWGD 57 QRLMEDICLPRWGCLWEDDE 58 QGLIGDICLPRWGCLWGRSV 59 QGLIGDICLPRWGCLWGRSVK 60 EDICLPRWGCLWEDD 61 RLMEDICLPRWGCLWEDD 62 MEDICLPRWGCLWEDD 63 MEDICLPRWGCLWED 64 RLMEDICLARWGCLWEDD 65 EVRSFCTRWPAEKSCKPLRG 66 RAPESFVCYWETICFERSEQ 67 EMCYFPGICWM

Advantageously, the use of albumin-binding peptides as a linker can increase the half-life of the polyspecific antibody molecule.

In one embodiment, when V ₁ is a scFv or dsscFv, there is a linker, eg, a suitable peptide for joining the variable domains of V _H and V _L from V ₁ .

In one embodiment, when V ₂ is a scFv or dsscFv, there is a linker, eg, a suitable peptide for joining the variable domains of V _H and V _L from V ₂ .

In one embodiment, the length of the peptide linker in the scFv or dsscFv is in the range of about 12 to 25 amino acids, such as 15 to 20 amino acids.

In one embodiment, when V ₁ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₁ has the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:68).

In one embodiment, when V ₂ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₂ has the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:68).

In one embodiment, when V ₁ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₁ has the sequence SGGGGSGGGGSGGGGS (SEQ ID NO:69).

In one embodiment, when V ₂ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₂ has the sequence SGGGGSGGGGSGGGGS (SEQ ID NO:69).

In one embodiment, when V ₁ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₁ has the sequence SGGGGSGGGGTGGGGS (SEQ ID NO:70).

In one embodiment, when V ₂ is scFv or dsscFv, the linker connecting the V _H and V _L variable domains of V ₂ has the sequence SGGGGSGGGGTGGGGS SEQ ID NO:70).

Accordingly, in one aspect, a polyspecific antibody molecule is provided, comprising:

a) polypeptide(th) chain(s) of formula ( I ): V _H -CH ₁ -X-V ₁ ; and

b) polypeptide(th) chain(s) of formula ( II ): V _L -C _L -YV ₂ ;

or consisting of a) and b), where:

V _H is a heavy chain variable domain;

CH ₁ is a heavy chain constant region domain, eg domain 1;

X is a bond or linker;

Y is a bond or linker;

V ₁ is dsFv, sdAb, scFv or dsscFv;

V _L is a light chain variable domain;

C _L is a domain from a light chain constant region such as Ccappa;

V ₂ is dsFv, sdAb, scFv or dsscFv;

wherein at least one of V ₁ or V ₂ is dsFv or dsscFv.

The present invention also provides sequences that are 80%, 90%, 91%, 92%, 93% 94%, 95% 96%, 97%, 98% or 99% similar to the sequence disclosed here.

As used herein, the term "identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences.

As used herein, the term "similarity" indicates that at any particular position in the aligned sequences, the amino acid residue is one of similar type between the sequences. For example, leucine can be replaced by isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:

- phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

- lysine, arginine and histidine (amino acids having basic side chains);

- aspartate and glutamate (amino acids having acidic side chains);

- asparagine and glutamine (amino acids having side chains with an amide group); as well as

- cysteine and methionine (amino acids with sulfur-containing side chains).

Degrees of identity and similarity can be easily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, BLAST™ software available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403- 410 Gish, W. & States, D. J. 1993, Nature Genet 3:266-272 Madden, T. L. et al., 1996, Meth Enzymol 266:131-141 Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, T.L. 1997, Genome Res. 7:649-656).

Antibodies for use in the polyspecific antibody constructs of the present invention may be generated by any suitable method known in the art.

Antibodies raised against an antigen polypeptide can be prepared, when immunization of an animal is needed, by administering the polypeptides to an animal, preferably a non-human animal, using well known and accepted protocols, see e.g. Handbook of Experimental Immunology, D. M. Weir (ed. ), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals such as rabbits, mice, rats, sheep, cows, camels or pigs can be immunized. However, mice, rabbits, pigs and rats are generally the most suitable.

Monoclonal antibodies can be prepared by any method known in the art, such as the hybridoma method (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma method, the human B-cell hybridoma method (Kozbor et al. 1983, Immunology Today, 4:72) and the EBV fusion method (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies can also be generated using single lymphocyte antibody techniques by cloning and expressing cDNA of immunoglobulin variable regions derived from single lymphocytes selected for specific antibody production using, for example, the methods described by Babcook, J. et al 1996, Proc. Natl. Acad. sci. USA 93(15):7843-7848l; WO92/02551; WO2004/051268 and WO2004/106377.

Antibodies for use in the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 187: 9-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982; WO95/20401; and US Pat. Nos. 5,698,426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743; 5969108, and WO20011/30305.

In one embodiment, polyspecific molecules in accordance with the present invention are humanized.

The term "humanized" (which includes CDR-grafted antibodies), as used herein, refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, for example, US Pat. No. 5585089; WO91/09967). It should be appreciated that it may only be necessary to transfer the specificity-determining residues of the CDR and not the entire CDR (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally include, in addition, one or more framework region residues derived from the non-human species from which the CDRs are derived.

The term "humanized antibody molecule" as used herein refers to an antibody molecule in which the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g., a mouse monoclonal antibody) grafted into the heavy and/or light chain variable region framework of an acceptor antibody (eg, a human antibody). For a review, see Vaughan et al. Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more of the specificity-determining residues from any of the CDRs described herein above is transferred to the framework region of a human antibody (see e.g. , Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment, only specificity-determining residues from one or more of the CDRs described herein above are transferred to the framework region of a human antibody. In another embodiment, only the specificity-determining residues from each of the CDRs described herein above are transferred into the framework region of a human antibody.

In the case of grafting CDRs or specificity-defining residues, any appropriate variable region framework acceptor sequence may be used, taking into account the class/type of donor antibody from which the CDRs are derived, including murine, primate, and human frameworks. Accordingly, a humanized antibody of the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.

Examples of human framework regions that can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al. supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain, and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences can be used; they are available at: http://www2.mrc-lmb.cam.ac.uk/vbase/list2.php.

In a humanized antibody molecule of the present invention, the acceptor heavy and light chains need not be derived from the same antibody, and may, if desired, include multiple chains having framework regions derived from different chains.

The framework regions do not have to have exactly the same sequence as the acceptor antibody. For example, uncommon residues can be replaced with more common residues for that class or type of acceptor chain. Alternatively, selected residues in the acceptor framework regions can be changed to match the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such substitutions should be kept to the minimum necessary to restore the affinity of the donor antibody. A protocol for selecting residues in acceptor frameworks that may need to be replaced is given in WO91/09967.

Framework region derivatives may contain 1, 2, 3, or 4 amino acids replaced with an alternative amino acid, such as the donor serine.

Donor residues are residues from the donor antibody, ie. the antibody from which the CDRs were originally derived. Donor residues can be replaced by a corresponding residue derived from the human receptor framework region (acceptor residues).

In one embodiment, the polyspecific antibodies of the present invention are fully human, in particular one or more of the variable domains are fully human.

Fully human molecules are those in which all of the variable regions and constant regions (if present) of both the heavy and light chains are of human origin, or are substantially identical to sequences of human origin, optionally from the same antibody. Examples of fully human antibodies may include antibodies produced, for example, using the phage display methods described above, and antibodies produced by mice in which the mouse immunoglobulin variable region and optionally constant region genes have been replaced with their human counterparts, for example, as described generally in EP0546073 US Patent No. 5545806, US Patent No. 5569825, US Patent No. 5625126, US Patent No. 5633425, US Patent No. 5661016, US Patent No. 5770429, EP0438474 and EP0463151.

In one embodiment, the multispecific antibody molecules of the present invention are capable of selectively binding two, three or more different antigens of interest.

In one embodiment, the antigens of interest bound by the antigen-binding domain formed by V _H /V _L or V ₁ or V ₂ are independently selected from a cell-associated protein, e.g., a cell surface protein on cells such as bacterial cells, yeast cells, T cells, B cells, endothelial or tumor cells, and soluble protein.

Antigens of interest can also be any medically relevant proteins, such as those proteins that are upregulated during disease or infection, such as receptors and/or their respective ligands. Specific examples of antigens include cell surface receptors such as T-cell or B-cell signaling receptors, costimulatory molecules, cell cycle checkpoint inhibitors, natural killer cell receptors, immunoglobulin receptors, TNFR family receptors, B7 family receptors, adhesion molecules , integrins, cytokine/chemokine receptors, GPCRs, growth factor receptors, receptors, kinases, tissue-specific antigens, cancer antigens, pathogen recognition receptors, complement receptors, hormone receptors, or soluble molecules such as cytokines, chemokines, leukotrienes, growth factors, hormones, or enzymes or ion channels, epitopes, fragments and their post-translationally modified forms.

In one embodiment, the polyspecific antibody molecule of the present invention can be used to functionally alter the activity of the antigen(s) of interest. For example, a protein fused to an antibody can neutralize the activity of the specified antigen, give rise to antagonism or agonism in relation to it, directly or indirectly.

In one embodiment, V ₁ and V ₂ are specific for the same antigen, eg, bind to the same or different epitope thereof. In one embodiment, V ₁ is specific for human serum albumin. In one embodiment, V ₂ is specific for human serum albumin. In one embodiment, V ₁ and V ₂ are specific for two different antigens.

In one embodiment, V ₁ and V ₂ are specific for the same antigens, eg, the same or different epitopes on the same antigen.

In one embodiment, the antigen of interest bound by V _H /V _L or V ₁ or V ₂ allows the mobilization of effector functions, such as activation of the complement activation pathway and/or recruitment of effector cells.

The mobilization of the effector function can be direct in the sense that the effector function is associated with the cell carrying the recruit molecule on its surface. Indirect mobilization can occur when binding of an antigen to an antigen-binding domain (e.g., V ₁ or V ₂ ) in a recruit polypeptide molecule of the present invention causes the release of, for example, a factor, which in turn can directly or indirectly mobilize an effector function, or may occur through signaling pathway activation. Examples include IL2, IL6, IL8, IFNγ, histamine, C1q, opsonin, and other members of the classical and alternative complement activation cascades such as C2, C4, C3 convertase, and C5-C9.

The term "recruit polypeptide" as used herein includes FcγR such as FcγRI, FcγRII and FcγRIII, a complement activation pathway protein such as, but not limited to, C1q and C3, a CD marker protein (cluster of differentiation marker) or a fragment thereof that retains the ability to mobilize a cell-mediated effector function, either directly or indirectly. The recruit polypeptide also includes immunoglobulin molecules such as IgG1, IgG2, IgG3, IgG4, IgE and IgA which have an effector function.

In one embodiment, the antigen-binding domain (eg, V ₁ or V ₂ or V _H /V _L ) in the multispecific antibody molecule of the present invention is specific for a complement activation pathway protein, with C1q being particularly preferred.

In addition, the polyspecific antibody molecules of the present invention can be used to form chelate complexes with radionuclides due to a single domain antibody that binds to a nuclide chelator protein. Such fusion proteins are used in imaging or therapeutic approaches that involve targeted delivery of radionuclides.

In one embodiment, an antigen-binding domain in a molecule according to the invention (e.g., V ₁ or V ₂ or V _H /V _L ) is specific for a serum carrier protein, a circulating immunoglobulin molecule, or CD35/CR1, for example, to provide an extended half-life for an antibody fragment with specificity for said antigen of interest by binding to said carrier protein in serum, a circulating immunoglobulin molecule, or CD35/CR1.

The term "serum carrier proteins" as used herein includes thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, fibrinogen, and albumin, or a fragment of any of these.

The term "circulating immunoglobulin molecule" as used herein includes IgG1, IgG2, IgG3, IgG4, sIgA, IgM and IgD, or a fragment of any of these compounds.

CD35/CR1 is a protein present on erythrocytes that has a half-life of 36 days (normal range 28 to 47 days; Lanaro et al., 1971, Cancer, 28(3):658-661).

