TITLE
Test cells for dogBiTEs FIELD
The present disclosure concerns cells suitable for use in the development of immunotherapeutic drugs for dogs, and bispecific molecules suitable for binding to both T-cells and target cells. BACKGROUND Until recently, the only drugs available to treat cancer in animals were those
approved for use in humans. However, a few drugs specifically intended for animals are now approved.
For example, the following drugs are approved or conditionally approved by FDA to treat cancer in dogs: ^
Palladia (toceranib phosphate), to treat mast cell tumors, FDA and EMAapproved in 2009; ^
Stelfonta (tigilanol tiglate injection), to treat mast cell tumors, FDA and EMA approved in 2020; ^
Tanovea-CA1 (rabacfosadine for injection), to treat lymphoma, conditionally FDA approved in 2016 and fully approved in 2021; and ^
Laverdia-CA1 (verdinexor tablets), to treat lymphoma, conditionally FDA approved in 2021
In humans, many advanced cancer drugs have been approved. Both tumor vaccines and cell therapies have been developed. However, cell therapies are quite
expensive, and they may be unaffordable for most dog owners. In contrast, tumor vaccines could be logistically simple and provide a relative affordable immunotherapy for dogs.
Checkpoint inhibitor therapy is a form of cancer immunotherapy based on monoclonal antibodies. Some cancers can protect themselves from the immune system by stimulating immune checkpoint targets. Checkpoint therapy can block
inhibitory checkpoints and thus restore the immune system function. The first anti- cancer drug targeting an immune checkpoint was ipilimumab, a CTLA4 blocker
approved by FDA in 2011.
Currently FDA-approved checkpoint inhibitor drugs target the molecules CTLA4, PD-1, or PD-L1. Examples of such drugs include Yervoy, Imjudo, Keytruda, Tecentriq, Bavencio, Imfinzi and Libtayo. Gilvetimab, a caninized monoclonal antibody against canine PD-, was recently (2024) conditionally approved by FDA to treat mast cell tumors and melanoma.
However, there is still a large unmet need concerning suitable immunotherapeutic targets in dogs. SUMMARY
The present disclosure provides cells useful during development of immunotherapeutic drugs intended for dogs. If the candidate compounds rely on binding to a T-cell receptor-complex (TCR-complex), the cells may be used as in vitro tools to validate immunotherapeutic candidate compounds for dogs. Instead of embarking on a cumbersome process for developing suitable canine model cells from scratch, the present disclosure provides a solution involving human cells. Said human cells are modified to represent a reliable and convenient canine model simply by expression of canine CD3-chains. It was found that canine CD3-chains could be successfully expressed in the cell membrane together with human T-cell
receptors, thus believed to form a canine-human hybrid TCR-complex in both T- cells and NK-cells. As demonstrated herein, this hybrid TCR-complex could then be
successfully used as a target to test bispecific T-cell engagers (BiTEs) for dogs without needing living dogs.
In a first embodiment, the present disclosure provides a human cell expressing a T- cell receptor (TCR) and at least two different canine CD3-chains in its cell membrane. In a first aspect, the TCR comprises a human Vα and a human Vβ, or a human α- chain and a human β-chain. In a second aspect, the at least two different canine CD3-chains are selected from the group consisting of CD3ζ, CD3γ, CD3δ and CD3ε. In a third aspect, the cell expresses canine CD3ε and canine CD3γ, or wherein the cell expresses canine CD3ε and canine CD3δ, or wherein the cell expresses canine CD3ε and canine CD3ζ, or wherein the cell expresses canine CD3ε, canine CD3γ and canine CD3δ.
In a fourth aspect, the cell expresses canine CD3ζ, canine CD3γ, canine CD3δ and canine CD3ε, which together with the TCR forms a functional TCR-complex in the cell membrane. In a fifth aspect, the cell expresses canine CD3γ, canine CD3δ and canine CD3ε, which together with the TCR forms a functional TCR-complex in the cell membrane. In a sixth aspect, the cell is a T-cell or a Natural killer-cell. In a seventh aspect, the cell is a cytotoxic T-cell.
In an eighth aspect, the cell is transduced with a nucleic acid encoding the polyprotein represented by SEQ ID NO: 1. In a second embodiment, the present disclosure provides a human cell-line comprising cells according to the first embodiment.
In a first aspect, the cell-line is derived from Jurkat or NK92. In a third embodiment, the present disclosure provides a recombinant nucleic acid encoding canine CD3ε and at least one canine CD3-chain selected from the group consisting of CD3γ and CD3δ.
In a first aspect, the recombinant nucleic acid encodes canine CD3ζ, canine CD3γ, canine CD3δ and canine CD3ε.
In a second aspect, the recombinant nucleic acid encodes at least one ribosomal skipping sequence between two CD3-chains.
In a third aspect, the recombinant nucleic acid encodes a ribosomal skipping sequences between each of the CD3-chains.
In a fourth aspect, the recombinant nucleic acid is the form of a polycistronic construct encoding the polyprotein represented by SEQ ID NO: 1. In a fourth embodiment, the present disclosure provides a protein comprising a first and a second Fv for binding to a first and a second target epitope, wherein each Fv comprises a VL and a VH, and the VL comprises three CDRs flanked by framework sequences, and the VH comprises three CDRs flanked by framework sequences,
wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a canine CD3-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on canine
or human target cells. In a first aspect, the first Fv also specifically binds, under physiological conditions, to an epitope located on the extracellular part of a human CD3-chain.
In a second aspect, the first and second Fv’s are scFv’s. In a third aspect, the first and second scFv’s are connected to each other by a linker, such as a peptide linker comprising 1 to 25 amino acid residues. Suitable examples include G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5 etc. and EAAAK, (EAAAK)2,
(EAAAK)3, (EAAAK)4. Alternatively, the scFv’s may be connected by other known technologies like knob-in-hole Fc’s.
