Multivalent antibody

Abstract

To provide, in the art for the design of a set of linkers including different characteristics of rigidity, flexibility and length that can be employed in a modular fashion depending on a combination of targets, while maintaining the stability, low immunogenicity and ease of developability; and new and useful formats for multivalent antibodies having three or more binding domains and linkers for production of antibodies which are widely applicable to rapid and robust generation of a broad range of antibodies which comprise three or more binding domains.SOLUTION: The invention relates to a multivalent antibody which comprises: a base antibody portion which comprises two binding domains; and at least one additional binding domain. The base antibody portion is connected by a linker to the at least one additional binding domain. Each binding domain of the base antibody portion and each of the at least one additional binding domain all have a common variable region. The linker comprises a hinge sequence or a sequence derived from a hinge sequence. The invention also relates to a multivalent antibody which comprises: a base antibody portion which comprises two binding domains; and at least one additional binding domain. The at least one additional binding domain comprises a CH1 region and is connected to the base antibody portion by the linker, linking the variable region of the base antibody portion and the CH1 region. The multivalent antibody binds to at least three different epitopes.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to multivalent antibodies having three or more binding domains and methods for making such multivalent antibodies. The present invention further relates to constituent polypeptides of the multivalent antibodies and to linkers that can be used to connect one or more binding domains of the multivalent antibodies. The present invention further relates to such multivalent antibodies, nucleic acids encoding linkers, and vectors comprising such nucleic acids, as well as host cells producing the multivalent antibodies. The present invention also relates to multivalent antibodies capable of simultaneously binding to three antigens or targets, including target antigens present on cancer cells or tumor cell antigens and targets involved in immune effector cells. The present invention also relates to pharmaceutical compositions comprising the multivalent antibodies, and to the multivalent antibodies for use in the treatment of humans or animals by therapy. The present invention also relates to methods for treating humans or animals using the antibodies. [Background technology]

[0002] Multivalent antibodies, such as bispecific antibodies capable of binding two antigens or two epitopes, are known in the art. Such multivalent binding proteins can be produced using a variety of techniques, including cell fusion, chemical conjugation, or recombinant DNA technology.

[0003] Antibodies are typically multimers composed of four proteins, including two identical heavy chains and two identical light chains, the heavy chains composed of a variable domain (VH) and three constant regions (CH1, CH2, CH3), and the light chains composed of a variable light domain (VL) and a constant region (CL). Typically, the light chains are paired with the heavy chains through the influence of a number of non-covalent interactions, as well as through disulfide bonds. The two heavy chains are paired at the hinge region connecting CH1 to CH2 and / or through amino acid interactions at the interface between the two CH3 domains. The pairing of VH and VL forms the antigen-binding domain, and typically variability is found in the three surface loop-forming regions within the VH and VL domains, which are the complementarity determining regions or CDRs.

[0004] Certain multivalent antibody formats are known in the art, such as antibodies with two different binding domains, such as bispecific antibodies, that can bind to two different antigens, or two different epitopes within the same antigen. In such formats, the multivalent antibody may allow for the use of coordinated binding, which allows selective targeting of cells or targets expressing two antigens or epitopes, such as tumor cells, without targeting normal cells expressing one antigen, or targeting such normal cells expressing one antigen at a lower expression level. Similarly, having two different binding domains on a multivalent antibody, such as a bispecific antibody, allows for binding of different antigens, which allows the multivalent antibody to be used to target both inhibitory and stimulatory molecules on a single cell or two interacting cells, thereby increasing the efficacy of the multivalent antibody. Multivalent antibodies may also be used to redirect cells, such as immunomodulatory cells, that can be redirected to tumors.

[0005] Incorporating three or more binding domains in a single antibody may allow for a wider variety of beneficial combinations of targets and efficacies. For example, a multivalent antibody with three or more binding domains may target the same antigen and epitope, allowing for specificity and / or saturation of a given target at a lower ratio of antibody to target. A multivalent antibody may contain two or more identical antigen binding domains to allow for high avidity binding to target cells. This can be used to specifically target antigens such as gangliosides that are overexpressed on tumor cells. These tumor-associated antigens are present on normal cells, but at very high density on tumor cells. A multivalent antibody containing several low affinity binding regions may allow for specific targeting to tumor cells while not reacting or reacting at a lower ratio with normal cells, while activating or blocking additional receptors. Finally, three or more binding regions are useful in such applications.

[0006] Although certain multivalent antibodies have been described in the art, the art needs new formats and new linkers that allow efficient production of multivalent antibodies, whose binding domains for many antigens can be easily produced and efficiently and stably converted into multivalent antibodies, and that can bind to a wide variety of antigens and epitopes.The engineering of antibodies that contain three or more binding domains has traditionally been time-consuming, inefficient, and expensive.In fact, there are many obstacles to the efficient production of high-quality, low-immunogenic multivalent antibodies that can be generated to target a variety of antigens.

[0007] For example, existing multivalent formats containing three or four binding domains rely on synthetic linkers such as Gly4Ser (G4S) repeat containing sequence domains that, firstly, tend to limit access to all binding domains in the molecule and, secondly, can be problematic for developability.

[0008] Existing multivalent antibody formats also rely on different heavy and light chains that are associated by disulfide bonds and amino acid interactions or, in the case of single-chain fragment variables (scFv), can be joined by a short linker. However, the need to use different variable chains (heavy and / or light chains) in each of the multiple variable domains used in multispecific formats of three or more binding specificities requires extensive engineering of such molecules to prevent mispairing of heavy and light chains. This invariably affects the complexity, stability, immunogenicity and production levels of these molecules.

[0009] Multivalent antibody formats may rely on the use of the same light chain for each binding domain, where one or more cognate variable regions that pair with the light chain are forced to pair by chemical modification, rather than the cognate chains forming and pairing with a common light chain in response to antigen exposure and the process of co-evolution that occurs during B-cell development.

[0010] A multivalent antibody format can also rely on a light chain from an existing monospecific antibody that binds one antigen, which is then used in a library to identify a heavy chain that can pair with the light chain and simultaneously bind a different epitope or antigen.

[0011] Such pseudo-common light chains are not preferred for use in the present invention. Preferred common light chains of the present invention are obtained, derived from or based on a common chain capable of pairing with diverse cognate chains and pairing with rearranged cognate chains encoded by DNA that has undergone somatic recombination, preferably somatic hypermutation in response to antigen exposure.

[0012] The pseudo-light chain approach limits the range of cognate chains available. Forcing pairing of heavy and light chains that do not form together in response to antigen exposure leads to loss of specificity and affinity, limiting its usefulness. Furthermore, allowing VH and VL shuffling and maintaining affinity and specificity is usually uncommon for any given antibody.

[0013] Forcing pairing of heavy and light chains, which have not co-evolved in the immune response, while maintaining the ability to bind antigen is not straightforward and may limit the flexibility of this approach.

[0014] Similarly, reusing the light chain of one antibody to identify a heavy chain capable of binding to it and also to a different antibody of interest limits the range of available heavy chains, increasing the likelihood of failing to identify suitable additional heavy chains, and more antigens or epitopes to be bound. That is, a light chain paired with a heavy chain may be used to form a Fab that binds to a given antigen to identify a subsequent heavy chain that can pair with the light chain and bind to a second antigen. However, it becomes increasingly rare to use the light chain a third time to identify a third heavy chain that can pair with the light chain while binding to a third antigen or epitope, and even rarer still, more heavy chains that can pair with the light chain while also binding to a different epitope or antigen are sought to be identified.

[0015] Preferred embodiments of the invention described herein use a common chain that pairs with diverse heavy chains in response to antigen, and do not require recycling of existing light chains of monospecific antibodies or forced pairing of light chains to their cognate chains by chemical modification.

[0016] The successful construction of a multivalent antibody depends on the appropriate selection of a protein linker between different domains, since direct fusion of two domains may impair biological activity. The biophysical properties of the linker or linkers, such as charge, rigidity, or flexibility, as well as the distance between binding domains and the spatial conformation between binding regions, may affect the accessibility of epitopes and the ability of a multivalent protein to bind to its target. Therefore, there is a need for a multivalent format that can use various linkers with different characteristics, allowing the construction of multivalent antibodies that are modular and allow simultaneous binding to different combinations of epitopes that may be located on different molecules and / or different cells. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] US Patent Application Publication No. 2010 / 0203056(A1) [Patent Document 2] International Publication No. 2011 / 066389 [Patent Document 3] International Publication No. 2013 / 079174 [Patent Document 4] International Publication No. 2013 / 181634 [Patent Document 5] International Publication No. 2016 / 149201 [Patent Document 6] International Publication No. 2017 / 034916 [Patent Document 7] International Publication No. 2013 / 173223 Summary of the Invention [Problem to be solved by the invention]

[0018] Thus, there is a need in the art to design a series of linkers that incorporate different rigidity, flexibility, and length characteristics that can be used modularly for different target combinations while maintaining stability, low immunogenicity, and ease of developability. Thus, there is a need for new and useful formats for multivalent antibodies with three or more binding domains and linkers for the production of antibodies that are broadly applicable for the rapid and robust generation of a wide range of antibodies containing three or more binding domains. [Means for solving the problem]

[0019] The present invention is based on novel modular formats of multivalent antibodies comprising three or more binding domains, which may be multispecific antibodies. In these formats, at least one binding domain is connected to a base antibody moiety, which comprises two binding domains. The additional binding domains may comprise variable regions, Fv domains, Fab domains, or modified Fab domains, or any functional fragments thereof. The base antibody moiety may be, for example, a full-length antibody or a fragment thereof, but in any case comprises two binding domains.

[0020] The one or more additional binding domains are connected to the base antibody moiety via a linker(s) to provide one or more binding moieties in addition to that of the base antibody moiety.

[0021] A linker is used to connect one or more additional binding domains to a base antibody moiety. The linker comprises a peptide region, for example, one or more hinge regions and / or one or more regions derived from a hinge region. The combination of the linker and the constant region (e.g., CH1) to which it is connected may be important in determining the properties of the multivalent antibody, allowing the correct function of the antibody and / or the orientation of one or more additional binding domains to the base antibody. Thus, if the linker sequence is based on a hinge of a given subtype, it may be preferable that the constant region of the additional binding domain to which the linker is attached is of the same subtype.

[0022] The one or more additional binding domain(s) may comprise a variable region, an Fv domain, a Fab domain, or a modified Fab domain.

[0023] Fab domains constitute valuable additional binding domains, in particular because they contain protein domains with predictable behavior that are useful for the production of multivalent molecules that are stable and can be easily produced.

[0024] To facilitate efficient production and developability of the multivalent antibodies of the invention, the multivalent antibodies may comprise a common variable region, which may be an immunoglobulin heavy chain variable region (VH) or light chain variable region (VL), but is typically a common light chain (cLC) variable region.

[0025] The consensus variable region is typically paired with a cognate variable region encoded by a nucleic acid that has undergone somatic recombination, preferably affinity maturation, or is based on or derived from a rearranged variable region encoded by a nucleic acid that has undergone the process of somatic recombination, preferably affinity maturation, and / or is based on or derived from known antibody generation techniques such as phage display, immunization of animals, including transgenic animals with humanized immune systems, and other techniques well known in the art.

[0026] The use of essentially identical common variable regions in each binding domain of a multivalent antibody of the invention facilitates the development and production of such antibodies.

[0027] Therefore, the selection of a common variable region for use in the multivalent antibodies of the present invention should be one that can be broadly used with a variety of cognate heavy or light regions.

[0028] Identical or substantially identical common variable regions, e.g., cLC variable regions, allow the use of complete or substantially complete Fab domains for all three or more binding regions without the need for extensive engineering as used in Crossmab technology, or linkers to prevent mispairing of heavy and light chains (as used in scFv domains). Also, the use of common variable regions in each of three or more Fab domains can result in reduced immunogenicity, since essentially germline-encoded, non-immunogenic common variable regions are known (see WO 2009 / 157771).

[0029] An additional advantage of the formats described herein for producing multivalent antibodies is the ability to use transgenic animals, preferably transgenic rodents, that have in their genome a common variable region that can pair with diverse cognate variable regions (e.g., a common light chain variable region paired with diverse heavy chain variable regions) (see WO 2009 / 157771), allowing DNA encoding cognate variable regions formed from exposure to different antigens to be introduced into a host cell along with DNA encoding the common variable region, which can each be expressed to generate multivalent antibodies.

[0030] For example, a transgenic mouse that comprises in its germline DNA encoding a common variable region and DNA encoding unrearranged immunoglobulin loci that can be rearranged to form cognate variable regions and that can undergo somatic recombination can be exposed to one or more antigens, such that the rearranged variable regions produced upon exposure to the three or more antigens can then be used to generate a multivalent antibody of the invention. The nucleic acid sequence encoding the common variable region and the three or more rearranged variable regions can be transformed into a host cell to express the multivalent antibody of the invention.

[0031] Thus, the formats described herein for producing multivalent antibodies utilize three or more binding domains that may all contain a common variable region, preferably a common light chain variable region.

[0032] The format of the present invention comprises one or more binding domains in addition to those of the base antibody moiety. Such additional binding domains may be Fv domains, Fab domains, or modified Fab domains, preferably comprising a CH1 domain and a variable domain, and are connected to the base antibody moiety via a linker. In modified Fab domains, the CH1 domain is not paired with a CL. A suitable CH1 domain may be engineered to remove one or more hydrophobic regions, or may be from a camelid or shark. Alternatively, the additional binding domain may comprise a CL domain and a variable domain, which are connected to the base antibody moiety via a linker. The CL domain may be either a Ckappa or Clamda domain.

[0033] Typically, the additional binding domain(s) is attached to one or both binding domains of the base antibody moiety at the N-terminal region of either the common variable portion or the rearranged variable region or both of the binding domains of the base antibody moiety.

[0034] Alternatively, the additional binding domain may be connected to the base antibody moiety via a linker that connects both the common chain and the reshaped variable domain of the binding domain of the base antibody moiety to the CH1 and CL regions of the additional binding domain. If the additional binding domain lacks a constant region, the novel linkers disclosed herein may connect both the common chain and the reshaped variable domain of the binding domain of the base antibody moiety to the common chain and / or the reshaped variable region of the additional binding domain.

[0035] Alternatively, the additional binding domain is connected to the base antibody portion by a single linker peptide, V H Area and V L Typically, additional binding domain(s) of this kind is / are attached to one or both binding domains of the base antibody moiety, either in the N-terminal region of the common variable portion or of the rearranged variable region of the binding domains of the base antibody moiety.

[0036] This format is surprisingly flexible when used with the inventive linkers disclosed herein, including different lengths, structures, and degrees of rigidity. Thus, the invention provides a repertoire of linkers with different properties for use in the disclosed multivalent antibody formats, facilitating development to combine three or more binding domains into a multivalent antibody.

[0037] With the linkers disclosed herein, the invention provides a modular approach combining the selection of an appropriate linker with the selection of a corresponding set of binding domains (e.g., Fab domains), thereby allowing these binding domains to function together for various purposes in a multivalent antibody.

[0038] The multivalent antibodies of the invention can be used therapeutically, in particular as so-called "engager" antibodies, whereby the antibodies are able to form a link between immune effector cells and tumor cells.

[0039] Therefore, according to the present invention, a base antibody portion comprising two binding domains; and at least one additional binding domain, the base antibody portion is connected to at least one additional binding domain by a linker; each binding domain of the base antibody portion and each of the at least one additional binding domain all share a common variable region; The linker comprises a hinge sequence or a sequence derived from a hinge sequence.

[0040] The present invention also provides a base antibody portion comprising two binding domains; and at least one additional binding domain, at least one additional binding domain comprises a CH1 region and is connected to the base antibody moiety by the linker to link the variable region and the CH1 region of the base antibody moiety; The multivalent antibody binds to at least three different epitopes.

[0041] Preferably, each binding domain of the base antibody moiety and each of the at least one additional binding domain may all share a common variable region.

[0042] A variety of one or more additional binding domains A preferred embodiment is a multivalent antibody, wherein one or more of the binding domains is an Fv domain comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0043] A further preferred embodiment is a multivalent antibody, wherein one or more of the binding domains is a Fab domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region of the Fab domain comprising a CH1 region (VH-CH1) and the light chain variable region of the Fab comprises a CL region (VL-CL). The Fab domain may contain a VL-CL that is either Vkappa-Ckappa, Vlambda-Clamda, Vlambda-Ckappa, or Vkappa-Clambda.

[0044] Another embodiment is a multivalent antibody, wherein the one or more additional binding domains are modified Fab domains consisting of VH-CH1 and VL. Alternatively, one embodiment is a multivalent antibody, wherein the one or more additional binding domains are modified Fab domains consisting of VL-CL and VH. In such modified Fab domains, a constant region CH1 or CL that is not paired with its cognate region is present, and / or a variable region VH or VL that is not paired with its cognate region is present.

[0045] common chain A preferred embodiment is a multivalent antibody, in which one or more additional binding domains comprise a Fab domain comprising a common rearranged variable region paired with a rearranged variable region that has undergone somatic rearrangement after exposure to an antigen, or are encoded by a nucleic acid obtained, derived or based on a sequence that is the result of a somatic rearrangement. Alternatively, the rearranged variable region may be obtained, derived or based on a synthetic repertoire in which diversity is introduced into the repertoire using molecular biology techniques known in the art, including the use of synthetic phage display libraries. Preferably, the Fab domain comprises a common light chain variable region that pairs with a corresponding rearranged heavy chain variable region. Preferably, the common light chain variable region is connected to a CL region and the rearranged heavy chain variable region is connected to a CH1 region. Preferably, the common light chain pairs with the heavy chain variable region through the binding of the CL and CH1 regions. Alternatively, the common chain is a heavy chain and the rearranged variable region is a light chain, and the chains may each comprise a CH1 and CL domain, or may pair through the binding of the CL and CH1 regions.

[0046] A preferred embodiment is a multivalent antibody, in which three or more binding domains each comprise the same common chain, but the three or more binding domains comprise different rearranged variable cognate chains, more preferably the same common chain is a common light chain.

[0047] A preferred embodiment is a multivalent antibody, in which three or more binding domains comprise rearranged variable regions that are obtained, derived from, or encoded by nucleic acids based on a nucleic acid sequence of a transgenic animal comprising a common light chain and an unrearranged heavy chain variable region that is exposed to an antigen and produces an antibody comprising a rearranged heavy chain variable region that pairs with a common light chain. Alternatively, in one embodiment of a multivalent antibody, three or more binding domains comprise rearranged variable regions that are obtained, derived from, or encoded by nucleic acids based on a nucleic acid sequence of a transgenic animal comprising a common heavy chain and an unrearranged light chain variable region that is exposed to an antigen and produces an antibody comprising a rearranged light chain variable region that pairs with a common heavy chain.

[0048] Linker Composition A preferred embodiment is said multivalent antibody, wherein said linker is a naturally occurring sequence or is based on a naturally occurring sequence. More specifically, said linker is a hinge sequence or comprises a sequence based on a hinge sequence. More specifically, said linker may comprise a hinge region based on an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region, or an IgG4 hinge region.

[0049] Alternatively, the linker may be: ESKYGPP (SEQ ID NO: 1) EPKSCDKTHT (SEQ ID NO:2) GGGGSGGGGS (SEQ ID NO: 3) ERKSSVESPPSP (SEQ ID NO: 4) ERKCSVESPPSP (SEQ ID NO:5) ELKTPLGDTTHT (SEQ ID NO:6) ESKYGPPSPSSP (SEQ ID NO: 7) ERKSSVEAPPVAG (SEQ ID NO: 8) ERKCSVEAPPVAG (SEQ ID NO: 9) ESKYGPPAPEFLGG (SEQ ID NO: 10) EPKSCDKTHTSPPSP (SEQ ID NO: 11) EPKSCDGGGGSGGGGS (SEQ ID NO: 12) GGGGSGGGGSAPPVAG (SEQ ID NO: 13) EPKSCDKTHTAPELLGG (SEQ ID NO: 14) ERKSSVESPPSPAPPVAG (SEQ ID NO: 15) ERKCSVESPPSPAPPVAG (SEQ ID NO: 16) ELKTPLGDTTHTAPEFLGG (SEQ ID NO: 17) ESKYGPPSPSSPAPEFLGG (SEQ ID NO: 18) EPKSCDKTHTSPPSPAPELLGG (SEQ ID NO: 19) ERKSSVEEAAAKEAAAKAPPVAG (SEQ ID NO: 20) ERKCSVEEAAAKEAAAKAPPVAG (SEQ ID NO: 21) ESKYGPPEAAAKEAAAKAPEFLGG (SEQ ID NO: 22) EPKSCDKTHTEAAAKEAAAKAPELLGG (SEQ ID NO: 23) ELKTPLGDTTHTEAAAKEAAAKAPEFLGG (SEQ ID NO: 24) or a peptide region comprising one or more of the sequences having at least about 85% sequence identity to any one of these.

[0050] Preferably, the multivalent antibody of the present invention comprises a linker that connects the base antibody moiety to one or more binding domains by comprising a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 24, or an amino acid sequence having at least about 85% sequence identity to any one of SEQ ID NOs: 1 to 24.

[0051] Table 1 shows how such linkers can be attached to the CH1 region.

[0052] A preferred multivalent antibody of the invention comprises a linker that is rigid. More preferably, said multivalent antibody comprises a linker that comprises a helix-forming sequence.

[0053] A preferred multivalent antibody of the invention comprises a linker which comprises a peptide sequence comprising an (EAAK)2 motif.

[0054] Preferred multivalent antibodies of the invention comprise linkers that are flexible.

[0055] A preferred multivalent antibody of the invention comprises a linker comprising three or more amino acid residues corresponding to the hinge region of the subtype of the constant region to which the multivalent antibody is connected.

[0056] A preferred multivalent antibody of the present invention comprises a linker comprising a sequence of SEQ ID NO: 1 to 24, which corresponds to the hinge region of the subtype of the constant region to which the multivalent antibody is linked.

[0057] Linker position / orientation A preferred embodiment is a multivalent antibody, in which a base antibody moiety is connected to one or more additional binding domains by a linker, which joins the N-terminus of the variable region of the base antibody moiety to the C-terminus of the one or more additional binding domains. Preferably, the base antibody moiety comprises a Fab domain, and the one or more additional binding domains comprise a Fab domain comprising a CH1 domain and a CL domain, and the linker connects the N-terminus of the variable region of the Fab of the base antibody moiety to either or both of the C-terminus of the CH1 domain and the CL domain of the Fab domain of the one or more additional binding domains.

[0058] A preferred embodiment of the invention is a multivalent antibody comprising a common chain for each binding domain of the base antibody moiety and for each of the one or more additional binding domains, and a linker connecting the N-terminus of the reconstituted variable region of the base antibody moiety to the C-terminus of the reconstituted variable region of the one or more additional binding domains. More preferably, the one or more additional binding domains comprise a Fab domain comprising a CH1 domain and a CL domain, and the linker connects the N-terminus of the variable region of the Fab domain of the base antibody moiety to the CH1 domain or the CL domain of the Fab domain of the one or more additional binding domains.

[0059] Pairing of regions containing additional binding domains Antibody assembly occurs through the association (pairing) of light and heavy chains, i.e., VH with VL and CH1 with CL, based on interacting residues at the interfaces between VH and VL and between CH1 and CL. Typically, pairing is further stabilized by covalently linking the light chain to the heavy chain through a disulfide bond between a cysteine ​​residue of the light chain in the CL and a cysteine ​​residue of the heavy chain in CH1 or in the hinge, depending on the subtype.

[0060] Thus, in the multivalent antibodies of the invention, one or more additional binding domain(s) are connected to the base antibody moiety via linker(s), where the one or more binding domain(s) comprise an Fv domain, a Fab domain, or a modified Fab domain, and where the corresponding immunoglobulin chains comprising the binding domains (usually heavy and light chain regions) pair together in a stable association.

