WO2019156566A1 - Bispecific molecules comprising gamma-delta tcr and t-cell or nk cell binding domain - Google Patents

Abstract

The invention provides a recombinant bispecific protein, comprising an extracellular domain of a gamma-delta T-cell receptor (TCR) which is fused to a T- cell- and/or Natural Killer (NK) cell-binding domain. The invention further provides a nucleic acid construct encoding a recombinant bispecific protein according to the invention and a cell expressing a recombinant bispecific protein according to the invention. Further provided is the recombinant bispecific protein according to the invention for use as a medicament, a pharmaceutical composition, comprising the recombinant bispecific protein according to the invention and a pharmaceutically acceptable excipient, and a method of producing the recombinant bispecific protein according to the invention.

Description

Title: Bispecific molecules comprising gamma-delta TCR and T-cell or NK cell binding domain

The invention relates to the field of oncology. More specifically, the invention relates to bispecific binding molecules in which the extracellular domain of a T cell receptor is fused to a domain that binds T-cells and/or Natural Killer cells. Such binding molecules are useful in methods for treatment of cancer and infectious diseases.

A novel opportunity for immunological interventions arises from the improved understanding of different layers of immune cells involved in daily cancer immune surveillance (Gentles et al., 2015. Nat Med 21:938-45). Among all immunological subtypes, gdT cells stand out in unbiased computational analyses to associate with an improved overall survival while e.g. NK cells rather associate with poor prognosis (Gentles et al., 2015. Ibid). gdT cells are innate like T lymphocytes that are present in both blood and tissue. gdT lymphocytes are known to be important for recognition of foreign pathogens, stress signatures of infected cells but also cancer cells (Bonneville et ah, 2010. Nat Rev Immunol 10: 467-78). In vitro, gdT lymphocytes display very potent and broad tumor recognition; they can target and lyse cancer cells of both hematological and solid origin (Scheper et ah, 2014. Leukemia 28:1181-90; Scheper et al., 2013. Leukemia 27:1328-38). gdT lymphocytes have also been described to participate in early cancer immune surveillance in vivo in mice (Dadi et ah, 2016. Cell 164: 365-77) and mice deficient for gdT cells are more prone to develop cancer (Girardi et ah, 2001. Science 294: 605-96). Tumor infiltrating gd lymphocytes (gdTI L) have been identified in patients. Ex vivo these cells efficiently recognized and lysed autologous cancer cells (Groh et ah, 1999. PNAS 96: 6879-84) and offer a new road to novel tumor antigens (Bouchie et al., 2017. Nat Biotech. 35: 322-33). However, the adoptive transfer of ex vivo expanded polyclonal gdT cells associates so far with little clinical responses (Deniger et ah, 2014. Front Immunol 5: 636), most likely because of an heavily underestimated diversity and many mechanisms of tolerance active in advanced cancer patients against this particular immune subset (Scheper et al., 2014. Front Immunol 5: 601).

To overcome limitations of gdT cell therapies fusing the clinically successful concept of CAR-T cell therapies (Smith et al., 2018. Blood 2017-08-752121;

Srivastava and Riddell, 2015. Trends Immunol 36: 494-502) with the improved molecular understanding of gdT cell receptors generated the model of aOT cells engineered to express a defined gdT cell receptor (TEG). TEGs combine the strong proliferation capacity of a6T cells which are active even in late stage cancer patients (Dudley et al., 2008. J Clin Oncol 26: 5233-9) with the broad tumor- reactivity of ydTCRs (Bouchie et al., 2017. Ibid ; Scheper et al., 2014. Ibid). A major drawback of engineered immune cells such as CAR-T and TEGs is however the rather challenging logistic for such advanced therapy medicinal products (ATMPs). Generating ATMPs is an individualized, cumbersome, and costly process that in most cases takes week and can associates with production failures (Levine et al., 2017. Mol Ther Methods Clin Dev 4: 92-101). In addition, patients had to be preselected due to a wide variability in in vitro cytotoxicity of patient gdT cells against autologous tumor tissue, and limited in vivo or ex vivo expansion potential of patient gdT cells (Scheper et al., Front Immunol 2014). Moreover, anti-tumor efficacy of gdT cells showed only marginal improvement over standard treatment options (Fisher et al., 2014. Oncoimmunol 3: e27572).

To avoid practical and socioeconomic challenges of ATMPs such as CAR-T (Chabannon et al., 2017. Bone Marrow Transplant 52: 1588-9) while still utilizing the immune system to attack cancer, an alternative strategy is currently employed for classical antigens like CD 19 by merging molecules that combine a tumortargeting domain with a T lymphocyte recruitment domain. Fusion antibodies combining a CD 19 binding domain with an anti-CD3 engager are now used in daily clinical practice (Batlevi et al., 2016. Nat Rev Clin Oncol 13: 25-40). An alternative strategy opening novel tumor-specific targets that are not expressed at the cell surface arose from utilizing the extracellular domain of an ali TCR directed against tumor- associated antigens as part of the bispecific molecule (Liddy et al., 2012. Nat Med 18: 980-7). MHC down-regulation is however observed as immune-escape mechanism in approximately 40 to 90% of all human tumors (Buhenik, 2003. Oncol Rep 10: 2005-8) thereby greatly limiting the applicability of therapies based on aBTCR mediated tumor recognition.

There is thus a clear need to develop novel tumor-targeting therapies.

The invention therefore provides a recombinant bispecific protein, comprising an extracellular domain of a gamma-delta T-cell receptor (TCR) which is fused to a T-cell- and/or Natural Killer (NK) cell-binding domain, preferably a high affinity T-cell- and/or Natural Killer (NK) cell-binding domain.

This combination provides low affinity y8TCR interaction with its ligand on tumor cells with high affinity interaction with T lymphocytes and/or with NK cells, preferably by binding to CD3 on T lymphocytes and/or CD 16 on NK cells. The currently used bispecific molecules, comprising aBTCRs, are based on the understanding that bi-specific molecules first need to find their target on the tumor cell and then bind to an effector cell. Therefore, these bispecific proteins critically depend on high affinity matured uBTCRs (Oates and Jakobsen, 2013. Oncoimmunol 2: e22891; Li et ah, 2005. Nat Biotechnol 23: 349-54), or high affinity antibodies binding to a tumor cell, followed by recruitment of a6T cells.

It has been reported that high affinity binding to tumor cells, when combined with high- affinity binding to CD3, reduces the efficiency of T cell stimulation and will target the bispecific reagent to T cells rather than to tumor cells (Bortoletto et ah, 2002. Eur J Immunol 32: 3102-7). Reducing the binding affinity to CD3, in spite of rapid dissociation, efficiently triggered T cell activation and cytotoxicity

(Bortoletto et al., 2002. Ibid).

In the bispecific molecules according to the invention, a different concept is employed. Low affinity of yfiTCR-ligand interaction is combined with a strong T cell and/or NK cell-binding domain such as an anti-CD3 binding domain. This allows first the hispecific molecule to bind to a T and/or NK cell and then later to recruit this cell to a tumor. This concept may be elaborated by generating trispecific molecules for which tumor binding depends on ySTCR and a second molecule like a checkpoint ligand. Although the affinity of, for example, PD1 for PDL1 is 8 micromolar (Cheng et ah, 2013. J Biol Chem 288: 11771-11785), which is 10-100 times better than the expected affinity of y8TCR, a“strong” T cell and/or NK cell- binding domain will be needed as even the combined binding strength of y8TCR and a checkpoint ligand is considerably less than that of matured aBTCRs (Holler et al., 2003. Nat Immunol 4: 55-62; Holler and Kranz, 2003. Immunity 18: 255-264; Holmberg et al., 2003. J Immunol 171: 2427-2434).

Fusion of the extracellular domain of a ybTCR as tumor binding domain to, for example, an anti-CD3 scFv can effectively target T cells to tumor cells without the need for engineering TEGs. It is demonstrated herein that yhTCR anti-CD3 bispecific molecules (abbreviated as GABs) can redirect CD 3+ effector cells towards several tumor cell lines of both hematologic and solid origin and preserve the mode of action of tumor recognition described for a particular y9S2TCR (Sebestyen et al., 2016. Cell Rep 15: 1973-85; Gu et al., 2017. PNAS 114: E7311-E20; Sandstrom et al., 2014. Immunity 40: 490-500), thereby opening a new universe of antigens to the bispecific format (Bouchie et al., 2017. Ibid).

The extracellular domain of a gamma-delta TCR and the T-cell- and/or Natural Killer (NK) cell-binding domain are preferably fused through a linking group which provides conformational flexibility so that the extracellular domain of a gamma-delta TCR can interact with its epitope, while the T-cell- and/or NK cellbinding domain can interact with its cognate epitope. A preferred linker group is a linker polypeptide comprising from 1 to about 60 amino acid residues, preferably from 5 to about 40 amino acid residues, most preferred about 15 amino acid residues such as 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues,

16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues or 20 amino acid residues. Some preferred examples of such amino acid sequences include Gly-Ser linkers, for example of the type (Gly_x Ser_y)_z such as, for example, (Gly4 Ser)3, (Gly4 Ser)7 or (Gly3 Ser2)3, as described in WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in, for example, WO

06/040153 and WO 06/122825, as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). A most preferred linker is a (Gly4 Ser)3 linker.

Said T-cell- and/or Natural Killer (NK) cell-binding domain is a domain that specifically binds to an antigen that is present on a human T cell or a human NK cell. Said antigen preferably is selected from cluster of differentiation 3 (CD3),

CD4, CD8, CD 16, CD56, CD103, CD134, CD154 and CD314. CD3 is present on the membranes of all mature T-cells, and on virtually no other cell type. CD3 consists of a protein complex and is composed of a CD3y chain, a C [)3ϋ chain, and two CD3e chains. Together with a T-cell receptor (TCR) and a z- chain, CD 3 constitutes a TCR complex.

