Patent Grant
12098203
This application contains a sequence listing submitted electronically via EFS-web, which serves as both the paper copy and the computer readable form (CRF) and consists of a file entitled “WRN11NP_seqlist.txt”, which was created on Oct. 25, 2019, which is 32,394 bytes in size, and which is herein incorporated by reference in its entirety.
This invention relates to a novel hetero-dimeric multi-specific format of multiple antibody variable domains comprising a core of two split variable domain pairs wherein both variable light domains and the two cognate variable heavy domains are positioned in tandem on two separate protein chains, respectively.
In the past forty years since the development of the first monoclonal antibodies [R17], antibodies have become an increasingly important class of biomolecules for research, diagnostic and therapeutic purposes.
Antibodies, as therapeutic agents, are evolving towards more rationally designed functionalities thus improving and expanding their inherent properties. Examples include the optimization of effector functions by glycoengineering [R18], specific localization like the transfer over the blood brain barrier [R19], or tuned half-life by e.g. increased binding to FcRn [R20].
A complementary approach of antibody functionalization is the combination of different target specificities in one molecule to generate bi- or multispecific antibodies or antibody fragments, thus allowing alternative mechanisms of action, like the retargeting of T cells, as exemplified by bispecific antibody Blinatumomab or the trispecific antibody Catumaxomab.
Despite the large number of different multispecific antibody formats that have been developed so far [R21], the current repertoire of bi- and multispecific antibody formats still leaves the industry with considerable technical challenges and little flexibility with only few formats that allow for tri- and multi-specific binding and even less formats supporting the formation of hetero-dimeric proteins.
Different multi-specific formats have been presented in the past. Conceptually these formats can be grouped into three categories: a) single-chain multi-specific formats, in which the different target binding domains are all located on one single protein chain, expressed from a single gene, b) homo-bi- and homo-multimeric formats, in which the different target-binding domains are located on identical protein chains that are assembled by the use of a multimerization domain resulting in bi-/multi-valent and optionally also multi-specific complexes, and c) hetero-dimeric formats in which the target-binding domains are located on different protein chains, and the assembly of the two protein chains is driven by a hetero-dimerization domain.
Hetero-dimeric multi-specific formats in principle offer the advantage that binding domains with different specificities and affinities can easily be tested in various combinations by simple permutation of the two hetero-dimerizing protein chains, thereby allowing for the screening for optimal combinations of specificities and affinities directly in the final format without the need for tedious cloning.
Such screening in the final product format is required in cases where the binding properties and/or potencies of the various domains need to be carefully matched to each other to achieve optimal potency of the bi-specific protein and at the same time minimize the risk for unspecific effects. In the clinical situation this would translate to optimal efficacy at minimal risk of adverse effects. Situations, where such optimal combinations are required, may for example be the concomitant blockade of two disease-driving cytokines that are produced in the course of the disease in different concentrations. In this situation, the therapeutic bi-specific protein should allow to effectively block both cytokines at one and the same therapeutic dose.
Another example, where the characteristics of the target-binding domains of a multi-specific molecule must be coordinated, is the therapy of cancer with a cytotoxic antibody targeting two cell surface targets on the tumor cells. While the two cell surface targets of the antibody in this situation may be co-expressed exclusively on cancer cells, they may be expressed individually in a variety of healthy tissues. In order to achieve best efficacy at lowest risk for adverse side effects in tumor therapy, the cytotoxic antibody should bind to a cell preferentially, when both targets are co-expressed, but should not bind to tissues expressing only one of the two targets. To achieve this, the affinities of the two target-binding domains need to be tuned such that on one hand the affinities of the individual domains to their target are too weak to result in cell lysis, and on the other hand the cooperative avidity resulting from concomitant binding of the bi-specific molecule to both targets on a cancer cell is sufficient to induce cell lysis. Due to geometrical constraints resulting from the simultaneous binding to different macromolecules immobilized on the cell surface, the combination of domains to achieve maximal cooperative binding is not only a function of affinities, but also of epitopes and may only be identified by testing different domain combinations in the actual product format.
The native IgG type antibody can be considered a homo-dimeric format.
In order to increase the number of specificities of the homo-dimeric antibody format employing the classic IgG architecture as a scaffold, additional binding moieties, such as single-chain Fvs [R15], Fvs [R16], single domains [e.g. Nanobodies: Huang et al., Expert Rev Mol Diagn. 10 (2010):777-85] or alternative scaffolds [e.g. Fynomers: Schlatter et al., MAbs. 4 (2012) 497-508] can be appended, either to the amino- or the carboxyl-terminus of both the heavy and the light chain. One advantage of this approach is that bi- to tri-specific constructs can be generated with a conventional IgG as core domain, which allows exploiting most of the manufacturing and modification technologies that have been established for conventional IgGs. Due to the homo-dimeric nature of conventional Fc regions, however, this approach will always result in at least two identical binding domains per molecule and consequently in bivalent binding to a certain target. This may not always be wanted, particularly not (a) if only cooperative binding to two targets shall result in the desired effect, of (b) if the molecular weight shall not be further increased. Furthermore, this approach oftentimes suffered from poor domain stabilities of the appended binding moieties rendering them unsuitable for pharmaceutical development.
The concept of fusing further binding domains to increase specificities can also be applied to Fab fragments [R14] or other antigen-binding fragments of IgGs [R23]. Due to the hetero-dimeric nature of the Fab, consisting of a heavy and a light chain, the Fab fragment can be used as a hetero-dimerization domain. The Fab fragment has for example been used to engineer the so-called Tribody. In this format scFv fragments are fused to the carboxyl-terminus of both the light and the heavy chain of a Fab resulting in a truly hetero-dimeric tri-specific molecule. The light chain-heavy chain association of the Fab is mainly driven by the interaction between CL-CH1, which in addition are connected though a covalent disulfide-bond [R2]. Challenges with this format are (a) the limitation of stability to the least stable component, which will most probably be the appended scFv, and (b) the limitation to maximally three target specificities.
As an approach to solve the limitations of homo-dimeric bi-specific formats, hetero-dimeric IgGs have been introduced [R31]. Simple co-expression of two different mAbs from one cell leads with very low probability to the assembly of hetero-dimeric bi-specific IgGs in which two different heavy chains will pair with each other, and the two different light chains will pair with their corresponding heavy chain [R24]. It will, however, also lead to A) the mismatch of heavy and light chains with different specificities and to B) mixtures of different heavy chain combinations resulting in mono- and bi-specific variants. To address these difficulties several approaches have been undertaken, which create an artificial asymmetry in the molecules. The “knob-into-holes” concept [R3, R4] uses engineering of the heavy chain/heavy chain or heavy chain/light chain interface to drive the association of the co-expressed chains towards the desired configuration. In another approach the CrossMab methodology [R5] allows selective pairing of an engineered light chain/heavy chain pair. A drawback of these methodologies is that any residual fraction of mismatched molecules is very difficult to separate from the product. Therefore other techniques focus on the separation problem by engineering differential binding properties for the mono- and bispecific binders [R22] and on the other hand tolerate the loss in yield caused by the stochastic distribution of variants.
A further limitation of the IgG-based hetero-dimeric formats is that they all necessarily comprise an Fc effector domain. A format in which hetero-dimerization would be driven by target binding domains directed to any target of choice would allow increasing the number of specificities/functionalities at the same or lower molecular weight. Molecules with lower molecular weights penetrate more efficiently into target tissues (e.g. solid cancers) and thus hold the promise for improved efficacy at the same or lower dose. However, smaller formats suffer from the disadvantage of having a shorter serum half-life. An alternative approach uses non-antibody fusion proteins to confer the desired multispecificity to, for example, scFv moieties. Examples of such fusion proteins are Dock-and-Lock [R25], barnase-barstar [R26], jun-fos [R27], TNF [R28], or HSA [R29]. These concepts have in common that at least one pair of domains is added that interact in a hetero-dimeric fashion to bring the bi- or multispecific binding domains together. These hetero-dimerization domains are not directly involved in target binding, nevertheless, they increase the molecular weight of the protein—similar to the constant region one (C1) in the Tribody format. Furthermore, they might come with the risk of increased immunogenicity by incorporating non-human epitopes and sequences.