[0061] In the case of the multivalent antibody of the invention containing additional binding domains, the binding domains are Fv or Fab domains, and cysteine ​​residues may be present or engineered into the heavy and light chain domains, which form disulfide bonds to stabilize the pairing between the heavy and light chains of the additional binding domain. If the multivalent antibody of the invention contains additional binding domains that include an IgG1 subclass, the upper hinge of the heavy chain IgG1 (EPKSC) connected upstream (n-terminal side) of an artificial linker such as (G4S)n can be used to provide a cysteine ​​that is covalently linked to the light chain of the additional binding domain. With regard to other subclasses used in the additional binding domains, the skilled person will recognize the ability to engineer cysteine ​​residues in the linker used to stabilize the pairing of the light and heavy chain domains of the additional binding domain and form disulfide bridges between the light and heavy chains used.

[0062] The wild type IgG1 hinge region has the sequence: EPKSCDKTHTCPPCPAPELLGG (SEQ ID NO: 42). The underlined C residue is Cys220 in the IgG1 heavy chain, which pairs with Cys214 of the light chain. When a multivalent antibody of the invention comprises such a hinge-based linker, preferably any Cys residue other than Cys220 is substituted with an amino acid residue that cannot form a disulfide bond, such as Ser.

[0063] The wild type IgG2 hinge region has the sequence: ERKCCVECPPCPAPPVAG (SEQ ID NO: 46). The underlined C residue is Cys219 in the IgG2-B heavy chain which pairs with Cys214 of the light chain. In IgG2-A, Cys127 of the heavy chain pairs with Cys214. When a multivalent antibody of the invention comprises such a hinge-based linker, preferably any Cys residue other than Cys215 is substituted with an amino acid residue that cannot form a disulfide bond, such as Ser.

[0064] The wild type IgG3 hinge region has the sequence: ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEFLGG (SEQ ID NO: 50). In IgG3, Cys131 of the heavy chain pairs with Cys214 of the light chain.

[0065] The IgG4 hinge region has the sequence: ESKYGPPCPSCPAPEFLGG (SEQ ID NO: 54). In IgG4, Cys131 of the heavy chain pairs with Cys214 of the light chain. When a multivalent antibody of the invention comprises such a hinge-based linker, preferably one or both of the Cys residues in the CPSPC region of the hinge are substituted with an amino acid residue that cannot form a disulfide bond, such as Ser.

[0066] As described herein, to generate multivalent constructs, including trivalent constructs, based on IgG structures capable of simultaneously binding three different epitopes, linkers based on IgG hinges of different subclasses are used to connect the binding domains of the base antibody moiety to the additional binding domains comprising the heavy chain constant region. To ensure stabilization of the covalent bond between the cysteines of the light and heavy chains of the additional binding domains, the present invention matches a linker comprising a hinge region or based on a hinge region of a particular subtype with the CH1 of the additional binding domain from the same subtype.

[0067] When a multivalent antibody comprises additional binding domains consisting of paired regions (e.g., VH-CH1 and VL-CL pairing, or VH and VL pairing), stabilizing the interface between the regions can be achieved in various ways in the present invention. When one or more additional binding domains are Fab domains consisting of a variable heavy chain region and a variable light chain region, CH1 can be connected to the variable heavy chain region. CH1 can be paired with CL by a covalent bond, typically a disulfide bridge connected to the variable light chain region. In addition, the heavy and light chains of the Fab domain are paired through non-covalent interactions. Alternatively, a linker that connects one or more additional binding domains to the base antibody portion can be further used to pair the variable heavy chain region with the variable light chain region of the additional binding domain by forming a peptide bond with either chain and a covalent bond with the corresponding chain. This can be achieved by engineering a cysteine ​​at or near the N-terminus of the linker and a cysteine ​​at or near the C-terminus of the variable heavy and / or variable light regions of the one or more additional binding domains, thereby forming a covalent bond between the linker and the variable heavy and / or variable light regions of the one or more additional binding domains. Other means of pairing domains, including one or more additional binding domains such as Fab domains, will be known to those of skill in the art and are described in further detail below.

[0068] A preferred embodiment is a multivalent antibody, comprising a base antibody moiety and one or more additional binding domains. When the one or more additional binding domains are Fv domains comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the linker of the invention connects the base antibody moiety to the Fv while pairing the heavy chain variable region and the light chain variable region of the Fv.

[0069] Alternatively, the binding domain is a Fab domain comprising a variable heavy region comprising a CH1 region and a variable light region comprising a CL region. The linker of the invention connects the base antibody moiety to the Fab domain at the CH1 region or the CL region or both, pairing the CH1 and CL regions of the Fab domain.

[0070] Base antibody moiety pairing Different techniques are known in the art for pairing the heavy chain constant regions (e.g., CH2 and CH3) of the base antibody moiety and causing their heterodimerization. For example, the use of DEKK mutations to cause heterodimerization of antibody heavy chains (WO 2013 / 157954 and De Nardis et al., J. Biol. Chem. (2017) 292(35) 14706-14717, incorporated herein by reference) further allows for efficient heterodimer formation, stable Fc regions, and ease of manufacture. This approach allows the Fc region of the molecule to function and engage with immune receptors such as Fc receptors, complement, and FcRn. Thus, certain multivalent antibody embodiments of the present invention preferentially heterodimerize the heavy chains of the base antibody moiety using DEKK modifications, or other Fc modifications known to those skilled in the art.

[0071] Furthermore, in certain embodiments of multivalent antibodies, it may be desirable to use the multivalent antibody to, for example, engage, stimulate, and / or co-stimulate T cells, but not engage effector functions of the immune system (e.g., to limit antibody-dependent cellular cytotoxicity, antibody-mediated phagocytosis, and / or cell-dependent cytotoxicity), in which case additional modifications to the Fc region may be used to eliminate or reduce effector function. Accordingly, certain multivalent antibody embodiments of the invention contain modifications to the heavy chain constant region of the base antibody moiety that eliminate or reduce effector function(s).

[0072] A further preferred embodiment of the present invention is a multivalent antibody comprising a base antibody portion consisting of two heavy chains lacking a CH2 or CH3 region, said heavy chains being joined together at the hinge region.

[0073] The invention also provides a method for preparing a multivalent antibody, the method comprising providing a cell comprising one or more nucleic acid sequences encoding polypeptides that can be assembled into a multivalent antibody of the invention. The cell can be cultured under conditions to provide for expression of the base antibody moiety, at least one additional binding domain, and at least one linker, and their assembly into the multivalent antibody of the invention.

[0074] The present invention also provides nucleic acids encoding the constituent proteins of the multivalent antibody of the present invention and the multivalent antibodies produced by them.

[0075] The present invention also provides a vector comprising a nucleic acid sequence of the present invention.

[0076] The invention also provides a host cell that expresses the nucleic acid and produces the multivalent antibody.

[0077] The invention also provides methods for generating multivalent antibodies, including by the use of transgenic animals that contain a common chain in their germline, which produce common chain antibodies with diversity in the cognate chains, wherein the multivalent antibodies comprise one or more binding domains with rearranged variable regions obtained, derived from, or encoded by nucleic acid based on one or more cognate chains of the common chain antibody expressed by the transgenic animal upon exposure to antigen.

[0078] The present invention also provides non-human transgenic animals comprising a common human light chain variable region capable of pairing with a variety of human heavy chain variable regions, wherein nucleic acids encoding the common light chain variable region and human heavy chain variable region are present in the endogenous variable region locus of the non-human transgenic animal (light and heavy chain, respectively, or vice versa) and / or are stably integrated elsewhere in the germline of the transgenic animal (e.g., the Rosa locus), and a series of rearranged human heavy chain variable regions that pair with the common light chain variable region are generated by contacting the transgenic animal with an antigen, and nucleic acid encoding the rearranged human heavy chain variable region is transformed into a host cell capable of producing a multivalent antibody of the invention, wherein the multivalent antibody comprises one or more binding domains comprising the rearranged human heavy chain variable region encoded by nucleic acid obtained, derived from, or based on one or more rearranged human heavy chain variable regions produced by the transgenic animal exposed to antigen.

[0079] The present invention also provides pharmaceutical compositions comprising an antibody of the invention and a pharma- ceutically acceptable carrier and / or diluent.

[0080] The present invention also provides an antibody of the invention for use in treatment of the human or animal body by therapy. The present invention also provides a method for treating a human or animal suffering from a medical indication, the method comprising administering to the human or animal a therapeutically effective amount of an antibody of the present invention.

[0081] For ease of reference, in Figures 15-28, when describing trispecific molecules, the following format is used: MFA x MFB: MFC or antigen A x antigen B: antigen C, whereby MFA or antigen A followed by x constitutes the "short arm", where x indicates dimerization, followed by MFB or antigen B describes the internal location of the long arm, followed by ":" indicates the linker, followed by MFC or antigen C, indicating the MFC or antigen C at the distal domain of the long arm. The term "mock", when used in the context of a multivalent molecule, refers to a binding domain of such a molecule that is capable of binding to an antigen not present in a given assay in which it is tested. Typically, mock binding domains as used herein bind to tetanus toxin (TT), fibrinogen (Fibri), or thyroglobulin (Thyro). [Brief description of the drawings]

[0082] [Figure 1a] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1b] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1c] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1d] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1e] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1f] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1g] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1h]1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1i] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1j] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1k] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1l] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1m] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1n] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1o] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1p] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1q] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1r] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1s] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1t] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 1u] 1 illustrates multivalent antibody formats of the present invention that include different binding domain structures, linkers, and base antibody moieties. [Figure 2a] 1 shows a schematic of the VH1-CH1-linker-VH2 insert used to clone the construct into vector MV1626. Although not shown, it is understood that the vector may also encode a CH3-CH2-CH1 region connected to VH2. [Figure 2b] A trispecific antibody is shown in which VH1 binds to a tetanus toxoid antigen, VH2 binds to a fibrinogen antigen, and VH3 binds to a thyroglobulin antigen. [Diagram 3] A schematic diagram of the MV1626 vector is shown. [Figure 4] A schematic diagram of the MG1025C377 expression vector is shown. [Diagram 5] An alignment of the sequences of the inserts used for cloning into MV1626 is shown. Note that the alignment only covers the CH1-linker for clarity purposes. [Figure 6] A schematic diagram of the MV1057 vector is shown. [Figure 7] Schematic diagram of the MV1260 vector. [Figure 8] Shown is an SDS-PAGE gel of IgG under non-reducing (top) and reducing (bottom) conditions. [Figure 9] Screening data for 24 multivalent constructs is shown. [Figure 10] Screening data for 18 multivalent constructs is shown. [Figure 11A] This is the consensus light chain amino acid sequence. [Figure 11B] DNA sequence and translation of the common light chain variable domain (IGKV1-39 / jk1). [Figure 11C] The DNA sequence and translation of the consensus light chain constant region. [Figure 11D] This is a translation of the IGKV1-39 / jk5 common light chain variable domain. [Figure 11E]The V region is IGKV1-39A. [Figure 12] Stability analysis of 18 multivalent IgG constructs and 4 control antibodies analyzed under 4 different conditions. [Figure 13] Bioinformatics modeling of eight linkers. [Figure 14] Two engager trispecific formats with a long arm internal immune cell binding domain and a short arm tumor cell antigen binding domain (14a) or a long arm distal tumor cell antigen binding domain and a short arm tumor cell antigen binding domain (14b). [Figure 15a] T cell activation (median EGFR expression) in BxPC3 cells by flow cytometry with expression of CD25 and CD69 as readout in the format of EGFR on the short arm (EGFRxCD3:TT in FIG. 15a). A bispecific antibody (EGFRxCD3) is used as a positive control. [Figure 15b] T cell activation (median EGFR expression) in BxPC3 cells by flow cytometry with expression of CD25 and CD69 as readout in the format of EGFR on the long arm (ThyroxCD3:EGFR in FIG. 15b). A bispecific antibody (EGFRxCD3) is used as a positive control. [Figure 16a] T cell cytotoxicity (median EGFR expression) in HCT116 cells was determined by measuring ATP levels assessed by CellTiterGlo for EGFRxCD3:TT (Figure 16a). The top graph ATP levels measured by luminescence on an Envision microplate reader show relative light unit (RLU) values ​​analyzed using GraphPad Prism, and the bottom graph correlates with the killing rate based on the following formula: Kill rate = (100 - (RLU of sample / RLU without IgG) x 100). [Figure 16b]ATP levels were determined by measuring ATP levels assessed by CellTiterGlo for both ThyroxCD3:EGFR (Figure 16b). The top graph ATP levels measured by luminescence on an Envision microplate reader show relative light unit (RLU) values ​​analyzed using GraphPad Prism, and the bottom graph correlates with the killing rate based on the following formula: Kill rate = (100 - (RLU of sample / RLU without IgG) x 100). [Figure 17a] Effect of linkers on T cell cytotoxicity in HCT116 cells (Figure 17a). [Figure 17b] A comparison of target cell lysis versus cytokine release for various linkers was demonstrated for IL-2 (Figure 17b). [Figure 17c] A comparison of target cell lysis versus cytokine release for various linkers was demonstrated for IFN-g (Figure 17c). [Figure 17d] A comparison of target cell lysis versus cytokine release for various linkers was demonstrated for TNF-A (Figure 17d). [Figure 18] This is the configuration of a CD3xPD-L1:EGFR trispecific T cell engager molecule in which the CD3 binding domain is located on the short arm. [Figure 19] This is the configuration of an EGFRxCD3:PD-L1 trispecific T cell engager molecule in which the CD3 binding domain is located in the internal region of the long arm. [Figure 20] T cell cytotoxicity data was provided against MDA-MB-231 cells by comparing trispecific molecules combining a CD3 binding domain and two tumor cell antigen binding domains with a trispecific control having one tumor cell antigen binding domain, a mock domain, and a CD3 binding domain, and the positive control described above. [Figure 21]T cell cytotoxicity data is provided against MDA-MB-231 cells for trispecific molecules combining a CD3 binding domain and two tumor cell antigen binding domains, compared to a trispecific control having one tumor cell antigen binding domain, a mock domain, and a CD3 binding domain, the trispecific molecules comprising tumor cell antigen binding domains with a range of affinities for targeting EGFR and PD-L1. [Figure 22] T cell cytotoxicity data is provided on HCT116 cells for trispecific molecules combining a CD3 binding domain and two tumor cell antigen binding domains, compared to a trispecific control having one tumor cell antigen binding domain, a mock domain, and a CD3 binding domain, the trispecific molecules containing tumor cell antigen binding domains with a range of affinities for targeting EGFR and PD-L1. [Figure 23a] FACS data on MDA-MB-231 cells are presented as area under the curve (AUC) for the PD-L1 affinity range versus the EGFR affinity range, demonstrating dual antigen binding that correlates with increased affinity of the tumor antigen binding domain. [Figure 23b] T cell cytotoxic activity data is provided against BxPC3 cells and shows a specific trispecific molecule with the format CD3xPD-L1:EGFR. Simultaneous dual antigen binding and immune cell engagement occurred with additive effects on cytotoxicity to single antigens and molecules that bind CD3 (either CD3xEGFR:Mock or CD3xMock:PD-L1). Specific heavy chain sequences are not shown. [Figure 24] This construct is an EGFRxfibrinogen:CD3 trispecific T cell engager molecule in which the CD3 binding domain is located in the distal region of the long arm. [Diagram 25] T cell activation data is provided for HT29 cells and shows T cell activation by various EGFRxFibrinogen:CD3 trispecific T cell engager molecules using different CD3 binding domains compared to the positive control EGFRxCD3 bispecific antibody used in FIG. 15. [Figure 26]Construction of an EGFRxCD3:EGFR bispecific trivalent molecule with the same EGFR binding domain (MF9891). [Figure 27] The T cell activation activity of HCT116 cells was measured for a series of bispecific trivalent EGFRxCD3:EGFR molecules carrying the same EGFR binding domain (MF9891) and different CD3 binding domains of different superclusters with various linkers. [Figure 28] The T cell activation activity of MDA-MB-231 cells was measured for a series of bispecific trivalent EGFRxCD3:EGFR molecules carrying the same EGFR binding domain (MF9891) and different CD3 binding domains of various superclusters with various linkers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] "Antibody" refers to a protein molecule belonging to the immunoglobulin class of proteins that contains one or more domains that bind to an epitope on an antigen, such domains being derived from or sharing sequence homology with the variable region of the antibody. Antibody binding has different properties including specificity and affinity. Specificity determines which antigen or its epitope is specifically bound by a binding domain. Affinity is a measure of the strength of binding to a particular antigen or epitope. It is worth noting that, as used herein, the "specificity" of an antibody refers to its selectivity for a particular antigen, while "affinity" refers to the strength of the interaction between the antigen-binding site of the antibody and the epitope to which it binds.

[0084] Thus, as used herein, "binding specificity" refers to the ability of an individual antibody binding site to react with an antigenic determinant. Typically, the binding site of the antibody of the present invention is located in the Fab domain and is constructed from the hypervariable regions of the heavy and / or light chains.

[0085] "Affinity" is the strength of interaction between a single antigen-binding site and its antigen. The single antigen-binding site of an antibody of the present invention for an antigen can be expressed as a dissociation constant (KD). Typically, antibodies for therapeutic use have a dissociation constant of up to 1×10 10 M or even higher affinity.

[0086] An "antigen" is a molecule that can induce an immune response in a host organism (to produce antibodies) and / or can be targeted by an antibody. At the molecular level, an antigen is characterized by its ability to be bound by the antigen-binding site of an antibody. A mixture of antigens can also be considered an "antigen", i.e., a person skilled in the art will recognize that while a tumor cell lysate or a virus particle may sometimes be referred to as an "antigen", a preparation of such a tumor cell lysate or virus particle will have many antigenic determinants. An antigen contains at least one epitope, and often many more.

[0087] An "epitope" or "antigenic determinant" is a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein (so-called linear and conformational epitopes, respectively). Epitopes formed from contiguous linear amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding typically lose structure on treatment with denaturing solvents. Epitopes may typically include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation.

[0088] The term "heavy chain" or "immunoglobulin heavy chain" includes any organism's immunoglobulin heavy chain constant region sequence, including the heavy chain variable domain, unless otherwise specified. The term heavy chain variable domain includes the three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has a variable domain (from N-terminus to C-terminus), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. A functional fragment of a heavy chain can specifically recognize an antigen and includes a fragment that includes at least one CDR.

[0089] The term "light chain" refers to an immunoglobulin light chain variable domain, or V L (or a functional fragment thereof), and an immunoglobulin constant domain, or a C L (or a functional fragment thereof). Unless otherwise specified, the term "light chain" may include a light chain selected from human kappa, lambda, and combinations thereof. L A V domain typically contains three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain contains a V domain containing, from N-terminus to C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. L Light chains that can be used in the present invention include, for example, those that do not selectively bind to the epitope selectively bound by the heavy chain.

[0090] Light chains suitable for use in the multivalent antibody invention include consensus light chains, such as those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or in silico), which do not substantially inhibit the affinity and / or selectivity of the epitope binding domains of the heavy chains, but are also suitable for pairing with a range of heavy chains. For example, suitable light chains include those derived from transgenic animals, such as transgenic rodents, that contain a consensus light chain integrated into their genome, which can be used to generate a large panel of consensus light chain antibodies with heavy chain diversity upon exposure to antigen.

[0091] The term "common light chain" according to the present invention refers to a light chain which may be identical or which may have some amino acid sequence differences, wherein the binding specificity of the antibody of the present invention is not affected, i.e. the differences do not substantially affect the formation of a functional binding region.

[0092] For example, it is possible to prepare or find non-identical but functionally equivalent variable chains within the definition of common chain used herein (e.g., by introducing and testing conservative amino acid changes, changes in amino acids in regions that do not contribute or only partially contribute to binding specificity when paired with a cognate chain, etc.). Such variants are therefore also capable of combining different cognate chains to form functional antigen-binding domains. Thus, as used herein, the term "common light chain" refers to a light chain that may be identical or may have some amino acid sequence differences while retaining the binding specificity of the resulting antibody after pairing with a heavy chain. The combination of a particular common light chain and such functionally equivalent variants is encompassed by the term "common light chain".

[0093] "Fv domain" means a binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL).

[0094] "Fab domain" refers to a binding domain comprising a variable region, typically comprising a paired heavy chain variable region and a paired light chain variable region. A Fab domain may comprise a constant region domain, comprising a CH1 and a VH domain, paired with a constant light domain (CL) and a VL domain. Such pairing may be, for example, covalently linked via disulfide bridges in the CH1 and CL domains.

[0095] "Modified Fab domain" refers to a binding domain comprising a CH1 and a VH domain, where the VH is paired with the VL domain and the CL domain is absent. Alternatively, the modified Fab domain is a binding domain comprising a CL domain and a VL domain, where the VL is paired with the VH domain and the CH1 domain is absent. It may be necessary to remove or reduce the length of the hydrophobic region so that the CH1 or CL region can exist in an unpaired form. The CH1 region of an animal species that naturally expresses single chain antibodies, for example a camelid such as a llama or camel, or a shark, may be used. Another example of a modified Fab domain is the presence of a constant region CH1 or CL that is not paired with its cognate region and / or the variable region VH or VL, which is not paired with its cognate region.

[0096] As used herein, the term "immune effector cell" or "effector cell" refers to a cell in the natural repertoire of cells in the mammalian immune system that can be activated to affect the viability of target cells. Immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells, but myeloid cells can also be considered immune effector cells, such as monocytes or macrophages, dendritic cells, and neutrophil granulocytes. Thus, the effector cell is preferably a NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell, or a neutrophil granulocyte.

[0097] When referring to nucleic acid or amino acid sequences herein, "percent (%) identity" is defined as the percentage of residues in a candidate sequence that are identical to the residues in a selected sequence after aligning the sequences for optimal comparison purposes. Gaps may be introduced in either of the two sequences being compared to optimize the alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments may be performed over shorter lengths, such as about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0098] The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be achieved using a mathematical algorithm. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percentage sequence identity between two amino acid or nucleic acid sequences can be determined using the Needleman and Wunsch algorithm for aligning two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program of the EMBOSS package is used to determine the percent identity of amino acid and nucleic acid sequences (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used as the substitution matrix. For DNA sequences, DNAFULL is used. The parameters used are a gap open penalty of 10 and a gap extension penalty of 0.5.

[0099] After alignment with the NEEDLE program as described above, the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences, after subtracting the total number of gaps in the alignment, is divided by the total length of the alignment.

[0100] As used herein, the term "connected" refers to domains that are joined together by peptide bonds in the primary amino acid sequence. For example, the heavy chain of a base antibody moiety that includes VH-CH1-CH2-CH3 can be connected to the heavy chain of an additional binding domain VH-CH1 (or an additional binding domain for the additional binding domain) via a linker (connecting the heavy chain of the additional binding domain to the VH region of the base antibody moiety at CH1), which together constitute one polypeptide chain. Similarly, the CH1 domain can be connected to the variable heavy region, and the CL domain can be connected to the variable light region.

[0101] "Pairing" refers to an interaction between the polypeptides constituting the multivalent antibody of the present invention such that they can multimerize. For example, the additional binding domain may comprise a heavy chain region (VH-CH1) paired with a light chain region (VL-CL), where CH1 and CL pair to form the binding domain. As described herein, the pairing of antibody domains (e.g., heavy and light chains) occurs through non-covalent interactions and through disulfide bonds and can be engineered by the techniques disclosed herein and methods known in the art.

[0102] Throughout this specification and the appended claims, the words "comprise," "include," and "having," as well as variations such as "comprises," "comprising," "includes," and "including," are to be interpreted inclusively, that is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where permitted by context.

[0103] The articles "a" and "an" are used herein to refer to more than one (i.e., to one or to at least one) of the grammatical object of the article. As an example, "an element" can mean one element or more than one element.