CD4 is a type I transmembrane protein found on T cells, but also on monocytes, macrophages and dendritic cells. CD4 is a member of the

immunoglobulin superfamily.

CDS is a cell surface glycoprotein that is composed of an alpha and a beta chain which are linked by two disulfide bonds. Both CDSalpha and CDSbeta are members of the immunoglobulin superfamily. CDS is highly expressed on cytotoxic T cells, but can also be found on natural killer cells, cortical thymocytes, and dendritic cells.

CD 16 is a transmembrane molecule that is present on natural killer cells, leukocytes, monocytes and macrophages. CD 16 is an Fc receptor that binds to the Fc portion of IgG antibodies which then activates the NK cell for antibody- dependent cell-mediated cytotoxicity (ADCC).

CD56, or Neural Cell Adhesion Molecule (NCAM), is a glycoprotein that is present on NK cells and on a subset of CD4+ and CD8+ T cells.

CD 103 is an integrin, alpha E molecule that is expressed on intraepithelial lymphocyte (IEL) T cells (both oB T cells and gd T cells) and on some peripheral regulatory T cells.

CD 134/0X40 is a transmembrane protein from the tumour necrosis factor receptor superfamily that is present selectively on activated T cells.

CD 154, also termed CD40 ligand or CD40L, is a member of the tumour necrosis factor super family and is primarily expressed on activated T cells.

CD314, also termed NKG2D, is a type II lectin-like receptor that is present on natural killer cells, T cells and some myeloid cells. CD314 recognizes proteins on the surface of stressed, malignant and infected cells. CD314 has been used for targeting effector cells (von Strandmann et ah, 2006. Blood 107: 1955-62).

Said T-cell- and/or Natural Killer (NK) cell-binding domain preferably is a CD3-binding domain. Said T-cell- and/or Natural Killer (NK) cell-binding domain preferably is an antibody, preferably a single heavy chain variable domain antibody such as a camelid VHH, a shark immunoglobulin-derived variable new antigen receptor, a scFv, a tandem scFv, a scFab, an improved scFab (Koerber et ab, 2015. J Mol Biol 427: 576-86), or an antibody mimetic such as a designed ankyrin repeat protein, a binding protein that is based on a Z domain of protein A, a binding protein that is based on a fibronectin type III domain, engineered lipocalin, and a binding protein that is based on a human Fyn SH3 domain (Skerra, 2007. Current Opinion Biotechnol 18: 295-304; Skrlec et ak, 2015. Trends

Biotechnol 33: 408-418).

If required, the heavy and light chain domains of, for example, a scFv antibody may be connected through a linking group which provides conformational flexibility so that the heavy and light chain can associate and bind to a target epitope. A preferred linker group is a linker polypeptide comprising from 1 to about 60 amino acid residues, preferably from 5 to about 40 amino acid residues, most preferred about 15 amino acid residues such as 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues,

18 amino acid residues, 19 amino acid residues or 20 amino acid residues. Some preferred examples of such amino acid sequences include Gly-Ser linkers, for example of the type (Glyx Sery)z such as, for example, (Gly4 Ser)3, (Gly4 Ser)7 or (Gly3 Ser2)3, as described in WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in, for example, WO 06/040153 and WO 06/122825, as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). A most preferred linker is a (Gly4 Ser)n linker such as a (Gly4 Ser) linker or (Gly4 Ser)3 linker.

Antibodies against CD3, CD8, CD56, CD 103, CD 134 and CD 154, including single chain antibodies, are known to a person skilled in the art. For example, a recombinant mouse antibody scFv fragment directed against human CD 134/0X40 is available from Creative Biolabs (Shirley, NY, USA). Further single chain antibodies against CD 134/0X40 are described in the published international patent application W02007/062245. Similarly, a recombinant mouse antibody scFv fragment which specifically reacts with human CD40L is available from Creative Biolabs, Antibodies, including single chain antibodies, against CDS are known in the art. For example, Liao et ah, 2000 (Liao et al., 2000. Gene Ther 7: 339-47) describes single-chain antibodies (scFv) against CD3 that are expressed on the plasma membrane of tumor cells. Further single chain antibodies against CDS are described in the published international patent application WO2001/051644. In addition, single chain antibodies against CD3 are commercially available, for example from Creative Biolabs as a recombinant anti-human CDSgamma VHH single domain antibody.

A preferred single chain antibody against CDS that is present in a

recombinant bispecific protein according to the invention comprises a single chain Fv anti-CD3 binding domain. Said single chain Fv anti-CD3 binding domain preferably is derived from a chimeric mouse-human OKT3 antibody, as is described in Arakawa et al., 1996. J Biochem 120: 657-62. A preferred scFv derived from the OKT3 antibody has been described (Adair et al., 1994. Human Antibodies 5: 41-47; Kipriyanov et al., 1997. Protein Engin Design Selection 10: 445-453).

The extracellular domain of the gamma and/or delta TCR may be fused to an extracellular domain of an immune checkpoint-related molecule, preferably an immune checkpoint inhibitor. The term“immune checkpoint related molecule” is clear to a person skilled in the art and refers to molecules that balance costimulatory and inhibitory signals of an immune response. Activated T cells are primary mediators of immune effector functions and as such, they express multiple co-inhibitory receptors. Such co-inhibitory receptors include the adenosine A2A receptor, programmed death 1 (PD-1) receptor, T-cell Immunoglobulin domain and Mucin domain 3, and V-domain Ig suppressor of T cell activation. Fusion of a gamma and/or delta TCR to an extracellular domain of an immune checkpoint- related molecule may further assist in targeting the gamma and/or delta TCR to cancer cells.

The extracellular domain of a gamma-delta TCR and the T-cell- and/or NK- cell binding domain may be fused to an extracellular domain of an immune checkpoint-related molecule through a linking group which provides

conformational flexibility so that the extracellular domain of a gamma-delta TCR as well as the extracelhdar domain of an immune checkpoint-related molecule can interact with their respective epitopes. A preferred linker group is a linker polypeptide comprising from 1 to about 60 amino acid residues, preferably from 5 to about 40 amino acid residues, most preferred about 15 amino acid residues such as 10 amino acid residues, 11 amino acid residues, 12 amino acid residues, 13 amino acid residues, 14 amino acid residues, 15 amino acid residues, 16 amino acid residues, 17 amino acid residues, 18 amino acid residues, 19 amino acid residues or 20 amino acid residues. Some preferred examples of such amino acid sequences include Gly-Ser linkers, for example of the type (Glyx Sery)z such as, for example, (Gly4 Ser)3, (Gly4 Ser)7 or (Gly3 Ser2)3, as described in WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in, for example, WO 06/040153 and WO 06/122825, as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678). A most preferred linker is a (Gly4 Ser)3 linker. Non limiting examples of fusions between a recombinant bispecific protein according to the invention to an extracellular domain of an immune checkpoint-related molecule are provided in Table 3 (SEQ ID NO:s 19-21).

Cancer cells often express suppressive immune checkpoint molecules in order to diminish a immune response against the tumor (Finn, 2012. Ann Oncol 23, viii6- viii9). Such suppressive immune checkpoint molecules interact with an immune checkpoint-related molecule in order to suppress an immune reaction against the cancer cells. For example, adenosine in the immune microenvironment results in activation of the immune suppressor A2a receptor. Programmed death 1 receptor, has two ligands, PD-L1 and PD-L2 of which especially PD-L1 is often upregulated in cancer cells. T-eell immunoglobulin domain and mucin domain 3 is expressed on activated human CD4+ T cells. Its ligand, galectin-9, is upregulated in, for example, oral cancer, pancreatic cancer and hematologic malignancies and induces apoptosis of mature TH1 cells. V-domain Ig suppressor of T cell activation (VISTA) may serve both as a ligand for antigen presenting cells, as well as a receptor for T cells. Although the exact binding partners are not known, activation of VISTA suppresses T-eell activation within especially solid cancers.

A gamma and/or delta TCR preferably is fused to the extracellular domain of programmed death 1 (PD-1) receptor. Said fusion may be at the N-terminus of the gamma and/or delta TCR, thereby positioning the extracellular domain of programmed death 1 (PD-1) receptor in front of the gamma and/or delta TCR, or at the C-terminus, thereby positioning the extracellular domain of programmed death 1 (PD-1) receptor behind the gamma and/or delta TCR. The extracellular domain of programmed death 1 (PD-1) receptor is preferably fused to a delta TCR, preferably a delta 2 TCR.

gd T cells may directly interact with cancer cells. The attraction of gd T cells to cancer cells results in inhibition of cancer cell growth by the production of specific chemokines and cytokines and lead to recruitment of monocytes and neutrophils to the cancer cells. In addition, gd T cells enhance cancer killing activity in some cancers, either by the Fas- or TRAIL-receptor pathway or through enhancement of antibody- dependent cellular cytotoxicity. Furthermore, gd T cells themselves may release granzymes and perforin that mediate cellular apoptosis.

The gamma TCR locus comprises at total of 14 V gene segments, which are followed by a J-C cluster consisting of three J segments and one C segment, and a J-C cluster of two J segments and one C segments. Some of the V gene segments are pseudogenes, resulting in a total of 7 active V gene segments (Hodges et ah, 2003. J Clin Pathol 56: 1-11).

The delta TCR locus comprises a total of eight V segments, three D segments, four J segments, and a single C segment. Although none of the eight V segments has been reported to be a pseudo gene segment, most gamma delta TCR are encoded by delta VI, V2 or V3 gene-segments, especially by V2 gene segments (Hodges et ah, 2003. Ibid).