In contrast to the interaction between CL and CH1 discussed above, the association of the paratope-forming VL-VH domains is generally regarded as weak. However, there are several hetero-dimeric antibody fragment concepts that are comprised exclusively of antibody variable domains. Approaches like diabodies [R6], DARTs [R10], and Tandabs [R7, R8], amongst others, offer elegant and minimalistic approaches to create homo- and hetero-dimeric bispecific and bi- to tetra-valent assemblies. The most important limitations of these formatting strategies are (a) the addition of further specificities by fusing e.g. an scFv to the amino- or the carboxyl-terminus of either chain of diabodies or DARTs could result in the intra-chain pairing of the variable light and variable heavy domains thereby rendering hetero-dimerization of the two protein chains very challenging, and (b) due to the weak domain interface binding between the variable light and the variable heavy chain often observed in the past, these formats suffered from low monomeric stability and poor producibility, so that further engineering such as the introduction of inter-domain disulfide bonds [R12] to stabilize the VL/VH interface was regarded as being necessary.
Aiming at constructing multi-specific single-chain tandem Fv antibodies, Kipriyanov et al [R30] suggested a design comprising two protein chains, each consisting of two split Fv domains arranged in the order VL-(linker1)-VH-(linker2)-VL-(linker3)-VH. For the construction of hetero-dimeric tetra-specific proteins, the hetero-dimer would consist of two protein chains with the following architecture. Chain 1: VLA-(linker1)-VHA-(linker2)-VLB-(linker3)-VHC, and chain B: VLD-(linker1)-VHD-(linker2)-VLC-(linker3)-VHB, wherein the assembly of FvB and FvC would drive hetero-dimerization of the two chains (see FIG. 10A of WO 2016/202457). In order to prevent intra-chain assembly resulting in a tandem single-chain Fv (scFv2)-like format, and to promote hetero-dimerization of two monomeric protein chains, shortened linkers at the positions “linker3” of maximally 10 amino acids have been suggested (EP1293514 A1) The proposed organization of the two split variable domains with a linker2 of at least 15 amino acids, however, results in the possibility of the second variable domains to fold back onto N-terminal domains, leading to a single-chain diabody (scDb)-like format consisting of non-matching VH/VL pairs, which in consequence would likely not be able to bind their target. In addition, there is also the potential for the formation of a hetero-dimer in which all variable heavy and light chains on protein chain 1 would pair with the variable light and heavy chains of protein chain 2, respectively, thereby preventing the formation of the terminal scFvs (scFvA and scFvD) and resulting in the pairing of non-cognate variable domains. The tandem scFv (scFv2) or scDb-type byproducts might be the reason for the very high fraction of protein observed at the apparent molecular weight of the non-multimerized protein chains [R30].
In theory the formation of scDb-like structures in the approach described above, could be further reduced by shortening also the second linker (linker2) between the two split variable domains. This would however, limit the flexibility of the construct, which in many cases would negatively impact on the range of accessible epitopes that allow for concomitant binding of two targets. These geometrical constraints are particularly limiting when two membrane proteins shall be bound at the same time.
Additionally, and most importantly however, both monomers might form homodimeric fragments (see
In summary, there is a well pronounced industry need for hetero-dimeric multi-specific formats that allow for simple permutation and subsequent characterization of different binding domains in the final format. Major challenges with such formats have been (a) the relatively poor efficiency of specific hetero-dimerization resulting in suboptimal production yields, and (b) the necessity to use either non target binding proteins as hetero-dimerization domains or engineered hetero-dimer Fc effector domains that come with poor flexibility in tuning serum half-life and that limit the flexibility in adding novel functionalities without increasing the molecular weight.
Thus, it was suggested that the optimal hetero-dimeric multi-specific format would exclusively consist of target binding domains and would allow for adjusting the geometry of the molecule for example by freely changing the linker lengths between the different binding domains to accommodate the geometrical constraints defined by the interaction partners (targets). As a solution to that problem, WO 2015/058861 and Egan et al., MAbs 9 (2017) 68-84 reported the development of a novel hetero-dimeric multi-specific format of multiple antibody variable domains, which comprises a core of two split variable domain pairs wherein both variable light domains and two cognate variable heavy domains are positioned in tandem on two separate protein chains, respectively, thereby driving hetero-dimerization of the two protein chains. This format has been termed “multispecific antibody-based therapeutics by cognate heterodimerization (MATCH)”. Up to two additional binding domains, particularly antibody-based binding domains, such as scFv fragments, are fused to the amino- and/or the carboxyl-terminus of either protein chain, resulting in an up to hexa-specific hetero-dimeric protein.
However, while it could successfully be shown that the underlying principle permitted the generation of such hetero-dimeric multi-specific proteins based on a core comprising a variable domain pair with binding specificity for human CD3 and a variable domain pair with binding specificity for a therapeutic target, constructs such as the MATCH constructs tested in WO 2015/058861 and in Egan et al. can be expected to require, on a case-by-case basis, the fine-tuning of the properties of the hetero-dimeric multi-specific proteins, such as by identifying suitable VL and VH domain frameworks, suitable linkers, and optionally, suitable positions for the cysteine residues for the formation of interchain disulfide bonds. Such fine-tuning is thought to be necessary since it does not appear to be possible to fully predict the heterodimerization propensity of such hetero-dimeric multi-specific proteins including a novel variable domain pair. Furthermore, it is known that the mechanism of action for such kind of multispecific molecules, comprising a CD3-binding domain and at least one domain binding to an antigen expressed on the target cells, is based on the lysis of target cells upon formation of an immunological synapse. Crosslinking and clustering of CD3 receptor on the T cell by interaction with a tumor associated antigen (TAA) on the target cell membrane will lead to T cell activation and subsequent release of cytokines and cytotoxic agents into the synapse. Without wishing to be bound by theory, it is expected that the formation of such an immunological synapse is driven, or at least fundamentally influenced by the geometry of the multispecific protein and the epitopes of the respective binding domains, so that again, it does not appear to be predictable that a novel combination of variable domain pairs forming said core will work as planned. Thus, there was still an unmet need to identify a more robust method for reliably generating such hetero-dimeric multi-specific proteins.
Furthermore, the present inventors realized, that in order to increase the specificity of target cell lysis, a T cell engaging molecule may comprise, in addition to the CD3-binding domain, at least two domains concomitantly binding to antigens expressed on the surface of the target cell. For the efficient identification of two binding domains, that together bind with optimal selectivity to the target cells, by permutation of expression plasmid combinations, each encoding one of the two protein chains of the MATCH complex, the two domains must each be located on a different protein chain of the heterodimeric MATCH complex. Therefore, there was the need for identifying a core domain, driving the heterodimerization of the two chains, that would comprise domains that are not involved in target cell binding.
The solution to this problem, i.e. the identification of a defined core of two fixed variable domain pairs which can be extended by fusing one or more additional targeting moieties to the N- and/or C-termini of one or both of the single-chain proteins, has hitherto neither been shown nor suggested in the prior art.
This invention relates to a novel hetero-dimeric multi-specific format of multiple antibody variable domains comprising a core of two split variable domain pairs wherein both variable light domains and two cognate variable heavy domains are positioned in tandem on two separate protein chains, respectively, thereby driving hetero-dimerization of the two protein chains, wherein the core is formed by a first variable domain pair with specificity for human CD3 and a second variable domain pair with specificity for human serum albumin. Up to two additional binding domains, particularly antibody-based binding domains, such as scFv fragments, are fused to the amino- and/or the carboxyl-terminus of either protein chain, resulting in an up to hexa-specific hetero-dimeric protein. In addition to forming constructs that are stable and well expressed and that can be expected to exhibit a long half-life in plasma, it could surprisingly be shown that such constructs exhibit a different pharmacodynamic profile by showing slower T cells activation kinetics and reduced cytokine release, without compromising on their maximal target cell lysis capacity, when compared to a single-chain diabody (scDb) format comprising the identical CD3-binding domain and the same domain binding to the target antigen expressed on the target cells. Such reduced cytokine release at comparable effect size holds the promise for reduced adverse effects due to cytokine release and therefore for a favorable risk-to-benefit profile.
Thus, in a first aspect the present invention relates to a hetero-dimeric protein comprising a first and a second single-chain protein,
wherein said first single-chain protein comprises a first amino acid sequence consisting of (from the N- to the C-terminus):
wherein said second single-chain protein comprises a second amino acid sequence consisting of (from the N- to the C-terminus):
wherein said first VL domain forms a first cognate pair of variable domains with specificity to a first target antigen with either said first or said second VH domain and said second VL domain forms a second cognate pair of variable domains with specificity to a second target antigen with the other of said VH domains, wherein one of said target antigens is human serum albumin and the other said target antigen is human CD3, and wherein at least one of said first or said second single-chain protein further comprises
In a second aspect, the present invention relates to one or two nucleic acid sequences encoding said first and second single-chain proteins.