[0104] The present invention relates to a base antibody portion comprising two binding domains; and at least one additional binding domain, the base antibody portion is connected to at least one additional binding domain by a linker; each binding domain of the base antibody portion and each of the at least one additional binding domain all share a common variable region; The linker comprises a hinge sequence or a sequence derived from a hinge sequence.

[0105] The present invention also provides a base antibody portion comprising two binding domains; and at least one additional binding domain, at least one additional binding domain comprises a CH1 region and is connected to the base antibody moiety by the linker to link the variable region and the CH1 region of the base antibody moiety; The multivalent antibody binds to at least three different epitopes.

[0106] In such a multivalent antibody, each binding domain of the base antibody moiety and each of the at least one additional binding domain may all share a common variable region.

[0107] Thus, the present invention typically provides multivalent antibodies capable of binding to a target or multiple targets via at least three binding domains (i.e., the antibody is a multivalent antibody). A multivalent antibody may optionally be a multispecific antibody. That is, an antibody of the present invention may bind to two or more different epitopes or two or more different antigens, for example two, three, four or more different epitopes or antigens.

[0108] Different formats of multivalent antibodies It should be noted that other features and aspects of the invention will become apparent from the detailed description in conjunction with the accompanying drawings, which illustrate by way of example the features according to embodiments of the invention. The drawings are exemplary and are not intended to limit or limit the scope of the invention as defined by the claims and the full scope of the detailed disclosure, but rather illustrate and enable the invention described herein. The multivalent antibody of the invention may comprise a base antibody portion and an additional binding domain, preferably a Fab domain comprising a VH-CH1 region paired with a VL-CL region. The multivalent antibody comprises three VH regions and three VL regions. Either the VH or VL may be a common variable region (VHc or VLc) paired with a rearranged variable region of a cognate chain. For example, the three VL regions may be a common chain (VLc), and each VH region (VH1-VH3) may comprise a rearranged variable region, and the VH1, VH2, and VH3 regions may bind the same epitope or three different epitopes. A multivalent antibody comprises a common light chain (VLc) and three heavy chain variable regions (VH1-VH3), and an additional Fab domain consisting of VH3-CH1 paired with VLc-CL may be connected to the base antibody via a linker disposed between the VH1 or VH2 region of the base antibody portion and the CH1 of the additional Fab domain. See, for example, FIG. 1a.

[0109] Alternatively, the additional Fab domain may be connected to the base antibody via a linker disposed between the common light chain region (VLc) of the base antibody and the CL region of the additional Fab domain. See, for example, FIG. 1b. In another embodiment of the present invention, the three VH regions may be a common chain (VHc), and each VL region may comprise a rearranged variable region, and the three VL regions may bind to the same or different epitopes (VL1 to VL3). The multivalent antibody comprises a common heavy chain (VHc) and three light chain variable regions (VL1 to VL3), and the additional Fab domain may be connected to the base antibody via a linker disposed between the VL1 or VL2 region of the base antibody moiety and the CL of the additional Fab domain. See, for example, FIG. 1c. Alternatively, the additional Fab domain may be connected to the base antibody via a linker disposed between the common heavy chain region (VHc) of the base antibody and the CH1 region of the additional Fab domain. See, for example, FIG. 1d.

[0110] Alternatively, the additional Fab domain may be connected to the base antibody via a linker located between both the heavy and light variable regions of the base antibody and the CH1 and CL regions of the additional Fab domain, regardless of whether the common chain is a heavy or light chain. See, for example, FIG. 1e.

[0111] The multivalent antibody of the present invention may comprise a base antibody moiety and two or more additional binding domains, for example two Fab domains. Either the VH or VL region of the multivalent antibody may be a common variable region (e.g., VHc or VLc) with cognate chains (e.g., VHc and VL1-VL4, or VH1-VH4 and VLc) comprising rearranged variable regions that bind to the same or different antigens or epitopes. The additional Fab domains may be connected to the base antibody moiety via a linker located between the common chain (VHc or VLc) of the base antibody moiety and the respective constant regions of the common variable regions of the additional Fab domains, or between the rearranged variable domains (VH1 and VH2, or VL1 and VL2) of the base antibody and the respective constant regions of the rearranged variable domains of the additional Fab domains. For example, FIG. 1f shows a multivalent antibody of the present invention comprising a base antibody and two additional Fab domains, the antibody comprising a common light chain (VLc) and four heavy chain variable regions (VH1-VH4), and a linker connecting the base antibody to the additional Fab domains at the rearranged heavy chain variable region (VH2 and VH3) of the base antibody and the CH1 region of the additional Fab domain. Alternatively, FIG. 1g shows a multivalent antibody of the present invention, the base antibody is connected to two additional Fab domains at the first rearranged heavy chain region (VH2) to the CH1 region of the first additional Fab domain, and the common light chain variable region (VLc) of the base antibody is connected to the CL region of the second additional Fab domain. Alternatively, FIG. 1h shows a multivalent antibody of the present invention, the base antibody is connected to two additional Fab domains via a linker connecting both common light chain regions (VLc) of the base antibody to the CL regions of the two additional Fab domains. Alternatively, Figure 1j shows a multivalent antibody of the invention, in which the base antibody is connected to a first additional Fab domain via a linker connecting both the rearranged heavy chain variable region (VH3) and the common light chain region (VLc) of the base antibody to the first additional Fab domain at CH1 and CL, respectively, and the second additional Fab domain is connected to a second rearranged heavy chain variable region (VH2) of the base antibody at the CH1 region of the second additional Fab domain via a linker.Alternatively, (FIG. 1i) the second additional Fab domain is connected to the CL region of the second additional Fab domain via a linker to the common light chain variable region (VLc) of the base antibody. Alternatively, (FIG. 1k) the second additional Fab domain is connected to both the second rearranged heavy chain variable region (VH2) and the common light chain (VLc) of the base antibody moiety via a linker in each of the CH1 and CL regions of the second additional Fab domain. The formats described herein and shown in FIGS. 1f-1k also apply when the common chain is a heavy chain (VHc) and the multivalent antibody comprises four rearranged light chain variable regions (VL1-VL4) comprising up to four different binding specificities.

[0112] Furthermore, two or more additional binding domains may be connected to only one binding domain of the base antibody moiety via a linker, such that a first Fab domain is connected via a linker to a second Fab domain, which is in turn connected to the base antibody moiety. That is, a first linker is disposed between the base antibody moiety and one of the additional Fab domains, and a second linker is disposed between the two additional Fab domains. The two linkers may be the same or different.

[0113] In another embodiment of the present invention, the individual proteins constituting the multivalent antibody can mix heavy and light chains within the same protein. For example, the multivalent antibody can consist of a first protein comprising an additional Fab domain linked to the base antibody in the order VLc-CL-VH2-CH1-CH2-CH3 from N-terminus to C-terminus, whereby the linker connects the VLc-CL region of the additional Fab domain to the base antibody moiety at VH2-CL. A second protein comprising VH1-CH1 pairs with the VLc-CL of the first protein. A third protein comprises VH3-CH1-CH2-CH3 from N-terminus to C-terminus, such that the third and first proteins pair under their respective CH1 regions. A fourth protein comprises VLc-CL from N-terminus to C-terminus, and pairs with the VH2-CH1 of the first protein and the VH3-CH1 of the third protein. See, for example, FIG. 1l.

[0114] Although the format described and illustrated in FIG. 1l shows the use of a common light chain and at least three rearranged heavy chain variable regions (VH1-VH3) with up to three different binding specificities, it should be understood that this format applies when the common chain is a heavy chain (VHc) and the multivalent antibody comprises three or more rearranged light chain variable regions (VL1-VL3) with up to three different binding specificities.

[0115] Another aspect of the invention is a multivalent antibody comprising four proteins, the common chain being a common light chain. The multivalent antibody is composed of four proteins in order from N-terminus to C-terminus, comprising a first protein of VH1-CH1-VLc-CL, where a linker connects CH1 to VLc, a second protein of VLc-CL, where VH1-CH1 pairs to form an additional Fab domain, a third protein comprising VH2-CH1-CH2-CH3, where the CH1 of the third protein pairs with the CL of the second protein, and a fourth protein comprising VH3-CH1-CH2-CH3, where the third and fourth proteins are paired under the CH1 region, and where the second protein (VLc-CL) pairs with the fourth protein at the CH1 region of the fourth protein. See, for example, FIG. 1m.

[0116] The multivalent antibody of the present invention may comprise a base antibody moiety and an additional Fab domain, where either the VH or VL region of the multivalent antibody may be a common variable region, and the additional Fab domain may be connected to the base antibody moiety via a linker located in either the VH (Figure 1n) or VL (Figure 1o) of the base antibody, which simultaneously connects the base antibody to the Fab domain and further pairs with the cognate chain of the Fab domain. In such an example, the Fab domain may optionally lack the CH1-CL domain, and a linker may be used to pair the variable domains of the Fab domain.

[0117] The multivalent antibody of the present invention may comprise a base antibody portion and an additional binding domain comprising paired VH and VL. The additional binding domain comprising VH and VL may be paired via a cysteine ​​bridge formed between VH and VL, which may not require the presence of a CH1 or CL region. See, for example, FIG. 1p. Note that although a cysteine ​​bridge is shown in FIG. 1p, one skilled in the art will understand that an additional cysteine ​​bridge is typically present at the CH1 / CL interface (not shown).

[0118] The multivalent antibody of the present invention may comprise a base antibody moiety and an additional modified Fab domain. The modified Fab domain may comprise a modified CH1, which eliminates the need for pairing with a CL. For example, the CH1 may be a Camelidae CH1, or may be based on a Camelidae CH1, or may be modified to lack hydrophobic residues via techniques known in the art. Each VH or VL may be a consensus or rearranged variable region. The additional modified Fab domain may be connected to the base antibody moiety via a linker located between the VH2 of the base antibody moiety and the CH1 of the modified Fab domain. The VH and VL of the modified Fab domain may be paired via a cysteine ​​bridge, or alternatively a non-covalent interaction. See, for example, FIG. 1q. Alternatively, the additional modified Fab domain may be connected to the base antibody moiety via a linker located between the VL of the base antibody moiety and the CH1 of the modified Fab domain. The VH and VL of the modified Fab domain may be paired via a cysteine ​​bridge. See, for example, FIG. 1r.

[0119] The multivalent antibody of the present invention may comprise a base antibody moiety and an additional modified Fab domain, which may comprise a modified CL, such that it is not necessary to pair with CH1. For example, the CL may be engineered to remove a hydrophobic region. Each VH or VL of the modified Fab domain may be a consensus or rearranged variable region. The additional modified Fab domain may be connected to the base antibody moiety via a linker located between the VL of the base antibody moiety and the CL of the modified Fab domain. The VH and VL of the modified Fab domain may be paired via a cysteine ​​bridge. See, for example, FIG. 1s.

[0120] The multivalent antibody of the present invention may comprise a base antibody moiety and an additional modified Fab domain, which may comprise a modified CL, such that it is not necessary to pair with CH1. For example, the CL may be engineered to remove a hydrophobic region. Each VH or VL of the modified Fab domain may be a consensus or rearranged variable region. The additional modified Fab domain may be connected to the base antibody moiety via a linker located between the VH2 of the base antibody moiety and the CL of the modified Fab domain. The VH and VL of the modified Fab domain may be paired via a cysteine ​​bridge. See, for example, FIG. 1t.

[0121] Base antibody moieties of the present invention Although Figures 1a-1u show a base antibody portion of a multivalent antibody comprising paired heavy chain constant regions comprising CH2 and CH3 regions, it should be noted that these regions are shown for illustrative purposes only and the invention is not limited to these embodiments. Different formats of base antibody portions and additional binding domains suitable for use in the disclosed antibodies are described herein.

[0122] The base antibody moiety of the multivalent antibody of the invention may be a full length immunoglobulin, such as a full length IgG, IgA, IgE, IgD or IgM moiety, but is preferably an IgG, more preferably an IgG1.

[0123] In any antibody of the invention, at least one of the additional binding domains, preferably Fab domains, may comprise a CH1 domain of an immunoglobulin subclass different from that of the CH1 domain(s) of the base antibody portion of the antibody and / or may have a light chain of a different class. For example, if the base portion of the antibody is a full-length IgG1, at least one of the additional binding domains may comprise a CH1 domain of subclass IgG2a, IgG2b, IgG3 or IgG4, and / or if the base portion of the antibody comprises a kappa light chain, at least one of the additional binding domains may comprise a lambda light chain.

[0124] The heavy chain of the base antibody may be designed to preferentially pair through techniques known to those skilled in the art, for example, engineering DEKK modifications in the CH3 region of the base antibody. See WO 2013 / 157954 and De Nardis Et al., J. Biol. Chem. (2017) 292 (35) 14706-14717, incorporated herein by reference, which show genetic engineering in the CH3 region to drive heterodimerization of heavy chains. Alternative approaches to drive heterodimerization that can be used in the present invention include the knob-in-hole format (WO 1998 / 050431), and the use of charge engineering (Gunasekaran, JBC 2010, vol 285, pp 19637-19646).

[0125] The Fc region mediates antibody effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). Depending on the multivalent antibody, it may be desirable to reduce or increase the effector function. A reduced effector function may be desired when an immune response is to be activated, enhanced, or stimulated, as in some embodiments of the present invention. Antibodies with reduced effector function may be used to target cell surface molecules, especially immune cells. An increased effector function may be desirable when the antibody is targeting harmful cells, thereby increasing the ability of immune effector cells or the complement cascade to eliminate or lyse such targets.

[0126] The effector function of the heavy chain Fc region can be reduced or eliminated by modifications known to those of skill in the art. Similarly, the effector function of the heavy chain Fc region can be enhanced by modifications known to those of skill in the art. For example, ADCC can be enhanced by genetic modification of the CH2 domain. See, e.g., Strohl, Curr. Opin. Biotechnol. 2009(6)685-91.

[0127] The multivalent antibody of the present invention may in one embodiment be afucosylated. The multivalent antibody of the present invention preferably comprises a reduced amount of fucosylation of N-linked carbohydrate structures in the Fc region when compared to the same antibody produced in normal CHO cells.

[0128] One embodiment of the multivalent antibody of the present invention also includes a base antibody lacking a CH2 or CH3 region, and the heavy chains of the base antibody moiety may be joined by a cysteine ​​covalent bridge below the CH1 region.

[0129] Embodiments of the invention involving variations of the base antibody moiety of the invention are shown in FIG.

[0130] Described herein is a repertoire of linkers that can be used to connect the base portion of an antibody of the invention with one or more binding domains. One or more binding domains, such as variable regions, Fv domains, Fab domains, or modified Fab domains, can be connected to the base antibody portion of an antibody of the invention.

[0131] The antibodies of the invention comprise a base antibody portion, linked via a linker or multiple linkers to one or more binding domains.

[0132] Multivalent antibodies comprising full-length IgG-based antibody moieties are preferred as such structures typically have beneficial properties such as favorable half-life, predictable biophysical behavior, and lower immunogenicity. The antibodies of the invention are typically suitable for therapeutic use and therefore comprise human sequences for use as human therapeutics. Alternatively, the antibodies have the sequence of the species in which the therapeutic is being used or are based on a consensus sequence within that given species, using techniques well known to those skilled in the art.

[0133] When the base antibody portion of the antibody of the invention is a full-length IgG, the full-length IgG may contain mutations that provide the desired properties. Such mutations are not deletions of significant portions of any region. However, full-length IgG portions in which one or several amino acid residues have been inserted, deleted, or substituted without essentially altering the binding properties of the resulting IgG portion are included in the term "full-length IgG". For example, such IgG portions may have one or more insertions, deletions, or substitutions of 1 to 10 amino acid residues, preferably in non-CDR regions, where the inserted, deleted, or substituted amino acids are not essential for the binding specificity of the IgG.

[0134] IgG1 may be preferred based on its long circulating half-life in humans, and to reduce immunogenicity in humans, it is preferred that the base antibody portion of the antibody according to the invention is a human antibody.

[0135] The base moiety of the antibody of the present invention may be a full length immunoglobulin, which is defined to include essentially a complete antibody. Such an essentially complete antibody may not necessarily have all of the functions of an intact antibody.

[0136] The full length base moiety of the antibody described herein comprises two heavy chains and two light chains. Each chain contains a constant (C) region and a variable (V) region, which are CH1, CH2, CH3, V for the heavy chain and CH4 for the light chain. H , and C for light chains. L , V L Antibodies can be broken down into domains designated as constant domains, Fc and Fc domains. Antibodies can interact with molecules and cells of the immune system via their constant domains, typically via the Fc portion.

[0137] The constant region of the antibody of the present invention, including bispecific or multispecific antibodies, is preferably a human constant region. This constant region may contain one or more, preferably no more than 10, preferably no more than 5 amino acids different from the constant region of a naturally occurring human antibody. The variable domains of the various antibodies produced herein are derived from a human antibody variable domain library. These variable domains are therefore human. The unique CDR regions may be of human origin, synthetic, or derived from another organism. The antibody or bispecific antibody of the present invention is preferably a human or humanized antibody. Suitable heavy chain constant regions are exemplified, but not limited to, in Table 21.

[0138] The antibodies of the present invention typically have an intact Fc region that maintains the half-life and stability of the multispecific antibody. The Fc may also allow interaction with immune effector molecules such as Fc receptors, complement, and FcRn. As will be appreciated by those skilled in the art, techniques are available to engineer the Fc region to prevent or reduce interaction with Fc receptors, or to enhance interaction with Fc receptors.

[0139] The base antibody moiety and one or more additional binding domains, e.g., Fab domains, are connected via one or more linkers. The at least one additional Fab domain may be of a given isotype or subclass, e.g., IgG1, 2a, 2d, 3, or 4, and the at least one additional Fab may be of a different subclass than the subclass of the Fab domain of the full-length IgG moiety, or may possess a light chain of a different class (kappa or lambda).

[0140] Linkers for use in multivalent antibody formats The antibodies of the invention comprise one or more linkers that connect one or more additional binding domains to the base antibody moiety. The linkers, together with the binding domains to which they are connected, at least in part determine the functionality of the multivalent antibody.

[0141] In the antibodies of the present invention, the peptide region of the linker may comprise a hinge sequence or may comprise a sequence based on a hinge sequence. Thus, the amino acid sequence of a suitable peptide region may comprise a naturally occurring sequence or may comprise a sequence based on a naturally occurring sequence. The use of such a sequence may aid in the developability of the multivalent antibodies of the present invention and may help ensure low immunogenicity.

[0142] The hinge region is a flexible amino acid stretch in the middle of the heavy chains of the IgG and IgA immunoglobulin classes (i.e., the part that connects the Fab to the Fc) that holds the two heavy chains together by disulfide bonds. It is rich in cysteine ​​and proline amino acids and has little similarity to any other immunoglobulin region.

[0143] Thus, suitable linkers for connecting one or more additional binding domains to a base antibody moiety for use in the multivalent antibodies of the invention may be derived from an IgG or IgA hinge sequence. The linker region may be based on an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region, or an IgG4 hinge region.

[0144] Typically, the type of hinge region used corresponds to the type of constant region, e.g. CH1, of the additional Fab domain to which the linker is connected, i.e. if the linker is based on the sequence or sequences of an IgG1 hinge region, the CH1 of the additional Fab domain to which the linker is connected is a CH1 from IgG1.

[0145] The linker of an antibody may be based on the upper, middle, or lower hinge region, or a subset of such regions.

[0146] The IgG1 hinge region has the sequence: EPKS C DKTHTCPPCPAPELLGG (sequence number 42).

[0147] The upper hinge region is defined as EPKSCDKTHT (SEQ ID NO:43).

[0148] The middle hinge region is defined as CPPCP (SEQ ID NO:44).

[0149] The lower hinge region is defined as APELLGG (SEQ ID NO:45).

[0150] Thus, in an antibody of the invention, the linker may comprise one or more of these sequences and / or a sequence based on one or more of these sequences.

[0151] The IgG2 hinge region has the sequence: ERK C CVECPPCPAPPVAG (sequence number 46).

[0152] The upper hinge region is defined as ERKCCVE (SEQ ID NO:47).

[0153] The central hinge region is defined as CPPCP (SEQ ID NO:48).

[0154] The lower hinge region is defined as APPVAG (SEQ ID NO:49).

[0155] Thus, in an antibody of the invention, the linker may comprise one or more of these sequences and / or a sequence based on one or more of these sequences.

[0156] The IgG3 hinge region has the sequence: ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEFLGG (SEQ ID NO:50).

[0157] The upper hinge region is defined as ELKTPLGDTTHT (SEQ ID NO:51).

[0158] The central hinge region is defined as CPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (SEQ ID NO:52).

[0159] The lower hinge region is defined as APEFLGG (SEQ ID NO:53).

[0160] The IgG4 hinge region has the sequence: ESKYGPPCPSCPAPEFLGG (SEQ ID NO:54).

[0161] The upper hinge region is defined as ESKYGPP (SEQ ID NO:55).

[0162] The central hinge region is defined as CPSCP (SEQ ID NO:56).

[0163] The lower hinge region is defined as APEFLGG (SEQ ID NO:57).

[0164] The middle region with the consensus sequence CXXC connects both IgG heavy chains and is rigid relative to wild-type IgG. These disulfide bridges are not essential for the current application, therefore the linker comprises a central hinge sequence, and preferably the other or both Cys residues in the CXXC consensus are replaced, for example, by Ser residues. Thus, in a preferred embodiment, CxxC may be SxxS.

[0165] A linker suitable for use in a multivalent antibody of the invention may be based on a middle hinge sequence, e.g. a sequence that includes a middle hinge sequence but does not include the lower and / or upper hinge sequence. A linker suitable for use in a multivalent antibody of the invention may be based on an upper hinge sequence, e.g. a sequence that includes an upper hinge sequence but does not include the lower and / or middle hinge sequence. A linker suitable for use in a multivalent antibody of the invention may not include a middle hinge sequence, e.g. a sequence that includes a combination of lower and upper hinge sequences.

[0166] Thus, the present invention provides a linker comprising one of the amino acid sequences of SEQ ID NOs: 3-5, 7-11, and 13-24.

[0167] Thus, in an antibody of the invention, the linker may comprise one or more of these sequences and / or a sequence based on one or more of these sequences. The peptide region may consist essentially of or be based on the middle region sequence, or may consist essentially of or be based on the upper and lower region sequences.

[0168] Linkers suitable for use in the antibodies of the invention can be defined by reference to a sequence comprising the amino acid sequence of any of the linker sequences described herein, in which 0-5 amino acid insertions, deletions, substitutions, or additions (or combinations thereof) are made. In some embodiments, the linker comprises an amino acid sequence that comprises 0-4, preferably 0-3, preferably 0-2, preferably 0-1, preferably 0 amino acid insertions, deletions, substitutions, or additions (or combinations thereof) with respect to the linker sequences described herein.

[0169] A suitable linker may be about 7 to about 29 amino acids in length, such as about 10 to about 20 amino acids in length. However, a suitable linker may also be a short linker, such as about 7 to about 10 amino acids in length, or a long linker, such as about 20 to about 29 amino acids in length.

[0170] The linker may comprise an Ig hinge region or may comprise a sequence based on an IgG hinge region connected to a CH1 region of the same subclass as the linker and may contain a cysteine ​​for covalent attachment of a common light chain.

[0171] Linkers suitable for use in the antibodies of the invention may be based on an IgG1 hinge region, an IgG2 hinge region, an IgG3 hinge region, or an IgG4 hinge region.

[0172] (G4S) n If a sequence is used, preferably it is used in combination with a hinge sequence derived from an isotype other than IgG or a subclass other than IgG1, and contains a CH1 region.

[0173] In the antibodies of the invention, the linker may be rigid or flexible, may contain charged sequences, and may be linear or curved.