The gamma TCR chain is synthesized during T cell development by a recombination event at the DNA level, joining a V segment with a J segment; the C segment is later joined by splicing at the RNA level. Recombination of many different V segments with several J segments provides a wide range of antigen recognition. Additional diversity is attained by junctional diversity, resulting from the random addition of nucleotides by terminal deoxynucleotidyl transferase. The delta TCR chain is synthesized during T cell development by VDJ recombination.

Vy9V52 T-cells, comprising V9 gamma and delta 2 V gene segments, are most abundant in Western populations. However, also non-Vo2+ gdT cells, such as V51+ gdT subtypes and V63+ gdT cells have been shown to target cancer cells and result in inhibition of cancer cell growth and lysis of cancer cells ((Seheper et ah, 2014. Leukemia 28, 1181-1190; Seheper et ah, 2013. Leukemia 27, 1328-1338; Willcox et aL, 2012. Nat Immunol 13, 872-879). In addition, VY9V62 T-cells as well as non- Vo2+ gdT cells, such as Vo 1 + gdT subtypes and V63+ gdT cells have been reported to target pathogenic organisms involved in diseases such as tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria (Chien et ah, 2014. Annual Reviews 32: 121-155).

As is shown herein, a VV'I Vo 5 GAB, derived from an endothelial protein C receptor-reactive TCR (Willcox et al., 2012. Nature Immunol 13: 872-879), was also found to be effective as a tumor-targeting GAB.

A preferred recombinant bispecific protein according to the invention comprises the extracellular domains of a gamma delta TCR, preferably gamma 9 delta 2 TCR. A preferred recombinant hispecific protein according to the invention comprises the extracellular domains of a TCR gamma chain, preferably gamma 9, that is coupled at it’s C-terminus to a CD3-binding domain, preferably a scFv derived from the OKT3 antibody as described Kipriyanov et al., 1997. Protein Engin Design Selection 10: 445-453, and an extracellular domain of a delta TCR, preferably a delta 2 TCR. The extracellular domain of gamma and/or delta TCR, preferably of the delta TCR, preferably of the delta 2 TCR, may he fused at the N- terminus or C-terminus to the extracellular domain of checkpoint-related molecule such as the extracellular domain of a PD-1 receptor. Non-limiting examples of such fusion products are provided in Table 3.

A further preferred recombinant bispecific protein according to the invention comprises the extracellular domains of a gamma delta TCR, preferably the extracellular domains of a gamma non-delta2 TCR, such as a gamma deltaS TCR.

A recombinant bispecific protein according to the invention may be tagged, preferably at its N- or C-terminus, with a specific tag by genetic engineering to allow the protein attach to a column specific to the tag and therefore be isolated from impurities. The purified protein is then exchanged from the affinity column with a decoupling reagent. The method is applied for purifying recombinant protein. Conventional tags for proteins, such as a polyhistidine tag (6 to 8 histidines), are used with an affinity column that specifically captures the tag (eg., a Ni-IDA column (Macherey-Nagel GmbH & Co. KG) for a Histidine tag) to isolate the protein from other impurities. The protein is then exchanged from the column using a decoupling reagent according to the specific tag (eg., immidazole for histidine tag). This method is more specific, when compared with traditional purification methods. Suitable further tags include c-myc domain (EQKLISEEDL), hemagglutinin tag (YPYDVPDYA), glutathione-S-transferase, maltose -binding protein, FLAG tag peptide, biotin acceptor peptide, streptavidin-binding peptide and calmodulin-binding peptide, as presented in Chatterjee, 2006. Cur Opin Biotech 17, 353-358). Methods for employing these tags are known in the art and may be used for purifying recombinant bispecific protein according to the invention.

The extracellular domains of gamma and/or delta TCR, preferably of the extracellular domain delta 2 TCR and/or of gamma 9 TCR, more preferred of the extracellular domain delta 2 TCR, of a preferred recombinant bispecific protein according to the invention comprises 12, 13, 14 or 15, or 16 or 17 amino acid residues between conserved cysteine 104 and phenylalanine 118 positions of the complementary determining region 3 (CDR3), whereby the positioning of the amino acid residues is determined according to IMGT nomenclature. The IMGT nomenclature is described in the online database at imgt.org, and in Lefranc et al., 2005 (Lefranc et al., 2005. Nucl Acids Res 33: D593-D597). Examples of such delta V2 and delta V5 TCR and gamma V9 and gamma V4 TCR are provided in Table 1. Please note that the numbering of amino acid residues in the CDR3 region according to the IMGT nomenclature allows additional positions at the top of the IMGT-CDR3 loop, indicated as 111.1, 112.2, 112.1 etc., in between amino acid residues 111 and 112, when necessary, Gammadelta T lymphocytes, and especially gamma9delta2 T lymphocytes, are non-conventional lymphocytes that do not depend on the expression of major histocompatibility complex molecules by target tumor cells. It has been shown that high variability in tumor recognition is predominantly regulated by the CDR3 domains of individual gamma 9/delta 2 TCRs (Griinder et al., 2012. Blood 120: 5153-5162).

Alanine-mutagenesis studies of the CDR3-domain of delta V2 TCR have shown that the amino acid sequence of the central part of this domain, from including amino acid residue number 108 to including amino acid residue number 117, is flexible (WO2013/147606).

Similarly, alanine-mutagenesis studies of the CDR3-domain of gamma V9 TCR have shown that the amino acid sequence of the central part of this domain, from including amino acid residue number 109 to including amino acid residue number 111, is flexible (WO2013/147606).

This flexibility is further reflected by the gamma V9 CDR3 sequences and delta V2 CDR3 sequences indicated in Table 1. An alignment of these sequences, as is shown herein below, indeed shows that this region is highly variable.

which all show functional binding in combination with SEQ ID NO:2 gamma CDR3.

In contrast, a 13 mer delta CDR3 amino acid sequence

CACV PLLAD TDKLI F (SEQ ID NO:3); a 16 mer delta CDR3 amino acid sequence

CACD TLGMGGEY TDKLI F (SEQ ID NO:5); and a 16 mer delta CDR3 amino acid sequence

CACD MGDASSWDTRQMF F (SEQ ID NO:33)

104 118

did not show functional binding in combination with TOR gamma sequences.

In addition, a 55y4-GAB, with a delta 5 CDR3 region comprising CAASSPIRGYTGSDKLI F (SEQ ID NO:24) and a gamma 4 CDR3 region comprising

CATWDGFYYKKLR (SEQ ID NO:25), was functional as a GAB according to the invention.

In a recombinant bispecific protein according to the invention, the

complementary determining region 3 of a delta TCR, preferably a delta 2 CDR3, comprises a consensus amino acid sequence A(X)_n, whereby n is an integer selected from 11, 12, 13, 14, 15, and 16, preferably selected from 12 and 13, said delta TCR preferably comprises a consensus amino acid sequence ACI)(X)_n, whereby n is an integer selected from 9, 10, 11, 12, 13, and 14, more preferably comprises a consensus amino acid sequence ACD(X),J , whereby n is an integer selected from 8, 9, 10, 11, 12 and 13, more preferably comprises a consensus amino acid sequence ACD(X)nLI , whereby n is an integer selected from 7, 8, 9, 10, 11 and 12, more preferably comprises a consensus amino acid sequence ACD(X)_nKLI , whereby n is an integer selected from 6, 7, 8, 9, 10, and 11, more preferably comprises a consensus amino acid sequence ACD(X)_nDKLI , whereby n is an integer selected from 5, 6, 7, 8, 9, and 10, more preferably comprises a consensus amino acid sequence ACD(X)_nTDKLI, whereby n is an integer selected from 4, 5, 6, 7, 8, and 9, in between the conserved cysteine 104 and phenylalanine 118 positions.

The 14 mer amino acid sequences of CDR3 of delta TCR all are very active, yet their sequence identity is only 57.1%. This indicates that a delta CDR3 length of 14 amino acids allows great variation in amino acid composition while still retaining GAB potency. Similarly, the 15 mer amino acid sequences of CDR3 of delta TCR are also active, despite their minimal sequence identity of only 33.3%.

Two GABs with 12 mer and 17 mer, respectively, amino acid sequences of CDR3 of delta TCR showed good activities. Their CDR3 sequences are included in the consensus sequence ACD(X)_nDKLI, whereby n is an integer selected from 5, 6,

7, 8, 9, and 10.

In a recombinant bispecific protein according to the invention, the

complementary determining region of delta TCR, preferably a delta 5 or delta 2 CDR3 region, comprises any one of SEQ ID NO:24, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO:32, as depicted in Table 1, in which five, four, three, two, or one amino acid residues has been altered in the region from amino acid residue number 106, preferably from amino acid residue number 107, more preferred from amino acid residue number 108, to the conserved

phenylalanine 118 position.

In a preferred recombinant bispecific protein according to the invention, the complementary determining region of delta 5 comprises SEQ ID NO:24, and a delta 2 CDR3 comprises any one of SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:9, SEQ II) NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ II) NO:31, SEQ ID NO:32, as depicted in Table 1.

In a recombinant bispecific protein according to the invention, the

complementary determining region of gamma TCR, preferably a gamma 9 TCR, comprises a consensus sequence A(X)_n, whereby n is an integer selected from 10,

11, 12, 13 and 14, preferably a consensus sequence ALWE(X)_nLGKKIKV, whereby n is an integer selected from 3 and 4, in between conserved cysteine 104 and phenylalanine 118 positions.

In a recombinant bispecific protein according to the invention, the

complementary determining region of gamma 9 TCR comprises any one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:25, as depicted in Table 1, in which three, two, or one amino acid residues has been altered in between amino acid residue number 106 and amino acid residue number 118, preferably in between amino acid residue number 108 and amino acid residue number 112.1.