In a third aspect, the present invention relates to one or two vectors comprising said one or two nucleic acid sequences.
In a fourth aspect, the present invention relates to a host cell or host cells comprising said one or two vectors.
In a fourth aspect, the present invention relates to a method for producing the first and second single-chain proteins, or the hetero-dimeric protein, of the present invention, comprising (i) providing a nucleic acid or nucleic acids according to the present invention, or a vector or vectors according to the present invention, expressing said nucleic acid or nucleic acids or said vector or vectors and collecting said first and second single-chain proteins, or said hetero-dimeric protein, from the expression system, or (ii) providing a host cell or host cells of the present invention, culturing said host cell or host cells, and collecting said first and second single-chain proteins, or said hetero-dimeric protein, from the cell culture.
In a fifth aspect, the present invention relates to a pharmaceutical composition comprising the hetero-dimeric protein of the present invention and a pharmaceutically acceptable carrier.
In a sixth aspect, the present invention relates to the hetero-dimeric protein of the present invention for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease, wherein at least one of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease. In a seventh aspect the present invention relates to a method for treating a patient suffering from a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease, comprising administering to a subject an effective amount of the hetero-dimeric protein of the present invention, wherein at least one of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease.
Particular embodiments of the present invention are set forth in the appended dependent claims.
Here we present a novel format exhibiting quantitative hetero-dimeric assembly of two protein chains containing multiple antibody variable domains. This format consists of a core of two split variable domain pairs (two Fv fragments), wherein both variable light domains and both variable heavy domains each are positioned on a separate protein chain, thereby driving hetero-dimerization of the two protein chains, wherein one pair of VL and VH domains is specific for human serum albumin and the other is specific for human CD3. Up to two additional binding domains, for example in the scFv format, with high intra- and inter-domain stability are fused to the amino- and/or the carboxyl-terminus of either peptide chain, resulting in an up to hexa-specific hetero-dimeric protein.
Thus, in a first aspect the present invention relates to a hetero-dimeric protein comprising a first and a second single-chain protein, wherein said first single-chain protein comprises a first amino acid sequence consisting of (from the N- to the C-terminus):
wherein said second single-chain protein comprises a second amino acid sequence consisting of (from the N- to the C-terminus):
wherein said first VL domain forms a first cognate pair of variable domains with specificity to a first target antigen with either said first or said second VH domain, and said second VL domain forms a second cognate pair of variable domains with specificity to a second target antigen with the other of said VH domains, wherein one of said target antigens is human serum albumin and the other said target antigen is human CD3,
and wherein at least one of said first or said second single-chain protein further comprises
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, composition or step or group of integers or steps, while any additional integer, composition or step or group of integers, compositions or steps may optionally be present as well, including embodiments, where no additional integer, composition or step or group of integers, compositions or steps are present. With respect to such latter embodiments, the term “comprising” thus includes the narrower term “consisting of”.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety to the extent possible under the respective patent law. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In the context of the present invention, the terms “VL domain” and “VH domain” refer to the variable light chain domain, and the variable heavy chain domain, respectively, of antibodies. In the context of the present invention, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e. molecules that contain an antigen binding site that specifically binds to an antigen, i.e. including antibody portions comprising at least an antigen-binding fragment of an antibody.
In the context of the present invention, an antibody, or any binding molecule in general, is considered to “specifically bind” to an antigen (in the case of an antibody), or to a cognate binding partner (in the case of a binding molecule in general), if it has a dissociation constant KD to said antigen/cognate binding partner as target of 100 μM or less, preferably 50 μM or less, preferably 30 μM or less, preferably 20 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 900 nM or less, more preferably 800 nM or less, more preferably 700 nM or less, more preferably 600 nM or less, more preferably 500 nM or less, more preferably 400 nM or less, more preferably 300 nM or less, more preferably 200 nM or less, even more preferably 100 nM or less, even more preferably 90 nM or less, even more preferably 80 nM or less, even more preferably 70 nM or less, even more preferably 60 nM or less, even more preferably 50 nM or less, even more preferably 40 nM or less, even more preferably 30 nM or less, even more preferably 20 nM or less, and even more preferably 10 nM or less. In the context of the present invention, the term “functional domains” refers to a proteinaceous domain having a predefined function, such as enzymatic activity or specific binding to a cognate ligand, wherein said proteinaceous domain is a structured domain having at least a secondary structure element. Methods for the determining the presence of secondary structure in polypeptides or proteins, such as X-ray crystallography, circular dichroism (CD), vibrational circular dichroism (VCD), NMR, or FT-IR, or for predicting the presence of secondary structure in polypeptides, such as PEP-FOLD (Shen et al., J. Chem. Theor. Comput. 10 (2014) 4745-4758) are well known to the practitioner in the art. In particular embodiments, said proteinaceous domain is a structured domain having a tertiary structure. In particular embodiments, said proteinaceous domain comprises at least about 20 amino acid residues (see Heitz et al., Biochemistry 38 (1999) 10615-25), particularly at least about 50 amino acid residues, more particularly at least about 100 amino acid residues. In particular embodiments, a functional domain is a proteinaceous domain that specifically binds to a cognate ligand. In particular embodiments, the functional domain is an antibody or an immunologically active portion of an antibody that specifically binds to an antigen.
In the context of the present invention, the term “polypeptide linker” refers to a linker consisting of a chain of amino acid residues linked by peptide bonds that is connecting two domains, each being attached to one end of the linker. In particular embodiments, the polypeptide linker has a continuous chain of between 2 and 30 amino acid residues (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues). In particular embodiments, the polypeptide linker is non-structured polypeptide. As mentioned above, methods for the determining the presence of secondary structure in polypeptides, such as X-ray crystallography, circular dichroism (CD), vibrational circular dichroism (VCD), NMR, or FT-IR, or for predicting the presence of secondary structure in polypeptides, such as PEP-FOLD (Shen et al., J. Chem. Theor. Comput. 10 (2014) 4745-4758) are well known to the practitioner in the art. In particular embodiments, a linker consists of amino acid residues selected from glycine and serine residues.
This invention is characterized by the following:
In a particular embodiment, the invention relates to a hetero-dimeric protein wherein said first or said second single-chain protein further comprises
In a particular embodiment, the invention relates to a hetero-dimeric protein wherein said first or said second single-chain protein further comprises
In a particular embodiment, the invention relates to a hetero-dimeric protein wherein said first or said second single-chain protein further comprises
In particular embodiments, said hetero-dimeric protein comprises said third and said fourth functional domain. In such embodiments, said hetero-dimeric protein is tetravalent, in particular embodiments, said hetero-dimeric protein is tetraspecific.
In particular embodiments, said hetero-dimeric protein comprises said third, said fourth, said fifth and said sixth functional domain. In such embodiments, said hetero-dimeric protein is hexavalent, in particular embodiments, said hetero-dimeric protein is hexaspecific.
In particular embodiments, said hetero-dimeric protein does not comprise a cognate pair of a first and a second immunoglobulin constant domain, wherein said first immunoglobulin constant domain is comprised in said first single-chain protein and wherein said second immunoglobulin constant domain is comprised in said second single-chain protein. In particular embodiments, at least one of said first and said second single-chain proteins does not comprise an immunoglobulin constant domain. In a particular embodiment, both said first and said second single-chain proteins do not comprise an immunoglobulin constant domain.
In particular embodiments, said hetero-dimeric protein does not comprise a cognate pair of a first proteinaceous interaction domain comprised in said first single-chain protein and a second proteinaceous interaction domain comprised in said second single-chain protein other than the cognate pairs of (i) said first VL domain and said first VH domain and (ii) said second VL domain and said second VH domain.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, triggers reduced cytokine levels at the time point of similar T cell activation as assessed by measuring Luciferase activity in Jurkat T cells expressing the Luciferase reporter gene under the control of NFAT in vitro in presence of physiological concentrations of HSA, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain.