In one embodiment, the antigen of interest for which V _H /V _L has specificity is a carrier protein in blood serum, such as a carrier in human serum, such as human serum albumin.

In one embodiment, the antigen of interest for which V ₁ has specificity is a carrier protein in blood serum, such as a carrier in human serum, such as human serum albumin.

In one embodiment, the antigen of interest for which V ₂ has specificity is a carrier protein in blood serum, such as a carrier in human serum, such as human serum albumin.

In one embodiment, only one of V _H /V _L , V ₁ or V ₂ has specificity for a serum carrier protein, such as a human serum carrier, such as human serum albumin.

In one embodiment, the binding site in the construct of the present invention includes 6 CDRs, for example, SEQ ID NO:71 for CDRH1, SEQ ID NO:72 for CDRH2, SEQ ID NO:73 for CDRH3, SEQ ID NO:74 for CDRL1, SEQ ID NO:75 for CDRL2 and SEQ ID NO:76 for CDRL3.

In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in the V _H /V _L position in the constructs of the present invention. In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in position V ₁ in the constructs of the present invention. In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in the V ₂ position in the constructs of the present invention. In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in position V ₁ and V ₂ in the constructs of the present invention. In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in position V _H /V _L and V ₁ in the constructs of the present invention. In one embodiment, said 6 CDRs, SEQ ID NOs: 71-76, are in position V _H /V _L and V ₂ in the constructs of the present invention.

In one embodiment, the albumin binding site in the construct of the present invention comprises a heavy chain variable domain selected from SEQ ID NO:77 and SEQ ID NO:78 and a light chain variable domain selected from SEQ ID NO:79 and SEQ ID NO:80, in particular SEQ ID NOs: 77 and 79 or SEQ ID NOs: 78 and 80 for the heavy chain and light chain, respectively. In one embodiment, these domains are in the V _H /V _L position in the constructs of the present invention. In one embodiment, these variable domains are in position V ₁ . In one embodiment, these variable domains are in position V ₂ . In one embodiment, these variable domains are at the V ₁ and V ₂ positions. In one embodiment, these variable domains are in position V _H /V _L and V ₁ in the constructs of the present invention. In one embodiment, these variable domains are at the V _H /V _L and V ₂ positions in the constructs of the present invention. When the variable domains are in two places in the constructs of the present invention, the same pair of variable domains can be in each place, or two different pairs of variable domains can be used.

In one embodiment, the polyspecific antibody molecules of the present invention are converted to provide increased affinity for the target antigen or target antigens. Such variants can be generated using a number of affinity maturation protocols, including CDR mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), the use of E. coli strain mutators (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol ., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sex-sham PCR (Crameri et al Nature, 391, 288- 291, 1998). Vaughan et al. (above) discuss these modes of affinity maturation.

Increased affinity, as used herein in this context, refers to an increase over the parent molecule.

If desired, the multispecific antibody construct for use in the present invention may be conjugated to one or more effector molecules. It should be appreciated that an effector molecule may include one effector molecule or two or more such molecules linked together to form a single component that can be attached to the antibodies of the present invention. If it is desired to obtain an antibody fragment associated with an effector molecule, it can be obtained using standard chemical techniques or recombinant DNA methods, in which the antibody fragment is associated either directly or with a crosslinking agent to the effector molecule. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al. Controlled Drug Delivery, ^2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol Rev., 62:119-58 and Dubowchik et al 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, those described in WO93/06231, WO92/22583, WO89/00195, WO89/01476 and WO03031581. Alternatively, if the effector molecule is a protein or polypeptide, linkage can be achieved using recombinant DNA techniques, for example as described in WO86/01533 and EP0392745.

As used herein, the term "effector molecule" includes, for example, biologically active proteins, such as enzymes, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof, such as DNA, RNA and fragments thereof, radionuclides, in in particular, radioiodine, radioisotopes, chelate metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR (EPR).

Other effector molecules may include chelated radionuclides such as 111In and 90Y, Lu177, Bismuth213, Californium252, Iridium192 and Tungsten188/Rhenium188; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids, and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas aeruginosa exotoxin or diphtheria toxin, protein such as insulin, interferon α, interferon β, nerve growth factor, platelet a growth factor or tissue plasminogen activator, thrombotic agent or anti-angiogenic agent such as angiostatin or endostatin, or a biological response modifier such as lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor ( GM-CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor, and immunoglobulins.

Other effector molecules may include detectable substances useful, for example, in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. See generally US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, fluorescein dichlorotriazinylamine, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; and suitable radionuclides include 125I, 131I, 111In and 99Tc.

In another embodiment, the effector molecule can increase the in vivo half-life of the antibody and/or decrease the immunogenicity of the antibody and/or improve delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO05/117984.

If the effector molecule is a polymer, it may generally be a synthetic or naturally occurring polymer, for example an optionally substituted straight or branched polyalkylene, polyalkenyl or polyoxyalkylene or a branched or straight chain polysaccharide, for example a homo- or heteropolysaccharide.

Specific optional substituents that may be present in the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted straight or branched polyethylene glycol, polypropylene glycol, polyvinyl alcohol or derivatives thereof, especially optionally substituted polyethylene glycol, for example methoxy polyethylene glycol or derivatives thereof.

Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

The term "derivatives" as used herein is intended to include reactive derivatives, for example, thiol selective reactive groups such as maleimides and the like. The reactive group may be linked to the polymer directly or via a linker segment. It will be appreciated that the remainder of such a group will in some cases form part of the product as a linking group between the antibody fragment and the polymer.

The size of the polymer can be varied as desired, but as a general rule it will generally be in the average molecular weight range of 500 Da to 50,000 Da, such as 5,000 to 40,000 Da, such as 20,000 to 40,000 Da. The size of the polymer may in particular be selected based on the intended use of the product, for example, the ability to localize to certain tissues such as tumors, or to increase the circulating half-life (for a review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, if the product is intended to leave the bloodstream and enter tissue, for example, for use in the treatment of a tumor, it may be preferable to use a low molecular weight polymer, for example, with a molecular weight of approximately 5000 Da. For applications where the product remains in the blood stream, it may be preferable to use a higher molecular weight polymer, for example having a molecular weight in the range of 20,000 Da to 40,000 Da.

Suitable polymers include polyalkylene such as polyethylene glycol or especially methoxypolyethylene glycol or a derivative thereof, and especially with a molecular weight in the range of from about 15,000 Da to about 40,000 Da.

In one embodiment, antibodies for use in the present invention are attached to polyethylene glycol (PEG) components. In one specific example, an antibody is an antibody fragment, and the PEG molecules can be attached through any available amino acid side chain or amino acid terminal functional group found in the antibody fragment, for example, any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may occur naturally in the antibody fragment, or may be engineered in the fragment using recombinant DNA techniques (see, for example, US Pat. No. 5,219,996; US Pat. No. 5,667,425; W098/25971, WO2008/038024). In one embodiment, an antibody molecule of the present invention is a modified Fab fragment, wherein the modification is the addition of one or more amino acids to the C-terminus of its heavy chain to allow attachment of an effector molecule. Accordingly, when the additional amino acids form a modified hinge region containing one or more cysteine residues to which an effector molecule can be attached. Multiple sites can be used to attach two or more PEG molecules.

Accordingly, when the PEG molecules are covalently linked through the thiol group of at least one cysteine residue found in the antibody fragment. Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulfur atom of the cysteine residue present in the fragment. The covalent bond will generally be a disulfide bond, or in particular a sulfur-carbon bond. If a thiol group is used as the point of attachment, suitably activated effector molecules, for example thiol-selective derivatives such as maleimides and cysteine derivatives, can be used. The activated polymer can be used as a starting material in the preparation of antibody fragments modified with the polymer as described above. The activated polymer can be any polymer containing a thiol reactive group, eg α-halocarboxylic acid or an ester, eg iodoacetamide, imide, eg maleimide, vinyl sulfone or disulfide. Such starting materials may be purchased (eg, from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or may be prepared from commercially available starting materials using conventional chemical techniques. Specific PEG molecules include 20K methoxy-PEG-amine (obtained from Nektar, formerly Shearwater; Rapp Polymere and SunBio) and M-PEG-SPA (obtained from Nektar, formerly Shearwater).

In one embodiment, the F(ab') ₂ , Fab or Fab' in the molecule is PEGylated, i. contains PEG (polyethylene glycol) covalently attached to it, for example, in accordance with the method described in EP0948544 or EP1090037 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, "Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one embodiment, the PEG is attached to the cysteine at the hinge region. In one example, the PEG-modified Fab fragment contains a maleimide group covalently linked to one thiol group in the modified hinge region. The lysine moiety may be covalently bonded to a maleimide group, and a methoxypolyethylene glycol polymer having a molecular weight of about 20,000 Da can be attached to each of the amine groups in the lysine moiety. Thus, the total molecular weight of the PEG attached to the Fab fragment may be approximately 40,000 Da.

Specific PEG molecules include 2-[3-(N-maleimido)propionamido]ethylamide N,N'-bis(methoxypoly(ethylene glycol) MW 20000)-modified lysine, also known as PEG2MAL40K (available from Nektar, formerly Shearwater).

Alternative sources of PEG linkers include NOFs that supply GL2-400MA2 (where m is 5 in the structure below) and GL2-400MA (where m is 2) and n is approximately 450:

That is, each of the PEGs is approximately 20,000 Da.

Other alternative PEG effector molecules are of the following type:

available from Dr. Reddy, NOF and Jenkem.

In one embodiment, an antibody molecule is provided that is a PEGylated (e.g., using PEG as described herein) molecule attached via a cysteine amino acid residue or approximately amino acid 226 in the chain, e.g., amino acid 226 of the heavy chain (according to continuous numbering).

In one embodiment, a polynucleotide sequence encoding a molecule of the present invention is provided, such as a DNA sequence.

In one embodiment, a polynucleotide sequence is provided encoding one or more, for example, two or more, or three or more, polypeptide components of a molecule of the present invention, for example:

a) a polypeptide chain of formula ( I ): V _H -CH ₁ -X-V ₁ ; and

b) a polypeptide chain of formula ( II ): V _L -C _L -YV ₂ ;

where:

V _H is a heavy chain variable domain;

CH ₁ is a heavy chain constant region domain, eg domain 1;

X is a bond or linker;

Y is a bond or linker;

V ₁ is dsFv, sdAb, scFv or dsscFv;

V _L is a light chain variable domain;

C _L is a domain from a light chain constant region such as Ccappa;

V ₂ is dsFv, sdAb, scFv or dsscFv;

wherein at least one of V ₁ or V ₂ is dsFv or dsscFv.

In one embodiment, a polynucleotide, such as DNA, is included in a vector.

The skilled artisan will appreciate that when V ₁ and/or V ₂ is a dsFv, the polyspecific antibody will include a third polypeptide encoding the corresponding free V _H or V _L domain that is not attached to X or Y. Accordingly, the polyspecific protein of the present The invention may be encoded by one or more, two or more, or three or more polynucleotides, and they may be included in one or more vectors.

General methods by which vectors can be constructed, transfection methods and culture methods are well known to those skilled in the art. Reference is made in this connection to "Current Protocols in Molecular Biology", 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning vectors or expression vectors comprising one or more DNA sequences encoding a polyspecific protein of the present invention. Any suitable host cell/vector system can be used to express DNA sequences encoding an antibody molecule of the present invention. Bacterial systems, such as E. coli and other microbial systems, may be used, or expression systems using eukaryotic host cells, such as mammalian cells, may also be used. Suitable mammalian host cells include CHO, myeloma cells, NSO myeloma cells and SP2 cells, COS cells or hybridoma cells.

The present invention also provides a method for producing a polyspecific protein according to the present invention, comprising culturing a host cell containing the vector of the present invention under conditions suitable to result in expression of the protein from DNA encoding the polyspecific protein of the present invention, and isolating the polyspecific protein .