A further preferred recombinant bispecific protein according to the invention comprises any random combination of gamma-delta amino acid sequences as depicted in Table 2 or Table 3, or a related protein that is at least 90% identical, preferably at least 91% identical, preferably at least 92% identical, preferably at least 93% identical, preferably at least 94% identical, preferably at least 95% identical, preferably at least 99% identical to said recombinant bispecific protein over its full length. Said related protein may have 20 or less, preferably 15 or less, preferably 10 or less, preferably 9 or less, preferably 8 or less, preferably 7 or less, preferably 6 or less, preferably 5 or less, preferably 4 or less, preferably 3 or less, preferably 2 or less, preferably one amino acid residues altered, preferably for an amino acid that has a related side group as described in Rajpal et ab, 2005 [Rajpal et al., 2005. PNAS 102: 8466-8471], Said related protein is able to bind tumor cells via the extracellular domain of a gamma-delta T-cell receptor (TCR), and able to bind T-lymphocytes and/or NK cells via the high affinity binding T-cell- and/or Natural Killer (NK) cell-binding domain.

Preferred sequences are provided by SEQ ID NO: 11 and SEQ ID NO:12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and by SEQ ID NO: 17 and SEQ ID NODS. However, as is indicated herein above, the gamma and/or delta extracellular sequences may be exchanged for other gamma and/or delta extracellular sequences; the scFv sequences may be exchanged for other sequences that bind to T-lymphocytes and/or NK cells. In addition, an extracellular domain of checkpoint-related molecule such as the extracellular domain of a PD-1 receptor, may be added to the N-terminus and/or the C-terminus of the

extracellular gamma and/or delta TCR sequences.

A further preferred recombinant bispecific protein according to the invention is a dimeric protein, or a higher multimer such as a trimer. It is possible that an increased avidity of binding GABs to tumor cells, as achieved with a dimeric GAB, helps to increase the activity of the GABs. For this, a recombinant bispecific protein of the invention, preferably comprises a dimerization or multi-merization motif. Alternatively, a bi- or multivalent recombinant bispecific protein may be generated by chemical cross-linking or by a heterologous dimerization or multimerization domain comprising, for example, a leucine zipper or a jun-fos interaction domain (Pack and Pluckthun, 1992. Biochemistry 31, 1579-1584; de Kruif and Logtenberg, 1996. JBC 271: 7630-7634).

Alternatively, or in addition, dimer formation of GABs may be stimulated by shortening of a linker between variably heavy (Vh) and light (VI) chains of a high affinity binding T-cell- and/or Natural Killer (NK) cell -bin ding domain, preferably of a scFv, as has been shown before (Lawrence et ak, 1998. FEBS Letters 425:479- 48). If the linker between the two variable domains is less than 15 amino acid residues, preferably less than 10 amino acid residues such as 9 amino acid residues, 8 amino acid residues, 7 amino acid residues, 6 amino acid residues, 5 amino acid residues, or 4 amino acid residues, the short linker prevents the correct association of the Vh and VI of the same chain and thereby prevents monomer formation (see also Figure 11). Said shortened linker will thus force dimerization of two different chains. As is indicated herein above, a preferred linker is a (Gly4 Ser)n linker such as a (Gly4 Ser) linker or (Gly4 Ser)3 linker.

The invention further provides a nucleic acid encoding a recombinant bispecific protein according to the invention. Said nucleic acid, preferably DNA, is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, from one or more constructs comprising nucleic acid sequences that encode the extracellular gamma and delta TCR domains, as is known to a skilled person. Alternatively, said nucleic acid is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person. Said nucleic acid is preferably codon-optimised to enhance expression of the recombinant bispecific protein, comprising an extracellular domain of a gamma- delta T-cell receptor (TCR) which is fused to a T-cell- and/or Natural Killer (NK) cell-binding domain, in a selected cell or cell line. Further optimization preferably includes removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that lead to unfavourable folding of the mRNA. The presence of an intron flanked by splice sites may encourage export from a nucleus of a selected host cell. In addition, the nucleic acid preferably encodes a protein export signal for secretion of the recombinant hispecific protein out of the cell into the periplasm of prokaryotes or into the growth medium, allowing efficient purification of the recombinant bispecific protein.

Further provided is a vector comprising a nucleic acid encoding a

recombinant hispecific protein according to the invention. Said vector preferably additionally comprises means for high expression levels such as strong promoters, for example of viral origin (e.g., human cytomegalovirus) or promoters derived from genes that are highly expressed in a cell such as a mammalian cell (Running Deer and Allison, 2004. Biotechnol Prog 20: 880-889; US patent No: 5888809). The vectors preferably comprise selection systems such as, for example, expression of glutamine synthetase or expression of dihydrofolate reductase for amplification of the vector in a suitable recipient cell, as is known to the skilled person.

The invention further provides a method for producing a recombinant hispecific protein according to the invention, comprising expressing at least one nucleic acid construct encoding a recombinant bispecific protein according to the invention in a host cell thereby producing the recombinant bispecific protein. The thus produced recombinant bispecific protein preferably is recovered from the cell. The nucleic acid, preferably a vector comprising the nucleic acid, is preferably provided to a cell by transfection or electroporation. The nucleic acid is either transiently, or, preferably, stably provided to the cell. Methods for transfection or electroporation of cells with a nucleic acid are known to the skilled person. A cell that expresses high amounts of the recombinant bispecific protein may

subsequently be selected. This cell is grown, for example in roller bottles, in fed- batch culture or continuous perfusion culture. An intermediate production scale is provided by an expression system comprising disposable bags and which uses wave-induced agitation (Birch and Racher, 2006. Advanced Drug Delivery Reviews 58: 671- 685). Methods for purification of recombinant bispecific proteins are known in the art and are generally based on chromatography, such as affinity chromatography using one or more of the protein tags, and ion exchange, to remove contaminants. In addition to contaminants, it may also be necessary to remove undesirable derivatives of the product itself such as degradation products and aggregates. Suitable purification process steps are provided in, for example, Berthold and Walter, 1994. Biologicals 22: 135- 150.

In a preferred method for producing a recombinant bispecific protein according to the invention, the recombinant bispecific protein is secreted into the growth medium of the host cell.

Further provided is a host cell comprising a nucleic acid or vector that encodes a recombinant bispecific protein according to the invention. Said host cell may be grown or stored for future production of a recombinant bispecific protein according to the invention.

The invention further provides a cell expressing a recombinant bispecific protein according to the invention. Said cell may be a bacterial cell, for example an Escherichia coli cell, or a eukaryotic cells such as a fungal cell including a yeast cell, for example Saccharomyces cerevisiae or a me thy lo trophic yeast such as Pichia pastoris, or a mammalian cell. Said eukaryotic cell preferably is a cell that can easily be infected and/or transfected using standard methods known to the skilled person, such as, for example, yeast cells and chicken fibroblast cells. Said eukaryotic cell preferably is an insect cell or a mammalian cell. Suitable insect cells comprise, for example, ovarian Spodoptera frugiperda cells such as Sf9 and Sf21, Drosophila Schneider 2 cells and Aedes albopictus C6/36 cells. Suitable mammalian cells comprise, for example, Baby Hamster Kidney cells, Human Embryonic Kidney cells such as HEK293 and freestyle HEK293F™ cells (ThermoFisher Scientific), VERO cells, MDCK cells, CHO cells, HeLa and PER.C6 cells (Fallaux, F. J. et al. 1998. Hum Gene Ther 9: 1909-1917). Preferred cells are Human Embryonic Kidney cells such as HEK293 and freestyle HEK293F™ cells.

The invention further provides the recombinant bispecific protein according to the invention, for use as a medicament. A recombinant bispecific protein of the invention is preferably used for prophylactic administration or therapeutic administration in humans that are suffering from a cancer or an infectious disease. Thus, a recombinant bispecific protein according to the invention may be administered to an individual that is suspected of suffering from a cancer or an infection, or may be administered to an individual already evidencing active infection in order to lessen signs and symptoms of said cancer or infection.

The administration of a recombinant bispecific protein according to the invention is preferably provided in an effective amount to an individual in need thereof. An effective amount of a recombinant bispecific protein of the invention is a dosage large enough to produce the desired effect in which the symptoms of the a cancer or an infection are ameliorated or the likelihood of a cancer or an infection is decreased. A therapeutically effective amount preferably does not cause adverse side effects. Generally, a therapeutically effective amount may vary with the individual's age, condition, and sex, as well as the extent of the disease and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician or veterinarian in the event of any complication. A therapeutically effective amount may vary from about 0.01 mg/kg to about 500 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days. Preferred is administration of the recombinant bispecific protein for 2 to 5 or more consecutive days in order to effectively treat a cancer and/or to avoid "rebound" of replication of an infectious agent. A recombinant bispecific protein according to the invention can be

administered by injection or by gradual infusion over time. The administration of a recombinant bispecific protein preferably is parenteral such as, for example, intravenous, intraperitoneal, intranasal, or intramuscular. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, aleoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishes (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The invention further provides a pharmaceutical composition comprising a recombinant bispecific protein according to the invention. A pharmaceutical composition preferably comprises a pharmaceutically acceptable carrier. A carrier, as used herein, means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient. The term

"physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The

characteristics of the carrier will depend on the route of administration.

Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts buffers, stabilizers, solubilizers, and other materials which are well known in the art.

The invention further provides the recombinant bispecific protein according to invention for use in a method for treatment of a cancer and/or an infection.