In particular embodiments, said cytokines are T cell derived cytokines associated with cytokine release syndrome, such as IL-2, IL-10, IL-6, TNF-alpha and/or interferon-gamma, preferably IL-2. In particular embodiments, said cytokine levels are at least two-fold, preferably three-fold, more preferably four-fold and most preferably five-fold lower, when compared to the scDb. In particular embodiments, the determination of Luciferase activity and of cytokine levels is performed as described in the Examples.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, demonstrates a slower kinetic to reach similar T cell activation as assessed by measuring Luciferase activity in Jurkat T cells expressing the Luciferase reporter gene under the control of NFAT in vitro in presence of physiological concentrations of HSA, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain. In particular embodiments, said kinetics are at least two-fold, preferably three-fold and most preferably four-fold slower compared to the scDb. In particular embodiments, the determination of Luciferase activity and of cytokine levels is performed as described in the Examples.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, leads to reduced cytokine levels at the time point of similar T cell activation as assessed by CD69 expression in vitro in presence of physiological concentrations of HSA, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain. In particular embodiments, said cytokines are T cell derived cytokines associated with cytokine release syndrome, such as IL-2, IL-10, IL-6, TNF-alpha and/or interferon-gamma, preferably IL-2. In particular embodiments, said cytokine levels are at least two-fold, preferably three-fold, more preferably four-fold and most preferably five-fold lower, when compared to the scDb. In particular embodiments, the determination of CD69 expression and of cytokine levels is performed as described in the Examples.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, leads to reduced cytokine levels at the time point of similar extent of target cell lysis in vitro in presence of physiological concentrations of HSA, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain. In particular embodiments, said cytokines are T cell derived cytokines associated with cytokine release syndrome, such as IL-2, IL-10, IL-6, TNF-alpha and/or interferon-gamma, preferably IL-2. In particular embodiments, said cytokine levels are at least two-fold, preferably three-fold, more preferably four-fold and most preferably five-fold lower, when compared to the scDb. In particular embodiments, the determination of cytokine levels is performed as described in the Examples.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, demonstrates a slower kinetic to reach similar extent of target cell lysis in vitro in presence of physiological concentrations of HSA, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain. In particular embodiments, said kinetics are at least two-fold, preferably three-fold and most preferably four-fold slower compared to the scDb. In particular embodiments, the determination of the kinetics of target cell lysis is performed as described in the Examples.
In particular embodiments, said hetero-dimeric protein, wherein at least one of said third, said fourth, said fifth and said sixth functional domains is binding to a target antigen expressed on the surface of a target cell, has the capacity to reach similar maximal target cell lysis, when compared to a single-chain diabody (scDb) comprising the same target antigen-binding domain and the same CD3-binding domain. In particular embodiments, the determination of target cell lysis is performed as described in the Examples.
In particular embodiments, said first polypeptide linker consists of from 5 to 20 amino acid residues, particularly from of 6 to 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4; and n being selected from 1, 2, 3, 4, and 5.
In particular other embodiments, said first polypeptide linker consists of from 11 to 20 amino acid residues, particularly from of 11 to 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4; and n being selected from 3, 4, and 5.
In particular embodiments, said second polypeptide linker consists of from 5 to 20 amino acid residues, particularly from of 6 to 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4; and n being selected from 1, 2, 3, 4, and 5.
In particular other embodiments, said second polypeptide linker consists of from 11 to 20 amino acid residues, particularly from of 11 to 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4; and n being selected from 3, 4, and 5.
In particular embodiments, said third, fourth, fifth and/or sixth polypeptide linkers independently consist of from 8 to 20 amino acid residues, particularly from of 10 to 15 amino acid residues. In particular embodiments, said polypeptide linkers independently have the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 4; and n being selected from 1, 2, 3, 4, and 5, particularly from 2 and 3.
In particular embodiments, said first VL domain (ia) and said first VH domain (ib) form a first cognate pair of variable domains with specificity to a first target antigen, and said second VL domain (iia) and said second VH domain (iib) form a second cognate pair of variable domains with specificity to a second target antigen. In such embodiment, said first and said second single-chain protein form said hetero-dimeric protein in a parallel arrangement of said single-chain proteins (see
In particular such embodiments, said first polypeptide linker consists of from 10 to 20 amino acid residues, particularly from of 12 to 17 amino acid residues, particularly of 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 4; and n being selected from 1, 2, 3, 4, and 5, particularly 3.
In particular such embodiments, said second polypeptide linker consists of from 10 to 20 amino acid residues, particularly from of 12 to 17 amino acid residues, particularly of 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 4; and n being selected from 1, 2, 3, 4, and 5, particularly 3.
In particular such embodiments, said third, fourth, fifth and/or sixth polypeptide linkers independently consist of from 10 to 20 amino acid residues, particularly from of 12 to 17 amino acid residues, particularly of 15 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 4; and n being selected from 1, 2, 3, 4, and 5, particularly 3.
In particular other embodiments, said first VL domain (ia) and said second VH domain (iib) form a first cognate pair of variable domains with specificity to a first target antigen, and said second VL domain (iia) and said first VH domain (ib) form a second cognate pair of variable domains with specificity to a second target antigen. In such embodiment, said first and said second single-chain protein form said hetero-dimeric protein in an anti-parallel arrangement of said single-chain proteins (see
In particular such embodiments, said first polypeptide linker consists of from 5 to 12 amino acid residues, particularly from of 5 to 10 amino acid residues, particularly of 6 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 2; and n being selected from 1, 2, 3, 4, and 5, particularly 2.
In particular such embodiments, said second polypeptide linker consists of from 5 to 12 amino acid residues, particularly from of 6 to 10 amino acid residues, particularly of 8 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 3; and n being selected from 1, 2, 3, 4, and 5, particularly 2.
In particular such embodiments, said third, fourth, fifth and/or sixth polypeptide linkers independently consist of from 10 to 20 amino acid residues, particularly from of 8 to 12 amino acid residues, particularly of 10 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GmS)n; with m being independently selected from 2, 3, and 4, particularly 4; and n being selected from 1, 2, 3, 4, and 5, particularly 2.
In another particular embodiment of the anti-parallel arrangement, said first and said second polypeptide linker each consists of from 10 to 20 amino acid residues comprising between 40 and 60% charged residues, particularly from of 12 to 16 amino acid residues comprising 50% charged residues, in each case, wherein the two linkers are able to interact by forming interchain pairs of positively and negatively charged residues. In particular embodiments, the charged residues on one of said first and second linkers are exclusively positively charged residues, and the charged residues on the other of said first and second linkers are exclusively negatively charged residues, particularly wherein said first and second linkers are selected from SEQ ID NOs. 16 and 17.
In particular embodiments, said third, fourth, fifth and/or sixth functional domains are independently selected from the list of: binding domains, toxins, enzymes, hormones, and signaling proteins.
In particular embodiments, said third, fourth, fifth and/or sixth functional domains are independently selected from binding domains.
In particular such embodiments, binding domains are independently selected from the list of: antibody-based binding domains including but not limited to scFv, Fab and single antibody variable domains, single domain antibodies based on the VNAR structure from shark, and binding domains based on alternative scaffolds including but limited to ankyrin-based domains, fynomers, avimers, anticalins, fibronectins, and binding sites being built into constant regions of antibodies (e.g. f-star technology; see, for example, Wozniak-Knopp et al., Protein Eng. Des. Sel. 23 (2010) 289-297).
In particular such embodiments, said binding domains are antibody-based binding domains selected from: single-chain Fv fragments and single antibody variable domains.
In certain such embodiments, the order of variable domain in such a single chain Fv fragment is selected from (from N-terminus to C-terminus) VL-(linker)-VH and VH-(linker)-VL. In certain embodiments, the order of variable domains is the same for all single-chain Fv fragments comprised in the hetero-dimeric protein. In certain embodiments, three VL domains are linked to each other by said first polypeptide linker and one of said third, fourth and fifth polypeptide linkers, respectively, for example where a single-chain Fv fragment in the order VL-(linker)-VH is C-terminal from said first amino acid sequence. In certain embodiments, three VH domains are linked to each other by said second polypeptide linker and one of said third, fourth and fifth polypeptide linkers, respectively, for example where a single-chain Fv fragment in the order VL-(linker)-VH is N-terminal from said second amino acid sequence (see
In certain other embodiments, the variable domain of any such antibody-based binding domain that is directly linked via the corresponding linker to the N- and/or the C-terminus of said first or second amino acid sequence is (a) a VH domain in case that it is fused to said first amino acid sequence, and (b) a VL domain in case that it is fused to said second amino acid sequence. Thus, a VH domain is fused to the N- and/or the C-terminus of a VL-linker-VL core region, and a VL domain is fused to the N- and/or the C-terminus of a VH-linker-VH core region (see, for example,
In particular embodiments, said third, fourth, fifth and/or sixth binding domains are single-chain Fv fragments.