[0174] Rigid sequences for the purposes of the present invention are those with a Karplus and Schulz flexibility prediction of about 1.015 or less. Partially flexible sequences are those with a Karplus and Schulz flexibility prediction of about 1.015 to about 1.04. Flexible sequences for the purposes of the present invention are those with a Karplus and Schulz flexibility prediction of at least about 1.015 (Karplus PA, Schulz GE. Flexibility prediction of chains in proteins - a tool for the selection of peptide antigens. Naturwissenschaften 1985; 72: 212-3; (http: / / tools.immuneepitope.org / bcell / ). Flexibility predictions are calculated over contiguous windows of seven residues along the sequence (in 1 residue increments), resulting in a predicted "flexibility" index for each window. The overall flexibility over the entire linker sequence is given as an average over the entire sequence.

[0175] Removal or substitution of Cys residues in the IgG hinge region can make the hinge-based linker more flexible, including replacing Cys residues with serine (Ser). Alternatively, the linker can be a rigid linker given the presence of a helix-forming sequence. Thus, the middle hinge region, e.g., the conserved CPPCP motif, can be replaced by a helix-forming sequence, e.g., (EAAAK)2, which results in a short, rigid helix in the linker. Thus, in the antibodies of the present invention, the linker can include a helix-forming sequence, e.g., including the amino acid sequence (EAAAK)2. The use of such a sequence can help add rigidity.

[0176] The linker of the present invention may preferably comprise an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 5, 7 to 11, or 13 to 24, or an amino acid sequence having at least about 90% sequence identity to any one of these, preferably at least about 95% sequence identity to any one of these, more preferably at least about 97% sequence identity to any one of these, more preferably at least about 98% sequence identity to any one of these, and more preferably at least about 99% sequence identity to any one of these.

[0177] For example, a linker suitable for use in a multivalent antibody of the invention may be defined by reference to a sequence comprising the amino acid sequence of any one of SEQ ID NOs: 1-24, in which 0-5 amino acid insertions, deletions, substitutions, or additions (or a combination thereof) are made. In some embodiments, the linker comprises an amino acid sequence having 0-4, preferably 0-3, preferably 0-2, preferably 0-1, preferably 0 amino acid insertions, deletions, substitutions, or additions (or a combination thereof) with respect to the sequence set forth in SEQ ID NOs: 3-5, 7-11, or 13-24.

[0178] A linker suitable for use in the multivalent antibody of the present invention can be defined by reference to a sequence that comprises the amino acid sequence of any one of SEQ ID NOs: 1-24, or an amino acid sequence having at least about 85% sequence identity to any one of these, such as at least about 90% sequence identity to any one of these, such as at least about 95% sequence identity to any one of these, such as at least about 98% sequence identity to any one of these, for example at least about 99% sequence identity to any one of these.

[0179] Table 1 shows how the linker sequences can be connected to the CH1 and VH2 regions. [Table 1]

[0180] Use of linkers in the region where additional binding domains are paired A linker as used herein may connect a base antibody moiety to at least one additional binding domain. In addition, if the at least one additional binding domain is a Fab domain or is composed of a pairing of a heavy chain variable region and a light chain variable region, the linker may pair the heavy and light chains via a covalent bond, typically via a disulfide bridge. The disulfide bridge may be formed between a cysteine ​​residue in the linker and the variable region of the additional binding domain(s). Such pairing caused by the linker may apply to additional binding domains including Fab domains comprising a common light chain and a corresponding rearranged heavy chain variable region, or Fab domains comprising a common heavy chain and a corresponding rearranged light chain variable region.

[0181] Multivalent and multispecific When the two binding domains of the base antibody of the multivalent protein of the invention bind different antigens, the first and second antigens can be two different molecules or moieties located on one cell or different cell types. Antibodies comprising two binding domains that mediate cytotoxicity by recruiting and activating endogenous immune cells are a new class of antibody therapeutics. This can be achieved by combining the antigen binding specificities of target cells (i.e., tumor cells) and effector cells (i.e., T cells, NK cells, and macrophages) in one molecule (see, for example, WO 2014 / 051433). The antibodies of the invention comprise at least three binding domains. The base antibody moiety typically comprises two different binding domains (although the two binding domains may have the same sequence and may bind to the same epitope). Multivalent antibodies comprising three or more binding domains may target one, two, three, or more tumor-associated antigens, allowing specific targeting of harmful cells relative to normal cells. For example, one binding domain or two binding domains of a multivalent antibody may bind to an antigen on an abnormal (tumor) cell, while the second or third binding domain of the multivalent antibody may bind to an antigen on an immune effector cell that may cause directed killing of tumor cells expressing one or more tumor-associated antigens. Alternatively, the two binding domains of the multivalent antibody may specifically bind to two different epitopes on the same or different antigens expressed on tumor cells, while the affinity of these arms is weakened to reduce binding to cells expressing only one antigen or when only one binding domain of the multivalent antibody is involved. Alternatively, the three binding domains of the multivalent antibody of the present invention may bind to three different antigens or the same antigen, but at different epitopes of immune effector cells.

[0182] Similarly, a multivalent antibody comprising three or more binding domains can bind to a functional target, such as a ligand or an enzyme, and induce a biological response or block the function of the target, resulting in an inhibitory or agonistic cellular activity. At least one binding domain of the multivalent antibody of the present invention is connected to the binding domain of the base antibody moiety via a linker. If the binding domain of the base antibody moiety is a Fab domain, it can take the form, for example, VH-CH1-linker-VH-CH1, where the linker connects the heavy chain of the base antibody moiety to at least one additional binding domain, preferably a Fab domain.

[0183] Alternatively, it may for example take the form VL-CL-linker-VL-CL, where the linker connects the light chain of the base antibody moiety to at least one additional binding domain, preferably a Fab domain.

[0184] The additional binding domains, such as Fab domains, may each be connected to each of the binding domains of the base antibody moiety via a separate linker. The two or more linkers connecting the additional binding domains to the base antibody moiety or to the additional binding domains may be the same or different. Furthermore, the linkers may allow pairing of cognate chains of the binding domains.

[0185] When an antibody of the invention comprises two or more linkers, those linkers may be the same or different, or a combination thereof. An example of the latter situation is where a multispecific antibody comprises three linkers, two of which are the same and the third linker is different (from the other two).

[0186] Furthermore, a binding domain connected to a binding domain of a base antibody moiety via a linker may itself be connected to a binding domain connected via a linker as described herein, and the base antibody moiety may be modularly extended by connecting it to an additional binding domain via a linker, which in turn may be connected to a second additional binding domain via a linker, etc.

[0187] In this way, the antibodies of the invention are capable of binding to three or more epitopes. Thus, the multispecific antibodies of the invention are capable of specifically binding to three or more epitopes.

[0188] The antibodies of the invention can bind to two, three or more antigens. Thus, the multispecific antibodies of the invention can specifically bind to two, three or more antigens.

[0189] An antibody of the invention may comprise two or more binding domains, such as two or more Fab domains capable of binding to different epitopes on an antigen.

[0190] Thus, an antibody of the invention comprises at least three binding domains, such as two or more different Fab domains.

[0191] Another embodiment of the invention includes multivalent antibodies that comprise at least three Fab domains and are therefore capable of binding to three epitopes that are typically all distinct from one another.

[0192] The antibody of the present invention may be multivalent. The antibody of the present invention may be multispecific. Multivalent indicates that the antibody has at least three binding domains and therefore at least three antigen-binding sites. Multispecific indicates that the antibody can bind to at least two different epitopes, for example, two different antigens or two epitopes on the same antigen. Trispecific indicates that the antibody can bind to three different epitopes. Tetraspecific indicates that the antibody can bind to four different epitopes, etc.

[0193] The antibody of the present invention can bind to target epitopes located on the same molecule.This can allow more efficient antagonism of the (biological) function of the target molecule compared to the situation where only one epitope is targeted.For example, the antibody of the present invention can simultaneously bind to two or more epitopes present on antigen cells, such as growth factor receptors, or soluble molecules that are important for tumor cell proliferation, thereby effectively blocking several independent signal transduction pathways that lead to uncontrolled proliferation.

[0194] Any combination of at least two antibodies of the invention may simultaneously bind to two, three, four or more epitopes present on a target molecule, such as a growth factor receptor or a soluble molecule.

[0195] The targeting moiety may be a soluble molecule, or it may be a membrane-bound moiety, or it may be a moiety present on the cell surface that is internalized upon binding.

[0196] The target epitopes may be located on different moieties, for example on two (i.e., two or more target epitopes on a first moiety and one or more target epitopes on a second moiety) or on three different moieties (i.e., at least one target epitope on each of the three moieties). In this case, each of the different target moieties may be either a soluble moiety or a membrane-bound moiety, or a moiety present on the cell surface that is internalized upon binding. In one embodiment, the different target moieties are soluble moieties. Alternatively, at least one target moiety is a soluble moiety while at least one target moiety is a membrane-bound moiety. In yet another alternative, all target moieties are membrane-bound moieties. In one embodiment, the different target moieties are expressed on the same cell, while in other embodiments, the different target moieties are expressed on different cells.

[0197] As non-limiting examples, any antibody of the invention, or any combination of an antibody of the invention with an additional antibody, may be suitable for simultaneously blocking multiple membrane-bound receptors, neutralizing multiple soluble molecules such as cytokines or growth factors of tumor cells, or neutralizing different viral serotypes or strains.

[0198] In the antibody of the present invention, at least one target epitope may be located on a tumor cell. Alternatively, or in addition, at least the target epitope may be located on the surface of an effector cell. This is suitable, for example, for the recruitment of T cells or NK cells for tumor cell killing. For example, the antibody of the present invention can recruit immune effector cells, preferably human immune effector cells, by specifically binding to a target molecule located on the immune effector cell. In a further embodiment, the immune effector cell is activated upon binding of the antibody of the present invention to the target molecule. The recruitment of effector mechanisms may include the redirection of immune-modulated cytotoxicity by administering an Ig-like molecule produced by the method according to the present invention, which can bind to a cytotoxic trigger molecule, such as a T cell receptor or an Fc gamma receptor, thereby activating downstream immune effector pathways or immune effector cells.

[0199] Immune Cell Engagers A multivalent multimer, such as an antibody, of the invention may be an immune effector cell engager antibody, i.e., a multivalent antibody of the invention may comprise at least one binding domain that specifically binds to an antigen on an immune effector cell, such as a T cell, or may comprise at least one binding domain that specifically binds to an antigen on an abnormal cell, such as a cancer or tumor cell.

[0200] Multivalent multimers of the invention, such as triabodies, may have three binding domains and may bring together three cells, including a tumor cell and two immune effector cells, into an engager complex.

[0201] Multivalent multimers of the invention, such as triabodies, can further have three binding domains and a soluble molecule that target two cells.

[0202] In the embodiments described herein, the Fc may be a wild-type Fc, may be enhanced for ADCC or Cq1 binding based on means known to those of skill in the art, or may be abrogated for such activities based on means known to those of skill in the art.

[0203] The components of such immune cell engaging multivalent antibodies can be arranged relative to each other in various configurations. Exemplary configurations are shown in Figures 1a-1u. In certain embodiments, the invention relates to immune cell engaging multivalent antibodies, in which a third binding domain is linked at the C-terminus of the Fab heavy chain to the N-terminus of the first or second binding domain of the base antibody.

[0204] In one embodiment, the immune cell-engaging multivalent antibody comprises three binding domains, i.e., a base antibody portion and one additional binding domain, whereby the multivalent antibody is trispecific.

[0205] One of the binding domains of the base antibody moiety may bind to an antigen on an immune effector cell. Alternatively, an additional binding domain may bind to an antigen on an immune effector cell. That is, the binding domain for the antigen on the immune effector cell may be at position 1, 2 or 3, which positions correspond to VH1, VH2 and VH3 shown in Figure 1a. Alternatively, when a common heavy chain is used, the binding domain for the antigen on the immune effector cell may be at position 1, 2 or 3, which positions correspond to VL1, VL2 and VL3, as shown in Figure 1c, for example.

[0206] In the immune effector cell engager antibodies of the invention, at least one of the binding domains can specifically bind to an antigen on an abnormal cell. Typically, at least two binding domains bind to an antigen on an abnormal cell, and typically at least two binding domains bind to at least two different antigens or epitopes on the antigen on the abnormal cell. In the immune effector cell engager antibodies of the invention, two or more binding domains may bind the same target, including antigens and epitopes, with different binding domains engaging immune effector cells.

[0207] In a preferred embodiment of the invention, a multivalent antibody of the invention specifically binds to an antigen on an immune effector cell and also specifically binds to two different antigens on an abnormal cell, such as a tumor cell.

[0208] In one embodiment of the present invention, the T cell engaging multivalent antibody can simultaneously bind to a target cell antigen, particularly a tumor cell antigen, and a surface antigen on human T cells. In one embodiment, the T cell engaging multivalent antibody can simultaneously bind to a target cell antigen, particularly a tumor cell antigen, and human CD3. In one embodiment, the T cell engaging multivalent antibody can crosslink the T cell and the target cell by simultaneously binding to the target cell antigen and CD3. In another embodiment, the simultaneous binding results in the lysis of the target cell, particularly a tumor cell. In one embodiment, the simultaneous binding results in the activation of the T cell. In another embodiment, the simultaneous binding results in a cellular response of the T lymphocyte, particularly a cytotoxic T lymphocyte, selected from the group of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. In one embodiment, the binding of the T cell engaging polypeptide to CD3 without simultaneous binding to the target cell antigen does not result in T cell activity, e.g., the remaining binding domain does not bind to the tumor cell antigen.

[0209] In one embodiment, the T cell-engaging multivalent antibody is capable of redirecting the cytotoxic activity of a T cell to a target cell. In one embodiment, the redirection is independent of MHC-mediated peptide antigen presentation by the target cell and / or the specificity of the T cell. In one embodiment, the T cell is a cytotoxic T cell. In another embodiment, the T cell is a CD4 + or CD8 + In another embodiment, the T cells are CD8 + T cells.

[0210] The T cell engaging multivalent antibody of the present invention comprises at least one antigen binding domain capable of binding to a surface antigen of a human T cell. In one embodiment, the binding domain binds to CD3 (also referred to herein as "CD3 antigen binding domain").

[0211] The term "CD3" (cluster of differentiation 3) refers to a protein complex composed of the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), the CD3 epsilon chain (SwissProt P07766), and the CD3 zeta chain homodimer (SwissProt P20963). CD3 epsilon is known under various aliases, some of which are "CD3e molecule, epsilon (CD3-TCR complex)", "CD3e antigen, epsilon polypeptide (TiT3 complex)"; T cell surface antigen T3 / Leu-4 epsilon chain; T3E; T cell antigen receptor complex, epsilon subunit of T3; CD3e antigen; CD3-epsilon 3; IMD18; TCRE. The Id for the CD3E gene is HGNC:1674; Entrez Gene:916; Ensembl:ENSG00000198851; OMIM:186830, and UniProtKB:P07766. These chains can associate with the T cell receptor (TCR) and zeta chain to form the TCR complex and generate activation signals in T lymphocytes by mitogenic signaling. CD3 is expressed on T cells and NK T cells. In this specification, when CD3 is mentioned, it refers to human CD3 unless otherwise specified.

[0212] In certain embodiments, the T cell engaging polypeptide comprises no more than two binding domains capable of specific binding to CD3. In one embodiment, the T cell engaging polypeptide provides monovalent binding to CD3. In one embodiment, the T cell engaging polypeptide comprises a member of a supercluster of CD3 binding binding domains. "Supercluster" is used herein to refer to a variable region that has tolerated amino acid changes, e.g., with respect to the heavy chain variable region comprising the VH or VL and / or CDRs of the present invention, without losing binding specificity to a particular antigen. More specifically, a "supercluster" is a group of clones that share the same VH V gene usage, have at least 70% sequence identity in HCDR3, and have the same HCDR3 length. Clones within a supercluster are expected to potentially bind to the same antigen with different affinities and / or different positions on the epitope.

[0213] CD3 binding domains can range in affinity, epitope, and other properties. Particular variable domains capable of binding to the extracellular portion of CD3 are those containing the amino acid sequences of the VH of MF8057, MF8058, MF8078 and the variable regions of this supercluster, MF8397 and the variable regions of this supercluster, MF8508 and the variable regions of this supercluster, and MF9249 and MF9267 and the variable regions of this supercluster.

[0214] The CD3 antigen-binding domain comprises at least one heavy chain complementarity determining region (CDR) selected from the group consisting of the VH of MF8057, MF8058, MF8078 and the variable regions of this supercluster, MF8397 and the variable regions of this supercluster, MF8508 and the variable regions of this supercluster, and MF9249 and MF9267 and the variable regions of this supercluster (SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:124, SEQ ID NO:133, SEQ ID NO:142, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:152, SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:135, SEQ ID NO:144 and / or SEQ ID NO:153) and at least one light chain CDR selected from the group consisting of SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256.

[0215] In one embodiment, the CD3 antigen binding domain comprises a heavy chain CDR1 of SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:124, SEQ ID NO:133, or SEQ ID NO:142, a heavy chain CDR2 of SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, or SEQ ID NO:152, a heavy chain CDR3 of SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:135, SEQ ID NO:144, or SEQ ID NO:153, a light chain CDR1 of SEQ ID NO:254, a light chain CDR2 of SEQ ID NO:255, and a light chain CDR3 of SEQ ID NO:256.

[0216] In one embodiment, the CD3 antigen binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:100, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:135, SEQ ID NO:145, and SEQ ID NO:154, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:37 and SEQ ID NO:40.

[0217] In one embodiment, the CD3 antigen binding domain comprises a heavy chain variable region of SEQ ID NO:100, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:127, SEQ ID NO:135, SEQ ID NO:145, or SEQ ID NO:154, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:40.

[0218] The positions of the binding domains in a multivalent antibody can be defined. If the binding or variable domains of the base antibody are called binding domains 1 and 2, the additional one or more binding domains can be called binding domains 3, 4, etc. The binding domains are also called BDs. BD3 may be linked to BD1 or BD2. If BD3 is linked to one of BD1 or 2, BD4, if present, is linked to the other of BD1 and 2.

[0219] The T cell engaging multivalent antibodies of the invention comprise at least two antigen binding domains capable of binding to a target cell antigen (also referred to herein as "target cell antigen binding domains" or "second" or "third" antigen binding domains). In certain embodiments, the T cell engaging multivalent antibody comprises two antigen binding domains capable of binding to a target cell antigen. In one embodiment, each of these antigen binding domains specifically binds to the same antigenic determinant. In another embodiment, the target cell antigen binding domains are identical. In one embodiment, the T cell engager polypeptide comprises no more than two target cell antigen binding domains capable of binding to a target cell antigen.

[0220] In one embodiment, the multivalent antibody comprises BD1, a CD3 binding domain, BD2, a binding domain that binds a first target cell antigen (hereafter referred to as TA1), and BD3, a binding domain that binds a second target cell antigen (hereafter referred to as TA2). In this embodiment, BD3 can be linked to BD1 or BD2. In one embodiment, TA2 that binds BD3 is linked to CD3 that binds BD1. In another embodiment, TA2 that binds BD3 is linked to TA1 that binds BD2.

[0221] In one embodiment, the multivalent antibody comprises a TA1 that binds BD1, a TA2 that binds BD2, and a CD3 that binds BD3. In this embodiment, BD3 can be linked to BD1 or BD2. In one embodiment, the CD3 that binds BD3 is linked to a TA1 that binds BD1. In another embodiment, the CD3 that binds BD3 is linked to a TA2 that binds BD2.

[0222] In one embodiment, the invention provides a multivalent antibody, where the base antibody comprises binding domains 1 and 2 (BD1 and 2), an additional binding domain 3 (BD3) is linked to binding domain 1 (BD1) and an optional additional binding domain 4 (BD4) is linked to binding domain 2 (BD2). In one embodiment, binding domain 1 is a CD3 binding domain and binding domains 2 and 3 bind different target cell antigens. In another embodiment, binding domain 2 is a CD3 binding domain and binding domains 1 and 3 bind different target cell antigens. In a further embodiment, binding domain 3 is a CD3 binding domain and binding domains 1 and 2 bind different target cell antigens.

[0223] In a preferred embodiment, the multivalent antibody comprises binding domains 4 that bind to further distinct target cell antigens.

[0224] The invention further provides a multivalent antibody as described herein, wherein the base antibody comprises binding domains 1 and 2, wherein additional binding domain 3 is linked to binding domain 1 and optional additional binding domain 4 is linked to binding domain 2. In one embodiment, binding domain 1 is a CD3 binding domain and binding domains 2 and 3 bind different target cell antigens. In another embodiment, binding domain 2 is a CD3 binding domain and binding domains 1 and 3 bind different target cell antigens. In a further embodiment, binding domain 3 is a CD3 binding domain and binding domains 1 and 2 bind different target cell antigens.

[0225] In the case where it comprises a binding domain 4, it is preferred that the domain further binds to a different target cell antigen.

[0226] In one embodiment, the first of the target cell antigen binding domains binds to PD-L1, EGFR, CD137, CLEC12A, fibrinogen, or thyroglobulin. In one embodiment, the first and second of the target cell antigen binding domains bind to an antigen selected from PD-L1, EGFR, CD137, CLEC12A, fibrinogen, and thyroglobulin. The first and second target cell binding domains preferably bind different antigens.

[0227] The CD3-binding domain preferably comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 of MF8057 or MF8058 or MF8078 or MF8397 or MF8508 or MF9249 or MF9267. The CD3-binding domain preferably comprises a heavy chain variable region having the amino acid sequence of VH of MF8057, MF8058, MF8078, MF8397, MF8508, MF9249, or MF9267, preferably having 0 to 10, preferably 0 to 5, amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDRs.

[0228] The target cell antigen-binding domain may be a PD-L1-binding domain. If present, the PD-L1-binding domain preferably comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 of MF5377, or MF5444, or MF5380. The PD-L1-binding domain preferably comprises a heavy chain variable region having the amino acid sequence of VH of MF5377, MF5444, or MF5380 with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDRs.

[0229] The target cell antigen-binding domain may be an EGFR-binding domain. When present, the EGFR-binding domain preferably comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 of MF8233, or MF9891, or MF9886, or MF9873, or MF9988. The EGFR-binding domain preferably comprises a heavy chain variable region having the amino acid sequence of VH of MF8233, MF9891, MF9886, MF9873, or MF9988, with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDRs.

[0230] The target cell antigen-binding domain may be a CLEC12A-binding domain. When present, the CLEC12A-binding domain preferably comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 of MF4327. The CLEC12A-binding domain preferably comprises a heavy chain variable region having the amino acid sequence of VH of MF4327, with 0-10, preferably 0-5, amino acid insertions, deletions, substitutions, additions, or combinations thereof at one or more positions other than the CDRs.

[0231] In one embodiment, the multivalent antibody comprises a CD3 binding domain, an EGFR binding domain, and a PD-L1 binding domain.

[0232] The binding domain having the heavy chain variable region shown comprises a light chain variable region. The light chain variable region preferably comprises CDR1, CDR2 and CDR3 regions comprising the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, i.e., the CDRs of IGKV1-39 (according to IMGT). The amino acid mutations, insertions, deletions, substitutions, additions or combinations thereof are preferably absent in the CDR3 region of the light chain variable region, and preferably absent in the CDR1 or CDR2 regions of the light chain variable region. In a preferred embodiment, the light chain variable region does not comprise deletions, additions or insertions relative to the sequence shown. In this embodiment, the light chain variable region may have 0-5 amino acid substitutions relative to the amino acid sequence shown. The amino acid substitutions are preferably conservative amino acid substitutions. The CDR1, CDR2 and CDR3 of the light chain of the antibody of the invention preferably comprise the amino acid sequences CDR1-QSISSY, CDR2-AAS, CDR3-QQSYSTP, respectively, i.e., the CDRs of IGKV1-39 (according to IMGT), as described elsewhere herein. The light chains of the binding domains with the heavy chain variable regions shown preferably all comprise the same light chain, preferably a common light chain as defined elsewhere herein.