The invention further provides a method of treating an individual suffering from a cancer and/or an infection, said method comprising providing a recombinant bispecific protein according to invention to an individual in need thereof to thereby treat the individual. The invention further provides use of a recombinant bispecific protein according to invention in the preparation of a medicament for treating an individual suffering from a cancer and/or an infection or suspected to suffer from a cancer and/or an infection.

Gamma-delta T cells, especially Vy9/V82 T cells, play an early and essential role in sensing 'danger' by invading pathogens as they expand dramatically in many acute infections and may exceed all other lymphocytes within a few days, e.g. in tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis and malaria. Gamma delta T cells, especially Vy9/V82 T cells, recognize a microbial compound (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) which is a natural intermediate of the non-mevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis. HMB-PP is an essential metabolite in most pathogenic bacteria including Mycobacterium tuberculosis and malaria parasites, but is absent from a human host. It has been shown that also non-V82 gd T cells are expanded in various infectious contexts involving intracellular bacteria ( Mycobacteria and Listeria) as well as extracellular bacteria, such as Borrelia burgdorferi and viruses (HIV, cytomegalovirus).

In addition, gamma-delta T cells, especially Vy9/V82 T cells, recognise isopentenyl pyrophosphate (IPP) that is overproduced in cancer cells as a result of dysregulated mevalonate pathway (Goher et ah, 2003. J Exp Med 197: 163-168). gd T cells may discriminate cancer cells from healthy cells by the upregulation of selfantigens like heat shock proteins (HSP), which are increased in cancer cells due to higher metabolism.

High affinity molecules directed against potential targets of y8TCR, such as anti-CD277 antibodies, have been reported to induce conformational changes, rather than recognize conformational changes on cells (Palakodeti et ah, 2012. J Biol Chem 287: 32780-32790; Sebestyen et ah, 2016. Ibid). The low affinity interaction of y8TCRs to their ligand and the complex recognition mechanisms of y8TCRs with their ligand is designed by nature to sense spatial and conformational changes at the cell membrane and not to induce them (Sebestyen et ah, 2016. Ibid).

The recombinant bispecific protein according to invention will attach to infected cells or cancer cells, as is indicated herein above, and couple to T-cells and/or Natural Killer (NK) cells to thereby elucidate an immune response against the infected cells or cancer cells that will reduce or even eliminate said cells. The small size of a recombinant bispecific protein according to invention, together with the non-eellular nature, render said recombinant bispecific protein as an ideal treatment tool for infectious diseases and cancer. These recombinant bispecific proteins provide great promise for treatment of cancer and infectious diseases.

Definitions

The term“antibody”, as used herein, refers to an antigen binding protein comprising at least a heavy chain variable region (Vli) that hinds to a target epitope. The term antibody includes monoclonal antibodies comprising

immunoglobulin heavy and light chain molecules, single heavy chain variable domain antibodies, and variants and derivatives thereof, including chimeric variants such as, for example, human-mouse chimeric antibodies, bispecific F(ab’)2, heterodimeric scFv, tandem scFv, diabodies, tandem diabodies, fos-jun zippered fragments, IgG and IgG derivatives, and CH3“knob-into-hole” IgG.

The term“recombinant antibody”, as used herein, refers to a non-naturally occurring antibody. The term recombinant excludes naturally occurring antibodies such as naturally occurring monoclonal antibodies.

As described herein, the amino acid sequence and structure of gamma and delta T-cell receptor extracellular domains comprise a variable region comprising four framework regions or‘FR, which are referred to in the art and herein as ‘Framework region 1’ or‘FRf;‘Framework region 2’ or’FR2’;‘Framework region 3’ or‘FR3’; and‘Framework region 4’ or‘FR4’, respectively. These framework regions are interrupted by three complementary determining regions or‘CDR’ s’, which are referred to in the art as‘Complementarity Determining Region G or ‘CDRF; as‘Complementarity Determining Region 2’ or‘CDR2’; and as

‘Complementarity Determining Region 3’ or‘CDR3’, respectively. The

extracellular domain further comprises a conserved region, and a connecting peptide that provides the connection to a transmembrane domain in a native T- cell receptor gamma or delta protein. The variable region may be preceded at the N-terminal end by a leader peptide. As described herein, the term“extracellular domain” of a gamma delta TCR comprises the variable (V) gamma, V delta and extracelullar part of the constant (C) gamma and C delta domains.

As described herein, the term“extracellular domain” of a gamma or delta TCR chain comprises the V gamma and extracelullar part of the C gamma domains, or the V delta and extracelullar part of the C delta domains.

The wild type variable domain of a germ line encoded gamma 9 TCR comprises 122 amino acids and is provided by UniprotKB entry code Q99603. The amino acid sequence this gamma 9 TCR is, in single letter code and excluding a leader peptide: (N-term)-AGHLEQPQIS STKTLSKTAR LECWSGITI

SATSVYWYRE RPGEVIQFLV SISYDGTVRK ESGIPSGKFE VDRIPETSTS TLTIHNVEKQ DIATYY CALW EV (SEQ ID NO:22). For the purpose of this patent application, amino acid residues 27-34 of gamma 9 TCR are defined as CDR1, amino acid residues 52-58 of gamma 9 TCR are defined as CDR2, and amino acid residues 98-102 of gamma 9 TCR are defined as (part of) CDR3. It is noted that positions of the amino acid residues in germline sequences are not equivalent to those in the rearranged IMGT sequences.

The wild type variable domain of a germ line encoded delta 2 TCR comprises 115 amino acids and is provided by UniprotKB entry code A0N8U5. The amino acid sequence this delta 2 TCR is, in single letter code and excluding a leader peptide: (N -term) - AIELVPEHQT VPVSIGVPAT LRCSMKGEAI

GNYYINWYRK TQGNTITFIY REKDIYGPGF KDNFQGDIDI AKNLAVLKIL APSERDEGSY YCACDT (SEQ ID NO:23). For the purpose of this patent application, amino acid residues 27-34 of delta 2 TCR are defined as CDR1, amino acid residues 52-54 of delta 2 TCR are defined as CDR2, and amino acid residues 93-96 of delta 2 TCR are defined as (part of) CDR3. It is noted that positions of the amino acid residues in germline sequences are not equivalent to those in the rearranged IMGT sequences.

The term hinding’ as used herein in the context of binding between an binding domain and an epitope on a T cell or NK cell, refers to the process of a non-covalent interaction between molecules. Preferably, said binding is specific. The terms‘specific’ or‘specificity’ or grammatical variations thereof refer to the number of different types of antigens or their epitopes to which a particular binding domain can bind. The specificity of a binding domain can be determined based on affinity. A specific binding domain preferably has a binding affinity Kd for its specific epitope of less than 10"⁷ M, preferably less than 10^-8 M.

The term affinity refers to the strength of a binding reaction between a binding domain and an epitope. It is the sum of the attractive and repulsive forces operating between the binding domain and the epitope. The term affinity, as used herein, refers to the apparent binding affinity, which is determined as the equilibrium dissociation constant (Kd). A high affinity binding domain preferably has a binding affinity Kd for its specific epitope of less than 10'⁸ M, preferably less than 10 ^iJ M. A low affinity binding domain preferably has a binding affinity Kd for its specific epitope of more than 10⁸ M, preferably more than lO ^" M.

The term epitope or antigenic determinant refers to a part of an antigen that is recognized by a binding domain. The term epitope includes linear epitopes and conformational epitopes, also referred to as continuous and discontinuous epitopes respectively. A conformational epitope is based on 3-D surface features and shape and/or tertiary structure of the antigen. A posttranslational modification, such as phosphorylation, glycosylation, methylation, acetylation and lipidation, may be relevant for an epitope for recognition by a specific binding domain.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

Figure legends

Figure 1. Staining of cell lines using TCR tetramers. Cell lines were stained with 25 nM tetramer, either streptavidin (SA) PE alone or containing ybTCR “sCl5” or uBTCR“sNEF”. Daudi cells were incubated with 100 mM pamidronate (+PAM condition) and K562 HLA A*24:02 cells were incubated with 5 mM NEF peptide, both for 2 h before tetramer staining. The stained cells were analyzed on a BD FACSCanto II (BD Bioscience). Figure 2. Design and expression of a GAB. A) Schematic representation of the design of the GABs, consisting of the extracellular ydTCR domain and anti- CD3 scFv B) SDS-PAGE and Coomassie staining of GAB after expression and purification, showing the VyCy-aCD3 scFV and the V5C6 chain.

Figure 3. GABs bind to CD3+ T lymphocytes. A) 0.2*10^L6 CD3+ T lymphocytes were stained with increasing CL5 GAB concentration, followed hy staining with anti- VyO-PE, VS2-FITC or pan gd -APG and measured by flow cytometry. B) 0.2*10^L6 T lymphocytes T were incubated with 90 pg/m I GAB, anti-panyd PE was used as secondary staining. C) 0.2*10^L6 T lymphocytes were incubated with 90 ug/ml GAB (left picture) or FACs buffer (right picture), and stained afterwards with anti-pany6-APC.

Figure 4. Activity of GABs critically depends on CDR3 domain. (A)

Differential GAB mediated redirection of CD3+ lymphocytes towards tumour cells. IFNy production of T lymphocytes was measured after co-culture with 90 pg/ml GAB and Daudi (+/- PAM 0.1 mM), SCC9, MZ 185 IRC, MM231 (+/- PAM 0.1 mM) and assessed by IFNy ELISPOT (B) Bulk ab T lymphocytes were coincubated with MZ1851RC target cells in the presence of PAM (0.1 mM) and an increasing concentration of 6_2 Gab (left) or AJ8 GAB (right). IFNy levels were measured by ELISA.