In particular such embodiments, the polypeptide linker connecting the variable domains of said single-chain Fv fragments consists of between 15 and 25 amino acid residues, particularly 20 amino acid residues. In particular embodiments, said polypeptide linker has the sequence (GGGGS)n, with n being selected from 3, 4, and 5, particularly 4.
In particular embodiments, the at least one of said antibody variable domains comprises CDR regions derived from a parental rabbit antibody, as evidenced by specific patterns inherent to rabbit CDRs.
In particular embodiments, at least one of said antibody variable domains comprises human framework regions.
In particular embodiments, said first single-chain protein and said second single-chain protein are cross-linked by at least one disulfide bond.
In particular embodiments, said disulfide bond is formed between a first cysteine residue flanking said first or said second VL domain and a second cysteine residue flanking said first or said second VH domain.
In particular embodiments, said disulfide bond is formed between a first cysteine residue comprised in a framework region of said first or said second VL domain and a second cysteine residue comprised in a framework region of said first or said second VH domain.
In particular embodiments, said first cysteine residue is located at position 141 of said first or said second VL domain and said second cysteine residue is located at position 51 of said first or said second VH domain.
In the context of the present invention, the numbering system used for antibody variable domains is based on the numbering system (“AHo numbering”) according to Honegger and Pluckthun (A. Honegger & A. Pluckthun. “Yet another numbering scheme for immunoglobulin variable domains: An automatic modeling and analysis tool”. J. Mol. Biol, 309 (2001)657-670).
In particular embodiments, said cognate pair of variable domains with specificity for human serum albumin comprises the three VL CDRs present in one of the VL protein sequences selected from SEQ ID NOs: 10, 12, and 14 in a human antibody VL framework, wherein the VL framework comprises VK frameworks FR1, FR2 and FR3, particularly VK1 frameworks, and a framework FR4, which is selected from a VK FR4, particularly VK1 FR4, and a VA framework 4, and the three VH CDRs present in one of the VH protein sequences selected from SEQ ID NOs: 11, 13, and 15 in a human antibody VH framework, particularly a VH3 framework.
In the context of the present invention, the assignment to Vκ, Vλ and/or VH frameworks is performed by alignment with the sequences of human antibodies shown in WO 97/08320. The definition of the frameworks and CDRs is used in accordance with Honegger & Pluckthun, loc. cit.
In particular such embodiments, at least one of said VL domains comprises (i) human VK framework regions FR1 to FR3, particularly human VK1 framework regions FR1 to FR3; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) a framework region IV, which is selected from
In a particular embodiment, said cognate pair of variable domains with specificity for human serum albumin comprises (i) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 14, and/or (ii) a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15.
In a particular embodiment, said cognate pair of variable domains with specificity for human serum albumin comprises (i) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 10, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to VH sequence according to SEQ ID NO: 11; or (ii) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 12, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 13; or (iii) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 14, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 15.
In a more particular embodiment, said cognate pair of variable domains with specificity for human serum albumin comprises
In a more particular embodiment, said cognate pair of variable domains with specificity for human serum albumin comprises a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 14, wherein said VL domain comprises CDR domains CDR1, CDR2 and CDR3 taken from the VL sequence according to SEQ ID NO: 14, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 15, wherein said VH domain comprises CDR domains CDR1, CDR2 and CDR3 taken from the VH sequence according to SEQ ID NO: 15, preferably wherein said VL domain comprises I2V, Q3V, K50Q and A51P (AHo numbering) and said VH domain comprises I55V, V103T, Y105F (AHo numbering).
In particular embodiments, said cognate pair of variable domains with specificity for human serum albumin comprises a VL domain comprising at least positions 5 to 140, particularly at least positions 3 to 145, of a protein sequence selected from SEQ ID NOs: 10, 12, and 14, and a VH domain comprising at least positions 5 to 140, particularly at least positions 3 to 145, of a protein sequence selected from SEQ ID NOs: 11, 13, and 15 (positions according to Honegger & Pluckthun, loc. cit.), particularly wherein said cognate pair of variable domains with specificity for human serum albumin comprises a VL domain selected from SEQ ID NOs: 10, 12, and 14, and a VH domain selected from SEQ ID NOs: 10, 12, and 14.
In particular embodiments, said cognate pair of variable domains with specificity for human CD3 comprises the three VL CDRs present in one of the VL protein sequence selected from SEQ ID NOs: 2, 4, 6 and 8 in a human antibody VL framework, wherein the VL framework comprises VK frameworks FR1, FR2 and FR3, particularly VK1 frameworks, and a framework FR4, which is selected from a VK FR4, particularly VK1 FR4, and a Vλ framework 4, and the three VH CDRs present in one of the VH protein sequences selected from SEQ ID NOs: 3, 5, 7 and 9 in a human antibody VH framework, particularly a VH3 framework.
In particular such embodiments, at least one of said VL domains comprises (i) human VK framework regions FR1 to FR3, particularly human VK1 framework regions FR1 to FR3; (ii) CDR domains CDR1, CDR2 and CDR3; and (iii) a framework region IV, which is selected from
In a particular embodiment, said cognate pair of variable domains with specificity for human CD3 comprises (i) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to a sequence selected from SEQ ID NOs: 2, 4, 6 and 8, and/or (ii) a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to a sequence selected from SEQ ID NOs: 3, 5, 7 and 9.
In a particular embodiment, said cognate pair of variable domains with specificity for human CD3 comprises (i) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 2, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 3; or (ii) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 4, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 5; or (iii) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 6, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 7; or (iv) a VL domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VL sequence according to SEQ ID NO: 8, and a VH domain exhibiting at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 percent, preferably at least 90 percent, sequence identity to the VH sequence according to SEQ ID NO: 9.
In a more particular embodiment, said cognate pair of variable domains with specificity for human CD3 comprises
In particular embodiments, said cognate pair of variable domains with specificity for human CD3 comprises a VL domain comprising at least positions 5 to 140, particularly at least positions 3 to 145, of a protein sequence selected from SEQ ID NOs: 2, 4, 6 and 8, and a VH domain comprising at least positions 5 to 140, particularly at least positions 3 to 145, of a protein sequence selected from SEQ ID NOs: 3, 5, 7 and 9 (positions according to Honegger & Pluckthun, loc. cit.), particularly wherein said cognate pair of variable domains with specificity for human CD3 comprises a VL domain selected from SEQ ID NOs: 2, 4, 6 and 8, and a VH domain selected from SEQ ID NOs: 3, 5, 7 and 9.
In particular such embodiments, said third, fourth, fifth and/or sixth binding domains are single-chain Fv fragments with specificity for a target selected from the list of: a cancer target, and a target present on immune effector cells.
In the context of the present application the term “target” refers to a cognate binding partner of a binding domain, such as an antigen of an antibody that is specifically bound by such binding domain.
In particular embodiments, said target is a cancer target, in particular an antigen or an epitope that is present on the surface of one or more tumour cell types or tumour-associated cells in an increased concentration and/or in a different steric configuration as compared to the surface of non-tumour cells. Particularly, said cancer target is present on the surface of one or more tumour or tumour stroma cell types, but not on the surface of non-tumour cells.
In other particular embodiments, said target is an antigen or epitope that is preferentially expressed on cells involved in autoimmune diseases. In other embodiments, said antigen or epitope is preferentially expressed on cells involved in an inflammatory disease.