To produce both heavy and light chain products, the cell line can be transfected with two vectors, the first vector encoding a light chain polypeptide and the second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, wherein the vector includes sequences encoding light chain and heavy chain polypeptides. In one example, a cell line can be transfected with two vectors, each of which encodes a polypeptide chain of an antibody molecule of the present invention. If V ₁ and/or V ₂ are dsFv, the cell line can be transfected with three vectors, each of which encodes the polypeptide chain of the polyspecific protein of the present invention.

In one embodiment, the cell line is transfected with two vectors, each encoding a different polypeptide selected from:

a) a polypeptide chain of formula ( I ): V _H -CH ₁ -X-V ₁ ; and

b) a polypeptide chain of formula ( II ): V _L -C _L -YV ₂ ;

where:

V _H is a heavy chain variable domain;

CH ₁ is a heavy chain constant region domain, eg domain 1;

X is a bond or linker;

Y is a bond or linker;

V ₁ is dsFv, sdAb, scFv or dsscFv;

V _L is a light chain variable domain;

C _L is a domain from a light chain constant region such as Ccappa;

V ₂ is dsFv, sdAb, scFv or dsscFv;

wherein at least one of V ₁ or V ₂ is dsFv or dsscFv.

In one embodiment, when V ₁ is dsFv and the V _H domain of V ₁ is fused to X, the cell line can be transfected with a third vector that encodes the V _L domain of V ₁ .

In one embodiment, when V ₁ is dsFv and the V _L domain of V ₁ is fused to X, the cell line can be transfected with a third vector that encodes the V _H domain of V ₁ .

In one embodiment, when V ₂ is dsFv and the V _H domain of V ₂ is fused to Y, the cell line can be transfected with a third vector that encodes the V _L domain of V ₂ .

In one embodiment, when V ₂ is dsFv and the V _L domain of V ₂ is fused to Y, the cell line can be transfected with a third vector that encodes the V _H domain of V ₂ .

In one embodiment, when both V ₁ and V ₂ are dsFv and the V _L domain of V ₂ is attached to Y and the V _L domain of V ₁ is attached to X, the cell line can be transfected with a third vector that encodes a common for both V ₁ and V ₂ V _H -domain.

In one embodiment, when both V ₁ and V ₂ are dsFv, and the V _H domain of V ₂ is attached to Y, and the V _H domain of V ₁ is attached to X, the cell line can be transfected with a third vector that encodes a common for both V ₁ and V ₂ V _H -domain.

It should be borne in mind that the ratio of each vector transfected into the host cell can be changed in order to optimize the expression of the product as a polyspecific antibody. In one embodiment, when two vectors are used, the ratio of vectors may be 1:1. In one embodiment, when three vectors are used, the ratio of vectors may be 1:1:1. It will be understood that the expert can find the optimal ratio by routinely checking the levels of protein expression after transfection. Alternatively or additionally, the expression levels of each polypeptide chain of a polyspecific construct from each vector can be controlled using the same or different promoters.

It will be appreciated that two or more, or if present, three of the polypeptide components may be encoded by a polynucleotide in a single vector. It should also be appreciated that when two or more, in particular three or more, of the polypeptide components are encoded by a polynucleotide in one vector, the relative expression of each polypeptide component can be altered by using different promoters for each polynucleotide encoding the polypeptide component of the present invention. .

In one embodiment, the vector comprises a single polynucleotide sequence encoding two or, if present, three polypeptide chains of a polyspecific antibody molecule of the present invention, under the control of a single promoter.

In one embodiment, the vector comprises a single polynucleotide sequence encoding two or, if present, three polypeptide chains of a polyspecific antibody molecule of the present invention, each polynucleotide sequence encoding each polypeptide chain being under the control of a different promoter.

Multispecific proteins in accordance with the present invention are expressed at good levels, based on host cells. Thus, the properties of antibodies and/or their fragments appear to be optimized and facilitate commercial production.

Advantageously, when the polyspecific antibody molecules of the present invention minimize the amount of aggregation observed after purification and maximize the amount of monomer in construct formulations at pharmaceutical concentrations, for example, the monomer may be present as 50%, 60%, 70%, or 75% or more, eg 80% or 90% or more, eg 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more of total protein. In one example, a purified sample of a multispecific antibody molecule of the present invention remains more than 98% or 99% monomeric after 28 days of storage at 4°C. In one example, a purified sample of a 5 mg/mL polyspecific antibody molecule of the present invention in phosphate buffered saline (PBS) remains more than 98% monomeric after 28 days at 4°C.

The antibody molecules of the present invention, and compositions comprising them, are useful in the treatment, for example, in the treatment and/or prevention of a disease state.

The present invention also provides a pharmaceutical or diagnostic composition comprising an antibody molecule of the present invention in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. Accordingly, the use of an antibody of the present invention in the treatment and manufacture of a medicament is contemplated, in particular for the indication disclosed herein.

The composition will usually be supplied as part of a sterile pharmaceutical composition, which will typically include a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable adjuvant.

The present invention also provides a method for preparing a pharmaceutical or diagnostic composition comprising adding and mixing an antibody molecule of the present invention together with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

The antibody molecule may be the sole active ingredient in the pharmaceutical or diagnostic composition, or may be supplemented with other active ingredients, including other antibody ingredients, such as anti-IL-1β, T-cell, IFNγ, or LPS antibodies, or non-antibody ingredients, such as xanthines. Other suitable active ingredients include antibodies capable of inducing tolerance, such as antibodies against CD3 or CD4.

In a further embodiment of the invention, an antibody, fragment or composition according to the present invention is used in combination with an additional pharmaceutically active agent.

Accordingly, when the pharmaceutical compositions contain a therapeutically effective amount of an antibody molecule of the present invention. The term "therapeutically effective amount" as used herein refers to the amount of a therapeutic agent necessary to treat, ameliorate, or prevent the target disease or condition, or to achieve a detectable therapeutic or prophylactic effect. For any antibody, a therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs, or primates. An animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine appropriate doses and routes of administration to humans.

The exact therapeutically effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the subject's age, weight and sex, diet, time and frequency of administration, drug combination(s), response sensitivity, and tolerance/response to ) therapy(s). This amount can be determined by routine experimentation and is within the judgment of the practitioner. Typically, a therapeutically effective amount will be from 0.01 mg/kg to 50 mg/kg, for example from 0.1 mg/kg to 20 mg/kg. Alternatively, the dose may be from 1 to 500 mg per day, for example 10 to 100, 200, 300 or 400 mg per day. Pharmaceutical compositions may conveniently be presented as unit dosage forms containing a predetermined amount of the active agent of the present invention.

The compositions may be administered to a patient individually, or may be administered in combination (eg, simultaneously, sequentially, or separately) with other agents, drugs, or hormones.

The dose at which an antibody molecule of the present invention is administered depends on the nature of the condition being treated, the degree of inflammation present, and whether the antibody molecule is used prophylactically or to treat an existing condition.

The frequency of dosing will depend on the half-life of the antibody molecule and the duration of its effect. If the antibody molecule has a short half-life (eg, 2 to 10 hours), it may be necessary to administer one or more doses per day. Alternatively, if the antibody molecule has a long half-life (eg, 2 to 15 days), it may only be necessary to dose once a day, once a week, or even once every 1 or 2 months.

The pharmaceutically acceptable carrier must not by itself induce the production of antibodies harmful to the individual receiving the composition and must not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles.

Pharmaceutically acceptable salts may be used, for example salts of inorganic acids such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, adjuvants such as wetting agents or emulsifiers or pH buffering agents may be present in such compositions. Such carriers make it possible to prepare pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by a patient.

Suitable administration forms include those suitable for parenteral administration, eg by injection or infusion, eg by bolus injection or continuous infusion. If the product is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it may contain auxiliary agents such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, to be reconstituted with an appropriate sterile liquid before use.

Once prepared, the compositions of the present invention may be administered directly to a subject. The subjects to be treated may be animals. However, in one or more embodiments, the compositions are adapted for administration to human subjects.

In one embodiment, in the compositions according to the present invention, the pH of the final preparation is not corresponding to the isoelectric point of the antibody or fragment thereof, so if the pH of the preparation is 7, then a pI of 8-9 or higher may be appropriate. Without wishing to be bound by any theory, it is believed that this may ultimately provide an end product with increased stability, eg, the antibody or fragment thereof remains in solution.

Pharmaceutical compositions of this invention may be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, transdermal (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral , topical, sublingual, intravaginal or rectal routes. Needleless syringes may also be used to administer the pharmaceutical compositions of the present invention. Typically, therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection may also be prepared. Preferably, the antibody molecules of the present invention are administered subcutaneously, by inhalation, or topically.

Direct delivery of the compositions will typically be by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or they will be delivered into the interstitial space of the tissue. The compositions may also be administered to the specific tissue of interest. In this case, the treatment regimen may be a single dose regimen or a multiple dose regimen.

It will be appreciated that the active ingredient in the composition will be an antibody molecule. As such, it will be subject to degradation in the gastrointestinal tract. Thus, if the composition is to be administered via the gastrointestinal tract, the composition will need to contain agents that protect the antibody from degradation, but which release the antibody after absorption from the gastrointestinal tract.

A detailed discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

In one embodiment, the composition is provided as a topical preparation, including inhalation.

Suitable inhalation preparations include inhalable powders, metered aerosols containing propellant gases, or inhalable solutions that do not contain propellant gases (such as spray solutions or suspensions). Inhalable powders according to the present invention containing an active substance may consist exclusively of the above-mentioned active substances or a mixture of the above-mentioned active substances with a physiologically acceptable excipient.

These inhalable powders may include monosaccharides (eg glucose or arabinose), disaccharides (eg lactose, sucrose, maltose), oligo- and polysaccharides (eg dextrans), polyalcohols (eg sorbitol, mannitol, xylitol), salts (eg sodium chloride, calcium carbonate) or mixtures thereof with each other. Accordingly, use mono- or disaccharides, lactose or glucose, in particular, but not exclusively, in the form of their hydrates.

Particles for deposition in the lung require a particle size of less than 10 µm, eg 1-9 µm, eg 0.1 to 5 µm, in particular 1 to 5 µm. The particle size of the active agent (such as an antibody or antibody fragment) is of paramount importance.

Propellant gases that can be used to prepare inhalation aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons such as n-propane, n-butane or isobutane and halogenated hydrocarbons such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The aforementioned propellant gases may be used alone or as mixtures thereof.

Particularly suitable propellant gases are halogenated alkane derivatives selected from TG11, TG12, TG134a and TG227. Of the above halogenated hydrocarbons, TG134a (1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

Propellant-containing inhalation aerosols may also contain other ingredients such as co-solvents, stabilizers, surfactants, antioxidants, lubricants, and pH adjusters. All of these ingredients are known in the art.

The propellant-containing inhalation aerosols according to the present invention may contain up to 5% by weight of the active substance. Aerosols according to the present invention contain, for example, 0.002 to 5% w/w, 0.01 to 3% w/w, 0.015 to 2% w/w, 0.1 to 2% w/w, from 0.5 to 2% by weight or from 0.5 to 1% by weight of the active substance.

Alternatively, topical administrations to the lungs can also take place by administering a liquid preparation in the form of a solution or suspension, for example, using a device such as a nebulizer, for example, a nebulizer connected to a compressor (for example, a Pari LC-Jet Plus(R) nebulizer, connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va).

In one embodiment, the drug is provided in the form of discrete ampoules containing a single dose for delivery by nebulization.

In one embodiment, the antibody is provided in lyophilized form, for reconstitution, or alternatively as a suspension composition.

The antibody of the present invention can be delivered dispersed in a solvent, for example, as a solution or suspension. It can be suspended in an appropriate saline solution, such as physiological saline, a pharmacologically acceptable solvent, or a buffered solution. Buffered solutions known in the art may contain 0.05 mg to 0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0 25 mg to 0.30 mg anhydrous citric acid and 0.45 mg to 0.55 mg sodium citrate per ml of water to achieve a pH of approximately 4.0 to 5.0. As mentioned above, a suspension can be prepared, for example, from a lyophilized antibody.