Figure 5. Differential GAB mediated redirection of CD3+ lymphocytes towards tumour cells. IFNy was determined after co-culture with 10 pg/ml GAB and different target cell lines (+/- PAM 100 mM). IFNy was measured after 16h hy Elispot and amount of spots were counted, error bars represent standard deviation N=2. *: separate spots were not countable. In these cases IFNy production is considered as maximum.

Figure 6. Varying IFNy production induced hy GABs differing in CDR36 length and amino acid composition. IFNy release from T lymphocytes was measured after 16h co-culture with 20 ug/ml GAB and RPMI 8226 target cell line (+/- PAM 100 pM).

Figure 7. CDR36 loop flexibility. The top 20 lowest energy loops modeled by the RCD+ server are shown for A) LM1. B) C15. C) G115. The TCRy chain is displayed in light gray and the TCRli chain is displayed in dark gray. Images were generated using PyMOL (Schrodinger LCC.). Figure 8. Activity of C132 GAB, derived from EPCR reactive Ug-1 Vo 5 TCR. A) Target cells were stained with primary EPCR antibody and secondary detection antibody (light grey) or secondary Ab only (dark grey) and measured by FACS analysis. B) IFNy production was measured after co-culture of target cells and T lymphocytes with three different C132 GAB concentrations.

Figure 9. GAB dimers are formed during expression. A) Size exclusion chromatography of GAB, 2 different size variants of the GAB appear in two separate peaks at retention time 7.2 and 8.2 min. B) SDS-page of the protein in the two SEC peaks. C) SEC-MALS experiment to determine molecular size of the two peaks, determining that the first peak corresponds to dimeric GAB (±1.77 x 1 OF 5 g/mol) while the second peak consists of GAB in a monomeric form

(±8.8xlOE4 g/mol).

Figure 10. GAB is more active in a dimeric form compared to monomeric GAB. A) IFNy release from T lymphocytes was measured after 16h co-culture with 10 m g/ rn 1 dimeric or monomeric AJ8 GAB and target cell lines (+/- PAM 100 pM). B) CDS + T cell degranulation was measured by staining for CD 107a after a 7 hour co-incubation of T lymphocytes and 2 different target cell lines in the presence and absence of monomeric or dimeric AJ8 GAB (10 pg/ml) and PAM (100 pM).

Figure 11. Increased dimer formation by shortened linker between OKT3 variable heavy and light chain. A) schematic representation of the most likely dimer conformation of the GAB, and the shortened linker between OKT3 Vh and VI. B) Size exclusion chromatography showing that shortening the linker to 5 amino acids results in a higher dimer peak (at 7.2 min) and trimer peak (at 6.8 min) compared to the 15 amino acid linker.

Table 1. GAB CDR3 sequences. Conserved Cys l04 and I die 1 18 are in italics.

0

3

O

Table 1. Continued O

' oJ\i

< oJ\\ o\

n H

Z

o o

<J\ o o

00

00

3

O

Table 2. GAB sequences, preceded by a leader sequence. The VbCb and VyCy amino acid sequences are underlined, linker sequences O are in italics, scFv sequences are in bold. The amino acid sequences end at the C-terminus with a tag. Individual“S””,“SA”,“VD” and 'Ji

Os “RT” amino acid residues are included because of restriction sites that were used. 'Ji

Os o\

n H

Z

o o o o

00

00

3

o

O

'Ji

Os

'Ji

Os o\

n H

Z

o

o

C/I o o

00

00

3

O

Table 3 GAB sequences with extracellular domain of a checkpoint-related molecule. PD-1 sequences are indicated in bold and O underlined. Individual“RT\ “VD”,“AT and“GS” amino acid residues are included because of restriction sites that were used. 'Ji

Os

'Ji

Os o\

n H

Z

o o o o

00

00

Examples

Example 1

Materials and methods

Cell lines

Daudi, HEK293FT and MDA-MB231 cells were obtained from ATCC,

MZ1851RC from Barbara Seliger (University Halle, Germany; see Seliger et al., 1996. Cancer Res 56: 1756-60). SCC9 was kindly provided by Niels Bovenschen (UMC Utrecht, The Netherlands; see (Grunder et al., 2012, Ibid). Freestyle 293-F cells were obtained from Thermo Fisher Scientific.

Daudi cells were cultured in RPMI, 10% FCS, 1% Pen/Strep, Freestyle 293-F in Freestyle expression medium (Gibco). All other cell lines in DMEM, 10% FCS,

1% Pen/Strep. T lymphocytes were expanded on a rapid expansion protocol. For assays either CDS and CD4 positive, or only CD4 positive cells were used.

Genera tion of Bispecific constructs

A customized pcDNA3 vector (Invitrogen), which allows consecutive expression of two genes of interest under their own CMV promoter, was a kind gift of Jan Meeldijk (LTI protein facility, UMC Utrecht). In short, a WPRE (Donello et al., 1998. J Virol 72: 5085-92) sequence was introduced at the 3^’ end of the multiple cloning site of pc,DNA3; a second CMV promotor followed by a multiple cloning site comprising nucleotides 208-1274 from pcDNA5/FRT (Invitrogen; cat no. V601020), flanked at the 5’ and 3’ ends by Sphl restriction sites were ligated in the Pcil restriction site of pcDNA3. For the generation of the final construct, the antiCD3- scFv (OKT3) (Arakawa et al., 1996. J Biochem 120: 657-62) gene was cloned into the first multiple cloning site of the vector. Besides the antiCD3-scFv gene the DNA fragment also contained bases encoding a (G4S):s flexible linker at the 5’end and poly histidine tag on the 3’ end. 5’ of the flexible linker a BsiWI restriction site was present for the subsequent introduction of the TCR gamma chain in the vector, resulting in the TCR gamma-CD3sc,Fv fusion gene. The TCR delta chain was cloned into the second multiple cloning site. TCR domain boundaries were used as in Allison et al., 2001. Nature 411: 820-824).

Expression and purification of Bispecifics

His tagged recombinant bispecific protein, comprising an extracellular domain of a gamma-delta T-cell receptor (TCR) which is fused to a T-cell- and/or Natural Killer (NK) cell-binding domain, (also termed ybTCR anti-CD3 bispecific molecules or“GAB”s) were expressed in 293 F cells. 293 F cells were cultured in Gibco Freestyle Expression medium, and transfected using 293Fectin (Invitrogen). Transfection was done using 293 F cells at a concentration of 1.10^L6 cells/ml mixed with 1 pg/ml DNA and 1.33 pl/ml 293Fectin. DNA and Fectin were pre-mixed in Optimem (1/30 of transfection volume), incubated for 20 minutes after which the mix was added dropwise to the cell cultures. The cultures were maintained shaking at 37 °C 5% C02. Cell culture supernatant was harvested after 5 days and filtered through a 0.22 mM filter top (Milipore). Supernatant was adjusted to 25 mM Tris, 150 mM NaCl and 15 mM Imidazole (pH 8). Supernatant was loaded on a 1 ml HisTrap FF column (GE healthcare) using the AKTA start purification system (GE Healthcare). Column was washed with IMAC loading buffer ( 25 mM Tris, 150 mM Nacl 15 mM Imidazole (pH 8), and protein was eluted using a linear imidazole gradient from 21 to 300 mM in 20 column volumes. Fractions containing the GAB were pooled, concentrated and buffer exchanged to TBS ( 25 mM Tris, 150 mM NaCl) using Vivaspin™ 6 spin columns (Sartorius) with a cutoff of 10 kDa.

Protein was diluted 100 times in IEX loading buffer (25 mM Tris pH 8), and loaded onto a HiTrap Q HP 1 ml column (GE Healthcare) using the AKTA start purification system, for a second purification step. Column was washed with 10 column volumes IEX loading buffer and protein was eluted using a linear NaCl gradient from 50 to 300 mM in 25 CV. Fractions containing the GAB were pooled, concentrated using Vivaspin 6 spin columns (Sartorius) and examined by SDS- PAGE and Coomassie Blue staining. Protein concentration was measured by absorbance on Nanodrop spectrophotometers (ThermoFisher Scientific) and corrected for the Extinction coefficients. Protein was snap frozen and stored at - 80°C and thawed before use.

Flow cytometry

Stainings were performed by incubating 0.2*10^L6 T lymphocytes with 90 pg/ml GAB in 20 pl FACS buffer ( PBS, 1% BSA, 1 % sodium azide) for 30 min at room temperature. Cells were washed once in FACS buffer and incubated with the appropriate secondary antibody mix for 30 min at room temperature. Cells were washed 2 times in FACS buffer and fixed in 1% paraformaldehyde in PBS. Data acquisition was done on FACS Canto and analyzed using FACS Diva software (both of Becton Dickinson). Antibodies that were used were pan-yBTCR-PE

(Beckman Coulter), pan-ySTCR APC (BD Pharmingen), V52-FITC (Biolegend) and Vy9-PE (BD Pharmingen).

IFNy Elispot

Effector cells were pre-incubated with or without 90 pg/nil GAB in 50 ul complete RPMI (10% FCS, 1% Pen/Strep), 30 min at room temperature after which they were diluted to 0.3 10^L6 cells/ ml in complete RPMI. Effector and target cells were incubated together at 3:1 target: effector ratio for 16 hours at 37 °C 5% C02. When pamidronate was applied, 0.1 mM pamidronate (pamidronate disodium salt; CAS 109552-15-0; Calbiochem Cat. No. 506600) was added to the cells. After 16 hours, cells were washed with PBS and stained for IFNy using mAbl-DlK (I, Mabtech) and mAb7-B6-l (II; Mabtech) following the manufacturer's recommended procedure. Results were analyzed using ELISPOT Scanner and ELI.Analyse software (A.EL.VIS, Hannover, Germany).