In certain embodiments, said first and said second single-chain protein are selected from the following list, wherein VLA, VLB, VHA, and VHB correspond to said first and second VL and VH domains, respectively, and VLC, VLD, VLE, VLF, VHC, VHD, VHE, and VHF are part of single-chain fragments with a linker corresponding to said third, fourth, fifth and/or sixth functional domain, respectively, linked via third, fourth, fifth and/or sixth linkers LINKER3, LINKER4, LINKER5 and LINKER6) to the core domain (in bold letters); all constructs are written in the direction N- to C-terminus:
A (Parallel; 6Fvs):
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB-(LINKER4)-VLD-(linker)-VHD
chain 2: VLE-(linker)-VHE-(LINKER5)-VHA-(LINKER2)-VHB-(LINKER6)-VLF-(linker)-VHF
B (Anti-Parallel 6Fvs):
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB-(LINKER4)-VLD-(linker)-VHD
chain 2: VLE-(linker)-VHE-(LINKER5)-VHB-(LINKER2)-VHA-(LINKER6)-VLF-(Iinker)-VHF
C1 (Anti-Parallel 4 Fvs) (See
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB
chain 2: VLD-(linker)-VHD-(LINKER4)-VHB-(LINKER2)-VHA
C2 (Anti-Parallel 4 Fvs) (See
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB
chain 2: VHB-(LINKER2)-VHA-(LINKER4)-VLD-(linker)-VHD
C3 (Anti-Parallel 4 Fvs):
chain 1: VLA-(LINKER1)-VLB-(LINKER3)-VLC-(linker)-VHC
chain 2: VLD-(linker)-VHD-(LINKER4)-VHB-(LINKER2)-VHA
C4 (Anti-Parallel 4 Fvs):
chain 1: VLA-(LINKER1)-VLB-(LINKER3)-VLC-(linker)-VHC
chain 2: VHB-(LINKER2)-VHA-(LINKER4)-VLD-(linker)-VHD
D1 (Parallel 4 Fvs) (See
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB
chain 2: VLD-(linker)-VHD-(LINKER4)-VHA-(LINKER2)-VHB
D2 (Parallel 4 Fvs):
chain 1: VLC-(linker)-VHC-(LINKER3)-VLA-(LINKER1)-VLB
chain 2: VHA-(LINKER2)-VHB-(LINKER4)-VLD-(linker)-VHD
D3 (Parallel 4 Fvs):
chain 1: VLA-(LINKER1)-VLB-(LINKER3)-VLC-(linker)-VHC
chain 2: VLD-(linker)-VHD-(LINKER4)-VHA-(LINKER2)-VHB
D4 (Parallel 4 Fvs):
chain 1: VLA-(LINKER1)-VLB-(LINKER3)-VLC-(linker)-VHC
chain 2: VHA-(LINKER2)-VHB-(LINKER4)-VLD-(linker)-VHD
In certain embodiments, the order of the VH and VL domains in one or more of the scFv fragments comprised in the constructs according to the heterodimeric formats A, B, C1 to C4, or D1 to D4 are in the reverse order (e.g. VHC-(linker)-VLC-(LINKER3)-VLA-(LINKER1)-VLB-(LINKER4)-VHD-(linker)-VLD in chain 1 of construct A).
In these formats the localization of two split variable heavy domains VHA and VHB on one protein chain and the two corresponding variable light domains VLA and VLB on the other protein chain (VH-VH/VL-VL) prevents the formation of intra-chain domain pairings resulting in inactive single-chain diabody (scDb)-like structures as it would be the case if the VH-VL/VH-VL orientation of the conventional diabody—similar to the design suggested by Kipriyanov et al—had been used to drive hetero-dimerization. In contrast, the VH-VH/VL-VL-orientation forces the formation of exclusively hetero-dimeric bi- to hexa-specific proteins.
There is the theoretical possibility that the VH/VL domain pairing of the target A and B binding VHA-VHB/VLA-VLB core domain would result in an inactive core domain due to the inappropriate pairing of VHA with VLB and VHB with VLA resulting in VHA-VLB and VHB-VLA pairs. Unexpectedly and surprisingly, such inactive variants have not been observed so far. Without wishing to be bound by theory, dimerization could be driven towards cognate pairing due to the more efficient packing of the CDRs of cognate pairs as opposed to potential packing interferences occurring in non-matching pairings.
In order to further drive the hetero-dimerization towards active pairing in the VH-VH/VL-VL core domain, the knob-into-hole or similar technologies could be applied in one or—if reciprocally applied—both VL/VH pairs of the VH-VH/VL-VL core domain. Thus, in certain embodiments, the active pairing in the VH-VH/VL-VL core domain of said hetero-dimeric protein is further supported by a technology selected from: knob-into-hole (Zhu et al., “Remodeling domain interfaces to enhance heterodimer formation”, Protein Sci 1997 Apr.; 6(4): 781-788.), and inter-chain cysteine bridges.
In a second aspect, the present invention relates to one or two nucleic acid sequences encoding said first and a second single-chain proteins.
In a third aspect, the present invention relates to one or two vectors comprising said one or two nucleic acid sequences.
In a fourth aspect, the present invention relates to a host cell or host cells comprising said one or two vectors.
In a fourth aspect, the present invention relates to a method for producing the first and second single-chain proteins, or the hetero-dimeric protein, of the present invention, comprising (i) providing a nucleic acid or nucleic acids according to the present invention, or a vector or vectors according to the present invention, expressing said nucleic acid or nucleic acids or said vector or vectors and collecting said first and second single-chain proteins, or said hetero-dimeric protein, from the expression system, or (ii) providing a host cell or host cells of the present invention, culturing said host cell or host cells, and collecting said first and second single-chain proteins, or said hetero-dimeric protein, from the cell culture.
In a fifth aspect, the present invention relates to a pharmaceutical composition comprising the hetero-dimeric protein of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers enhance or stabilize the composition, or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
In a sixth aspect, the present invention relates to the hetero-dimeric protein of the present invention for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease. In particular embodiments, at least one of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease.
The terms “treatment”, “treating”, “treat”, “treated”, and the like, as used herein, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease or delaying the disease progression. “Treatment”, as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) inhibiting the disease, e.g., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease.
In a seventh aspect the present invention relates to a method for the treatment of a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease, comprising the step of administering the hetero-dimeric protein of the present invention, wherein at least one of said third, fourth, fifth, or sixth functional domains is able to specifically interact with a target of therapeutic relevance in the corresponding disease. In particular, the present invention relates to a method for treating a subject suffering from a disease selected from cancer, an inflammatory and an autoimmune disease, comprising administering to said subject an effective amount of the hetero-dimeric protein of the present invention, wherein at least one of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease.
The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted the terms “patient” or “subject” are used herein interchangeably.
The term “effective amount” or “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent, the disease and its severity and the age, weight, etc., of the subject to be treated.
In an eights aspect the present invention relates to use of the hetero-dimeric protein of the present invention in a manufacture of a medicament for use in the treatment of a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease, wherein at least one of said cognate pairs of VL and VH domains, or of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease.
In a ninth aspect the present invention relates to use of the hetero-dimeric protein of the present invention in the treatment of a disease, particularly a human disease, more particularly a human disease selected from cancer, an inflammatory and an autoimmune disease, wherein at least one of said cognate pairs of VL and VH domains, or of said third, fourth, fifth, or sixth functional domain is able to specifically interact with a target of therapeutic relevance in the corresponding disease.
Construct Design, Expression and Purification
The heterodimeric MATCH molecules were designed to contain the specificity for CD3ε and HSA in the split-variable domains of the heterodimeric core assembly. An IL23R binding scFv was attached to the N-terminus of each of the heterodimerization domains. In order covalently associate the two peptide chains of the MATCH and to confirm the correct assembly of the corresponding domains in the heterodimerization core an interchain disulfide (described in [11]) was introduced in the VL/VH interface of either the anti-CD3 or the anti-HSA domain.