Therapeutic preparations in the form of suspensions or solutions may also contain one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol and glycerin. Solutions or suspensions can be encapsulated in liposomes or biodegradable microspheres. The formulation will typically be provided in a substantially sterile form using sterile manufacturing processes.

These may include manufacturing and filter-sterilizing the buffered solvent used for the formulation, aseptically suspending the antibody in a sterile buffered solvent, and dispersing the drug into sterile vials by methods well known to those of ordinary skill in the art.

The nebulizable formulation of the present invention may be provided, for example, in single dose packs (eg, sealed plastic containers or vials) packaged in foil envelopes. Each vial contains a unit dose volume, eg 2 ml, of a solvent buffer solution.

The antibodies of the present invention are believed to be suitable for nebulization delivery.

It is also contemplated that the antibody of the present invention can be introduced using gene therapy. To achieve this, the DNA sequences encoding the heavy and light chains of the antibody molecule are administered to the patient under the control of the respective DNA components, so that the antibody chains are expressed from the DNA sequences and assembled in situ.

The pathological condition or disorder may, for example, be selected from the group consisting of infections (viral, bacterial, fungal and parasitic), infection-associated endotoxin toxic shock, arthritis such as rheumatoid arthritis, asthma such as severe asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, celiac disease, gallbladder disease, pilonidal disease, peritonitis, psoriasis, vasculitis, surgical adhesions, stroke , type I diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory diseases of the central and peripheral nervous system, such as multiple sclerosis, lupus (eg, systemic lupus erythematosus) and Guillain-Barr syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Graves' disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune diseases, pancreatitis, trauma (surgery), graft versus host disease, graft rejection, disease heart, including ischemic diseases such as myocardial infarction, as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochloridria.

The present invention also provides a multispecific antibody molecule according to the present invention for use in the treatment or prevention of pain, especially pain associated with inflammation.

Thus, a multispecific antibody according to the present invention is provided for use in treatment and methods of treatment using it.

In one embodiment, a method for purifying a multispecific antibody (particularly an antibody or fragment according to the present invention) is provided.

In one embodiment, a method is provided for purifying a multispecific antibody (particularly an antibody or fragment according to the present invention) comprising the steps of: performing anion exchange chromatography in a non-binding mode such that impurities are retained on the column and the antibody is retained in an unbound fraction. This step may, for example, be carried out at a pH of about 6-8.

The method may further include an initial capture step using cation exchange chromatography performed, for example, at a pH of about 4-5.

The process may further include additional chromatographic step(s) to ensure the product and process related impurities are properly removed from the product stream.

The purification process may also include one or more ultrafiltration steps, such as a concentration step and a diafiltration step.

As used above, the term "purified form" is intended to refer to at least 90% purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% by weight or greater purity.

The term "substantially free of endotoxin" is generally intended to refer to an endotoxin content of 1 endotoxin unit per mg of antibody product or less, such as 0.5 or 0.1 endotoxin unit per mg of product.

The term "substantially free of host cell protein or DNA" is generally intended to refer to a host cell protein and/or DNA content of 400 micrograms per mg of antibody product or less, e.g., 100 micrograms per mg or less , in particular 20 micrograms per mg, as appropriate.

The antibody molecule of the present invention can also be used in diagnostics, such as in vivo diagnostics and imaging of disease states.

In one embodiment, a method for selecting a polyspecific protein construct according to the present invention is provided, comprising:

a) determining the yield of a monomeric polyspecific protein when either V ₁ or V ₂ is dsscFv for a particular pair of variable regions;

b) determining the yield of a monomeric polyspecific protein when that of V ₁ or V ₂ that was tested in step (a) is dsFv, for the same particular pair of variable regions; and

c) comparing the monomer yield determined in steps (a) and (b) and selecting the polyspecific protein with the highest monomer yield.

Typically, the "variable region pair" in steps (a) and (b) of this method is a V _H and V _L pair. Typically, V _H and V _L together form an antigen-binding domain, V ₁ or V ₂ . The method thus allows the pairing of V _H and V _L to be tested as both dsscFv and dsFv in the constructs of the present invention, and the most monomeric construct is selected in step (c).

Typically, in steps (a) and (b) of this method, the yield is determined after purification, for example after affinity chromatography. The monomer yield can be determined using any suitable method such as gel permeation chromatography.

"Comprising (comprising)" in the context of the present description is intended to mean including. Where technically feasible, embodiments of the present invention may be combined. Embodiments are described herein as including certain features/elements. The present invention also extends to individual embodiments consisting or essentially consisting of these features/elements.

Technical references such as patents and applications are incorporated herein by reference. Any embodiments specifically and expressly set forth herein may form the basis of a disclaimer, either alone or in combination with one or more additional embodiments.

The present invention is further described by way of illustration only in the following examples, which refer to the accompanying drawings, in which:

Brief description of the drawings

In FIG. 1 shows the Fab-2xdsscFv and Fab-dsscFv-dsFv constructs of the present invention.

In FIG. 2 shows SDS-PAGE analysis of protein-G purified, HEK293-expressed Fab-1xdsscFv, 1xscFv, and Fab-2xdsscFv proteins.

In FIG. 3 shows G3000 analysis of SE-HPLCs of protein-G purified, HEK293-expressed Fab-1xdsscFv, 1xscFv, and Fab-2xdsscFv proteins.

In FIG. 4 shows SDS-PAGE analysis of purified HEK293-expressed Fab-2xdsscFv#3 proteins.

In FIG. 5 shows S200 SE-HPLC analysis of purified HEK293-expressed Fab-2xdsscFv#3 proteins.

In FIG. 6 shows SDS-PAGE analysis of various protein-G purified Fab-dsscFv-dsFv constructs of the present invention. (A) Fab-dsscFv-dsscFv samples. (B) Fab-dsscFv-dsFv samples.

In FIG. 7 shows G3000 SE-HPLC analysis of protein-G purified Fab-dssFv-dsFv constructs of the present invention.

In FIG. 8 shows SDS-PAGE analysis of various constructs in accordance with the present invention under reducing conditions.

In FIG. 9 shows the G3000 SE-HPLC time dependence analysis of the Fab-1xdsscFv-1xscFv and Fab-2xdsscFv monomer formats.

In FIG. 10 shows SDS-PAGE analysis of protein-G purified, EXPiHEK expressed Fab-2xscFv and Fab-2xdsscFv formats.

EXAMPLES

Antibody fragments against antigen 1 are designated #1.

Antibody fragments against antigen 2 are designated #2.

Antibody fragments against antigen 3 are designated #3.

Antibody fragments against antigen 4 are designated #4.

Example 1: Fab-2xdsscFv

Construction of plasmids for expression in mammalian cells

The expression plasmids Fab#2-(HC)dsscFv#3,(LC)dsscFv#4 and Fab#2(LC)dsscFv#3,(HC)dsscFv#4 (see Fig. 1) were constructed by fusing scFv#3 and scFv#4 with the C-terminus of the human kappa constant region of light chain Km3 allotype #2 using the flexible linker SGGGGSGGGGS [also referred to here as S, 2xG4S] (SEQ ID NO:2), or by fusion of scFv#3 and scFv#4 to the C-terminus of the CH ₁ constant region of the human gamma-1 heavy chain γ1 isotype #2 using a flexible linker SGGGGTGGGGS [also referred to herein as S, G4T, G4S] (SEQ ID NO:1). In addition, point mutations were introduced in DNA sequences at selected residues in the framework region of both VL#3/VL#4 and VH#3/VH#4. Mutations were introduced (at G44C in the heavy chain and G10°C in the light chain) to create an interchain disulfide bond between the Fv#3 heavy and light chains. Mutations were introduced (at G44C in the heavy chain and Q10°C in the light chain) to create an interchain disulfide bond between the Fv#4 heavy and light chains.

Gene fragments encoding scFv#4 and dsscFv#4 (with vHvL and vLvH orientation) were chemically generated and fused to Fab#2 as described above to create:

Plasmids e1: Light#2-(SGGGGSGGGGS [SEQ ID NO:2])-dsvL#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-dsvH#4;

Plasmids e2: Light#2-(SGGGGSGGGGS [SEQ ID NO:2])-vH#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-vL#4;

Plasmids f2: Light#2-(SGGGGSGGGGS [SEQ ID NO:2])-dsvH#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-dsvL#4;

Plasmids g1: Heavy#2-(SGGGGTGGGGS [SEQ ID NO:1])-vL#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-vH#4;

Plasmids h1: Heavy#2-(SGGGGTGGGGS [SEQ ID NO:1])-dsvL#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-dsvH#4;

Plasmids g2: Heavy#2-(SGGGGTGGGGS [SEQ ID NO:1])-vH#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-vL#4; and

Plasmids h2: Heavy#2-(SGGGGTGGGGS [SEQ ID NO: 1])-dsvH#4-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO: 68])-dsvL#4.

Plasmid pND1 (Fab#2 Heavy-(SGGGTGGGGS SEQ ID NO:1)-dsvH#3-(GGGGSGGGGSGGGGSGGGGS SEQ ID NO:68)-dsvL#3) [plasmid i] was already available. The gene fragment encoding dsHLscFv#3 was excised from pND1 and fused to light chain #2 as described above to create: Light#2-(SGGGGSGGGGS SEQ ID NO:2)-dsvH#3-(GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 68)-dsvL#3 [plasmids j].

All Fab fusion formats were cloned into mammalian expression vectors under the control of the HCMV-MIE promoter and the SV40E polyA sequence.

Expression in HEK293 of Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

HEK293 cells were transfected with the appropriate plasmids using the 293-fectin transfection reagent from Invitrogen according to the manufacturer's instructions. Plasmids were mixed as shown in Table 4 to express different constructs:

Table 4

Antibody design Plasmid used Fab#2-(HC)dsHLscFv#3,(LC)HLscFv#4 1. Plasmid e2
2. Plasmid i Fab#2-(HC)dsHLscFv#3,(LC)dsHLscFv#4 1. Plasmid f2
2. Plasmid i Fab#2-(HC)dsHLscFv#3,(LC)LHscFv#4 1. Plasmid e1
2. Plasmid i Fab#2-(HC)dsHLscFv#3,(LC)dsLHscFv#4 1. Plasmid f1
2. Plasmid i Fab#2-(LC)dsHLscFv#3,(HC)HLscFv#4 1. Plasmid g2
2. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)dsHLscFv#4 1. Plasmid h2
2. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)LHscFv#4 1. Plasmid g1
2. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)dsLHscFv#4 1. Plasmid h1
2. Plasmid j

The ratio of plasmids used for transfections was 1:1. A total of 50 µg of plasmid DNA was incubated with 125 µl of 293-fectin + 4.25 ml of OptiMEM medium for 20 min at room temperature. The mixture was then added to 50 ml of HEK293 cells in suspension at a concentration of 1×10 ⁶ cells/ml and incubated with shaking at 37°C. Supernatants were collected on day 10 by centrifugation at 1500×g to remove cells, and the supernatant was passed through a filter with a pore size of 0.22 μm. Expression level was determined by HPLC using protein-G.

Table 5 shows the results of HPLC analysis using protein-G. As can be seen, the expression levels of all constructs were comparable to each other, spanning the range of 11-23 μg/ml. Constructs marked with an asterisk (*) were expressed using a separate transfection, so the absolute expression levels of LC-scFv#3,HC-scFv#4 cannot be compared with HC-scFv#3,LC-scFv#4.