IFNy Elisa

IFNy ELISA was performed using the ELISA-Ready-Go! Kit (eBiosciences) following the manufacturer's instructions. Effector and Target cells (E:T 1:1) were incubated for 16 hours in the presence of pamidronate (0.1 mM) and GAB (0.1 to 30 μg/ml) in complete RPMI.

Results

Low affinity ofgdTCRs as compared to aflTCR

In order to formally confirm that y8TCRs have a substantial lower affinity than aBTCRs, we engineered multimers consistent of aBTCRs specific for the HIV peptide NEF and gdTCR comprised of y9 and 62TCR chains of clone 5 (Grander et ah, 2012. Blood 120: 5153-62). In line with the assumption that y982TCR have a lower affinity, aBTCRs where able to bind to their target such as peptide loaded K562 HLA-A*24:02 cells but y9B2TCRs did not bind to their ligand (See Figure 1).

Production of highly pure GABs

In order to study whether the limitations of current bispecifics with high affinity uBTCR linked to anti-CI)3scFv (Liddy et ah, 2012. Nat Med 18: 980-7) can be overcome, we assessed whether low affinity yBTCRs are able to mediate anti- tumor reactivity in a bi-specific format, when linked to a strong anti-CD3 binder. For this, GdTCR Anti-CD3 Bispecific molecules (GABs) were cloned with an anti- CDSscFv derived from the anti-CD3e antibody OKT3 linked to the C terminus of the gamma chain of a soluble yhTCR using a flexible (G4S)3 linker (Figure 2A). CDR3 domains of both the g and 8TCR chains have been reported to be key in modulating functional avidity of T cells (Grunder et ah, 2012. Ibid ; Starick et ah, 2017. Eur J Immunol 47: 982-92) and could therefore potentially impact also activity of GABs. Therefore we generated six GABs in total. Five GABs were derived from different tumor reactive Vy9V52 TCRs, a medium affinity yhTCR namely G115 (Allison et ah, 2001. Ibid: Grunder et ah, 2012. Ibid ) and high affinity yhTCR namely CL5 as well as a so far not further characterized CL13 TCR mediating strong anti-tumor reactivity (Grunder et ah, 2012. Ibid). In addition we utilized two random sequences identified recently by high throughput sequencing from healthy donors which also showed anti-tumor activity when expressed within the TEG format (unpublished data), namely AJ8 and 6_2 TCRs to create the other two GABs. As potential negative control, a g952 TCR was used where the CDR3 region of the h chain was replaced by a single alanine and reported to be nonfunctional once expressed in uBT cells, thus the TEG format (TEG-LM1) (Grunder et ah, 2012. Ibid).

GABs were expressed in mammalian freestyle 293 F cells as secreted proteins and purified from the culture supernatant using His-tag purification followed by a second ion exchange purification step, to ensure a highly pure protein product. After these purification steps the protein was run on SDS-PAGE, and visualized by Coomassie blue staining. As expected the two different chains of the GAB, ectoGamma-CDSscFv and ectoDelta, are both clearly visible on gel (see Figure 2B). This indicates that during expression the two separate chains of the GAB associate properly, resulting in a heterodimeric bispecific molecule.

GABs bind to CDS positive T lymphocytes

To further address proper folding of GABs we employed a flow cytometry based analysis. T lymphocytes were incubated with different, concentrations of GABs, followed by a secondary staining using fluorochrome labeled antibodies against Vb2, Vy9 or panyb-TCR. A strong and specific staining could by observed with all three antibodies at 10 m g/m 1 (Figure 3A). 10 m g/m I GAB was consequently used for the loading of immune cells with GABs. Comparing binding of all engineered GABs on T cells by counterstaining with a panyd-TCR showed equivalent signals between all constructs (Figures 3A and B), suggesting correctly folded ecto-ySTCR domains for all GABs. Next the expression levels of the ecto- ydTCR domain of GABs was compared to the expression level of ydTCRs when retrovirally expressed within the TEG format at the cell surface of u6T cells (Sebestyen et ah, 2016. Ibid; Grander et al., 2012. Ibid; Straetemans et ak, 2015. Clin Cancer Res 21: 3957-68; Marcu-Malina et al., 2011. Blood 118: 50-9).

Therefore TEGs have been further discarded of poorly and non engineered immune cells by a6T cell depletion as reported (Straetemans et al., 2015. Ibid) and expanded. In addition non-engineered uBT cells expanded within the same protocol have been incubated after expansion with GAB and both cell populations stained with a panyd-TCR. Expression of GABs on coated uBT cells was identical in maximal expression of ydTCR when compared to TEGs. However, in contrast to TEGs, who showed approximately 24% of cells with reduced y6TCR expression despite previous selection for high TCR expression, 99% of GAB coated cells were strongly positive for the ecto-y5TCR domain (Figure 3C).

GAB induced specific activation of T lymphocytes

Vy9Vd2 T lymphocytes and TEGs are known to recognize Daudi cells, a Burkitt’s lymphoma cell line (Grander et al., 2012. Ibid). This recognition can be greatly enhanced by treating the target cells with pamidronate (Grander et ah, 2012. Ibid). Pamidronate-depended enhancement of recognition relies on an inside out mechanism involving RhoB and a conformational and spatial change in CD277 (CD277J) (Sebestyen et al., 2016. Ibid; Gu et al., 2-017. Ibid). To determine whether the GABs can mimic the mode of action described for Vy9V62 TCR chains expressed in T cells, uBT cells and Daudi target cells were co-incubated in the presence or absence of pamidronate with GABs. abT cells activation was determined by measuring IFNy production using an ELISPOT assay. Presence of CL5, AJ8 and 6_2 GAB in the co-culture induced a robust IFNy production in a pamidronate- dependent manner (Figure 4A), indicating that GAB mediated recognition of target cells is similar to the described mode of action for y962TCR chains. Surprisingly, although the CDR3 region is known to he key for the activity of y952TCR chains expressed in T cells (Grander et ah, 2012. Ibid; Starick et al., 2017. Ibid) GAB-mediated activities of containing CDS domains of G115 and CL13 GAB which have been reported to by highly active in gdT cells (Grunder et ah,

2012. Ibid; Marcu-Malina et al., 2011. ibid) and TEGs (Grunder et al., 2012. Ibid) showed only little or no specific T cell activation upon co-culture with pamidronate treated Daudi target cells. In addition, LM1 GAB, although derived from a nonfunctional Vy9Vd2 TCR within the TEG format induced some IFNy production in this analysis, which could again not been further be enhanced by adding pamidronate. These data suggest that only GABs equipped with CDR3 domains from clone 5, 6_2 and AJ8 fully mimicked the mode of action usually active through ydTCR when expressed in T cells when interacting with tumor cells. To further characterize activity of GABs, 6_2 and AJ8 GABs were titrated in a co-culture of T lymphocytes with MZ1851RC tumor cells (Figure 4B). Increasing concentration of both GABs increases IFNy production by the T cells, with a plateau being reached around 10 to 30 pg/ml.

Analysis of the CDR3 region of non-functional and functioned. GABs

The lacking or very weak and partially unspecific activity of GABs engineered with CDR3 regions derived from clone FM1, G115, and clone 13 was highly unexpected. G115 engineered TEGs have been despite its somewhat lower activity when compared to clone 5 reported to be highly functional within the TEG format in vitro and in vivo (Marcu-Malina et al., 2011. Ibid). Also clone 13 has been extracted from a highly active g9d2 T cell clone (Grunder et al., 2012. Ibid) and LM1 mutants have been reported to be non-functional (Grunder et al., 2012. Ibid). Therefore we further compared sequences of utilized GABs and focused on the CDR36 residues between the conserved TCR residues Cysl04 and Phell8 according to the IMGT numbering. These analyses suggested that GABs are substantially more susceptible to variations in sequence and lengths of the CDR3 region of the 8TCR chain when compared to TEGs.

Broad tumor reactivity of GABs

Next, two strong GABs, namely AJ8 and 6_2 GAB were tested against a broader range of tumor cell lines that are known targets for several g9o2T( 'Its within the TEG format. LM1 GAB was included as negative control and had in these experiments a negligible activity to all targets (Figure 5). IFNy production was induced to different extend by both GABs against all seven cell lines. Indicating that the GABs can redirect T cells towards multiple tumor types, similar to recognition by TEGs.

Example 2

Materials and methods

See Example 1, unless otherwise specified.

Results

CDR38 lengths and amino acid compositions

To further explore the impact of CDR36 length and amino acid composition, we selected 8 additional 62 chains of tumor reactive g962 TCRs that, are functional in the TEG format, to create GABs (Table 1). The 8 new GABs were expressed and purified as described herein above. Tumor reactivity of these different GABs was determined by an IFNy release assay against the tumor cell line RPMI 8226 at a concentration of 20 pg/rnl. AJ8 GAB was used as additional positive control (Fig. 6).

The GABs with a CDR36 length of 14 amino acids, A4 and C4, were both very potent despite having different amino acid composition to CL5 GAB. The CDR36 of C15 contains 2 positively charged amino acids while the CDR36 of A4 contains a negatively charged amino acid at one of these positions and the CDR36 of C4 contains hydrophobic amino acids. The CDR36 of A4 and C4 have 71.4% sequence identity to C15, while A4 and C4 only have 57.1% sequence identity, indicating that a CDR36 length of 14 amino acids allows great variation in amino acid composition while still retaining GAB potency.

Cl GAB, which has a CDR36 length of 15 amino acids, was less potent than AJ8 GAB, which also comprises a 15 amino acid CDR36, however still more potent than most of the GABs containing 16 amino acid CDR36. All 3 GABs with a CDR38 of 15 amino acids are active and again the CDR36 sequences have broad amino acid usage. The CDR36 of Cl and 6_2 have only 33.3% sequence identity to AJ8, while Cl and 6_2 have 53.3% sequence identity of their CDR36.