A number of different embodiments were generated varying parameters like, MATCH arrangement (parallel or anti-parallel), CD3 binding domains used (clones 28-21-D09 or 09-24-H09), HSA binding domain used (clones 19-01-H04 or 23-13-A01) and different core linkers (SEQ 16-20). The two possible arrangements (parallel or anti-parallel) of the MATCH format have been described in detail (WO2016202457) the antiparallel arrangement (ap): Chain A (VL1-VH1-VH2-VH3) and Chain B (VL4-VH4-VL3-VL2); and the parallel arrangement (p): Chain A (VL1-VH1-VH2-VH3) and Chain B (VL4-VH4-VL2-VL3). Other than the variation of the arrangement also different choices for the core domains, the scFv modules and core linkers shown in Tables 1 and 2 have been tested:
GSHVNTYYANWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCATSGSSVLYF
KFWGQGTLVTVSS ggggsggggs
LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGSYACSSADCYVFGCGTKV
SGPTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYFCARGGWTGTSHSN
IWGQGTLVTVSS
LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLGSYACSSADCYVFGTGTKV
SGPTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYFCARGGWTGTSHSN
IWGQGTLVTVSS
vkgrftisrdnskntvylqmnslraedtavyycarrhynregypigigdlwgqgtlvtvss
asgvpsrfsgsgsgtdftltisslqpedfatyycqgeftcsnadcftfgtgtkvtvlg
swykgrftisrdnskntvylqmnslraedtavyycarrhynregypigigdlwgqgtlvt
LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGFSSSSDTAFGGGTKLTV
GFTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYFCARDRHGGDSSGAF
YLWGQGTLVTVSS
LASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGFSSSSDTAFGCGTKLTV
FTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYFCARDRHGGDSSGAFY
LWGQGTLVTVSS
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQCTYIDSNFGAFGGGTKLTVLG
TGDGTTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTATYFCARPVSVYYYG
MDLWGQGTLVTVSS
SGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGGGTKLTVLG
GYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDY
To generate the constructs outlined in above the amino acid sequences for the Fv domains and linkers were back-translated into corresponding nucleic sequences, which were de novo synthesized. The coding sequences were assembled and cloned by standard molecular biology techniques (e.g. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual) into a suitable expression vector (e.g. pcDNA3.1, Invitrogen) for recombinant protein secretion.
The MATCH protein was produced in CHO-S cells (Thermo Fisher) by transient transfection using CHOgro® expression kit (Mirus Bio® LLC) according to supplier's protocol. Protein fraction was purified from CHO-S supernatant which was harvested by centrifugation as soon as cell viability decreased below 80% (after 6 days of incubation with orbital shaking at 37° C. and 8% CO2). Purification was done by protein L-affinity purification, capturing variable domains with Capto® L resin (GE Healthcare) in a column affixed to an ÄKTA Pure® FPLC system (GE Healthcare) and eluted with 0.1 M Citric acid, pH 2.0, followed by the rapid adjustment of sample pH with the addition of ⅓ (v/v) 2M tris(hydroxymethyl)aminomethane hydrochloride (Tris™-HCl), pH 7.5. Protein solutions were then buffer exchanged with 1xPBS pH7.4 (supplemented with 300 mM Arginine) using a dialysis membrane (3.5 kDa MWCO, Spectrum Laboratories, Inc.) and finally concentrated using a Vivaspin® Protein Concentrator Spin Column (5 kDa MWCO, GE Healthcare).
The reference proteins in the single-chain diabody (scDb) format were designed as described previously [10]. In short, the variable domains as listed in Table 1 were arranged in an VLA-S1-VHB-L1-VLB-S2-VHA fashion, where S1 and S2 are short GS4 linkers and L1 is a long (GS4)4 linker. The molecules generated for reference purposes included the identical Fv fragments as the MATCH construct.
All tested proteins were analyzed by size-exclusion high-performance liquid chromatography (SE-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for purity and UV/Vis spectroscopy for protein content. In case of the MATCH construct the quantitative disulfide linking of the two heterodimers was confirmed by non-reducing SDS-PAGE.
The SDS-PAGE analysis of the MATCH construct variants shows in
→Due to the position of the designated interchain disulfide bridge in the interface of a VL/VH pair a mispairing of the chains is highly unlikely, especially in combination with the conserved affinity of the core domains in the SPR experiment
Affinity Measurement by Surface Plasmon Resonance (SPR)
Binding affinities of individual target binding domains in the single-chain Fv (scFv) format as well as of the purified hetero-dimeric MATCH constructs to recombinant target proteins human IL-23 receptor ECD (IL23R), human CD3 gamma-epsilon single-chain (CD3) and human serum albumin (HSA) were measured by surface plasmon resonance (SPR) using a Biacore® T200 SPR apparatus (General Electric®) or a MASS-1 (Sierra Sensors®) device. In brief, recombinant proteins were directly immobilized by amine coupling chemistry on a CM5 (Biacore®, General Electric®) or high capacity sensor chip (Sierra Sensors®). Different concentrations of target binding domains were injected as analyte and the binding response (in response units, RU) was measured. After each injection regeneration procedure was performed. Obtained binding data were double-subtracted (zero analyte injection, reference flow channel) and analyzed using the respective software package.
Affinities of hetero-dimeric MATCH constructs to each of the targets was generally very similar to the affinities of the individual binding domains measured in the scFv or scDb format. Notably the apparent affinity of the MATCH construct to IL23R appears increased in comparison to the scDb reference molecule, which can be explained by the avidity effect resulting from the incorporation of two IL23R binding domains in each MATCH molecule.
These data demonstrate maintained binding activity for each variable domain in the MATCH constructs and confirms correct assembly of the cognate variable domain pairs irrespective of the choice of core linkers, location of the interchain disulfide, the anti-CD3, anti-HSA domains used or attached scFv modules.
Certain differences in the affinities to the targets of the core-domain were observed for the various constructs. For the constructs containing a disulfide bond in the VL/VH interface of the CD3 domain, the charged core linker combination (comprising SEQ16 and SEQ17) used in PRO746 showed best affinity.
T-Cell Activation by NFAT Reporter Gene Assay
NFAT Assay
A CHO-K1 cell line stably expressing the human IL23R IL12Rbeta 1 heterodimer under control of a CMV promoter was generated by lentiviral transduction of the parental CHO-K1 cell line. These cells were used as target cells in the NFAt reporter gene assay while the parental CHO-K1 cell line was uses as control. 25,000 viable target cells diluted in 50 μl assay medium (RPMI 1640, 10% FCS) containing 25 g/L human serum albumin (HSA) were plated in white flat bottom 96-well plates. Then, 25 μl of 4 times concentrated test proteins diluted in assay medium containing HSA (25 g/L) were added to appropriate wells. Finally, 25 μl of assay medium with HSA containing 50,000 Jurkat cells was added to each well and plates were incubated at RT for 10 min with gentle agitation. One plate was made for each time point such as 5 h, 22 h or 30 h corresponding to the incubation times at 37° C., 5% CO2. In order to detect luciferase activity, one step luciferase assay kit (Amsbio) was used according to manufacturer's instructions. Briefly, at the end of the incubation times, luciferase reagent substrate was mixed with the luciferase reagent buffer and 50 μl were added to each well and plates were incubated for 15 min in the dark at RT. Plates were read with the TopCount™ analyzer (PerkinElmer©).25,000 viable target cells diluted in 50 μl assay medium (RPMI 1640, 10% FCS) were plated in white flat bottom 96-well plates. Then, 25 μl of 4 times concentrated test proteins diluted in assay medium were added to appropriate wells. Finally, 25 μl of assay medium containing 50,000 Jurkat cells was added to each well and plates were incubated at RT for 10 min with gentle agitation. One plate was made for each time point such as 5 h, 22 h or 30 h corresponding to the incubation times at 37° C., 5% CO2 either containing physiological concentrations of human serum albumin or not. In order to detect luciferase activity, one step luciferase assay kit (Amsbio) was used according to manufacturer's instructions. Briefly, at the end of the incubation times, luciferase reagent substrate was mixed with the luciferase reagent buffer and 50 μl were added to each well and plates were incubated for 15 min in the dark at RT. Plates were read with the TopCount™ analyzer (PerkinElmer®).
IL-2 Quantification by ELISA
100 μl supernatants were collected at different time points during the NFAT reporter gene assay from the wells containing 250 nM of the test molecules. IL-2 quantification was done using the IL-2 ELISA MAX™ Standard kit (Biolegend®) according to manufacturer's instructions. Briefly, 100 μl of capture antibody diluted in dilution buffer (PBS, 1% BSA, 0.2% polysorbate 20 (Tween© 20) was coated on 96-well plates Maxisorb (Nunc) over night at 4° C. Next day, plates were washed 3 times with wash buffer (PBS, 0.005% polysorbate 20 (Tween® 20). Wells were blocked with 300 μl dilution buffer for 1 h at RT, then washed 3 times with wash buffer. Next, 100 μl supernatant of the tested samples as well as 100 μl of each concentrations of the standard curve were added to the appropriate wells and plates were incubated for 2 h at RT with shaking. Plates were washed 3 times with wash buffer prior to incubation with the detection antibody for 1 h at RT with shaking. Plates were washed again 3 times with wash buffer and 100 μl Avidin-HRP was added to each well and incubated at RT for 30 min with shaking. Tree final washes were performed before addition of 100 μl TMB substrate solution. Plates were incubated for 15 minutes in the dark and reaction was stopped by adding 100 μl of Stop solution to each well. Absorbance was read at 450 nm and 570 nm. Absorbance at 570 nm was subtracted from the absorbance at 450 nm.