Table 5

Antibody design Expression level (µg/ml) Fab#2(HC)dsHLscFv#3,(LC)HLscFv#4 23 Fab#2(HC)dsHLscFv#3,(LC)dsHLscFv#4 eighteen Fab#2(HC)dsHLscFv#3,(LC)dsHLscFv#4 21 Fab#2(LC)dsHLscFv#3,(LC)dsLHscFv#4 twenty Fab#2(LC)dsHLscFv#3,(HC)HLscFv#4 13* Fab#2(LC)dsHLscFv#3,(HC)dsHLscFv#4 12* Fab#2(LC)dsHLscFv#3,(HC)LHscFv#4 12* Fab#2(LC)dsHLscFv#3,(HC)dsLHscFv#4 eleven*

Protein-G purification of HEK293-expressed Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

Approximately 50 ml of HEK293 cell supernatants were concentrated ˜25 times to ˜2 ml using centrifugal concentrators with a molecular weight cutoff=10 kDa. The concentrated supernatants were applied to a 1 ml HiTrap Protein-G FF column (GE Healthcare) equilibrated in 20 mM phosphate, 40 mM NaCl, pH 7.4. The column was washed with 20 mM phosphate, 40 mM NaCl, pH 7.4 and the bound material was eluted with 0.1 M glycine/HCl pH 2.7. The elution peak was collected and the pH was adjusted to ~pH 7.0 with 2M Tris/HCl pH 8.5. The pH-adjusted eluates were concentrated and the buffer changed to PBS pH 7.4 using centrifugal concentrators with molecular weight cutoff=10 kDa.

Analysis by SDS-PAGE of protein-G purified, HEK293 expressed Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

Samples (2 μg) were diluted using PBS to a volume of 9.75 μl, to which was added 3.75 μl 4xLDS sample buffer and 1.5 μl 100 mM N-ethylmaleimide (unreduced samples) or 1.5 μl 10xNuPAGE reducing agent ( restored samples). Samples were shaken, incubated at 70 ^° C for 10 min, cooled and centrifuged at 12,500 rpm for 30 seconds. The prepared samples were applied to a 4-20% acrylamide gel in Tris/glycine with SDS, and electrophoresis was performed for ~100 minutes at 125V, constant voltage. The SeeBluePlus2 molecular weight marker (Life Technologies) was used. The gels were stained with Instant Blue protein stain (Expedeon) and decolorized with distilled water.

The expected band sizes after electrophoresis in SDS-PAGE under reducing and non-reducing conditions are shown in Table 6.

Table 6:

Expected band sizes after SDS-PAGE (kDa) (H=heavy chain, L=light chain, +/- reducing agent) -Red +Red -Red +Red Fab-1xdsscFv,1xscFv ~100 H~50 L~50 Fab-2xdsscFv ~100 H~50 L~50

For all proteins, the non-reducing gel should show a band corresponding to M.m. ~100 kDa, while reducing gels should have shown a doublet corresponding to M.m. ~50 kDa, with the same coloration in both bands.

For all Fab-1xdsscFv-1xscFv and Fab-2xdsscFv proteins, SDS-PAGE gels under reducing conditions showed band patterns that indicated correct expression of the constructs in terms of migration position and staining intensity, with a doublet corresponding to M.m. ~50 kDa (Fig. 2B, D). The additional uppermost minor band corresponds to unrecovered full-length protein. As expected, disulfide-linked multimers are observed in the non-reducing gel (FIG. 2A,C), which disappear under reducing conditions (FIG. 2B,D). Minor bands corresponding to M.m. ~50 kDa, in a non-reducing gel (FIG. 2A,C) may correspond to incomplete disulfide bond formation between the heavy and light chains in the Fab fragment.

G3000 analysis of SE-HPLCs of protein-G purified, HEK293 expressed Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

10 μg of purified protein samples (100 μl of 0.1 mg/ml stock solution diluted in PBS) were injected onto a TSK Gel G3000SWXL, 7.8×300 mm column (Tosoh) 3 days after purification and separated using a 200 isocratic gradient mM phosphate pH 7.0 at a rate of 1 ml/min. Signal detection was performed by absorption at 280 nm.

The results are shown in FIG. 3. As can be seen from Figure 3, after protein-G purification, the Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats were 83-89% monomers.

Although all samples have similar monomer levels in the study, those samples containing scFv without Fv disulfide are in dynamic equilibrium. This means that the % monomer determined in the assay for these samples depends on the concentration of the sample, more concentrated samples give a higher % monomer and less concentrated samples give a lower % monomer. In contrast, samples in which both scFvs contain disulfide are stable and are not in dynamic equilibrium. Therefore, the % monomer determined in the study does not change with the concentration of the sample.

Because the monomers and multimers in the Fab-2xdsscFv formats are stable and not in dynamic equilibrium, samples can easily be further purified to increase the % monomer. Purified monomeric samples will also remain monomeric even when the concentration of the construct in a given formulation is increased, thereby making the constructs very suitable for use in pharmaceutical formulations. In contrast, Fab-2xscFv can, after being purified as a monomer, undergo an intermolecular dynamic domain exchange between the vL and vH scFv domains. For this reason, in addition to an increased risk of dimer, trimer, higher order structures and aggregates formation, such molecules are difficult to observe as non-aggregated forms can decompose into monomers after the dilution steps that are used during analytical methods. Therefore, Fab-2xdsscFv bring additional clarity in the analysis of pharmaceutical compositions.

Expression in CHOS of Fab-1xdsscFv-lxscFv and Fab-2xdsscFv formats

CHOS cells were transfected with the appropriate plasmids by 1 L scale electroporation methods. Plasmids were mixed as shown in Table 7 for protein expression. Cultures were grown in CD-CHO medium supplemented with 2 mM GlutaMAX and incubated at 37°C with 8% CO ₂ at 140 rpm for 24 h and then incubated for another 13 days at 32°C. On the 4th day after transfection, sodium butyrate at a final concentration of 3 mm was added to the culture. On day 14 post-transfection, culture supernatants were collected by centrifugation and sterilized through a 0.22 µm filter. Expression titers were measured by HPLC using Protein-G (Table 8).

Table 7:

Antibody design Plasmid used Fab#2-(LC)dsHLscFv#3,(HC)HLscFv#4 3. Plasmid g2
1. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)dsHLscFv#4 3. Plasmid h2
1. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)LHscFv#4 3. Plasmid g1
1. Plasmid j Fab#2-(LC)dsHLscFv#3,(HC)dsLHscFv#4 3. Plasmid h1
1. Plasmid j

Table 8:

Antibody design Expression level (µg/ml) Fab#2-(LC)dsHLscFv#3,(HC)HLscFv#4 fifteen Fab#2-(LC)dsHLscFv#3,(HC)dsHLscFv#4 17 Fab#2-(LC)dsHLscFv#3,(HC)LHscFv#4 eighteen Fab#2-(LC)dsHLscFv#3,(HC)dsLHscFv#4 16

Protein-G purification of CHO-expressed Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

1 L of CHO supernatants was concentrated ˜25-fold to ˜40 ml using an Amicon agitated cuvette with a molecular weight cutoff=10 kDa. Concentrated supernatants were applied to an AKTA Express Purification system with a protein G column and PBS as running buffer. The bound material was eluted using 0.1 M glycine/HCl pH 2.7 and the pH was adjusted to ~pH 7.0 with 2 M Tris/HCl pH 8.5. The eluted material was then concentrated using concentrators with molecular weight cutoff=10 kDa and the resulting concentrate was loaded onto a Superdex (GE Healthcare) gel filtration column with PBS as running buffer. Individual elution peaks were collected and analyzed by size exclusion HPLC to determine the monomer fraction. The final monomeric protein was concentrated to 5 mg/ml in PBS and stored at 4°C.

SDS-PAGE analysis of protein-G purified, CHOS-expressed monomeric fractions of Fab-1xdsscFv-1xscFv and Fab-2xdsscFv formats

Samples (2 μg) were diluted using PBS to a volume of 9.75 μl, to which was added 3.75 μl 4xLDS sample buffer and 1.5 μl 100 mM N-ethylmaleimide (unreduced samples) or 1.5 μl 10xNuPAGE reducing agent ( restored samples). The samples were shaken, incubated at 70°C for 10 min, cooled and centrifuged at 12500 rpm for 30 seconds. The prepared samples were applied to a 4-20% acrylamide gel in Tris/glycine with SDS, and electrophoresis was performed for ~100 minutes at 125V, constant voltage. The molecular weight marker Markl2 (Life Technologies) was used. The gels were stained with Instant Blue protein stain (Expedeon) and decolorized with distilled water.

The expected band sizes after electrophoresis in SDS-PAGE under reducing and non-reducing conditions are shown in Table 9.

Table 9:

Expected band sizes after SDS-PAGE (kDa) (H=heavy chain, L=light chain, +/- reducing agent) -Red +Red -Red +Red Fab-1xdsscFv,1xscFv ~100 H~50 L~50 Fab-2xdsscFv ~100 H~50 L~50

For all proteins, the non-reducing gel should show a band corresponding to M.m. ~100 kDa, while reducing gels should have shown a doublet corresponding to M.m. ~50 kDa, with the same coloration in both bands.

For all Fab-1xdsscFv-1xscFv and Fab-2xdsscFv proteins, SDS-PAGE gels under reducing conditions showed band patterns indicating that the constructs are monomeric and correctly expressed in terms of migration position and staining intensity, with a doublet corresponding to M.m. ~50 kDa (Fig. 8B,D). An additional uppermost minor band corresponds to unreduced full-length protein (FIG. 8B,D). For all proteins, the non-reducing gel showed a band corresponding to M.m. ~100 kDa, indicating a full-length protein (Fig. 8A, a). Minor bands corresponding to M.m. ~50 kDa, in a non-reducing gel (FIG. 8A, b) may correspond to incomplete disulfide bond formation between the heavy and light chains in the Fab fragment.

G3000 SE-HPLC Time Dependence Analysis of Fab-1xdsscFv-lxscFv and Fab-2xdsscFv Monomer Formats

5 mg/ml of purified antibody format monomer was stored at 4° C. in PBS and analyzed on day 4, day 14 and day 28 after purification. Samples were diluted in PBS to 10 μg and applied to a TSK Gel G3000SWXL, 7.8 x 300 mm column (Tosoh) and separated using an isocratic gradient of 200 mM phosphate pH 7.0 at a rate of 1 ml/min. Signal detection was performed by absorption at 280 nm. The results are shown in FIG. 9. On day 4 after purification, analysis of Fab-2xdsscFv showed that most of the protein was monomeric at the beginning of the experiment (solid lines) and remained monomeric and stable over time, with <1% defined as multimers (dimers, trimers , higher-order structures, and large aggregates). On the other hand, for Fab-1xdsscFv-1xscFv, more multimerization events were detected, which was observed to increase linearly over time (dashed lines). Indeed, the occurrence of multimerization was more pronounced in the case of formats containing scFv not stabilized with a disulfide bond in the V _L /V _H orientation. However, it appears that formats that contain scFv without Fv disulfide are in dynamic equilibrium and are more prone to multimerization upon storage than scFv that are stabilized with a disulfide bond. Indeed, formats that contain scFvs, both of which are stabilized by a disulfide bond, have been shown to remain monomeric even when the protein concentration in a given preparation is increased. This makes constructs in which both scFvs contain Fv disulfide ideally suited for pharmaceutical applications where it is critical that the risk of higher order multimers or large aggregates being formed during storage periods is negligible.

Example 2: Fab-2xdsscFv (bivalent with two identical dsscFv)

Construction of plasmids for expression in mammalian cells

Plasmids for the expression of Fab#2-(HC)dsscFv#3,(LC)dsscFv#3 (see Figure 4) were constructed by fusing sscFv#3 to the C-terminus of the human kappa constant region of the light chain Km3 allotype #2 using a flexible linker SGGGGSGGGGS [also referred to here as S, 2xG4S] (SEQ ID NO: 2), or by fusion of scFv#3 to the CH ₁ C-terminus of the heavy chain γ1 isotype γ1 human gamma-1 constant region #2 using a flexible linker SGGGGTGGGGS [also referred to here as S, G4T, G4S] (SEQ ID NO:1). Point mutations were introduced in DNA sequences at selected residues in the framework region of both vL#3/vL#1 and vH#3/vH#1. Mutations were introduced (at G44C in the heavy chain and G10°C in the light chain) to create an interchain disulfide bond between the Fv#3 heavy and light chains.