[0233] The amino acid insertions, deletions, substitutions, additions, or combinations thereof are preferably not in the CDR3 region of the heavy chain variable region, and preferably not in the CDR1 and / or CDR2 regions of the heavy chain variable region. In a preferred embodiment, the heavy chain variable region does not contain deletions, additions, or insertions relative to the sequence shown. In one embodiment, the heavy chain variable region can have 0-10, preferably 0-5, amino acid substitutions relative to the amino acid sequence shown. In a preferred embodiment, the heavy chain variable region contains 0-9, 0-8, 0-7, 0-6, 0-5, 0-4, preferably 0-3, preferably 0-2, preferably 0-1, and preferably 0 amino acid insertions, deletions, substitutions, additions, or combinations thereof relative to the amino acid sequence shown at positions other than the CDRs. A combination of insertions, additions, deletions, or substitutions is a claimed combination if the aligned sequences do not differ at more than 10 positions (preferably less than 5). A gap in one of the aligned sequences corresponds to the same number of amino acids skipped in the other sequence. The amino acid substitutions, if any, are preferably conservative amino acid substitutions.

[0234] In one embodiment, the target cell antigen binding domain is a Fab molecule. In one embodiment, the target cell antigen binding domain is a Fab molecule that binds to a specific antigenic determinant and can direct the T cell-engaged multivalent antibody to a target site, for example, a specific type of tumor cell that bears the antigenic determinant.

[0235] In a particular embodiment, the target cell antigen binding specifically binds to Programmed Cell Death 1 protein (PD-L1), preferably human PD-L1 (SEQ ID NO: 257).

[0236] PD-L1 is a type 1 transmembrane protein that plays a role in suppressing immune responses during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis. Binding of PDL1 to PD-1 or B7.1 (CD80) transmits an inhibitory signal, which reduces the proliferation of PD-1 expressing T cells. It is believed that PD-1 can control the accumulation of foreign antigen-specific T cells via apoptosis. PD-L1 is expressed by various cancer cells, and its expression is believed to be at least partially involved in dampening the immune response against cancer cells. PD-L1 is a member of the B7 family of proteins and is known by various other names, such as CD274 molecule, CD274 antigen, B7 homolog 1, PDCD1 ligand 1, PDCD1LG1, PDCD1L1, B7H1, PDL1, programmed cell death 1 ligand 1, programmed cell death ligand 1, B7-H1, and B7-H, the external Ids for CD274 are HGNC: 17635, Entrez Gene: 29126; Ensembl: ENSG00000120217; OMIM: 605402; UniProtKB: Q9NZQ7.

[0237] PD-L1 binding domains can range in affinity, epitope, and other properties. Particular variable domains capable of binding to the extracellular portion of PD-L1 are variable domains comprising the amino acid sequence of the VH of MF5377, MF5444, or MF5380.

[0238] The PD-L1 antigen-binding domain comprises at least one heavy chain CDR selected from the group consisting of the VH of SEQ ID NO:160, SEQ ID NO:169, SEQ ID NO:178, SEQ ID NO:161, SEQ ID NO:170, SEQ ID NO:179 SEQ ID NO:162, SEQ ID NO:171, and SEQ ID NO:180, and at least one light chain CDR selected from the group of SEQ ID NO:254, SEQ ID NO:255, and SEQ ID NO:256.

[0239] In one embodiment, the PD-L1 antigen-binding domain comprises a heavy chain CDR1 of SEQ ID NO: 160, SEQ ID NO: 169, or SEQ ID NO: 178, a heavy chain CDR2 of SEQ ID NO: 161, SEQ ID NO: 170, or SEQ ID NO: 179, a heavy chain CDR3 of SEQ ID NO: 162, SEQ ID NO: 171, or SEQ ID NO: 180, a light chain CDR1 of SEQ ID NO: 254, a light chain CDR2 of SEQ ID NO: 255, and a light chain CDR3 of SEQ ID NO: 256.

[0240] In one embodiment, the PD-L1 antigen-binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:163, SEQ ID NO:172, and SEQ ID NO:181, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:37 and SEQ ID NO:40.

[0241] In one embodiment, the PD-L1 antigen-binding domain comprises a heavy chain variable region of SEQ ID NO: 163, SEQ ID NO: 172, or SEQ ID NO: 181, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 40.

[0242] In certain embodiments, the PD-L1 antigen-binding domain comprises heavy and light chain variable regions of a PD-L1 antibody comprising the amino acid sequence MPDL3280A, RG7446 (see U.S. Patent Publication No. 2010 / 0203056(A1)), MEDI-4736 (see WO 2011 / 066389), MSB-0010718C (see WO 2013 / 079174), STI-1014 (see WO 2013 / 181634), CX-072 (see WO 2016 / 149201), KN035 (Zhang et al., Cell Discov. 7:3 (March 2018) 2017)), LY3300054 (see, e.g., WO 2017 / 034916), and CK-301 (see, Gorelik et al., AACR: Abstract 4606 (Apr 2016)), and 12A4 or MDX-1105 (see, e.g., WO 2013 / 173223).

[0243] In certain embodiments, the PD-L1 antigen binding domain is selected from the group consisting of the PD-L1 antibodies MPDL3280A, RG7446 (see US2010 / 0203056 A1), MEDI-4736 (see WO 2011 / 066389), MSB-0010718C (see WO 2013 / 079174), STI-1014 (see WO 2013 / 181634), CX-072 (see WO 2016 / 149201), KN035 (see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (see, e.g., WO 2017 / 034916), and CK-301 (Gorelik et al., AACR: Abstract 4606 (Apr 2016)), and 12A4 or MDX-1105 (see, e.g., WO 2013 / 173223).

[0244] In certain embodiments, the PD-L1 antigen binding domain is selected from the group consisting of the PD-L1 antibodies MPDL3280A, RG7446 (see US2010 / 0203056 A1), MEDI-4736 (see WO 2011 / 066389), MSB-0010718C (see WO 2013 / 079174), STI-1014 (see WO 2013 / 181634), CX-072 (see WO 2016 / 149201), KN035 (see Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (see, e.g., WO 2017 / 034916), and CK-301 (Gorelik et al., AACR: Abstract 4606 (Apr 2016)), and 12A4 or MDX-1105 (see, e.g., WO 2013 / 173223), which compete for binding to PD-L1 with the heavy and light chain variable regions of 12A4 or MDX-1105.

[0245] In certain embodiments, the target cell antigen binding specifically binds human epidermal growth factor receptor (EGFR) (SEQ ID NO: 258). "ErbB1" or "EGFR" is a member of a family of four receptor tyrosine kinases (RTKs) designated Her- or cErbB-1, -2, -3 and -4. EGFR has an extracellular domain (ECD) composed of four subdomains, two of which are involved in ligand binding and one of which is involved in homodimerization and heterodimerization. Reference numbers used in this section refer to the numbering of the enumeration entitled "References Cited herein". EGFR integrates extracellular signals from various ligands to obtain diverse intracellular responses. The major signaling pathway activated by EGFR consists of the Ras mitogen-activated protein kinase (MAPK) mitogenic signaling cascade. Activation of this pathway is initiated by the recruitment of Grb2 to tyrosine phosphorylated EGFR. This leads to activation of Ras via the Grb2-bound Ras guanine nucleotide exchange factor Son of Sevenless (SOS). In addition, the PI3-kinase-Akt signaling pathway is also activated by EGFR, but this activation is much stronger in the presence of co-expression of Her3. EGFR is involved in several human epithelial malignancies, especially cancers of the breast, bladder, non-small cell lung, colon, ovary, head and neck, and brain. Activating mutations in this gene, as well as overexpression of the receptor and its ligands, have been found, resulting in an autocrine activation loop. Therefore, this RTK is widely used as a target for cancer therapy. Both small molecule inhibitors directed against the extracellular ligand-binding domain of the RTK and monoclonal antibodies (mAbs) targeting the RTK have been developed, which have shown some clinical success, so far, mostly for select groups of patients. The database accession number for the human EGFR protein and the gene encoding it is (GenBank NM_005228.3).This accession number is provided primarily to provide an additional method of identifying the EGFR protein as a target, and the actual sequence of the EGFR protein bound by the antibody may vary due to mutations in the encoding gene, such as those that occur in some cancers. References to EGFR herein refer to human EGFR unless otherwise specified. The antigen binding site that binds to EGFR binds to EGFR and various mutants, such as those expressed in some EGFR-positive tumors.

[0246] EGFR binding domains can range in affinity, epitope, and other properties. Particular variable domains capable of binding to the extracellular portion of EGFR are those comprising the amino acid sequence of VH of MF8233, MF9891, MF9886, MF9873, MF9988.

[0247] The EGFR antigen binding domain comprises at least one heavy chain CDR selected from the group consisting of SEQ ID NO:187, SEQ ID NO:196, SEQ ID NO:205, SEQ ID NO:214, SEQ ID NO:223, SEQ ID NO:188, SEQ ID NO:197, SEQ ID NO:206, SEQ ID NO:215, SEQ ID NO:224 SEQ ID NO:189, SEQ ID NO:198, SEQ ID NO:207, SEQ ID NO:216, and SEQ ID NO:225, and at least one light chain CDR selected from the group of SEQ ID NO:254, SEQ ID NO:255, SEQ ID NO:256.

[0248] In one embodiment, the EGFR antigen binding domain comprises a heavy chain CDR1 of SEQ ID NO: 187, SEQ ID NO: 196, SEQ ID NO: 205, SEQ ID NO: 214, or SEQ ID NO: 223, a heavy chain CDR2 of SEQ ID NO: 188, SEQ ID NO: 197, SEQ ID NO: 206, SEQ ID NO: 215, or SEQ ID NO: 224, a heavy chain CDR3 of SEQ ID NO: 189, SEQ ID NO: 198, SEQ ID NO: 207, SEQ ID NO: 216, or SEQ ID NO: 225, a light chain CDR1 of SEQ ID NO: 254, a light chain CDR2 of SEQ ID NO: 255, and a light chain CDR3 of SEQ ID NO: 256.

[0249] In one embodiment, the EGFR antigen binding domain comprises a heavy chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:190, SEQ ID NO:199, SEQ ID NO:208, SEQ ID NO:217, and SEQ ID NO:226, and a light chain variable region sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO:37 and SEQ ID NO:40.

[0250] In one embodiment, the EGFR antigen binding domain comprises a heavy chain variable region of SEQ ID NO: 190, SEQ ID NO: 199, SEQ ID NO: 208, SEQ ID NO: 217, or SEQ ID NO: 226, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 40.

[0251] In certain embodiments, the EGFR antigen binding domain comprises the heavy and light chain variable regions of the EGFR antibody, cetuximab (C225, Erbitux®, Lilly) or panitumumab (Vectibix, Amgen).

[0252] In certain embodiments, the EGFR antigen binding domain binds to the same epitope as the heavy and light chain variable regions of the EGFR antibody, cetuximab (C225, Erbitux®, Lilly) or panitumumab (Vectibix, Amgen).

[0253] In certain embodiments, the EGFR antigen binding domain competes for binding to EGFR with the heavy and light chain variable regions of the EGFR antibody, cetuximab (C225, Erbitux®, Lilly) or panitumumab (Vectibix, Amgen).

[0254] Common variable region The multivalent antibodies of the invention preferably use a common chain in each of the three or more binding domains. As described, the base antibody moiety of the multivalent antibody of the invention preferably has a first heavy chain variable region / light chain variable region (VH / VL) combination that binds one antigen and a second VH / VL combination that binds a second antigen. Each additional binding domain attached to the base antibody moiety may comprise an additional VH / VL combination that binds to an additional epitope on the antigen.

[0255] The base antibody moiety of the present invention preferably comprises two heavy chains (one or both of which comprises one or more additional CH1 and VH domains) and a light chain paired with each CH1 and VH domain. Preferably, the two heavy chains have compatible heterodimerization domains, and preferably the light chain is a common light chain. Alternatively, the base antibody moiety of the multivalent antibody of the present invention comprises two light chains (one or both of which comprises one or more additional CL and VL domains) and a heavy chain variable region paired with each CL and VL domain, the heavy chain variable region comprising a common heavy chain variable region.

[0256] Embodiments of the invention include multivalent antibodies comprising a common light chain, which is expressed in a host cell comprising DNA encoding two or more heavy chain variable regions, and which is capable of pairing with available heavy chains (or CH1-VH1 regions), thereby forming at least three functional antigen-binding domains.

[0257] A functional antigen-binding domain is capable of specifically binding to an epitope on an antigen. Preferably, the common light chain used in the multivalent antibody of the invention is capable of pairing with all heavy chains (or CH1-VH1 regions) produced by the method according to the invention, thereby forming a functional antigen-binding domain, such that mispairing of mismatched heavy and light chains is avoided or is produced in a significantly lower proportion than in multivalent antibodies.

[0258] It is a preferred embodiment of the invention that the multivalent antibodies of the invention have a common light chain (variable region) that can combine with a set of heavy chain variable regions to form an antibody with a functional antigen-binding domain (WO 2004 / 009618, WO 2009 / 157771).

[0259] The common light chain (variable region) for use in the multivalent antibody of the present invention is preferably a human light chain (variable region). The common light chain (variable region) preferably has a germline sequence. Preferred germline sequences are light chain variable regions that are frequently used in the human repertoire and have good thermodynamic stability, yield, and solubility. A preferred germline light chain is O12. The common light chain is preferably a rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 (FIG. 11A; SEQ ID NO: 35). The common light chain variable region is preferably a rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * 01 variable region (FIG. 11A; SEQ ID NO: 35). The common light chain preferably comprises a light chain variable region as shown in FIG. 11B or 8D (SEQ ID NO: 37 or 40, respectively) with 0-5 amino acid insertions, deletions, substitutions, additions, or a combination thereof. The common light chain preferably further comprises a light chain constant region, preferably a kappa light chain constant region. The nucleic acid encoding the common light chain may be codon optimized for the cell system used to express the common light chain protein. The encoding nucleic acid may deviate from the germline nucleic acid sequence.

[0260] The common light chain (variable region) for use in the multivalent antibody of the present invention may be a lambda light chain, which is also provided within the context of the present invention, but is preferably a kappa light chain. The common light chain of the present invention may comprise the constant region of a kappa or lambda light chain. This is preferably the constant region of a kappa light chain, and preferably the common light chain is a germline light chain, preferably a rearranged germline human kappa light chain comprising an IgVKI-39 gene fragment, such as the rearranged germline human kappa light chain IgVKl-39. * 01 / IGJKI* 01 (Figure 11). Rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * The terms IGKV1-39 / IGKJ1, huVκ1-39 light chain, or simply huVκ1-39 for short, or simply 1-39, are used interchangeably throughout this application. Those skilled in the art will recognize that "common" also refers to functional equivalents of light chains that are not identical in amino acid sequence. Many variants of the light chain exist, and mutations (deletions, substitutions, additions) exist that do not substantially affect the formation of a functional binding region.

[0261] IgVκ1-39 is a contraction for the immunoglobulin variable kappa 1-39 gene. This gene is also known as immunoglobulin kappa variable 1-39, IGKV1-39, IGKV1-39, O12a, or O12. The external Ids for this genetic element are HGNC:5740, Entrez Gene:28930, Ensembl:ENSG00000242371. A preferred amino acid sequence for IgVκ1-39 is shown in FIG. 11, which lists the sequence of the V region. The V region can be combined with one of five J regions. FIG. 11 describes two preferred sequences for IgVκ1-39 combined with a J region. The combined sequences are shown as IGKV1-39 / jk1 and IGKV1-39 / jk5, with alternative names being IgVκ1-39. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 * 01 (Worldwide Web nomenclature of the IMGT database at imgt.org).

[0262] The common light chain variable region is preferably linked to a kappa light chain constant region. In a preferred embodiment, the light chain variable region used in the multivalent antibody of the present invention is the kappa light chain IgVκ1-39. * 01 / IGJκ1 * 01 or IgVκ1-39 * 01 / IGJκ5 *In a preferred embodiment, the common light chain in the multivalent antibody is IgVκ1-39. * 01 / IGJκ1 * The number is 01.

[0263] Cells producing common light chains include, for example, rearranged germline human kappa light chain IgVκ1-39 * 01 / IGJκ1 * Light chains can be produced that comprise the variable regions of the aforementioned light chains fused to 01 and lambda constant regions. Where germline sequences are referred to herein, it is preferred that the variable regions are germline sequences.

[0264] A preferred common light chain for use in the multivalent antibody of the invention is that which comprises the sequence set forth in SEQ ID NO:29.

[0265] The common chain for use in the multivalent antibody of the present invention may be a heavy chain, which is also provided in the context of the present invention. Common heavy chains have been used in the art to generate bispecific antibodies and can be used herein in generating multivalent antibodies comprising three or more binding domains, two or more of which comprise a common heavy chain as known in the art. For example, the use of antibody libraries in which the heavy chain variable domains are the same for all library members, and thus the diversity is based on the light chain variable domains. Such libraries are described, for example, in PCT / US2010 / 035619 and PCT / US2010 / 057780, each of which is incorporated herein by reference in its entirety. These and other techniques for generating binding domains with a common heavy chain can be made by those skilled in the art and can be used in the present invention to produce multivalent antibodies with the novel formats disclosed herein.

[0266] Production of multivalent antibodies Multivalent antibodies of the invention can be produced by co-transfecting individual cells with one or more genetic constructs that together encode three or more proteins that form a multimer comprising a multivalent antibody as described above, including Figures 1a-1u. For example, a host cell can be co-transfected with nucleic acid encoding three or more heavy chain variable regions and a common light chain variable region to produce a multivalent antibody. Alternatively, multivalent antibodies of the invention can be produced by co-transfecting individual cells with one or more genetic constructs that together encode three or more light chain variable regions and a common heavy chain.

[0267] Several methods have been published to favor the production of antibodies that are heterodimers. In the present invention, it is preferred that the cells prefer the production of heterodimers over the production of the respective homodimers. This is typically achieved by modifying the constant region of the heavy chain to prefer heterodimerization (i.e., dimerization in which one heavy chain is bound to a second heavy chain) over homodimerization. In a preferred embodiment, the antibody of the present invention comprises two different immunoglobulin heavy chains with compatible heterodimerization domains.

[0268] This compatible heterodimerization domain is preferably a compatible immunoglobulin heavy chain CH3 heterodimerization domain. If wild-type CH3 domains are used, co-expression of two different heavy chains (A and B) with a common light chain will result in three different antibody species, AA, AB and BB. AA and BB are designations for two homodimeric antibodies, and AB is designation for a heterodimeric antibody. To increase the proportion of the desired heterodimeric product (AB), CH3 engineering can be used, or in other words, heavy chains with compatible heterodimerization domains can be used, as defined below. The art describes various ways in which such heterodimerization of heavy chains can be achieved.

[0269] As used herein, the term "compatible heterodimerization domain" refers to a protein domain that has been engineered such that engineered domain A' preferentially forms heterodimers with engineered domain B', and vice versa, with reduced homodimerization between A'-A' and B'-B'.

[0270] In US Patent Application Publication No. 13 / 866,747 (now issued as US Patent No. 9,248,181), US Patent Application Publication No. 14 / 081,848 (now issued as US Patent No. 9,358,286), WO 2013 / 157953, and WO 2013 / 157954, methods and means for producing multivalent antibodies using compatible heterodimerization domains are disclosed. These means and methods can be suitably employed in the present invention. In particular, the antibodies of the present invention preferably contain mutations for producing essentially only bispecific full-length IgG molecules. Preferred mutations are amino acid substitutions L351K and T366K (EU numbering) in the first CH3 domain or corresponding positions ("KK-mutant" heavy chain), and amino acid substitutions L351D and L368E in the second domain or corresponding positions ("DE-mutant" heavy chain) (or vice versa). It has been previously demonstrated in our U.S. Patents Nos. 9,248,181 and 9,358,286, and in WO 2013 / 157954, that DE- and KK-mutants preferentially pair to form heterodimers (so-called "DEKK" bispecific molecules). Homodimerization of DE-mutant heavy chains (DEDE homodimers) or KK-mutant heavy chains (KKKK homodimers) rarely occurs due to repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0271] In a preferred host cell of the invention capable of expressing proteins that multimerize to form a multivalent antibody, the host cell is transformed with nucleic acid encoding three proteins, in order from N-terminus to C-terminus, the encoded proteins comprise a first protein comprising VH1-CH1-VH2-CH1-CH2-CH3, a linker connecting VH2 and CH1 on the first protein, a second encoded protein comprising VLc-CL, and a third encoded protein comprising VH3-CH1-CH2-CH3, CH1 of the first and third encoded proteins pairing with CL of the second encoded protein, and the encoded CH3 regions of the first and third proteins respectively encoding amino acids L351K and T366K (EU numbering) at or corresponding positions in the first CH3 protein, or encoding amino acids L351D and L368E at or corresponding positions in the third protein, or vice versa. Alternatively, the first and third proteins contain other compatible heterodimerization domains that cause efficient pairing of the respective CH3 domains of these proteins.

[0272] The nucleic acids encoding the three proteins may be present on one or more vectors to produce the multivalent antibodies of the invention. Similarly, a host cell may encode and produce more than three proteins for each of the multivalent antibodies described above, including those of Figures 1a-1u.

[0273] The nucleic acids encoding the three proteins may be stably integrated into the genome of the host cell, preferably in a chromosomal region known for high expression and the absence or reduced gene silencing.

[0274] Thus, according to the present invention there is provided a method for preparing a multivalent antibody, the method comprising the steps of: Providing a cell comprising one or more nucleic acid sequences encoding polypeptides that can be assembled into a multivalent antibody according to the invention; and culturing the host cells under conditions that provide for the expression of the polypeptides and their assembly into the multivalent antibody.

[0275] The host cells of the present invention are capable of producing a multivalent antibody of the present invention with a purity of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% based on total expressed immunoglobulin.

[0276] A host cell of the invention is capable of producing multivalent antibodies, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% of the multivalent antibodies produced contain variable rearrangement regions paired with cognate common chains for all binding sites.

[0277] A host cell of the invention is capable of producing multivalent antibodies, wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% of the expressed common chains are paired into a multivalent antibody and are not free unassociated proteins.

[0278] Suitable cells for producing antibodies are hybridoma cells, Chinese hamster ovary (CHO) cells, NS0 cells, or PER-C6 cells. In a particularly preferred embodiment, the cells are CHO cells. The cells for producing the antibodies disclosed herein are also referred to as host cells.

[0279] Various institutions and companies have developed cell lines for the large-scale production of antibodies, e.g., for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. At least some of these cells are also used for other purposes, such as for the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. In a preferred embodiment, the present invention provides industrial cell lines producing the antibodies of the present invention.

[0280] In one embodiment, the present invention provides a cell (host cell) comprising an antibody according to the invention and / or a nucleic acid according to the invention. The cell is preferably an animal cell, more preferably a mammalian cell, more preferably a primate cell, most preferably a human cell. For the purposes of the present invention, a suitable cell, a suitable host cell, is any cell that contains and is preferably capable of producing an antibody according to the invention and / or a nucleic acid according to the invention.

[0281] The invention further provides a cell comprising an antibody according to the invention. Preferably, the cell (typically an in vitro, isolated or recombinant cell) produces the antibody. In a preferred embodiment, the cell is a hybridoma cell, a Chinese Hamster Ovary (CHO) cell, an NS0 cell, or a PER-C6 cell. In a particularly preferred embodiment, the cell is a CHO cell. A cell culture comprising a cell according to the invention is further provided. Various institutions and companies have developed cell lines for large-scale production of antibodies, e.g., for clinical use. Non-limiting examples of such cell lines are CHO cells, NS0 cells, or PER.C6 cells. These cells are also used for other purposes, such as for the production of proteins. Cell lines developed for industrial-scale production of proteins and antibodies are further referred to herein as industrial cell lines. Thus, in a preferred embodiment, the invention provides the use of a cell line developed for large-scale production of antibodies for the production of a multivalent antibody of the invention. The invention further provides a cell for producing a multivalent antibody comprising another nucleic acid molecule that is encoded alone or together with the claimed multivalent antibody.