Functional analysis of the MATCH construct variants in the NFAT reporter gene assay after 5 h of incubation showed the potency to activate T-cells. For comparison across different plates the data was normalized to an on-plate reference.
For the further in-depth characterization, the highest affinity MATCH construct (for CD3) PRO746, which also showed the best potency to activate T-cells, was used. In addition, constructs PRO821 and PRO824 were used a) to evaluate the functional activity of the HSA/CD3 core domain in the context of a different targeting domain (in this case anti-Her2), and b) to evaluate the alternative positioning of the disulfide bond in the HSA-binding domain instead of the anti-CD3 domain.
The potency of the molecules to activate T-cells in the presence of antigen-bearing target cells and physiological concentrations of human serum albumin (HSA) was determined over multiple timepoints and molecule concentrations (see
The plot of the T-cell activation for the different molecules in the presence of cells devoid of the target protein (
The plot of the plateau values of the dose response signals at different timepoints (
The plot of the EC50 values of the dose response curves for the scDb and MATCH molecule (
The release of T-cell activation marker IL-2 was quantified for the samples of the NFAT reporter gene assay of the molecules PRO624 (scDb) and PRO746 (MATCH) (see
In summary, our findings indicate that the MATCH format comprising the HSA/CD3 core-domain has the same capacity to activate T cells, with a slower activation kinetic, though. This is interesting in the light of the fact, that side effects associated with cytokine release with other T cell engaging bispecifics (e.g. blinatumomab/blincyto) are strongest during the early times of dosing. In fact, only low doses of blinatumomab/blincyto are tolerated at the beginning of the therapy, while considerably higher doses are tolerated afterwards. A probable explanation for this is the strong cytokine burst that occurs immediately after dosing that may lead to the so-called cytokine release syndrome (CRS). Therefore, the MATCH presented here, due to its slow T cell activation kinetics and consequential reduced cytokine release is likely to lead to reduced toxicities, whereas it has the capacity to reach the same level of T cell activation over time. In addition, the reduction of cytokine release even at similar levels of T-cell activation is an even more surprising feature of PRO746, which suggests to reduce occurrence of the frequently observed cytokine release syndrome associated with T-cell recruiting therapies further.
Cytotox Assay (T-Cell Driven Target Cell Depletion)
Blood Cells Fractionation
Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood of healthy volunteers using the lymphocyte separation medium Lymphoprep® (Stemcell Technologies®) according to manufacturer's instructions. Briefly, blood was diluted 1:1 with isolation buffer in 50 ml centrifuge tubes (PBS, 2% FCS, 2 mM EDTA) and applied to separation tubes (Leucosep™) containing recommended amount of Lymphoprep® medium. LeucoSep™ tubes were centrifuged 30 min at 800 g without brakes at RT. Then, the cell layer containing PBMCs was collected and washed twice with isolation buffer and red blood cells were lysed using red blood cells lysis buffer for 5 min at RT. Cells were then washed once with isolation buffer and once with assay medium (RPMI-1640, 10% FCS). After platelet removal, isolated PBMCs were resuspended in assay medium containing 25 g/ml HSA at a density 3×106 viable cells per ml.
Flow Cytometry-Based In Vitro Cytotoxicity Assay and CD8+ T Cells Activation
A CHO-K1 cell line stably expressing the human IL23R IL12Rbeta 1 heterodimer under control of a CMV promoter was generated by lentiviral transduction of the parental CHO-K1 cell line. These cells were used as target cells in the cytotoxicity assay while the parental CHO-K1 cell line was uses as control. Additionally, a CHO-K1 cell line stably expressing human HER2 was as well generated by lentiviral transduction of parental CHO-K1 cells with full-length HER2 cDNA. Expression levels of HER2 and the IL23R were determined by flow-cytometry. HER2 levels at the cell surface are much higher compared to IL23R levels. 5,000 viable target cells previously labelled with PKH67 and diluted in 75 μl of assay medium (RPMI-1640, 10% FCS) containing 25 g/L human serum albumin (HSA) were added to 96-well plates. Next, 25 μl of 6 times concentrated tested proteins diluted in assay medium with HSA were added to appropriate wells. Then, in order to have an E:T ratio of 30:1, 150,000 viable effector cells (PBMCs) diluted in 50 μl assay medium containing HSA were added to each well and plates were mixed on a nutating mixer at RT prior to their incubation at 37° C., 5% CO2. After 16 h, cells were trypsinized, resuspended in staining buffer (PBS, 2% BCS, 2 mM EDTA) and transferred into non-binding plates.
Cells were stained for different markers as CD69, CD8, CD4, CD11c and Annexin-V. For analysis, the focus is on apoptotic and dead target cells and activated CD8+ T cells. Thereby, target cells are identified by green fluorescence (PKH67) and their viability is analyzed by Annexin-V APC. Effector cells (CD8+ cells) were identified by detecting CD8 on their surface (anti-CD8 PerCP-Cy5.5). Activation of CD8+ T cells is finally detected by quantification of CD69 expression (anti-CD69 PE). CD4 is used to better discriminate CD8+ and CD4+ T cells. CD11c is used to mark monocytes and dendritic cells and exclude them. For each marker except Annexin-V antibodies are incubated 30 minutes at RT under gentle agitation. Cells are washed once with staining buffer, once with Annexin binding buffer and Annexin-V staining is carried on for 30 minutes at RT under agitation. Cells are washed once with Annexin-V binding buffer and flow cytometry analysis was done on a Novocyte® Flow Cytometer.
The percentage of specific target cells lysis is calculated according to the following equation:
The percentage of activated CD8+ T cells correspond to the proportion of CD69+CD8+ T cells.
IL-2 Quantification by Flow Cytometry
IL-2 quantification in supernatant was performed using the cytometric bead array human Th1/Th2 cytokine kit II (BD® Biosciences) according to manufacturer's protocol. Briefly, 50 μl of the mixed Capture Beads were added to each supernatant analyzed as well as to the IL-2 standard dilutions. After 3 h hours incubation at RT in the dark, beads were washed 3 times with wash buffer and analyzed by flow cytometry on a Novocyte® instrument.
The cell lysis data shows the specific potency and specificity of both molecules PRO746 (MATCH-4 anti-TAA2xCD3xHSA) and PRO624 (scDb; anti-TAAxCD3) to induce depletion of target-bearing cells, while the negative control PRO811 (scDb; anti-CD3xHSA) fails to induce target cell lysis (see
The quantification of the T-cell activation in the dose-response of the three molecules is consistent with the observations of cell lysis (see
The concentrations of IL-2 in the wells were measured (
The tetraspecific assemblies were generated to show the potential of the MATCH assemblies with a CD3/HSA heterodimerization core to be used to combine two antigen specificities of tumor associated antigens of different expression profiles (see
This application is a 371 national phase of International Patent Application No. PCT/EP2018/064633 filed Jun. 4, 2018, which claims priority to U.S. Provisional Patent Application No. 62/515,293 filed Jun. 5, 2017, the content of which applications is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/064633 | 6/4/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/224443 | 12/13/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 11236150 | Meyer | Feb 2022 | B2 |
| 11365260 | Cui | Jun 2022 | B2 |
| 20160368987 | Urech | Dec 2016 | A1 |
| 20200115449 | Gunde | Apr 2020 | A1 |
| 20200325214 | Gunde | Oct 2020 | A1 |
| 20220403028 | Gunde | Dec 2022 | A1 |
| Number | Date | Country |
|---|---|---|
| 2014206561 | Dec 2014 | WO |
| 2015172800 | Nov 2015 | WO |
| 2016184570 | Nov 2016 | WO |
| 2016202457 | Dec 2016 | WO |
| Entry |
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| ClinicalTrials.gov Identifier: NCT06299163, pp. 1-8; Apr. 9, 2024. |
| Egan et al., “Novel multispecific heterodimeric antibody format allowing modular assembly of variable domain fragments,” MABS 9(1):68-84 (Oct. 27, 2016). |
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| Number | Date | Country | |
|---|---|---|---|
| 20200181263 A1 | Jun 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62515293 | Jun 2017 | US |