Plasmid pND1 (Fab#2 Heavy-(SGGGGTGGGGS [SEQ ID NO:1)-dsvH# 3-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-dsvL#3) [plasmid i] was already available. The gene fragment encoding dsHLscFv#3 was excised from pND1 and fused to light chain #2 as described above to create: Light#2-(SGGGGSGGGGS [SEQ ID NO:2])-dsvH#3-(GGGGSGGGGSGGGGSGGGGS [SEQ ID NO:68])-dsvL#3 [plasmids j].

Fab fusion formats were cloned into vectors for expression in mammalian cells under the control of the HCMV-MIE promoter and the SV40E polyA sequence.

Expression in HEK293 Fab-2xdsscFv#3

HEK293 cells were transfected with the appropriate plasmids using the 293-fectin transfection reagent from Invitrogen according to the manufacturer's instructions. Plasmids were mixed as shown in Table 10 for protein expression:

Table 10

Antibody design Plasmid used Fab#2-(HC)dsHLscFv#3,(LC)dsHLscFv#3 1. Plasmid j
2. Plasmid i

The ratio of plasmids used for transfections was 1:1. A total of 50 µg of plasmid DNA was incubated with 125 µl of 293-fectin + 4.25 ml of OptiMEM medium for 20 min at room temperature. The mixture was then added to 50 ml of HEK293 cells in suspension at a concentration of 1×10 ⁶ cells/ml and incubated with shaking at 37°C. Supernatants were collected on day 10 by centrifugation at 1500×g to remove cells, and the supernatant was passed through a filter with a pore size of 0.22 μm. Expression level was determined by HPLC using protein-G. Table 11 shows the results of HPLC analysis using protein-G. The expression level was 2 μg/ml.

Table 11

Antibody design Expression level (µg/ml) Fab#2-(HC)dsHLscFv#3,(LC)dsHLscFv#3 twenty

Purification of HEK293-expressed Fab-2xdsscFv#3

Approximately 50 ml of HEK293 cell supernatants were concentrated ˜25 times to ˜2 ml using centrifugal concentrators with a molecular weight cutoff=10 kDa. The concentrated supernatants were purified and further concentrated, and the buffer was changed to PBS pH 7.4 using centrifugal concentrators with molecular weight cutoff=10 kDa.

Analysis by electrophoresis in SDS-PAGE purified using protein-G expressed in HEK293 Fab-2xdsscFv#3

Samples (2 μg) were diluted using PBS to a volume of 9.75 μl, to which was added 3.75 μl 4xLDS sample buffer and 1.5 μl 100 mM N-ethylmaleimide (unreduced samples) or 1.5 μl 10xNuPAGE reducing agent ( restored samples). The samples were shaken, incubated at 70°C for 10 min, cooled and centrifuged at 12500 rpm for 30 seconds. The prepared samples were applied to a 4-20% acrylamide gel in Tris/glycine with SDS, and electrophoresis was performed for ~100 minutes at 125V, constant voltage. The SeeBluePlus2 molecular weight marker (Life Technologies) was used. The gels were stained with Instant Blue protein stain (Expedeon) and decolorized with distilled water. The expected band sizes after electrophoresis in SDS-PAGE under reducing and non-reducing conditions are shown in Table 12.

Table 12:

Expected band sizes after SDS-PAGE (kDa) (H=heavy chain, L=light chain, +/- reducing agent) -Red +Red Fab-2xdsscFv#3 ~100 H~50 L~50

The non-reducing gel should have shown a band corresponding to M.m. ~100 kDa, while the reduction gel should have shown a doublet corresponding to M.m. ~50 kDa, with the same coloration in both bands.

SDS-PAGE gels under reducing conditions showed band patterns that indicated correct expression of the constructs in terms of migration position and staining intensity, with a doublet corresponding to M.m. ~50 kDa (FIG. 4B), however, some additional minor species were also observed, possibly due to a sub-optimal purification scheme, in contrast to the standard protein-G purification method that is typically used to purify Fab. In the non-reducing gel, disulfide-linked multimers are observed (FIG. 4A), which disappear under reducing conditions (FIG. 4B).

Analysis with S200 SE-HPLC Purified HEK293 Expressed Fab-2xdsscFv#3

10 μg of purified protein samples (100 μl 0.1 mg/ml stock solution diluted in PBS) were injected onto a Superdex 200 10/300 GL Tricorn column (GE Healthcare) 3 days after purification and separated using a PBS pH 7 isocratic gradient ,4 at a rate of 1 ml/min, with continuous absorption detection at 280 nm. The results are shown in FIG. 5. As can be seen from FIG. 5, after purification, the Fab-2xdsscFv#3 format was 81% monomer.

Example 3: Fab-(HC)dsscFv-(LC)dsFv

Construction of plasmids Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and Fab#2(HC)dsscFv#3(LC)dsFv#1 (with LC-vL linkage) for expression in mammalian cells

Fab#2 fusion proteins for expression of Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL linkage) (see FIG. 6) were constructed by fusing either dsscFv#1 (dsvH-4xG4S-dsvL) or dsvL#1 to the C-terminus of the human kappa constant region of light chain Km3 allotype #2 using a flexible linker SGGGGSGGGGSGGGGS (SEQ ID NO:69 ), for creating:

Light#2-(SGGGGSGGGGSGGGGS [SEQ ID NO:69])-dsscFv#1 [plasmids b] and

Light#2-(SGGGGSGGGGSGGGGS [SEQ ID NO:69])-dsvL#1 [plasmid c]; and

by fusing HLdsscFv#3 (dsvH-4xG4S-dsvL) to the CH ₁ C-terminus of the human gamma-1 constant region of heavy chain γ1 isotype #2 using a flexible linker SGGGGSGGGGTGGGGS (SEQ ID NO: 70), to create:

Heavy#2-(SGGGGSGGGGTGGGGS [SEQ ID NO:70])-dsscFv#3 [plasmids a].

Point mutations (at G44C in the heavy chain and G10°C in the light chain) were introduced in DNA sequences at selected residues in the framework region vL#3, vH#3, vL#1 and vH#1 to create an interstrand disulfide bond between heavy and light chains Fv#3 and Fv#1. Fab heavy and light chain fusion genes were cloned into vectors for expression in mammalian cells under the control of the HCMV-MIE promoter and the SV40E polyA sequence. Genes encoding:

Heavy#2-(SGGGGSGGGGTGGGGS [SEQ ID NO:70])-dsscFv#3 [plasmid a] and

free domain dsvH#1 [plasmid d]

were obtained chemically and cloned separately into vectors for expression in mammalian cells under the control of the HCMV-MIE promoter and the SV40E polyA sequence.

The genes encoding dsscFv#1 and dsvL#1 were chemically generated and fused to the C-terminus of light chain #2 to create:

Light#2-(SGGGGSGGGGSGGGGS [SEQ ID NO:69])-dsscFv#1 [plasmid b] and

Light#2- (SGGGGSGGGGSGGGGS [SEQ ID NO:69])-dsvL#1 [plasmid c]

and the complete constructs were cloned into a mammalian expression vector under the control of the HCMV-MIE promoter and the SV40E polyA sequence.

Expression in HEK293 :

Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and

Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL link)

HEK293 cells were transfected with the appropriate plasmids using the 293-fectin transfection reagent from Invitrogen according to the manufacturer's instructions. Plasmids were mixed as follows to express different constructs as shown in Table 13 below:

Table 13

Antibody design Plasmid used Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 1. Plasmid a
2. Plasmid b Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL link) 1. Plasmid a
2. Plasmid c
3. Plasmid d

For the combination of two plasmids, the ratio of plasmids used for transfections was 1:1, while for the combination of 3 plasmids, the ratio was 1:1:1. A total of 50 µg of plasmid DNA was incubated with 125 µl of 293-fectin + 4.25 ml of OptiMEM medium for 20 min at room temperature. The mixture was then added to 50 ml of HEK293 cells in suspension at a concentration of 1×10 ⁶ cells/ml and incubated with shaking at 37°C. Supernatants were collected on day 10 by centrifugation at 1500xg to remove cells, and the supernatant was passed through a filter with a pore size of 0.22 μm. Expression level was determined by HPLC using protein-G.

The results are shown in Table 14. As can be seen, the expression levels of both constructs were comparable to each other (16-18 μg/ml). There have been reports in the literature that expression of Fv fragments that lack either a linker between vL and vH or a dimerization motif to bring vL and vH closer together have significantly lower levels of expression than linked Fvs. Surprisingly, this was not noted in these data, in which there were no significant differences noted between the level of expression of each type of construct.

Table 14

Antibody design Expression level (µg/ml) Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 eighteen Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL link) 16

Protein-G purification of HEK293-expressed Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL linkage) )

Approximately 50 ml of HEK293 cell supernatants were concentrated ˜25 times to ˜2 ml using centrifugal concentrators with a molecular weight cutoff=10 kDa. The concentrated supernatants were applied to a 1 ml HiTrap Protein-G FF column (GE Healthcare) equilibrated in 20 mM phosphate, 40 mM NaCl, pH 7.4. The column was washed with 20 mM phosphate, 40 mM NaCl, pH 7.4, and the bound material was eluted using 0.1 M glycine/HCl pH 2.7. The elution peak was collected and the pH was adjusted to ~pH 7.0 with 2M Tris/HCl pH 8.5. The pH-adjusted eluates were concentrated and the buffer was changed to PBS pH 7.4 using centrifugal concentrators with molecular weight cutoff=10 kDa.

Analysis by SDS-PAGE of Purified Protein-G Expressed in HEK293 Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and Fab#2-(HC)dsscFv#3-(LC) dsFv#1 (with LC-vL link)

Samples (2 μg) were diluted using PBS to a volume of 9.75 μl, to which was added 3.75 μl 4xLDS sample buffer and 1.5 μl 100 mM N-ethylmaleimide (unreduced samples) or 1.5 μl 10xNuPAGE reducing agent ( restored samples). The samples were shaken, incubated at 70°C for 10 min, cooled and centrifuged at 12500 rpm for 30 seconds. The prepared samples were applied to a 4-20% acrylamide gel in Tris/glycine with SDS, and electrophoresis was performed for ~100 minutes at 125V, constant voltage. The SeeBluePlus2 molecular weight marker (Life Technologies) was used. The gels were stained with Instant Blue protein stain (Expedeon) and decolorized with distilled water.

The results are shown in FIG. 6. For Fab#2-(HC)dsscFv#3-(LC)dsscFv#1, the non-reducing gel should have shown a band corresponding to M.m. ~100 kDa, while the reduction gel should have shown a doublet corresponding to M.m. ~50 kDa, with approximately the same coloration in both bands. For Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL bond), the non-reducing gel should have shown a band corresponding to M.m. ~100 kDa, while the reduction gel should have shown 3 bands corresponding to M.m. ~50, ~36 and ~13 kDa, with approximately 3:2:1 staining from top to bottom band.

SDS-PAGE gels under reducing conditions showed band patterns that indicated correct expression of the constructs in terms of both migration position and stain intensity. SDS-PAGE gels under non-reducing conditions showed significant >200 kDa band patterns for Fab-2xdsscFv, consistent with multimerization (FIG. 6A, lane 2), but fewer high molecular weight molecules were noted in the Fab-dsscFv-dsFv sample (FIG. 6B). , track 2).

G3000 Analysis of Protein-G Purified SE-HPLCs Expressed in HEK293 Fab#2-(HC)dsscFv#3-(LC)dsscFv#1 and Fab#2-(HC)dsscFv#3-(LC)dsFv #1 (with LC-vL connection)

10 μg of purified protein samples (100 μl of 0.1 mg/ml stock solution diluted in PBS) were injected onto a TSK Gel G3000SWXL, 7.8×300 mm column (Tosoh) 3 days after purification and separated using a 200 isocratic gradient mM phosphate pH 7.0 at a rate of 1 ml/min. Signal detection was performed by absorption at 280 nm.