An additional 3 GABs were tested using a CDR36 of 16 amino acids. While A1 and C3 GABs were able to activate T cells in presence of tumor cells, they were less potent than the GABs using CDR36 of 14 or 15 amino acid length. The other GAB with a 16 amino acid CDR36, C7, did not show any tumor reactivity at all. Interestingly, two GABs with C 1)1135 lengths of 12 and 17 amino acids, A3 and C5 respectively, showed very potent induction antitumor reactivity.

These data show that for g9d2 TCRs based GABs the use of CDR36 with a length of 14 and 15 amino acids, with large sequence variability, results in bispecific molecules able to potently induce antitumor reactivity in T cells, notwithstanding the fact that there are CDR35s of different length that can also induce antitumor reactivity though not all GABs using aCDR38 of other lengths allow always potent activity.

To gain more insight in the observed CDR35 length preference in the GAB format we used the RCD+ fast loop modeling server (Lopez-Blanco et al., 2016. Nucleic Acids Research 44: W395-W400) to predict energetically favorable loop conformations. This server can accurately predict native loop conformations of a large number of benchmark proteins.

The CDR38 loops of LM1 (11 amino acids), C15 (14 amino acids) and G115 (16 amino acids) were predicted, based on the crystal structure of G115 (Allison et al., 2001. Nature volume 411: 820-824) to see whether there were large differences in loop conformation. The short CDR35 loop of LM1 didn^’t show any variation in loop conformation as all predicted loops in the top20 of lowest energy loops adopted a similar conformation, meaning that the Ca atoms of the amino acids deviated less than 1 A between the different loops (Fig. 7a). Multiple conformations were modeled for the CDR35 loop C15 (Fig. 7b). Interestingly there were 2 loop conformations found that were predicted multiple times in the top20, one cluster containing 8 of the top 20 loops and the second cluster containing 5 of the top 20 loops. For G115 the top 20 CDR38 loops had hardly any convergence, 3 of the 20 loops had a conformation that just fell within the 1 A cut off value (Fig. 7c), whereas the clusters of C15 had a mean deviation below 0.4 A. This implies that CDR38 loop flexibility might play an important role in the activation potential; too rigid as LM1 is not preferred, as is too flexible like G115.

Example 3

Materials and methods

Same as in Example 1.

Degranulation assay Target cells were incubated with T lymphocytes at E:T ratio of 1:3 with and without AJ8 GAB mono-/dimer (10 ug/ml) and Pamidronate (3-amino-l- hydroxypropylidenebisphosphonate pentahydrate, Sigma-Aldrich; 100 uM) in the presence of CD107a-PE (Becton Dickinson) for 7 hours, after 2 hours Golgistop (Becton Dickinson) was added. After 7 hours cells were washed in FACs buffer and stained with aCD3-eFluor450( eBioscience) and aCD8-PerCP-Cy5.5 (Biolegend). Cells were washed 2 times in FACS buffer and fixed in 1% paraformaldehyde in PBS. Data acquisition was done on FACS Canto and analyzed using FACS Diva software (BD)

Size exclusion chromatography and multi angle light scattering

Size exclusion chromatography was perfomed on a Yarra 3 uM SEC 3000 column (Phenomenex) using the high Performance Liquid Chromatography system

(Shimadzu). Column was washed with SEC running buffer (100 mM Sodium Phosphate, 150 mM NaCl, pH=6.8). Protein samples were 5x diluted in SEC running buffer and filtered through a 0.22 uM centrifugal filter (Milipore) before loading on the column. Fractions corresponding to the peaks were pooled and used for further analysis. For mass determination the MINIDAWN TREOS® multiangle light scattering detector and software were used (Wyatt Technology Corp.). Results

Vy4Vd5 EPCR reactive GAB

In addition to the Vy9 Vo 2 GABs described in Example 1, also a Vy-l Vo 5 GAB was created. This Vy4V85 GAB was derived from an endothelial protein C receptor (EPCR)-reactive TCR (Willcox et ah, 2012. Nature Immunol 13: 872-879), and termed C132 GAB. This GAB was tested against cell lines with varying EPCR expression. MZ 185 IRC cells are renal cell carcinoma cells that moderately express EPCR. HEK 293T cells hardly express endogenous EPCR, which was exogenously introduced in HEK293T-EPCR cells (Figs 8A and B). An IFNy release assay was performed by co-culturing T lymphocytes with EPCR-expressing cell lines and different C132 GAB concentrations (see Fig. 8B). C132 GAB could efficiently induce IFNy production, which was dependent on EPCR expression level on the target cell, as well as on the concentration of the GAB (Fig. 8B).

GAB dimer formation during expression After expression and purification, size exclusion chromatography (SEC) was used to further characterize the produced GAB protein. The resulting SEC

chromatogram (Fig. 9A) shows that the GAB has two major size variants, visible as peak 2 and 3 on the chromatogram. Peak 2 accounts for 24% of the loaded protein, while the majority of the GAB, 74.5%, resides in peak 3. Analysis on SDS-PAGE shows no apparent differences between the two different samples (Fig. 9B). To determine the molar mass of the different protein peaks, size exclusion

chromatography with multi angle light scattering was performed (SEC-MALS).

The MAES analysis provided precise molar masses for both peaks: peak 2 consist of a protein with a molar mass 176.7xlOE3 g/mol and peak 3 of a protein with a molar mass of 88.45xlOE3 g/mol, corresponding to dimeric and monomeric GAB, respectively (Fig. 9C). This indicates that during expression around 25% of the protein folds as a dimer.

To test whether there are functional differences between the dimer and monomer GABs, the two peaks were collected and used in an IFNy release assay (Fig. 10A). GAB dimer induces more IFNy production when co-cultured with T cells and target cells compared to the monomer, while the negative target cell line HF60 is not recognized by both GAB fractions. This result was also confirmed by a degranulation assay. CD 107 expression, which is a marker for degranulation of CD8+ T cells, was measured by flow cytometry after co-culture of T cells with target cells and monomeric or dimeric GAB (Fig. 10B) Again, dimeric GAB results in more degranulation of CD8+ T cells compared to the monomeric GAB.

Increased dimer formation

It was hypothesized that the dimer is formed by dimerization of the CD3scFv of two different GABs. That scFVs can cause proteins to dimerize has been shown before (Fawrence et ah, 1998. FEES Fetters 425: 479-48), this is mainly mediated by the length of the linker between the variably heavy (Vh) and light (VI) chain of the scFv. If the linker between the two domains is too short it prevents the correct association of the Vh and VI of the same chain and thereby monomer formation, instead scFv of two different chains pair which results in the formation of dimers. To test if CDSscFv dimerization can cause the formation of GAB dimers and if we can further increase this, we shortened the linker between the OKT3 Vh and VI from 15 amino acids to 5 amino acids (Fig. 11A). After production and purification, a sample of this shortened linker GAB was run on a SEC column (Fig. 12B). The resulting chromatogram shows that with the shorter linker, 56% of the GAB protein is dimerized while only 36% is still in the monomeric form. Furthermore, the first peak of the chromatogram which corresponds to trim eric GAB, also increases form approximately 1 to 10% of the protein fraction.

Claims

Claims

1. A recombinant bispecific protein, comprising an extracellular domain of a gamma-delta T-cell receptor (TCR) which is fused to a high affinity T-cell- and/or Natural Killer (NK) cell-binding domain.

2. The recombinant bispecific protein according to claim 1, wherein the extracellular domain of the gamma TCR is fused to a CD3-binding domain.

3. The recombinant bispecific protein according to claim 1 or claim 2, wherein the extracellular domain of a gamma TCR is fused to a single chain Fv anti-CD3 binding domain, preferably a chimeric mouse-human OKT3 antibody.

4. The recombinant bispecific protein according to any one of claims 1-3, wherein the extracellular domain of gamma and/or delta TCR is fused to an extracellular domain of a checkpoint-related molecule, preferably programmed cell death 1 (PD1).

5. The recombinant bispecific protein according to any one of claims 1-4, comprising the extracellular domains of gamma 9 and delta 2 TCR.

6. The recombinant bispecific protein according to any one of claims 1-5, wherein the complementary determining region of delta TCR comprises 14, 15 or 16 amino acid residues between conserved cysteine 104 and phenylalanine 118 positions, according to IMGT nomenclature.

7. The recombinant bispecific protein according to any one of claims 1-6, wherein the complementary determining region of delta TCR comprises any one of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:24, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32.

8. The recombinant bispecific protein according to any one of claims 1-7, comprising any combination of gamma-delta amino acid sequences as indicated in Table 2, or a protein that is at least 90% identical thereto.

9. The recombinant bispecific protein according to any one of claims 1-8, which is a dimer or a higher multimer such as a trimer.

10. A nucleic acid construct encoding a recombinant bispecific protein according to any one of claims 1-9.

11. A cell expressing a recombinant bispecific protein according to any one of claims 1-9.

12. Method of producing the recombinant hispecific protein according to any one of claims 1-9, comprising expressing at least one nucleic acid construct encoding a recombinant bispecific protein according to any one of claims 1-9 in a host cell thereby producing the recombinant bispecific protein.

13. The method according to claim 12, whereby the recombinant hispecific protein according to any one of claims 1-9, is secreted into the growth medium of the host cell.

14. A pharmaceutical composition, comprising the recombinant hispecific protein according to any one of claims 1-9, and a pharmaceutically acceptable excipient.

15. The recombinant hispecific protein according to any one of claims 1-9, for use as a medicament, preferably for use in a method for treatment of a cancer and/or an infection.