[0282] The present invention also provides a method for producing two or more antibodies by the same cell, at least one of which is a multivalent antibody as described herein. This embodiment is exemplified by the DE / KK heterodimerization system described above. However, the present invention is not limited to a particular method for enabling heterodimerization of heavy chains. As described above, DE-mutants and KK-mutants preferentially pair to form heterodimers (so-called "DEKK" bivalent / multivalent molecules). Homodimerization of DE-mutant heavy chains (DEDE homodimers) or KK-mutant heavy chains (KKKK homodimers) rarely occurs due to repulsion between charged residues at the CH3-CH3 interface between identical heavy chains. Introducing additional heavy chains with either DE- or KK-mutant heavy chains allows the production of additional DEKK bivalent / multivalent molecules. The newly introduced DE-heavy chain (DE2) can associate with the existing KK heavy chain. Thus, the cell produces two double / multivalent antibodies, DE1KK and DE2KK bivalent antibodies. If a new KK heavy chain (KK2) is introduced instead of the new DE heavy chain, a bivalent antibody with a combination of DEKK1 and DEKK2 is produced. The levels at which different antibodies can be produced by the cell are typically optimally regulated by adjusting the relative expression of the DE1 / 2 and KK1 / 2 chains relative to each other. The light chain is typically produced sufficiently to reduce the level of a single heavy chain, and the level at which one chain is produced is typically sufficient to allow effective pairing with the complementary chain. In DE1 / 2, the DE1 and DE2 heavy chains, which are KK examples, are preferably produced at a level that matches the level of the KK heavy chain. The level of each antibody can be regulated by adjusting the level at which DE1 and DE2 are produced in conjunction with each other. In the case of the KK1 / 2 DE variant, the situation is of course similar, but in the case of the KK1 and KK2 chains, which are linked to each other. Depending on the number of binding or variable domains associated with each of the heavy chains, this method allows the production of a variety of different bivalent / multivalent antibodies. Some non-limiting examples are now described.In this example, the heavy chain DE1 has one heavy chain variable region with a common light chain for all binding domains of the binding or variable domains that bind antigen V, and the heavy chain DE2 has two heavy chain variable regions with a common light chain that form two binding or variable domains that bind antigens W and X. The heavy chain KK has one heavy chain variable region with a common light chain that forms a binding or variable domain that binds antigen Y. When these heavy and light chains are produced in a cell, the antibody DE1KK and the antibody DE2KK are produced, where the antibody DE1KK is a bivalent antibody that binds antigens V and Y. The antibody DE2KK is a multivalent antibody that binds antigens W, X, and Y. In the above example, if DE1 also has two heavy chain variable regions that form two binding or variable domains with a common light chain, two multivalent antibodies of the invention are produced. The KK heavy chain can also provide additional heavy chain variable regions, thereby adding further binding domains of the same or different antigen binding specificities. Combinations of two or more different heterodimerization domains, such as the DE / KK described above, and knob-in-hole domains, can add further diversity in oligoclonal antibody production. For example, the addition of two heavy chains, one with a knob and the other with a complementary hole, allows the production of independent bivalent / multivalent antibodies containing knob heavy chains and hole heavy chains. Depending on the number of heavy chain variable regions associated with each heavy chain, and whether they are the same or different, additional monospecific antibodies or additional bivalent or multivalent antibodies are produced.

[0283] According to the present invention, there is provided a composition comprising two or more antibodies, at least one of which may be a multivalent antibody of the present invention. Such a composition of the present invention may comprise two or more multivalent antibodies of the present invention. Such a composition may comprise three, four, five or more antibodies, at least one of which may be a multivalent antibody of the present invention. Such a composition may comprise three, four, five or more antibodies, all of which may be multivalent antibodies of the present invention.

[0284] In such compositions, one or more of the antibodies present in the composition may have one heavy chain in common.

[0285] A host cell of the invention may express or be capable of expressing two or more antibodies, at least one of which may be a multivalent antibody of the invention. A host cell of the invention may express or be capable of expressing two or more multivalent antibodies of the invention. Such a host cell may express or be capable of expressing three, four, five or more antibodies, at least one of which may be a multivalent antibody of the invention. Such a host cell may express or be capable of expressing three, four, five or more antibodies, all of which may be multivalent antibodies of the invention.

[0286] Thus, according to the invention there is provided a method for preparing a composition comprising two or more antibodies, the method comprising: providing a cell comprising one or more nucleic acid sequences encoding polypeptides capable of assembling into two or more antibodies, at least one of which is a multivalent antibody according to the invention; Culturing the host cell under conditions that provide for the expression of the polypeptide and for their assembly into two or more antibodies, at least one of which is a multivalent antibody according to the invention.

[0287] The invention also provides methods for producing two or more of said antibodies by the same cell, at least one of which is a multivalent antibody as described herein.

[0288] The present invention relates to a method for producing a composition comprising two or more antibodies, at least one of which is a multivalent antibody as claimed, the method comprising: a nucleic acid encoding a first heavy chain having a heavy chain variable region that, together with a common light chain, forms a binding domain or variable domain that binds to a first antigen; a nucleic acid encoding a second heavy chain having a heavy chain variable region that is associated with the common light chain and that forms a variable domain that binds a second antigen, and a heavy chain variable region that is associated with the common light chain and that forms a variable domain that binds a third antigen; a nucleic acid encoding a third heavy chain having a heavy chain variable region that, together with the common light chain, forms a variable domain that binds a fourth antigen; and a nucleic acid encoding a polypeptide comprising the common light chain, Two or more of the nucleic acids may or may not be physically linked, each of the nucleic acids further comprising an expression control sequence allowing expression of the encoded heavy and light chains in the cells, the method further comprising culturing the cells to allow expression of the heavy and light chains, and optionally harvesting the two or more antibodies. In one embodiment, the first and second heavy chains have compatible heterodimerization domains, preferably DE / KK heterodimerization domains. In a preferred embodiment, the third heavy chain comprises one of the portions of the compatible heterodimerization domains, resulting in the production of two antibodies. In one embodiment, the method further comprises providing a collection of cells having the nucleic acids, and selecting from the collection a cell having a desired ratio of expression of each of the heavy and light chains. In a preferred embodiment, the two or more antibodies are two or more multispecific antibodies. In a preferred embodiment, the cells produce essentially equimolar amounts of the two or more antibodies. In some embodiments, the cells produce more of one antibody than the other of the two or more antibodies.

[0289] Non-human animals The synthesis and expression of multivalent binding proteins is problematic, in part due to issues associated with identifying suitable light chains that can be expressed in conjunction with two or more different heavy chains, and in part due to separation issues. Furthermore, the art lacks a range of linkers that allow for diverse antibody valencies, flexibility with stability, and low immunogenicity.

[0290] The methods and compositions described herein allow for the production of suitable multivalent binding proteins with binding domains obtained, derived or based on suitable methods. Suitable methods can include phage display methods (including modification of germline sequences generated in phage display systems) and other in vitro methods known in the art. A particularly useful method is to allow a genetically modified non-human animal to generate suitable heavy chain variable domains that can be expressed in association with a common light chain by the natural process of somatic recombination and affinity maturation.

[0291] In one embodiment, the variable domains used in the multivalent antibodies of the invention are obtained, derived or based on the heavy and light chain variable regions of a non-human transgenic animal that contains an unrearranged heavy chain variable locus in the germline and expresses a single rearranged human light chain variable domain, e.g., a common light chain mammal, such as a rodent. Such a non-human transgenic animal, upon exposure to antigen, expresses diverse heavy chain variable regions paired with a common light chain, which can then be used to develop nucleic acid sequences encoding heavy chain variable regions obtained, derived or based on the transgenic animal that can be efficiently transformed into host cells for the production of multivalent antibodies.

[0292] In particular, human variable region sequences from suitable B cells of immunized common light chain animals engineered to express human light chain variable domains derived from no more than one, or no more than two, human VL gene segments, can be used to construct potential VLs of multivalent antibodies of the invention. HThe B cells of the animal are immunized with one or more antigens of interest, which in various embodiments are antigens to which the multivalent antibody binds. The B cells of the animal are immunized with one or more antigens of interest, which in various embodiments are antigens to which the multivalent antibody binds. The cells, tissues, or serum, spleen, or lymphatic material of the animal are screened to obtain heavy chain variable domains (or B cells expressing them) that exhibit the desired properties for the antigen of interest, such as high affinity, low affinity, blocking ability, activation, internalization, or other properties. Substantially all of the heavy chain variable domains generated in response to antigenic stimulation in the transgenic animal have less than one, or less than two V L Because they are made in conjunction with the expression of human immunoglobulin light chains derived from gene segments, the heavy chain variable regions are capable of expressing and associating with the consensus light chain domain expressed in the transgenic animal.

[0293] In one aspect there is provided an epitope binding protein as described herein, L and V H The sequences are encoded by nucleic acids based on nucleic acids obtained from B cells of the transgenic mice described herein and / or the transgenic animals disclosed in WO 2009 / 157771, which are incorporated herein by reference, that have been immunized with an antigen containing the epitope of interest.

[0294] Nucleic Acid Sequences, Polypeptides, Vectors, and Cells The invention further provides nucleic acid sequences encoding polypeptides or linkers that may be used in the assembly of multivalent antibodies of the invention, vectors comprising such nucleic acid sequences, cells capable of producing the multivalent antibodies of the invention, and methods of preparing such multivalent antibodies using such cells.

[0295] Multivalent antibodies according to the invention are typically produced by cells expressing nucleic acid sequences encoding polypeptides which assemble together to form the antibodies of the invention.

[0296] Thus, the present invention provides a linker comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3 or 5 to 24, or a polypeptide having at least about 85% sequence identity to any one of them, such as at least about 85% sequence identity to any one of them, for example at least about 90% sequence identity to any one of them, such as at least about 95% sequence identity to any one of them, for example at least about 98% sequence identity to any one of them, for example at least about 99% sequence identity to any one of them.

[0297] The present invention further provides a polypeptide comprising VH1-CH1-hinge based linker-VH2-CH1.

[0298] In certain embodiments, VH1 and VH2 bind to the same epitope. In certain embodiments, VH1 and VH2 bind to the same antigen but different epitopes. In certain embodiments, VH1 and VH2 bind to distinct epitopes and antigens.

[0299] Also provided by the invention are nucleic acid sequences encoding such linkers or polypeptides, and vectors containing such nucleic acid sequences.

[0300] The nucleic acid sequences employed to produce the described polypeptides may be placed into any suitable expression vector, and in appropriate circumstances may be placed into two or more vectors within a single host cell.

[0301] Generally, nucleic acid sequences encoding the variable domains are cloned with suitable linkers and / or constant regions and the sequences are placed in operably linked to a promoter in a suitable expression construct in a suitable cell line for expression.

[0302] Thus, the present invention also provides a method for preparing an antibody, the method comprising the steps of: providing a cell comprising one or more nucleic acid sequences encoding polypeptides capable of assembling into a multivalent antibody of the invention; and culturing the host cells under conditions that provide for the expression of the polypeptides and their assembly into the multivalent antibody.

[0303] Expression of multivalent antibodies The expression of antibodies in recombinant host cells has been described in the art. The nucleic acid molecules encoding the light and heavy chains of the antibody of the present invention may be present as extrachromosomal copies and / or stably integrated into the host cell chromosome. The latter is preferred, in which case they may target loci known to lack gene silencing.

[0304] It is well known to those skilled in the art that in order to obtain expression of a nucleic acid sequence encoding a polypeptide to be assembled into an antibody of the present invention, sequences capable of driving such expression can be operably linked to the nucleic acid sequence encoding the polypeptide. Operative linkage is meant to describe that the nucleic acid sequence encoding the polypeptide or its precursor is linked to sequences capable of driving expression such that these sequences can drive the expression of the polypeptide or its precursor. Useful expression vectors are available in the art, for example, the pcDNA vector series from Invitrogen. When the sequence encoding the polypeptide of interest is appropriately inserted with reference to sequences that regulate the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing the polypeptide of interest, which is called expression. Sequences that drive expression can include promoters, enhancers, and the like, and combinations thereof. They must be able to function in the host cell, thereby driving the expression of the nucleic acid sequence operably linked to them. Promoters can be constitutive or regulated and can be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or artificially designed.

[0305] The expression of the nucleic acid sequence of the present invention may be from a native promoter or a derivative thereof, or from a completely heterologous promoter. Some well-known and frequently used promoters for expression in eukaryotic cells include promoters from viruses, e.g., adenovirus, e.g., E1A promoter, promoters from cytomegalovirus (CMV), e.g., CMV immediate early (IE) promoter, promoter from Simian Virus 40 (SV40). Suitable promoters may also be derived from eukaryotic cells, e.g., metallothionein (MT) promoter, elongation factor la (EF-la) promoter, actin promoter, immunoglobulin promoter, heat shock promoter, etc. Any promoter or enhancer / promoter capable of driving the expression of the nucleic acid sequence of the present invention in a host cell is suitable in the present invention. In one embodiment, the sequence capable of driving expression comprises a region of the CMV promoter, preferably the region comprising nucleotides -735 to +95 of the CMV immediate early gene enhancer / promoter. The skilled artisan will recognize that the expression sequences used in the present invention may be suitably combined with elements capable of stabilizing or enhancing expression, such as insulators, matrix attachment regions, STAR elements, etc. This may improve the stability and / or level of expression.

[0306] Any cell suitable for expressing recombinant nucleic acid sequences can be used to produce the antibodies of the invention. Preferably, the cells are adapted for suspension growth.

[0307] The multivalent antibodies of the present invention can typically be expressed in host cells by culturing suitable cells of the present invention and harvesting the antibodies from the culture. Preferably, the cells are cultured in serum-free medium. The antibodies of the present invention can be recovered from the cells or, preferably, from the cell culture medium by methods commonly known to those skilled in the art.

[0308] Further provided is an antibody obtainable by the method for producing an antibody according to the invention. The antibody is preferably purified from the culture medium.

[0309] After harvesting, the antibodies can be purified from the culture using methods known in the art, including precipitation, centrifugation, filtration, size exclusion chromatography, affinity chromatography, cation and / or anion exchange chromatography, hydrophobic interaction, chromatography, etc. Linker sequence-based affinity chromatography can be used as a means to separate the multivalent antibodies of the present invention.

[0310] Pharmaceutical Compositions and Methods of Use Also provided by the present invention is a pharmaceutical composition comprising an antibody of the present invention and a pharma- ceutically acceptable carrier and / or diluent.

[0311] Thus, the present invention provides a multispecific antibody as described herein for use in the treatment of the human or animal body by therapy.

[0312] Further provided by the invention is a method for treating a human or animal suffering from a medical condition, the method comprising administering to the human or animal a therapeutically effective amount of an antibody as described herein.

[0313] The amount of the antibody according to the present invention to be administered to a patient typically falls within the therapeutic window, meaning that a sufficient amount is used to obtain a therapeutic effect, but does not exceed a threshold that results in an unacceptable degree of side effects. The smaller the amount of antibody required to obtain the desired therapeutic effect, the larger the therapeutic window will typically be. Thus, antibodies according to the present invention that exert sufficient therapeutic effects at low doses are preferred.

[0314] The reference herein to a patent document or other matter offered as prior art should not be construed as an admission that the document or matter was known or that the information it contains was part of the common general knowledge at the priority date of any of the claims.

[0315] The disclosure of each reference mentioned herein is incorporated herein by reference in its entirety.

[0316] Although features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the invention may include embodiments having all or a partial combination of the described features.

[0317] The following examples illustrate the invention. For ease of reference, for Examples 8-15, the following format is used when describing the trispecific molecules: MFA x MFB: MFC or antigen A x antigen B: antigen C, such that MFA or antigen A followed by x constitutes the "short arm", where x indicates dimerization, followed by MFB or antigen B describes the internal location of the long arm, followed by ":" to indicate the linker, followed by MFC or antigen C to indicate the MFC or antigen C in the distal domain of the long arm. EXAMPLES

[0318] Example 1: Cloning of variable domains and linkers for the generation of vectors capable of expressing multispecific antibodies The 24 linker constructs were cloned in pools according to size detailed in Table 2 into the MV1626 vector (see FIG. 3) containing KK residues (L351K, T366K) in the CH3 region for the generation of IgG heavy chain heterodimers (WO 2013 / 157954 and WO 2013 / 157953). The constructs were cloned into the vector MV1626 using the restriction enzymes SfiI and XhoI. All constructs contain consecutively the VH gene of MF1337, the CH1 domain, the linker sequence whose translation is listed in Table 2, and the VH gene of MF1122. As an example, the DNA sequence of the construct MF1337xIgG4 UHxMF1122 is provided below in Table 3. Schematically the constructs are shown in FIG. 2a. The constructs are based on both the CH1 and linker sequences of the IgG isotypes indicated in the name of the constructs. Translations of all 24 CH1 regions combined with the linker sequences are provided in Figure 5. Translations of all three VH genes and the common light chain gene are provided in Table 4 below. [Table 2] [Table 3] [Table 4]

[0319] The insert and vector were digested using SfiI restriction enzyme for 2 hours at 50°C, followed by digestion with XhoI enzyme for 2 hours at 37°C. The digested DNA was loaded onto a 0.8% agarose gel and run at 100 volts for 2 hours. The digested vector and insert were then isolated from the gel using a Qiagen QIAquick gel extraction kit before being ligated overnight at 16°C with T4 DNA ligase in a 1 / 5 ratio (w / w vector / insert). 50 μL of DH5α-T1R competent E. coli was transformed in the presence of 5 μL of ligation mix for 30 minutes on ice, followed by a heat shock procedure at 42°C for 2 minutes and on ice for 2 minutes. The transformed bacteria were plated on LB agar supplemented with ampicillin and incubated overnight at 37°C. A single colony was picked, mixed with 100 μL of sterile deionized water, and used for colony PCR using primers DO_2130 and DO_1056 to confirm the presence of the insert, followed by sequencing PCR with BigDye® Terminator v1.1 Cycle Sequencing Kit (Thermofisher) for clone confirmation using primer DO_2130.

[0320] A single colony of a confirmed clone was used to inoculate 4 mL of LB-Amp. Overnight cultures at 37°C were prepared in 24-well format minipreps using the QIAGEN Plasmid Mini Kit according to the manufacturer's manual. After elution from the column, the purified DNA was precipitated by adding 0.7 volumes of room temperature isopropanol. The DNA pellet was washed with 1 ml of 70% ethanol, air-dried under sterile conditions, and resuspended in sterile Tris-EDTA buffer before storage at -20°C. The final construct was dideoxy sequenced using the BigDye® Terminator v1.1 Cycle Sequencing Kit with primers DO_1488, DO_1056, and DO_2130 for the insert and primers DO_0182 and DO_0091 for the CH2 / CH3 region.

[0321] All primer sequences are shown in Table 5.

[0322] Sequencing showed that all constructs were successfully prepared. [Table 5]

[0323] Example 2: Transfection and IgG purification The expression vector generated in Example 1 is combined with vector MG1025C377 (Figure 4) expressing the second heavy chain of the base antibody part of a multispecific antibody, carrying the L351D-L368E mutations in the CH3 region (WO 2013 / 157954 and WO 2013 / 157953) and the thyroglobulin Fab gene of antibody MF1025 (see Example 2 of WO 2013 / 157953). Expression of the two heavy chains together with a common light chain results in the production of a trispecific antibody, as shown in Figure 2b.

[0324] FreeStyle 293-F cells (Thermofisher) were used for expression of the designed antibodies in a 24-well plate format. Two days prior to transfection, FreeStyle 293-F cell stock was split 1:1 into 293-F medium and incubated overnight at 37°C, 8% CO2 with an orbital shaking speed of 155 rpm. The day before transfection, cells were diluted to 5x10 5The cells were diluted to a density of 10000 cells / mL. 4 ml of suspension cells were seeded into 24 deep-well plates, covered with a breathable seal, and incubated at 37 °C, 8% CO2, and an orbital shaking speed of 285 rpm. On the day of transfection, 4.8 ml of 293-F medium was mixed with 240 μg of polyethyleneimine (PEI) linear (MW 25,000). For each IgG produced, 200 μL of 293F culture medium-PEI mixture was added to 8 μL of DNA (4 μL of DNA encoding each heavy chain in the case of IgG heterodimers) as detailed in Table 6. The mixture was incubated at room temperature for 20 minutes before gently adding it to the cells. The day after transfection, penicillin-streptomycin (Pen Strep) diluted in 500 μL of 293F medium was added to each well. Plates were incubated at 37 °C and 8% CO2 with an orbital shaking speed of 285 rpm until harvesting 7 days after transfection. Plates were centrifuged for 5 min at 500 g and supernatants containing IgG were filtered using 10-12 μm melt-blown polypropylene filter plates and stored at -20 °C prior to purification.

[0325] Various control antibodies were also expressed, namely: Bivalent anti-tetanus toxoid antibody using Fab MF1337 (using vector MG1337C057) Bivalent anti-thyroglobulin antibody using Fab MF1025 (using vector MG1025C059) Bivalent anti-fibrinogen antibody using Fab MF1122v (using vector MG1122C057)

[0326] MG1337C057 denotes a construct expressing the VH region of MF1337 from vector MV1057. MG1025C059 denotes a construct expressing the VH region of MF1025 from vector MV1059. MG1122C057 denotes a construct expressing the VH region of MF1122 from vector MV1057. MV1057 (Figure 6) and MV1059 are vectors expressing monospecific bivalent human IgG1 molecules. MV1057 and MV1059 are essentially identical vectors and result in the expression of the same IgG1 molecule.

[0327] Bispecific anti-thyroglobulin x anti-tetanus toxoid antibody combining Fab MF1337 and Fab MF1025 (using MG1025C377 x MG1337C260)

[0328] Bispecific anti-thyroglobulin x anti-fibrinogen antibody combining Fab MF1122 and Fab MF1025 (using MG1025C377 x MG1122C260)

[0329] MG1025C377 (Figure 4) expresses the heavy chain variable domain of antibody MF1025 in the context of a human IgG1 heavy chain containing L351D, L368E (DE) mutations. MG1337C260 (Figure 7) expresses the heavy chain variable domain of antibody MF1337 in the context of a human IgG1 heavy chain containing L351K, T366K (KK) mutations. MG1122C260 expresses the heavy chain variable domain of antibody MF1122 in the context of a human IgG1 heavy chain containing L351K, T366K (KK) mutations. [Table 6]

[0330] After harvesting, antibodies were purified in a 24-well format as follows: the supernatant was mixed with 50 μL 1M Trizma (pH 8) and 100 μL ProteinA Sepharose CL-4B beads (50% v / v, GE Healthcare Life Sciences) and incubated for 2 hours at 25°C with orbital shaking at 600 rpm. The beads were vacuum filtered and washed twice with 3 mL PBS pH 7.4. Elution of the antibodies was performed by adding 200 μL citrate buffer 0.1 M, pH 3, followed by neutralization with 300 μL 1M Trizma pH 8. The purified IgG fraction was immediately buffer exchanged into PBS pH 7.4. The IgG samples were transferred to a 30 kDa 96-well filter plate and the polyethersulfone membranes were centrifuged at 1500 g at 4°C until a volume of 10 μL remained per well. 200 μL of PBS was added to each well and samples were mixed at 500 rpm for 3 min before IgG was collected for storage at 4° C. IgG concentrations were measured by Octet and Protein A biosensors (Pall ForteBio). Human IgG was used as a standard in seven two-fold dilutions starting from 192 μg / ml down to 3 μg / ml. The concentrations of IgG samples were measured in duplicate.