The results are shown in FIG. 7. After protein-G purification, Fab#2-(HC)dsscFv#3-(LC)dsFv#1 (with LC-vL bond) was 91% monomer while Fab#2-(HC)dsscFv# 3-(LC)dsscFv#1 was 30% monomer.

dsscFv#1 is known to be particularly prone to multimerization. Given that the multimers are physically linked by a dsscFv linker (vL1#1 or vH#1 is paired with vH#1 or vL1#1 from a different polypeptide chain), when replacing dsscFv#1 with dsFv#1 (which does not contain the scFv linker), a significant increase in the percentage of monomer produced was observed.

Thus, it will be apparent to one skilled in the art that, depending on the tendency of dsscFv to multimerize, it would be advantageous in some cases to use dsFv instead of dsscFv.

Example 4 Biacore Affinity and Demonstration of Co-Binding of Target Antigens

Affinities and kinetic parameters for Fab#2-(HC)dsHLscFv#3(LC)dsHLscFv#4 interactions were determined by surface plasmon resonance (SPR) conducted on a BIAcore T100 or BIAcore 3000 using CM5 sensor chips (GE Healthcare Bio-Sciences AB) and running buffer HBS-EP (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA 0.05% v/v P20 surfactant). All experiments were carried out at 25°C. Antibody samples were captured onto the surface of the sensor chip using either a human F(ab') ₂ specific goat Fab (Jackson ImmunoResearch) or a proprietary anti-human CH ₁ monoclonal antibody. Covalent immobilization of the captured antibody was achieved by standard amine coupling chemistry to a level of 6000-7000 response units (RU). Antigen #2, #3 or #4 was titrated separately on the captured antibody. Each assay cycle consisted of a first capture of an antibody sample using a 1 min injection, to an association phase consisting of a 3 min antigen injection, after which dissociation was observed for 10 min. After each cycle, the capture surface was regenerated with 2 x 1 min injections of 40 mM HCl followed by a 30 sec injection of 5 mM NaOH. The flow rates used were 10 µl min ^-1 for capture, 30 µl min ^-1 for the association and dissociation phases, and 10 µl min ^-1 for regeneration. Kinetic parameters were determined by simultaneous complex fitting of the obtained sensograms to the standard binding model in a 1:1 ratio using the BIAcore T100 Evaluation v2.0.1 or BIAcore 3000 BIAEvaluation v3.2 software. The results are shown in Table 15. The antibody showed the expected affinities in the pM-nM range for the tested antigens.

Table 15:

analyte ka(1/Msec) kd(1/sec) KD(M) KD(pM) Antigen#2 5.06e+06 3.80e-05 7.51e-12 7.51 Antigen#3 5.54e+06 6.43e-05 1.16e-11 11.6 Antigen#4 7.75e+04 1.35e-04 1.74e-09 1740

The ability of Fab#2-(HC)dsHLscFv#3(LC)dsHLscFv#4 to simultaneously bind to all 3 antigens was assessed by capturing a trispecific antibody on a sensor chip using immobilized anti-human IgG F(ab') ₂ . Each antigen or antigen #2, #3 and #4 mixed solution was titrated against the captured antibody over 3 min injections. The binding responses observed for the independent injections and the pooled responses are shown in Table 16. The binding response for the pooled antigens #2/#3/#4 solution was equal to the sum of the responses for the independent injections. This confirms that Fab#2-(HC)dsHLscFv#3(LC)dsHLscFv#4 is capable of binding all 3 tested antigens simultaneously.

Table 16:

analyte Binding (RU) Antigen#2 58 Antigen#3 38 Antigen#4 47 Antigen#2+#3+#4 130(143)

Example 5 Comparison of Monomer Yield Between Fab-2xscFv and Fab-2xdsscFv Formats

EXpiHEK cells were transfected with the appropriate plasmids by electroporation techniques at a 50 ml scale. Plasmids were mixed as shown in Table 17 for protein expression. Cultures were grown in EXpiHEK expression medium and incubated at 37°C with 8% CO ₂ at 120 rpm for 16-18 h before adding enhancer 1 and 2. Cultures were then incubated for another 4 days at 37°C. The culture supernatant was collected by centrifugation and sterilized by passing through a filter with a pore size of 0.22 μm. Expression titers were measured by HPLC using Protein-G (Table 18).

Table 17:

Antibody design Plasmid used Fab#4-(LC)HLscFv#5,(HC)HLscFv#6 1. Plasmid k1
2. Plasmid l1 Fab#4-(LC)dsHLscFv#5,(HC)dsHLscFv#6 1. Plasmid k2
2. Plasmid l2 Fab#4-(LC)HLscFv#7,(HC)HLscFv#8 1. Plasmid m1
2. Plasmid n1 Fab#4-(LC)dsHLscFv#7,(HC)dsHLscFv#8 1. Plasmid m2
2. Plasmid n2 Fab#4-(LC)HLscFv#9,(HC)LHscFv#10 1. Plasmid o1
2. Plasmid p1 Fab#4-(LC)dsHLscFv#9,(HC)dsLHscFv#10 1. Plasmid o2
2. Plasmid p2 Fab#4-(LC)HLscFv#7,(HC)LHscFv#10 1. Plasmid q1
2. Plasmid r1 Fab#4-(LC)dsHLscFv#7,(HC)dsLHscFv#10 1. Plasmid q2
2. Plasmid r2

Table 18:

Antibody design Expression (µg/ml) Fab#4-(LC)HLscFv#5,(HC)HLscFv#6 62 Fab#4-(LC)dsHLscFv#5,(HC)dsHLscFv#6 31 Fab#4-(LC)HLscFv#7,(HC)HLscFv#8 195 Fab#4-(LC)dsHLscFv#7,(HC)dsHLscFv#8 34 Fab#4-(LC)HLscFv#9,(HC)LHscFv#10 298 Fab#4-(LC)dsHLscFv#9,(HC)dsLHscFv#10 55 Fab#4-(LC)HLscFv#7,(HC)LHscFv#10 226 Fab#4-(LC)dsHLscFv#7,(HC)dsLHscFv#10 58

Protein-G purification of EXPiHEK-expressed Fab-2xscFvFv and Fab-2xdsscFv formats

The supernatants were concentrated ˜25 times to ˜2 ml using centrifuge concentrators with a molecular weight cutoff=10 kDa. Concentrated supernatants were purified by protein-G HPLC using phosphate buffer, pH 7.4. The bound material was eluted using 0.1 M glycine/HCl pH 2.7 and the pH was adjusted to ~pH 7.0 with 2 M Tris/HCl pH 8.5. The eluted material was concentrated using concentrators with a molecular weight cutoff=10 kDa and the buffer was changed to PBS. The purified protein was concentrated to ~4-5 mg/mL in PBS and stored at 4 ^° C.

SDS-PAGE analysis of protein-G purified, EXPiHEK expressed Fab-2xscFv and Fab-2xdsscFv formats

Samples (2 μg) were diluted using PBS to a volume of 9.75 μl, to which was added 3.75 μl 4xLDS sample buffer and 1.5 μl 100 mM N-ethylmaleimide (unreduced samples) or 1.5 μl 10xNuPAGE reducing agent ( restored samples). The samples were shaken, incubated at 70°C for 10 min, cooled and centrifuged at 12500 rpm for 30 seconds. The prepared samples were applied to a 4-20% acrylamide gel in Tris/glycine with SDS, and electrophoresis was performed for ~100 minutes at 125V, constant voltage. The Seeblue2 molecular weight marker (Life Technologies) was used. The gels were stained with Instant Blue protein stain (Expedeon) and decolorized with distilled water.

The expected band sizes after electrophoresis in SDS-PAGE under reducing and non-reducing conditions are shown in Table 19.

Table 19:

Expected band sizes after SDS-PAGE (kDa) (H=heavy chain, L=light chain, +/- reducing agent) -Red +Red -Red +Red Fab-2xscFv ~100 H~50 L~50 Fab-2xdsscFv ~100 H~50 L~50

For all proteins, the non-reducing gel should show a band corresponding to M.m. ~100 kDa, while reducing gels should have shown a doublet corresponding to M.m. ~50 kDa, with the same coloration in both bands.

For all Fab-2xscFv and Fab-2xdsscFv proteins, reducing SDS-PAGE gels showed band patterns that indicated correct expression of the constructs in terms of migration position and staining intensity, with a doublet corresponding to M.m. ~50 kDa (Fig. 10B, a). An additional uppermost minor band corresponds to unreduced full-length protein (Fig. 10B, b). For all proteins, the non-reducing gel showed a band corresponding to M.m. ~130 kDa, indicating a full-length protein (Fig. 10A, a). Bands corresponding to M.m. ~50 kDa, in a non-reducing gel (FIG. 10A, b) may correspond to incomplete disulfide bond formation between the heavy and light chains in the Fab fragment.

G3000 SE-HPLC analysis of Fab-2xscFv and Fab-2xdsscFv formats

Purified antibody proteins at a concentration of ~5 mg/ml were stored at 4°C in PBS for 24 hours before analysis. Samples equivalent to a concentration of 25 μg were applied to a TSK Gel G3000SWXL, 7.8×300 mm column (Tosoh) and separated using an isocratic gradient of 200 mM phosphate pH 7.0 at a rate of 1 ml/min. Signal detection was performed by absorption at 280 nm. The results are shown in Table 20. Analysis showed that all Fab-2xdsscFv proteins were >90% monomeric. On the other hand, for all Fab-2xscFv, more multimerizations were found. Clearly, formats that contain scFv with Fv disulfide are more monomeric than scFv without Fv disulfide. This points to the advantage of using scFv formats that contain Fv disulfides in terms of selecting therapeutic molecules with stable properties as well as characteristics that would be beneficial in the manufacturing process.

Table 20:

Format % monomer Fab#4-(LC)HLscFv#5,(HC)HLscFv#6 51.3 Fab#4-(LC)dsHLscFv#5,(HC)dsHLscFv#6 96.8 Fab#4-(LC)HLscFv#7,(HC)HLscFv#8 80.8 Fab#4-(LC)dsHLscFv#7,(HC)dsHLscFv#8 98.8 Fab#4-(LC)HLscFv#9,(HC)LHscFv#10 84.9 Fab#4-(LC)dsHLscFv#9,(HC)dsLHscFv#10 94.3 Fab#4-(LC)HLscFv#7,(HC)LHscFv#10 53.2 Fab#4-(LC)dsHLscFv#7,(HC)dsLHscFv#10 90.8

--->

SEQUENCE LIST

<110> UCB Biopharma SPRL

<120> Multispecific antibody constructs

<130> G0228_WO

<160> 80

<170> Patent in version 3.5

<210> 1

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 1

Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10

<210> 2

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Peptide linker

<400> 2

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 3

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 3

Asp Lys Thr His Thr Cys Ala Ala

fifteen

<210> 4

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 4

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10

<210> 5

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 5

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro Pro Cys

1 5 10 15

ProAla

<210> 6

<211> 25

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 6

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Thr Cys Pro Pro Cys

1 5 10 15

Pro Ala Thr Cys Pro Pro Cys Pro Ala

20 25

<210> 7

<211> 30

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 7

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys Pro Thr Leu

1 5 10 15

Tyr Asn Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr

20 25 30

<210> 8

<211> 31

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 8

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Gly Lys Pro Thr His

1 5 10 15

Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr

20 25 30

<210> 9

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 9

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

1 5 10 15

<210> 10

<211> 26

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 10

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

1 5 10 15

Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala

20 25

<210> 11

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Articulated linker

<400> 11

Asp Lys Thr His Thr Cys Pro Ser Cys Pro Ala

1 5 10

<210> 12

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 12

Ser Gly Gly Gly Gly Ser Glu

fifteen

<210> 13

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 13

Asp Lys Thr His Thr Ser

fifteen

<210> 14

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 14

Ser Gly Gly Gly Gly Ser

fifteen

<210> 15

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 15

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 16

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 16

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 17

<211> 21

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 17

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Gly Ser

twenty

<210> 18

<211> 26

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 18

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 19

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 19

Ala Ala Ala Gly Ser Gly Gly Ala Ser Ala Ser

1 5 10

<210> 20

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<220>

<221> different sign

<222>(7)..(7)

<223> Xaa may be a naturally occurring amino acid.

<400> 20

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

1 5 10 15

<210> 21

<211> 21

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<220>

<221> different sign

<222>(7)..(7)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (12)..(12)

<223> Xaa may be a naturally occurring amino acid.

<400> 21

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Gly Ala Ser Ala Ser

twenty

<210> 22

<211> 26

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<220>

<221> different sign

<222>(7)..(7)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (12)..(12)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (17)..(17)

<223> Xaa may be a naturally occurring amino acid.

<400> 22

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

20 25

<210> 23

<211> 31

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<220>

<221> different sign

<222>(7)..(7)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (12)..(12)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (17)..(17)

<223> Xaa may be a naturally occurring amino acid.

<220>

<221> different sign

<222> (22)..(22)

<223> Xaa may be a naturally occurring amino acid.

<400> 23

Ala Ala Ala Gly Ser Gly Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser

1 5 10 15

Xaa Gly Gly Gly Ser Xaa Gly Gly Gly Ser Gly Ala Ser Ala Ser

20 25 30

<210> 24

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<220>

<221> different sign

<222>(7)..(7)

<223> Xaa may be a naturally occurring amino acid.

<400> 24

Ala Ala Ala Gly Ser Gly Xaa Ser Gly Ala Ser Ala Ser

1 5 10

<210> 25

<211> 28

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 25

Pro Gly Gly Asn Arg Gly Thr Thr Thr Thr Thr Arg Arg Pro Ala Thr Thr

1 5 10 15

Thr Gly Ser Ser Pro Gly Pro Thr Gln Ser His Tyr

20 25

<210> 26

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 26

Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr

1 5 10

<210> 27

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 27

Ala Thr Thr Thr Gly Ser

fifteen

<210> 28

<211> 21

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 28

Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Ser Pro Pro Ser Lys Glu

1 5 10 15

Ser His Lys Ser Pro

twenty

<210> 29

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 29

Gly Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

1 5 10 15

<210> 30

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 30

Gly Gly Gly Gly Ile Ala Pro Ser Met Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 31

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 31

Gly Gly Gly Gly Lys Val Glu Gly Ala Gly Gly Gly Gly Gly Ser

1 5 10 15

<210> 32

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 32

Gly Gly Gly Gly Ser Met Lys Ser His Asp Gly Gly Gly Gly Ser

1 5 10 15

<210> 33

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 33

Gly Gly Gly Gly Asn Leu Ile Thr Ile Val Gly Gly Gly Gly Ser

1 5 10 15

<210> 34

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 34

Gly Gly Gly Gly Val Val Pro Ser Leu Pro Gly Gly Gly Gly Ser

1 5 10 15

<210> 35

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 35

Gly Gly Glu Lys Ser Ile Pro Gly Gly Gly Gly Ser

1 5 10

<210> 36

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 36

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

1 5 10 15

ArgPro

<210> 37

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 37

Tyr Pro Arg Ser Ile Tyr Ile Arg Arg Arg His Pro Ser Pro Ser Leu

1 5 10 15

Thr Thr

<210> 38

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 38

Thr Pro Ser His Leu Ser His Ile Leu Pro Ser Phe Gly Leu Pro Thr

1 5 10 15

Phe Asn

<210> 39

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 39

Arg Pro Val Ser Pro Phe Thr Phe Pro Arg Leu Ser Asn Ser Trp Leu

1 5 10 15

ProAla

<210> 40

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 40

Ser Pro Ala Ala His Phe Pro Arg Ser Ile Pro Arg Pro Gly Pro Ile

1 5 10 15

Arg Thr

<210> 41

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 41

Ala Pro Gly Pro Ser Ala Pro Ser His Arg Ser Leu Pro Ser Arg Ala

1 5 10 15

Phe Gly

<210> 42

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 42

Pro Arg Asn Ser Ile His Phe Leu His Pro Leu Leu Val Ala Pro Leu

1 5 10 15

Gly Ala

<210> 43

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 43

Met Pro Ser Leu Ser Gly Val Leu Gln Val Arg Tyr Leu Ser Pro Pro

1 5 10 15

Asp Leu

<210> 44

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 44

Ser Pro Gln Tyr Pro Ser Pro Leu Thr Leu Thr Leu Pro Pro His Pro

1 5 10 15

Ser Leu

<210> 45

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 45

Asn Pro Ser Leu Asn Pro Ser Tyr Leu His Arg Ala Pro Ser Arg

1 5 10 15

Ile Ser

<210> 46

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 46

Leu Pro Trp Arg Thr Ser Leu Leu Pro Ser Leu Pro Leu Arg Arg Arg

1 5 10 15

Pro

<210> 47

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 47

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

1 5 10 15

Pro Pro

<210> 48

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 48

Val Pro Pro Ala Pro Val Val Ser Leu Arg Ser Ala His Ala Arg Pro

1 5 10 15

Pro Tyr

<210> 49

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 49

Leu Arg Pro Thr Pro Pro Arg Val Arg Ser Tyr Thr Cys Cys Pro Thr

1 5 10 15

Pro

<210> 50

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 50

Pro Asn Val Ala His Val Leu Pro Leu Leu Thr Val Pro Trp Asp Asn

1 5 10 15

Leu Arg

<210> 51

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 51

Cys Asn Pro Leu Leu Pro Leu Cys Ala Arg Ser Pro Ala Val Arg Thr

1 5 10 15

PhePro

<210> 52

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 52

Gly Ala Pro Ala Pro Ala Ala Pro Ala Pro Ala

1 5 10

<210> 53

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> Rigid linker

<400> 53

Pro Pro Pro Pro

one

<210> 54

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 54

Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp

1 5 10

<210> 55

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 55

Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10

<210> 56

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 56

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Gly Asp

1 5 10 15

<210> 57

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 57

Gln Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Glu Asp Asp Glu

twenty

<210> 58

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 58

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val

twenty

<210> 59

<211> 21

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 59

Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp

1 5 10 15

Gly Arg Ser Val Lys

twenty

<210> 60

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 60

Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 61

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 61

Arg Leu Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 62

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 62

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp

1 5 10 15

<210> 63

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 63

Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp

1 5 10 15

<210> 64

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 64

Arg Leu Met Glu Asp Ile Cys Leu Ala Arg Trp Gly Cys Leu Trp Glu

1 5 10 15

Asp Asp

<210> 65

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 65

Glu Val Arg Ser Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys

1 5 10 15

Pro Leu Arg Gly

twenty

<210> 66

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 66

Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu Thr Ile Cys Phe Glu

1 5 10 15

Arg Ser Glu Gln

twenty

<210> 67

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 67

Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met

1 5 10

<210> 68

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> Albumin binding peptide

<400> 68

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 69

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 69

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 70

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Flexible linker

<400> 70

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10 15

<210> 71

<211> 10

<212> Protein

<213> Artificial

<220>

<223> CDRH1 dAbH1

<400> 71

Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn

1 5 10

<210> 72

<211> 16

<212> Protein

<213> Artificial

<220>

<223> CDRH2 dAbH1

<400> 72

Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly

1 5 10 15

<210> 73

<211> 13

<212> Protein

<213> Artificial

<220>

<223> CDRH3 dAbH1

<400> 73

Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu

1 5 10

<210> 74

<211> 12

<212> Protein

<213> Artificial

<220>

<223> CDRL1 dAbL1

<400> 74

Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

1 5 10

<210> 75

<211> 7

<212> Protein

<213> Artificial

<220>

<223> CDRL2 dAbL1

<400> 75

Glu Ala Ser Lys Leu Thr Ser

fifteen

<210> 76

<211> 11

<212> Protein

<213> Artificial

<220>

<223> CDRL3 dAbL1

<400> 76

Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr

1 5 10

<210> 77

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Anti-albumin antibody heavy chain variable domain (not ds)

<400> 77

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 Ile Asp Leu Ser Asn Tyr

20 25 30

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

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 78

<211> 121

<212> Protein

<213> Artificial

<220>

<223> Anti-albumin heavy chain variable domain (ds)

<400> 78

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 Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

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

65 70 75 80

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

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 79

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Anti-albumin antibody light chain variable domain (not ds)

<400> 79

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

<210> 80

<211> 112

<212> Protein

<213> Artificial

<220>

<223> Anti-albumin (ds) light chain variable domain

<400> 80

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn

20 25 30

Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile

85 90 95

Ser Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105 110

<---

Claims (27)

1. A trispecific antibody molecule, comprising: a) a polypeptide chain of formula ( I ): V _H -CH ₁ -X-V ₁ ; and b) a polypeptide chain of formula ( II ): V _L -C _L -YV ₂ ; or consisting of a) and b), where: V _H is a heavy chain variable domain; CH ₁ is a heavy chain constant region domain, eg domain 1; X is a bond or linker; Y is a bond or linker; V ₁ is dsscFv; V _L is a light chain variable domain; C _L is a domain from a light chain constant region such as Ccappa; V ₂ is dsscFv; where each of VH/VL, V ₁ and V ₂ forms an antigen-binding site, and where only one of V _H /V _L , V ₁ and V ₂ has specificity for a carrier protein in serum, and where each of V _H /V _L , V ₁ or V ₂ binds a different antigen. 2. The trispecific antibody molecule according to claim 1, wherein said serum carrier protein is selected from the list consisting of albumin, thyroxine-binding protein, transthyretin, α1-acid glycoprotein, transferrin, and fibrinogen, or a fragment of any of them. 3. A trispecific antibody molecule according to claim 1 or 2, wherein said serum carrier protein is human serum albumin, and wherein said V _H /V _L or V ₁ or V ₂ having specificity for serum carrier protein blood, form an albumin-binding site. 4. The trispecific antibody molecule according to claim 3, wherein said albumin-binding site comprises SEQ ID NO: 71 as CDRH1, SEQ ID NO: 72 as CDRH2, SEQ ID NO: 73 as CDRH3, SEQ ID NO: 74 as CDRL1 , SEQ ID NO: 75 as CDRL2 and SEQ ID NO: 76 as CDRL3; or a heavy chain variable domain selected from SEQ ID NO: 77 and SEQ ID NO: 78, and a light chain variable domain selected from SEQ ID NO: 79 and SEQ ID NO: 80. 5. A trispecific antibody molecule according to claim 4, wherein said 6 CDRs with sequences of SEQ ID NOs: 71-76 or variable domains with sequences of SEQ ID NOs: 77-80 are in position V _H /V _L or V ₁ or V ₂ . 6. A trispecific antibody molecule according to claim 4 or 5, wherein the heavy chain variable domain of SEQ ID NO: 78 and the light chain variable domain of SEQ ID NO: 80 are at position V ₂ , and wherein said light chain variable domain or the heavy chain variable domain from V ₂ is attached to Y, for example, via a peptide bond. 7. A trispecific antibody molecule according to any one of paragraphs. 1-6, wherein the light chain and heavy chain variable domains from V ₁ and/or the light chain and heavy chain variable domains from V ₂ are disulfide bonded between two engineered cysteine residues, wherein the position of said pair of cysteine residues is V _H 44 and V _L 100 (numbering according to Kabat). 8. A trispecific antibody molecule according to any one of paragraphs. 1-7, where X and/or Y is a peptide linker, e.g. SEQ ID NOS: 1, 2, 69 and 70. 9. A polynucleotide encoding a trispecific antibody molecule according to any one of paragraphs. 1-8. 10. An expression vector comprising a polynucleotide as defined in claim 9. 11. A host cell for expression of a trispecific antibody molecule according to any one of paragraphs. 1-8, comprising one or more polynucleotides or vectors according to claim 9 or 10, respectively. 12. A host cell for expression of a trispecific antibody molecule according to any one of paragraphs. 1-8, comprising two vectors, where the first vector includes a polynucleotide encoding a polypeptide chain of formula (I) of a trispecific antibody molecule according to any one of paragraphs. 1-8, and the second vector comprises a polynucleotide encoding a polypeptide chain of formula (II) of said trispecific antibody molecule. 13. A method of obtaining a trispecific antibody molecule according to any one of paragraphs. 1-8, including the expression of a trispecific antibody molecule from a host cell, as defined in paragraph 11 or 12.

Images