[0331] Reducing and non-reducing SDS-PAGE was performed for all 30 produced IgGs, including the control. The results are shown in FIG. 8. Under NR conditions, the expected product sizes of about 200 kDa were observed for the trispecific multivalent antibody and about 150 kDa for the control monoclonal IgG and Biclonics®. Under R conditions, product sizes of about 25 kDa (LC), about 50 kDa (HC / 1VH), and about 75 kDa (HC / 2VH) were observed for the trivalent antibody. The band sizes of about 25 kDa (LC) and about 50 kDa (HC) for the control IgG were as expected. The results also showed a band of about 150 kDa for the trispecific construct. These are homodimers resulting from the association of DE heavy chains, which may be a result of the higher expression levels of the DE containing short DE heavy chains than the longer KK-containing heavy chains.

[0332] Example 3: Binding activity of Fab domains at VH1, VH2, and VH3 positions measured in ELISA The binding activity of the three Fab domains in each construct was confirmed by ELISA using tetanus toxoid, fibrinogen, and thyroglobulin antigens, and huEGFR-Fc antigen as negative controls (see Table 7 for coating conditions, suppliers, and catalog numbers).

[0333] Each multispecific IgG sample was initially diluted to 10 μg / ml in PBS and analyzed in four 5-fold dilutions from 10 to 0.08 μg / ml in titrations of fibrinogen, tetanus toxoid, and thyroglobulin. All 30 samples were analyzed with huEGFR-Fc at 10 μg / mL. The appropriate amount of antigen in PBS was prepared. 50 μl of diluted antigen solution was added per ELISA plate well and coated at 4°C. Plates were washed twice with wash buffer (PBS / Tween). Wells were blocked with 300 μl / well blocking buffer (PBS / 2% BSA) for 1 hour at room temperature. During incubation, appropriate IgG dilutions were made in blocking buffer. Plates were emptied by inversion over a sink followed by tapping on a tissue. 50 μl of diluted IgG samples and controls were added to the wells of the blocking plate, covered with a seal, and incubated at room temperature for 60 minutes. Plates were washed 3 times with wash buffer (PBS / 0.05% Tween). Detection antibody (mouse anti-human IgG HRP conjugate; Becton Dickinson, Cat. No. 555788) diluted 1 / 2000 in blocking buffer was added at 50 μL / well. Plates were covered with a seal and incubated at room temperature for 60 minutes. Plates were washed 3 times with wash buffer. TMB substrate solution (BD, OptEIA™ Cat. No. 51-2606KC) was made by mixing reagents A and B in a 1:1 ratio and adding 50 μl / well and allowed to develop for 10 minutes (max). 50 μl of 1M H2SO4 was added to each well to stop the staining reaction.

[0334] Plates were read at A450 using a BioTek Elx808 ELISA plate reader. Binding curves were plotted using GraphPad Prism 7 and the area under the curve (AUC) calculated for each antigen ELISA for each construct are listed in Table 8. Small variations in AUC are seen for binding to 12% and 8% thyroglobulin (VH1, on the DE arm, see Figure 1) and tetanus toxoid (VH3, on the tip of the KK arm, see Figure 1). Larger variations are seen for the fibrinogen-arm (which is VH2, Figure 1). This indicates that the accessibility or affinity of the Fab domain in the VH2 position depends on the linker connecting the Fab in the VH2 position to the Fab in the VH3 position. All linkers provide a functional VH2. [Table 7] [Table 8]

[0335] Example 4: Stability of binding activity The stability of the binding activity of the three Fab domains in the trivalent antibody construct was analyzed following four accelerated stress conditions: Samples were diluted to 10 μg / ml in PBS and incubated at 4° C. for one month. Samples were diluted to 10 μg / ml in D10F medium and incubated at 40° C. for seven days. Samples were diluted to 10 μg / ml in D10F medium and incubated at 50° C. for two days. Samples were diluted in PBS and subjected to five freeze-thaw cycles (5XFT).

[0336] Following these accelerated stress conditions, the binding activity to the antigen recognized by the three Fab domains was analyzed by ELISA as described above. Areas under the curve were calculated and tabulated.

[0337] Stress applied at 40°C only significantly affected the binding of Fab at the VH2 position, which binds to fibrinogen to different extents in the different constructs tested. Stress at 50°C and 4°C with 5xFT affects the binding of all three Fab domains to different extents in the different constructs tested.

[0338] The binding activities were ranked for each antigen and stress condition, and the 16 most optimal constructs under each stress condition were identified for each Fab position. The number of times a construct was among the 16 optimal constructs was summed and used to rank all constructs based on the conservation of binding activity of the three Fabs under accelerated stress conditions.

[0339] The results are depicted in FIG. 9 and show that there is a variable stability of the different constructs under accelerated stress conditions. The stability of the binding activity of the three Fab domains in the 21 produced trivalent antibody constructs was analyzed according to the four accelerated stress conditions. The ELISA data (AUC) are tabulated. The binding activity was ranked and 16 optimal constructs under each stress condition were identified for each Fab position. The number of times a construct was among the 16 optimal constructs was summed and used to rank all constructs based on the conservation of the binding activity of the three Fabs under accelerated stress conditions.

[0340] All antibodies are stable, some more stable than others.

[0341] Example 5: Large-scale transfection and IgG purification Eighteen constructs were selected for large-scale production for further analysis as follows: IgG1 MH, IgG1 H, IgG1 R, IgG1 G4S, IgG1 UH, IgG3 R, IgG3 UH, ** IgG2A R, IgG2A MH, IgG3 ULH, IgG2B R, IgG4 MH, IgG4 UL, IgG2A H, IgG2B H, * IgG1 UL, * IgG4H, *IgG4 R. As controls the following products were included: bispecific anti-thyroglobulin x anti-tetanus toxoid (using MG1025C377 x MG1337C260 as described above in Example 2) and bispecific anti-thyroglobulin x anti-fibrinogen (using MG1025C377 x MG1122C260 as described above).

[0342] DNA for these constructs was prepared as described above. Multispecific IgG was transfected as described above by co-transfection of the constructs listed in Table 6. Selected constructs were produced on a larger scale. Two days prior to transfection, FreeStyle HEK293-F cell stocks were split 1:1 into 293-F medium in a final volume of 100 ml per 500 ml culture flask and incubated at 37°C and 8% CO2 with an orbital shaking speed of 155 rpm. One day prior to transfection, 5.0x10 IgG was prepared by diluting the cells in 293-F culture medium. 5 Cells were counted in cell suspension with a density of 100 cells / ml. Cells were then seeded at 100 ml of cell suspension per T500 flask and incubated at 37° C. and 8% CO2 with an orbital shaking speed of 155 rpm. The next day, cells were transfected. A mixture of 293-F medium, PEI, and DNA was prepared by mixing 7.5 ml of 293-F medium, 187.5 μl of PEI stock at 1 μg / μl, and 150 μl of DNA at 0.5 μg / μl. This was incubated at room temperature for 20 minutes and then added to the cells, which were then incubated at 37° C. and 8% CO2 with an orbital shaking speed of 155 rpm for 7 days.

[0343] The supernatant containing the antibody protein was centrifuged at 1000 g for 10 min to remove cells. The supernatant was filtered using a 0.45 μm filter. IgG was purified from the supernatant using an AKTAexplorer 100 system (GE Health Care) and protein A affinity chromatography, followed by desalting. HiTrap MabSelect SuRe 5 ml columns and HiTrap 5 ml desalting columns (GE Health Care) were used according to the manufacturer's instructions. IgG concentrations were determined by OD280 absorbance. 0.8-4.9 mg of IgG was obtained in PBS for all constructs. The produced proteins were analyzed by SDS-PAGE (reduced and non-reduced) as previously described in Example 2. The data confirms the data found in Example 2 and is not provided here.

[0344] HP-SEC was performed to confirm the expression ratio between the two heavy chains that constitute the base antibody portion of the multispecific antibody. Due to the size difference of the two heavy chains in the trivalent construct, the half-bodies and homodimers can be identified and quantified in high performance size exclusion chromatography (HP-SEC). HP-SEC was performed using a Dionex HPLC system equipped with a TSK guard column SWXL (Tosoh Bioscience Catalog No. 08543) and a TSK-gel column G3000SWXL (Tosoh Bioscience Catalog No. 08541). For each analysis, 20 μg of protein sample in PBS was injected onto the column, which was run at a flow rate of 1 mL / min at 4° C. using 200 mM sodium phosphate, 50 mM NaCl as the running buffer. The chromatograms were analyzed for retention time and relative peak area based on the UV280 results using Chromeleon 6.80 software. The ratio of the amount of trivalent IgG / DEDE homodimer was calculated and is shown in Table 9 below. Constructs with above-average trivalent / DEDE ratios are presented in italics. [Table 9]

[0345] The stability of large-scale purified IgG (including two controls) was evaluated after the different stress conditions described above, after 5 freeze-thaw cycles in PBS, after 1 week of incubation at 40° C. in D10F medium, after 1 week of incubation at 50° C. in D10F medium, and after 1 week of incubation at 50° C. in PBS. The performance of the stressed samples was compared with that of the same samples after 1 week of incubation at 4° C. (control). For that purpose, purified IgG from 100 ml of production was diluted to 0.2 mg / mL in PBS and split into two batches: one for stability testing at 0.2 mg / mL in PBS (4° C., 3xFT, and 50° C.) and one diluted to 0.1 mg / mL in D10F for stress testing at 4° C., 40° C., and 50° C. as described above. For sample IgG1 H, a concentration of 0.194 mg / mL was used.

[0346] Following these accelerated stress conditions, the binding activity to the antigen recognized by the three Fab fragments was analyzed by ELISA as described above. The area under the curve was calculated and tabulated (see FIG. 10). The percentage of remaining binding activity after stress compared to the activity of the control sample stored at 4° C. was calculated for each binding activity. For each binding activity to each target in each stress condition, the samples were ranked based on these percentages. The percentage above the average is displayed and the number of times each sample showed above the average was summed and listed in the last column of the table in FIG. 10. This shows that there is a variety of stability of the constructs as measured by the binding activity of the three Fab arms after stress.

[0347] Example 6: Stability analysis of 18 multivalent IgG constructs Stability analysis was performed as described above in Example 5 on the 18 multivalent constructs identified in Figure 10. In addition, four control antibodies were used with heavy chain binding domains including MF6744 (SEQ ID NO: 91), MF1337 (SEQ ID NO: 28 and SEQ ID NO: 288), and MF1122 (SEQ ID NO: 26 and SEQ ID NO: 286), which are previously described in WO 2018 / 056821(A1), which is incorporated by reference, and which are bound to cLC, i.e. Monospecific anti-CD137 antibody using Fab MF6744 / cLC, Monospecific anti-fibrinogen antibody using Fab MF1122 / cLC, Monospecific anti-tetanus toxoid antibodies using Fab MF1337 / cLC, and The DEKK bispecific control is a bispecific anti-fibrinogen x anti-tetanus toxoid antibody containing Fab MF1122 / cLC and Fab MF1337 / cLC.

[0348] A list of samples tested for stability analysis is provided in Table 10 below. [Table 10]

[0349] The stability of 18 multivalent constructs and 4 control antibodies was analyzed according to 4 different conditions. Thus, 22 samples (18 trivalent + 4 controls) were diluted to 0.2 mg / ml in PBS, At 4°C (T0) for 1 month (used as reference), 50℃ for 7 days, 5X freeze-thaw (FT) cycles at -80°C, or Shake at 400 rpm for 4 hours at room temperature.

[0350] After each of these four conditions, stability was analyzed using seven different methods: UV-Vis Absorption Spectroscopy: After subtraction of background buffer absorption and light scattering by aggregates, the absorbance is examined at 350 nm to provide information on the aggregation state of the sample, as described in Eckhardt, 1994: Mulinacci, 2011b and Peters, 2013. 90° Light Scattering Spectroscopy: In solution, the scattering intensity of light can be affected by different factors such as protein concentration, refractive index, particle size and shape, and the wavelength of the incident light. This method is used to study protein aggregation as reported in Cappelle, 2005; Demeule, 2007a and b; Mulinacci, 2011a and 2013; Luca, 2010; Patois, 2011 and 2012; Peters 2013.

[0351] Tryptophan specific fluorescence emission expressed as % change compared to T0: Changes in the hydrophobicity and rigidity of the environment can be measured by tryptophan fluorescence emission (Capelle, 2005; Demeule, 2007a & b & 2009; Mulinacci, 2011a & b; Luca, 2010; Patois, 2011; Peters, 2013).

[0352] 1,8-ANS fluorescence emission is expressed as the percentage change compared to T0:As a small, uncharged, hydrophobic fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) becomes fluorescent in water upon binding to electrostatic pockets of proteins, protein aggregates, detergent micelles, exudates, membranes and cellular components and can therefore be used to study membrane surfaces and proteins (Demeule, 2009; Mulinacci, 2011a and b; Luca 2010).

[0353] Nile Red fluorescence emission expressed as the percentage change compared to T0: As an uncharged, small, hydrophobic fluorescent probe, Nile Red is sensitive to the polarity of its environment and can be used to analyze protein degradation, protein aggregation, lipid structure, and protein unfolding (Sackett and Wolff, 1987).

[0354] Nile Red Fluorescence Microscopy to measure number of particles / μl: Nile Red is used to stain samples to visualize protein aggregates for fluorescence microscopy using a Leica DMi8 microscope (Demeule, 2007a and b, and 2009; Mulinacci, 2011b and 2013; Patois 2011).

[0355] Dynamic Light Scattering, expressed as % change of monomer compared to T0 (intensity calculation): Dynamic Light Scattering is measured using a NanoFlex instrument. A laser passing through an optical fiber is scattered and reflected from the particles towards a detector that measures the scattered light intensity to determine the particle size distribution profile.

[0356] The readouts of these methods relate to protein aggregation, fragmentation, and unfolding, which are measures of protein stability. The results showed that shaking at 400 rpm for 4 hours had the most impact on the stability of the antibodies. The control monospecific PG1337 / MF1337 antibody was the most affected by the stress conditions. Therefore, all results were normalized with PG1337 set as the threshold, since all 18 multivalent constructs and the bispecific control contained PG1337 / MF1337 Fab.

[0357] The stability of the molecules was calculated by combining the scores of all seven methods between the four different conditions. The results can be seen in Figure 12 and reveal that the constructs have a range of stability, with the various trispecific molecules showing superior stability to the control bispecific IgG.

[0358] Example 7: Bioinformatics Linker Characterization Eight linkers, described in Table 11 below and shown in FIG. 13, were further characterized. A flexibility prediction was obtained for each of these sequences using the Karplus and Schulz flexibility prediction method (calculating the average of the flexibility index of each amino acid in the sequence). The flexibility index is derived from the average properties of each amino acid in the protein structure as described in Karplus PA, Schulz GE. Flexibility Prediction of Chains in Proteins - A Tool for the Selection of Peptide Antigens Naturwissenschaften 1985;72:212-3;(http: / / tools.immuneepitope.org / bcell / ). Table 11 denotes linkers as rigid (R) if the KS score is less than or equal to 1.015 and as partially flexible if the KS score is between about 1.015 and 1.04. A flexible sequence for the purposes of the present invention is a sequence with a Karplus and Schulz flexibility prediction of greater than 1.04. [Table 11]

[0359] A second bioinformatics prediction of these linkers was obtained using Rosetta local structure prediction. The Rosetta fragment picker was used herein to provide local structure predictions as described in Gront D, Kulp DW, Vernon RM, Strauss CEM, Baker D (2011) Generalized Fragment Picking in Rosetta: Design, Protocols and Applications. PLoS ONE 6(8): e23294. https: / / doi.org / 10.1371 / joural.pone.0023294. A minimum of 40 residues is preferred when using prediction tools in this way, therefore glycine residues were introduced at the ends of the linkers to create each 40 residue long sequence with a linker sequence in the middle. These linkers were sequenced through the Rosetta fragment pipeline, which finds matches of nearby local sequences in structures in the ProteinDataBank, and the matches between these sequences are used to predict local structures, to characterize the secondary structure. The central fragment was then visualized for each of the eight sequences in FIG.

[0360] A summary of the results is shown in Table 12 below: F=flexible, M=intermediate, R=rigid, C=coil=flexible, H=helical=rigid, E=stranded=intermediate, which indicates general agreement with the Karplus and Schultz scores based on predicted secondary structure. [Table 12]

[0361] Example 8: Generation of anti-CD3, PD-L1, and EGFR binding domains Mice used for immunization. For the generation of human antibodies that bind to CD3, EGFR, and PD-L1, transgenic mice with a human common light chain and a human heavy chain (HC) minilocus (including a selection of human V gene segments, all human D and all human J) (see WO2009 / 157771, incorporated herein by reference) can be immunized with either DNA or recombinant DNA encoding the proteins found below. These mice are referred to as "MeMo®" mice. For the specific heavy chain variable regions or trivalent multimers having the sequences disclosed herein, these can be produced by any means known to those of skill in the art.

[0362] Protein immunization "MeMo®" mice were immunized by subcutaneous injection with recombinant protein and Gerbu adjuvant MM (Gerbu Biotechnik c#3001). Recombinant huPDL1-His (SinoBiological; Cat. No. 10084-H08H) protein was used for immunization. Mice were immunized with 40 μg of recombinant protein in PBS mixed with 40 μl of adjuvant in a total volume of 100 μl. Subsequently, on days 14 and 28, mice were boosted with 20 μg of recombinant protein in PBS mixed with 20 μl of adjuvant in a total volume of 50 μl. On day 35, mouse serum was collected and serum titers were determined. Mice with low serum titers underwent further cycles of booster immunizations and serum analysis. Each cycle consisted of two weekly immunizations with 20 μg of recombinant protein in 50 μl of PBS, followed by serum collection for titer analysis one week later. Mice showing high serum titers against human and macaque targets received a final boost consisting of daily injections of 20 μg of recombinant protein in 50 μl PBS for three consecutive days. Mouse lymphoid tissues were harvested one day after the final injection.

[0363] DNA immunization MeMo® mice were immunized by DNA tattoo using a micropigmentation device. DNA tattoo immunization was performed with 20 μg of plasmid DNA encoding the target antigen. Mice were immunized with DNA encoding the human target PD-L1. For PD-L1 immunization, Treg cells were depleted 4 days before the start of immunization by injecting mice with 0.5 mg of anti-CD25 antibody PC61.5 to break tolerance. Mice were immunized on days 0, 3, 6, 14, 17, 28, and 31. Mouse serum was collected on day 35 and serum titers were determined. Mice with low seroreactivity to human and / or macaque targets were subjected to further cycles of booster immunization with human, rat, or macaque DNA antigens and serum analysis was performed. Each cycle consisted of two weekly DNA immunizations followed by serum collection for titer analysis one week later. Mice showing seroreactivity to cells expressing human and macaque targets received a final booster immunization after which lymphoid tissues were harvested 3 days later.

[0364] Lymphoid tissue collection Spleens and draining lymph nodes were removed from all normally immunized mice. Single cell suspensions were generated from both splenic and inguinal lymph nodes, and these tissues were subsequently dissolved in Trizol LS reagent and stored at -80°C until use. "Immune" phage antibody repertoires were generated by RT-PCR cloning of VH genes from normally immunized mice, and inguinal lymph nodes were used for the construction of "immune" phage antibody repertoires. For this purpose, RNA was extracted from TRIZOL LS-lysed lymph tissues, and 1 μg of total RNA was used in a RT reaction with IgG-CH1 specific primers. The resulting cDNA was then used to amplify a polyclonal pool of VH-encoding cDNAs using in-house developed VH specific primers, essentially as described by Marks et al. (J Mol Biol. 1991 Dec 5; 222(3): 581-97). The resulting PCR products were cloned into a phagemid vector for display of the Fab fragments on phage as described by de Haard et al (J Biol Chem. 1999 Jun 25;274(26):18218-30), except that the light chain was the same for all antibodies and was encoded by the vector. After ligation, the phagemid was used to transform E. coli TG1 bacteria and the transformed bacteria were plated on LB-agar 105 plates containing ampicillin and glucose. All phage libraries were cultured at 10 6 The plasmid contained more than 100 transformants and had an insert frequency of over 80%. Bacteria were harvested after overnight growth and used to prepare phage according to established protocols (de Haard et al., J Biol Chem. 1999 Jun 25;274(26):18218-30).

[0365] targeting antibody EGFR and PD-L1 cLC antibodies were derived from phage antibody repertoires generated from successfully targeted immunized MeMo® mice using methods previously described. Additionally, methods for generating antibody variable domain VH chains for EGFR antibodies, including synthetic human anti-EGFR antibodies, are also described in pending applications WO2015 / 130173(A1), WO2015 / 130172(A1), which are incorporated herein by reference.

[0366] Immunization of Memo® mice bearing CDR3 For the generation of human antibodies that bind to CD3, mice transgenic for human common light chain and human heavy chain (HC) minilocus (including a selection of human V gene segments, all human D and all human J) (see WO2009 / 157771, incorporated herein by reference) were immunized with TCR / CD3-containing lipoparticles (Intergral Molecular). These mice are referred to as "MeMo®" mice. For the specific heavy chain variable regions or trivalent multimers having the sequences disclosed herein, these can be produced by any means known to those of skill in the art.

[0367] MeMo® mice were immunized with Hek293T-derived human 5D5M TCR / CD3 containing lipoparticles followed by immunization with human T cells for generation of anti-TCR / CDR3 immune responses and generation of an anti-TCR / CD3 antibody panel.

[0368] Lipoparticles concentrate structurally intact membrane proteins directly from the cell surface, allowing these complex proteins to be manipulated as soluble, highly abundant proteins for antibody immunization and screening.

[0369] The lipoparticles used for immunization in this study contain the 5D5M TCRαβ combination. Amino acid sequences (SEQ ID NO: 289 and SEQ ID NO: 290)

[0370] Hek293T-derived TCR / CD3 lipoparticles of the 5D5M TCRαβ combination were synthesized, cloned, and used to generate lipoparticles containing this TCR / CD3 combination by transient transfection into HEK293T cells (Intergral Molecular).

[0371] 5D5M TCRα (SEQ ID NO: 289) MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLFWYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDSASYLCAVMDSNYQLIWGAGTKLIIKPDIQNPDP AVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0372] 5D5M TCRβ (SEQ ID NO: 290) MRIRLLCCVAFSLLWAGPVIAGITQAPTSQILAAGRRMTLRCTQDMRHNAMYWYRQDLGLGLRLIHYSNTAGTTGKGEVPDGYSVSRANTDDFPLTLASAVPSQTSVYFCASSEAGGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEIS HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF

[0373] MeMo® mice were used for immunization with TCR / CD3 lipoparticles and primary human T cells.

[0374] The immunization schedule included time points on days 35, 56, 77, and 98, and antigen-specific IgG serum titers were determined by ELISA using 3SDX TCR / CD3 positive and negative lipoparticles from QTG using anti-mouse IgG detection, and by ELISA using CD3c5E-Fc fusion protein as a positive control. Reactivity was observed in serum collected on day 35 to determine which mice developed relevant anti-TCR / CD3 responses.

[0375] For all immunized mice, lymphoid material for antibody discovery was collected and stored as follows: The titer is 1 / 300 in human TCR / CD3 (in an ELISA using lipoparticles) or Titers were less than 1 / 300 and greater than 1 / 100 in human TCR / CD3 and did not increase during the final booster immunization.

[0376] Priming immunisation with lipoparticles To prime the humoral immune response in MeMo® mice for TCR / CD3, lipoparticles containing the human 5D5M TCRαβ combination were used for immunization. The lipoparticles were used with Gerbu adjuvant for the first and second injections.

[0377] Booster immunization with polyclonal T cells Mice were immunized by subcutaneous injection of a cell suspension. The first booster immunization (day 28) contained a mixture of cells and adjuvant in PBS, and all subsequent injections consisted of cells in PBS alone. Mice that developed serum IgG titers of 1 / 300 against human TCR / CD3 (measured by ELISA using lipoparticles) on day 35 were boosted with cells on days 42, 43, and 44. Mice that failed to meet these criteria were boosted with cells (days 42 and 49). All subsequent immunizations were given as subcutaneous injections of cells in PBS. After the final immunization, mice were sacrificed and serum and spleens and left inguinal lymph nodes were collected.

[0378] Screening of sera from immunized mice in ELISA Provisional serum IgG titers were screened by ELISA using TCR / CD3-containing and "null" lipoparticles. Serum IgG titers were measured using anti-mouse IgG staining, which was shown to be the most sensitive.

[0379] The CD3-binding variable domain was prepared using the amino acid sequences of the heavy chain variable region of the CD3 MF in its CDR regions as shown in SEQ ID NOs: 92-154.

[0380] Recloning of VH-encoding cDNA from a phagemid vector into an IgG expression vector The VH-encoding cDNA of all target-specific clones were sequenced, and then selection of unique clones based on sequence identity and cluster analysis was recloned into different IgG expression vectors using Sfi1-BstEII or SfiI / XhoI digestion and ligation of the pool of digested cDNA into the IgG expression plasmid, following standardized molecular biology techniques.

[0381] Purification of antibodies from culture supernatant The medium containing the antibody is collected and centrifuged to remove cell debris. Protein A Sepharose beads are then added to the medium. The medium and Protein A Sepharose beads are incubated with the antibody to allow binding.

[0382] After incubation, the beads are isolated from the medium and washed by vacuum filtering. The antibodies are eluted from the beads by incubation with an elution buffer.

[0383] Optionally, the purified IgG is buffer exchanged / desalted.

[0384] buffer exchange To desalt the purified antibody, the antibody fraction is centrifuged using a filter plate or filter column. The plate or column is centrifuged to reduce the volume of the antibody fraction. PBS or the required buffer is then added to the fraction to replace the buffer with a low salt buffer. Optionally, this centrifugation step followed by the addition of buffer is repeated to further desalt the antibody storage buffer.

[0385] Example 9: Generation of trispecific antibodies with tumor cell antigens on the short or long arms. Triabodies were generated by transient co-transfection of two plasmids encoding IgGs with different VH domains using CH3 engineering technology for efficient heterodimerization and formation of triabodies. A common light chain is also co-transfected into the same cell, either on the same plasmid or on a separate plasmid. Co-pending applications of the present invention (e.g. WO 2013 / 157954 and WO 2013 / 157953, incorporated herein by reference) disclose methods and means for producing multispecific antibodies from a single cell, thereby providing a means to favor the formation of multispecific antibodies over the formation of monospecific antibodies. These methods can also be suitably used in the present invention for the generation of multivalent multimers, including triabodies.

[0386] Specifically, the preferred mutations for generating predominantly trispecific full-length IgG molecules are amino acid substitutions relative to the human wild-type sequence at positions 351 and 366 in the first CH3 domain, e.g., L351K and T366K (numbered according to EU numbering) ("KK-mutant" heavy chain), and amino acid substitutions at positions 351 and 368 in the second CH3 domain, e.g., L351D and L368E ("DE-mutant" heavy chain), or vice versa. It has been previously demonstrated by the co-pending application of the present invention that negatively charged DE-mutant heavy chains and positively charged KK-mutant heavy chains preferentially pair to form heterodimers (so-called "DEKK" molecules). Homodimerization of DE-mutant heavy chains (DE-DE homodimers) or KK-mutant heavy chains (KK-KK homodimers) rarely occurs due to the strong repulsion between charged residues at the CH3-CH3 interface between identical heavy chains.

[0387] According to the present invention, the immune cell engaging binding domain or the tumor antigen binding domain can be located anywhere on the multivalent molecule, including at distal or internal positions on the long or short arms, and heterodimerization techniques can be utilized to advantageously generate trispecific molecules over monospecific, bivalent homodimers, or tetraspecific homodimers.

[0388] First, it was demonstrated that the tumor cell antigen-binding domain could be located in either the distal or internal regions of the long or short arms.

[0389] 3.0x10 for each of the trispecific and / or trivalent antibodies described herein 6 Expression is achieved via suspension-growth adapted 293 cells, cultured in T125 flasks in shaker plates to a density of 0.3–0.5x10 cells / ml. Cells are plated in each well of a 24-deep well plate at 0.3–0.5x10 cells / ml. 6The cells were seeded at a density of 10000 viable cells / ml. The cells were transiently transfected with a mixture of two plasmids encoding different antibodies and cloned into a proprietary vector system. Seven days after transfection, the cell supernatant was harvested and filtered through a 0.22 μM filter. The sterile supernatant was stored at 4°C until purification of the trispecific antibodies.

[0390] For an example of a trispecific molecule with an immune-engaging binding domain at the inner position of the long arm and a tumor cell antigen at the short arm, DNA encoding the VH gene of the CD3 binding domain (MF8078), the linker of the invention, and the tetanus toxoid (TT) binding domain (MF1337) is cloned into a vector encoding a positively charged CH3 domain (KK), and then DNA encoding the VH gene of the EGFR binding domain (MF8233) is cloned into a vector encoding a negatively charged CH3 domain (DE) to encode a trispecific molecule of EGFRxCD3:TT. The heavy chain variable regions of the three binding domains are listed in Table 13, and the activities of these trispecific molecules are listed in Figures 15a and 16a. In the case of these trispecific molecules, each heavy chain variable region is paired with a common light chain (SEQ ID NO: 29). [Table 13]

[0391] For an example of a trispecific antibody with immune-engaging binding domains at the medial position of the long arm and tumor cell antigens at the distal position, DNA encoding the VH gene of the CD3 binding domain (MF8078), a linker of the invention, and DNA encoding the VH gene of the EGFR binding domain (MF8233) are cloned into a vector encoding a positively charged CH3 domain (KK), in which case DNA encoding the VH gene of the thyroglobulin binding domain ("Thyro") (MF1025) is cloned into a vector encoding a negatively charged CH3 domain (DE) encoding a ThyroxCD3:EGFR trispecific molecule. Figure 14b. The heavy chain variable regions of the three binding domains are listed in Table 14, and the activities of these trispecific molecules are listed in Figures 15b and 16b. In these trispecific molecules, each heavy chain variable region is paired with a common light chain (SEQ ID NO: 29). [Table 14]

[0392] Example 10: Effect of placing tumor cell antigen binding domains on the short or long arms of T cell engaging trivalent molecules. cell line BxPC3 is a human pancreatic cancer cell line. HCT-116 is a human colon cancer cell line.

[0393] The above series of trispecific IgGs incorporating 11 different linkers into the trispecifics containing an anti-EGFR binding domain on the short arm (Figure 14a and Table 13) and an anti-EGFR binding domain on the long arm (Figure 14b and Table 14), as well as a series of control antibodies, were generated in 24-well production. These molecules have been evaluated in cytotoxicity assays for their ability to trigger T cell activation.

[0394] Resting T cells were isolated from whole blood of healthy donors using Ficoll and EasySep human T cell isolation kits according to standard procedures, confirmed >95% T cell purity with anti-CD3 antibodies using flow cytometry analysis, and then cryopreserved. For cytotoxicity assays, cryopreserved T cells were thawed and used if viability was >90% upon thawing as measured by standard trypan blue staining. Briefly, thawed resting T cells and BxPC3 (Figure 15) or HCT116 (Figures 16 and 17) target cells were co-cultured for 48 hours at an E:T ratio of 5:1. For trivalent antibodies, a six-step 3-fold dilution series was used starting at a concentration of 4 μg / ml. EGFRxCD3 bispecific antibody was used as a positive control; mock x mock:mock, mock xCD3:mock, and EGFRxmock:mock trivalent antibodies were used for specificity controls. T cell activation was quantified using flow cytometry; CD4 and CD8 T cells were gated based on CD4 and CD8 expression and subsequently analyzed for their activation status by measuring CD25 and CD69 expression on T cells. Target cell lysis was determined by measuring the percentage of viable cells by measuring ATP levels assessed by CellTiterGlo (Promega). ATP levels measured by luminescence on an Envision microplate reader yielded relative light unit (RLU) values ​​that were analyzed using GraphPad Prism.

[0395] The target cell lysis for each sample was calculated as follows: % Kill=(100-(RLU sample / RLU no IgG)×100).

[0396] These data on T cell activation (see FIG. 15) and cytotoxicity (FIG. 16) indicate that the trispecific antibodies are functional. As shown in FIGS. 15 and 16, it is demonstrated that the MockxCD3:EGFR and EGFRxCD3:Mock trispecific molecules are capable of inducing EGFR target-specific T cell activation and cytotoxicity. When the anti-EGFR Fab is placed distally within the long arm, the MockxCD3:EGFR trispecific showed enhanced activity in both T cell activation (FIG. 15) and cytotoxicity (FIG. 16) relative to both the bispecific EGFRxCD3 and the trispecific with the EGFR binding domain on the short arm and the CD3 and Mock TT (MF1337) binding domains on the long arm (see FIG. 14a).

[0397] Furthermore, based on the activity of trispecific antibodies with EGFR and CD3 on the long arm, the linkers can be linked to those associated with relatively high cytokine production (IgG1 UH (SEQ ID NO: 2), IgG1 MH (SEQ ID NO: 11), IgG2A MH (SEQ ID NO: 4), and IgG1 G4S (SEQ ID NO: 12)), as well as those associated with relatively low cytokine production (IgG1 UL (SEQ ID NO: 14), IgG2A H (SEQ ID NO: 15), IgG2B R (SEQ ID NO: 21), IgG2A R (SEQ ID NO: 20), IgG1 H (SEQ ID NO: 19), IgG1R (SEQ ID NO: 23)), as shown in Figures 17b-d. The effect of changing the linker usage on cytotoxicity was less pronounced (Figure 17a).

[0398] Example 11: Generation of trispecific antibodies with binding domains on the short or long arms and immune cell engaging. According to the present invention, the immune cell engaging binding domain can be located anywhere on the multivalent molecule, including at distal or internal positions on the long or short arms, and heterodimerization techniques can be utilized to suitably generate trivalent molecules. See Figure 18 (CD3 binding domain on the short arm), Figure 19 (CD3 binding domain on the internal long arm), and Figure 25 (CD3 binding domain on the distal long arm).

[0399] For example, the immune engagement domains are placed in the short arms, DNA encoding the VH gene of the CD3 binding domain (MF8078) is cloned into a vector encoding the positively charged CH3 domain (KK), and DNA encoding the VH gene of the EGFR binding domain (MF9988 (SEQ ID NO:218) or MF9891 (SEQ ID NO:191)), linker IgG2A MH, and PD-L1 binding domain (MF5380 (SEQ ID NO:173) or MF5444 (SEQ ID NO:164)) is cloned into a vector encoding the negatively charged CH3 domain (DE). Figure 18 (CD3xPD-L1:EGFR). [Table 15]

[0400] Alternatively, the immune engagement domain is located at the medial position of the long arm, in which case DNA encoding the VH gene of the CD3 binding domain (MF8078), linker IgG2A MH (SEQ ID NO: 4), and the VH gene of the PD-L1 binding domain (MF5444 (SEQ ID NO: 164)), MF5380 (SEQ ID NO: 173), MF5377 (SEQ ID NO: 155)) are cloned into a vector encoding the positively charged CH3 domain (KK), and DNA encoding the VH gene of the EGFR binding domain (MF9886 (SEQ ID NO: 200), MF9988 (SEQ ID NO: 218), MF9891 (SEQ ID NO: 191), or MF9873 (SEQ ID NO: 209)) is cloned into a vector encoding the negatively charged CH3 domain (DE). Figure 19 (EGFRxCD3:PD-L1). [Table 16]

[0401] Alternatively, the immune engagement domain is placed at the distal position of the long arm, in which case DNA encoding the CD3 binding domain (MF8078 (SEQ ID NO:110), MF8508 (SEQ ID NO:128) or MF8057 (SEQ ID NO:92)), the VH gene for the linker IgG 1H (SEQ ID NO:19), and the VH gene for the fibrinogen binding domain ("Fibri") (MF1025) are cloned into a vector encoding the positively charged CH3 domain (KK), in which case DNA encoding the VH gene for the EGFR binding domain (MF8233) is cloned into a vector encoding the negatively charged CH3 domain (DE). Figure 24 (EGFRxFibri:CD3). [Table 17]

[0402] For each of the above trispecific molecules, each heavy chain variable region is paired with a common light chain, as depicted in Figures 18, 19, and 24. SEQ ID NO:29.

[0403] As described further below, the configuration of each CD3 binding domain has been demonstrated to be effective in generating T cell cytotoxicity or activation against cells expressing one or more extracellularly exposed tumor cell antigens.

[0404] Example 12: Effective dual tumor antigen binding and T cell engagement via CD3 for the EGFRxCD3:PD-L1 trispecific format. Cell line: MDA-MB-231 cells (ATCC® HTB-26) are breast cancer cells, derived from a metastatic site.

[0405] Trispecific antibodies were produced according to the format of Figure 19 and the ability of such molecules was analyzed to simultaneously achieve tumor antigen targeting and T cell engagement through cytotoxicity. These antibodies were generated by the techniques described above.

[0406] Four anti-EGFR Fabs (MF9886, MF9988, MF9891, MF9873) with varying affinities from low to high were used on the short arm and combined with different anti-PD-L1 Fabs (MF5444, MF5380, and MF5377) that further contained varying distal affinities from low to high on the long arm. The anti-CD3 Fab and linker were kept constant using MF8078 and linker IgG2 AMH (SEQ ID NO: 4).

[0407] EGFR and PD-L1 affinity rankings were based on the binding data in Tables 18 and 19, respectively; rankings were based on binding to the reference anti-EGFR and anti-PD-L1 antibodies listed below. [Table 18]

[0408] The affinity ranking of the EGFR heavy chains is based on the relative ability of these monospecific bivalent antibodies to bind to the EGFR expressing cells as described above, compared to a positive control of a monospecific bivalent antibody with heavy chain MF8233. [Table 19]

[0409] The relative affinity ranking of PD-L1 heavy chain arms is based on their ability to bind human PD-L1 in ELISA. To this end, ELISA plates were coated with huPD-L1-His (Sinobiological) in an eight-step, three-fold titration dilution range starting at 10μg / ml. The binding of each PD-L1 Fab was then assessed as PD-L1xTT IgG at 5μg / ml. The EC50 for binding was determined and normalized to the binding EC50 determined for anti-PD-L1 RG7446 MPDL3280A. See US Patent Application Publication No. 2010 / 0203056 present on each ELISA plate.

[0410] These trispecific EGFRxCD3:PD-L1 molecules were then tested for their ability to induce cytotoxicity against two cell lines (HCT116 and MDA-MB-231) with different antigen densities for tumor cell antigens based on the methods previously described herein. The expression profiles of these cell lines were determined using FACS staining by using control antibodies (cetuximab for EGFR and MPDL3280A for PD-L1) and expression of the antigen of interest was considered positive if the mean fluorescence intensity (MFI) was 3-fold higher than the background signal. PD-L1 was repeated three times and EGFR four times, and the results were reported as MFI as shown below. [Table 20]

[0411] This study investigating the cytotoxicity of the trispecific molecules against these cell lines shows that all three binding domains of the EGFRxCD3:PD-L1 trispecific molecule can simultaneously bind two tumor cell antigens and CD3, thereby contributing both tumor antigen binding domains of the trispecific molecule to cytotoxicity upon T cell engagement. Figures 20 and 21 (against MDA-MB-231 cells) and Figure 22 (against HCT116 cells). These trispecific molecules generally show higher functional activity than the bispecific EGFRxCD3xMock and MockxCD3xPD-L1 controls, with the trispecific molecule 9873x8078:5377 showing the highest lysis rate (Figure 21). The trispecifics tested here are more potent against MDA-MB-231 cells, which have relatively higher target antigen levels compared to HCT116 cells (Figures 21 and 22).

[0412] Example 13: CD3xPD-L1: Efficient dual tumor antigen binding and T cell engagement via CD3 for a trispecific format of EGFR Simultaneous tumor antigen targeting is further demonstrated when trispecific antibodies are produced according to the format of Figure 18, with the immune engagement domain present on the short arm. These antibodies were generated by the techniques described above. In this format, dual antigen target correlation binding was demonstrated, as with increasing PD-L1 affinity there was a continued increase in target cell binding as measured by FAC of MDA-MB-231 cells observed with CD3xPD-L1:EGFR molecules. Figure 23a. In the case of certain of these trispecific molecules with the format CD3xPD-L1:EGFR, simultaneous dual antigen binding and immune cell engagement was shown to have an additive effect on BxPC3 cell cytotoxicity in molecules that bind a single antigen and CD3 (heavy chain sequence not shown), confirming the ability of these molecules to simultaneously engage all three binding arms. Figure 23b. The cytotoxicity assay protocol for these data is described above.

[0413] Example 14: Efficient T cell activation via CD3 on the distal long arm with EGFRxfibrinogen:CD3 trispecific format. Jurkat-NFAT-RE-luc2 cells (Promega) are an engineered Jurkat T cell line that expresses a luciferase reporter driven by the NFAT response element (NFAT-RE).

[0414] HT29 (ATCC HTB-38) are human colon carcinoma cells.

[0415] Studies were performed using a trivalent molecule of EGFR (MF8233) x fibrinogen (MF1122):CD3 (MF8078) to place the CD3 binding domain in the distal region of the long arm. Figure 24. T cell activation assay performed on Jurkat-NFAT-RE-luc2 cells against target cells HT29 to establish the functional T cell activation capacity of this format. Briefly, reporter assay: Jurkat effector T cells were co-incubated with target cells in the presence of a concentration range of trivalent and control antibodies. After 5 hours of incubation, luciferase activity of reporter cells was measured as a readout for T cell activation using the Bio-Glo Luciferase Assay System (Promega). Luminescence activity was measured on an Envision microplate reader to obtain relative light unit (RLU) values ​​and analyzed using GraphPad Prism.

[0416] As shown in FIG. 25, T cell activation by the EGFRxFibri:CD3 trispecific molecule was shown to be at levels equal to or greater than the positive control, which was an EGFRxCD3 antibody previously shown to cause T cell activation in FIG. 15a.

[0417] Example 15: Effective tumor antigen binding and T cell engagement via CD3 for a series of CD3 binding domains and linkers A panel of EGFRxCD3:EGFR bispecific trivalent molecules was generated (Figure 26) to show the efficacy of tumor targeting and T cell engagement for a variety of different CD3, immune cell engagement binding domains, and eight different linkers. Each trivalent molecule contained two identical anti-EGFR binding domains (MF9891) located on the short arm and distal long arm, and a CD3 binding domain located on the internal position of the long arm. In this study, Jurkat-NFAT-RE-luc2 cells and reporter cell lines of target cells HCT116 (intermediate EGFR expression) and MDA-MB-231 were used to measure T cell activation by the method described above. For negative controls, trivalent molecules using CD3 binding domains of different superclusters were produced MockxCD3xMock or bispecific (EGFR(MF8233)xTT(MF1337)) (4,000ng / ml). The readout was dependent on the reporter activity after 5 hours of incubation.

[0418] Each CD3 tested from the various superclusters showed reporter activity using HCT116 cells, with the CD3 binding domain from supercluster 7 showing the lowest relative reporter activity, while displaying a spectrum of activity based on the linker arm. In contrast, two CD3 binding domains from supercluster 1 (MF8058 and MF8078) and a CD3 binding domain from supercluster 4 (MF8508) showed relatively consistent activity regardless of linker arm. Finally, one CD3 binding domain from supercluster 1 (MF8057) showed relatively low reporter activity, resulting in some differences associated with the different linkers. See Figure 27. A review of these data indicates that trivalents containing IgG1 MH linkers consistently appear to be the most potent across the superclusters.

[0419] Similarly, each CD3 binding domain tested from the various superclusters exhibited reporter activity using MDA-MB-231 cells, with the CD3 binding domain from supercluster 7 exhibiting relatively low reporter activity with a spectrum of activity based on the linker used. In contrast, the three CD3 binding domains from supercluster 1 (MF8057, MF8058, and MF8078) and the CD3 binding domain from supercluster 4 exhibited relatively similar activity regardless of the linker used. See Figure 28. [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27]

Claims

1. a full length base antibody portion comprising two Fab binding domains; and at least one additional binding domain, the full length base antibody portion is connected to the at least one additional binding domain by a linker; each binding domain of the base antibody portion and each of the at least one additional binding domain all share a common variable region; A multivalent antibody, wherein the linker comprises an IgG hinge sequence.

2. The multivalent antibody of claim 1 , wherein the at least one additional binding domain comprises an Fv domain, a Fab domain, or a modified Fab domain.

3. The multivalent antibody of claim 1 or 2, wherein the at least one additional binding domain is a Fab domain comprising a CH1 region and is connected to the full-length base antibody portion by the linker, linking the variable region and the CH1 region of the full-length base antibody portion.

4. The multivalent antibody of claim 1 , wherein the linker comprises an IgG1 hinge sequence.

5. The multivalent antibody of claim 4 , wherein the linker comprises an IgG1 upper hinge sequence.

6. The multivalent antibody of claim 4 or 5, wherein the linker comprises an IgG1 central hinge sequence.

7. The multivalent antibody of claim 4 , wherein the linker comprises an IgG1 lower hinge sequence.

8. The multivalent antibody of claim 1 , wherein the linker comprises an IgG2 hinge sequence.

9. The multivalent antibody of claim 8 , wherein the linker comprises an IgG2 upper hinge sequence.

10. The multivalent antibody of claim 8 or 9, wherein the linker comprises an IgG2 middle hinge sequence.

11. The multivalent antibody of claim 8 , wherein the linker comprises an IgG2 lower hinge sequence.

12. The multivalent antibody of claim 1 , wherein the linker comprises an IgG3 hinge sequence.

13. The multivalent antibody of claim 12, wherein the linker comprises an IgG3 upper hinge sequence.

14. The multivalent antibody of claim 12 or 13, wherein the linker comprises an IgG3 central hinge sequence.

15. The multivalent antibody of claim 12 , wherein the linker comprises an IgG3 lower hinge sequence.

16. The multivalent antibody of claim 1 , wherein the linker comprises an IgG4 hinge sequence.

17. The multivalent antibody of claim 16 , wherein the linker comprises an IgG4 upper hinge sequence.

18. The multivalent antibody of claim 16 or 17, wherein the linker comprises an IgG4 central hinge sequence.

19. 19. The multivalent antibody of claim 16, wherein the linker comprises an IgG4 lower hinge sequence.

20. A multivalent antibody according to any one of claims 1 to 19, capable of binding to at least two different antigens.

21. 21. A multivalent antibody according to any one of claims 1 to 20, wherein at least one of the binding domains specifically binds to an antigen on an immune effector cell.

22. The multivalent antibody of claim 21 , wherein the immune effector cell is a T cell.

23. The multivalent antibody of claim 22, wherein the antigen on the T cell is CD3.

24. 24. A multivalent antibody according to any one of claims 21 to 23, wherein at least one of the binding domains specifically binds to an antigen on an abnormal cell.

25. 25. The multivalent antibody of claim 24, wherein at least two binding domains specifically bind to an antigen on an abnormal cell.

26. The multivalent antibody of claim 24 or 25, wherein the abnormal cell is a tumor cell.

27. 1. A method for preparing a multivalent antibody, the method comprising: Providing a cell comprising one or more nucleic acid sequences encoding polypeptides capable of assembling into a multivalent antibody according to any one of claims 1 to 26; and culturing said cells under conditions that provide for the expression of said polypeptides and their assembly into multivalent antibodies.

28. 27. A cell comprising one or more nucleic acid sequences encoding polypeptides capable of being assembled into a multivalent antibody according to any one of claims 1 to 26.

29. 27. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 26 and a pharma- ceutically acceptable carrier and / or diluent.

30. 27. A multivalent antibody according to any one of claims 1 to 26 for use in the treatment of the human or animal body by therapy.

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