In a fourth aspect, the linker is a glycine-serine linker comprising 5 to 50, 10 to 50, 15 to 50 or 20 to 50 amino acid residues. In a fifth aspect, the first Fv specifically binds, under physiological conditions, to the extracellular part of both human and canine CD3ε.
In a sixth aspect, the second Fv specifically binds, under physiological conditions, to an epitope on a Tumor Associated Antigen located on a cancer cell.
In a seventh aspect, the second Fv specifically binds, under physiological conditions, to an epitope on Tumor Associated Antigens located on both human and canine cancer cells.
In an eighth aspect, the first Fv comprises, a VL with the following three CDRs; SEQ ID NO: 10 (CDR1), SEQ ID NO: 11 (CDR2) and SEQ ID NO: 12 (CDR3), and a VH with the following three CDRs; SEQ ID NO: 7 (CDR1), SEQ ID NO: 8 (CDR2) and SEQ ID NO: 9 (CDR3).
In a nineth aspect, the second Fv is a scFv represented by SEQ ID NO: 14 or 15. In a tenth aspect, the second Fv specifically binds, under physiological conditions, to an epitope on ALPL-1 on an osteosarcoma cell, to an epitope of CD22 or to an epitope of HER2.
In an eleventh aspect, the framework sequences in the first Fv are human, murine or canine.
In a twelfth aspect, the framework sequences in the second Fv are human, murine or canine.
In a fifth embodiment, the present disclosure provides a recombinant nucleic acid encoding the protein according the fourth embodiment. In a first aspect, the nucleic acid is in the form of linear or circular cDNA, mRNA or a circular DNA expression vector.
In a sixth embodiment, the present disclosure provides the use of the cells according to the first embodiment for in vitro testing of immunotherapeutic candidate compounds for dogs.
In a seventh embodiment, the present disclosure provides an in vitro method for testing immunotherapeutic candidate proteins for dogs, comprising the steps; a) providing a cell population comprising human cells according to the first embodiment in a suitable medium, b) incubating the human cell population in step a) with an immunotherapeutic candidate protein according to the fourth embodiment, as well as incubating the human cell population in step a) with canine cancer target cells, and then
c) measuring the canine cancer cell mortality, viability or proliferation. BRIEF DESCRIPTION OF THE FIGURES Figure 1 visualizes the generation of the cells expressing a TCR and canine CD3-
chains. Human NK and T-cells can be converted by transferring the polycistronic nucleic acid encoding the canine CD3 complex including CD3^^^CD3^^^CD3^^and^CD3^. The resulting T-cell will express both human and canine CD3 proteins in complex with the endogenous (human)TCR. NK cells will be transduced with a second gene encoding for a human/canine TCR and the complex will be sent to the membrane. Both cells will become “caninized” for their CD3. This canine CD3 complex will be recognized by a dogBiTE and stimulate the
cell through its TCR. Notably, this mechanism involves interaction between a human TCR and canine CD3-chains.
Figure 2 visualizes, in a simplified manner, a conventional TCR-complex comprising an α-chain with three CDRs and a β-chain with three CDRs together forming an αβTCR. The variable domain of the α-chain is called Vα. The variable domain of the β-chain is called Vβ. The TCR-complex comprises two CD3ζ-chains,
two CD3ε-chains, one CD3γ-chain and one CD3δ-chain. The cell membrane is also represented.
Figure 3a visualizes, in a simplified manner, a conventional antibody comprising two heavy chains and two light chains. Each light chain is connected to a heavy chain by a cysteine bridge, and the heavy chains are connected to each other by two cysteine bridges. The Variable domains forms two identical Fv’s. The N-terminal of the chains is represented by an N. The C-terminal of the chains is represented by a C. Each Fv comprises a VL and a VH with three CDRs (see Figure 3b). The VL and VH can be connected from C-terminal to the N-terminal by a peptide linker represented by a diagonal line, to form a scFv. The two orientations VL-linker-VH and VH-linker-VL are visualized in Figure 3c and 3d.
Figure 4 visualizes, in a simplified manner, some suitable formats for BiTEs. The smallest BiTEs will contain a first scFv connected to a second scFv by a linker (see Figure 4a). However, the first Fv may be embedded in an antibody while the second Fv may be a scFv connected to the antibody by a linker (see Figure 4b and 4c). Accordingly, the bispecific proteins may comprise more than one Fv with the first specificity and more than one Fv with the second specificity.
Figure 5 visualizes the design of a DogBiTE expression vector, here as a retroviral vector. The long terminal repeat (LTR) sequences will delimitate the region of interest for the transfer, and also serve as promoter sequence for the production. The
anti-cancer scFv is cloned at the beginning of the construct. The scFv is fused to a leader peptide (not shown) for ER-targeting and further exocytosis from the producing cell on the N-terminal part. On the C-terminal partt, a peptide linker connects it to the scFv recognizing the canine extracellular domain of the CD3ε protein which contains on its C-terminal part an His-tag for purification and detection. The sequence encoding the BiTE does not contain a STOP but a 2A ribosome skipping sequence followed by a green fluorescent protein (GFP) coding sequence. The resulting product is a BiTE comprising 2 scFv’s separated by a linker and His tagged, and a separated cytosolic GFP . The latter will be used to detect
expressing cells. 2061 and 2063 are the code name for the CD19 targeting BiTE constructs. 2062 and 2064 are the code name for the TP3 targeting BiTEs
constructs. These constructs differ in the orientation L-H or H-L of the anti-canine CD3 scFv.
2061 pMP71-CD19BiTE-cCD3_scFv(H-L)-GFP 2062 pMP71-TP3BiTE-cCD3_scFv(H-L)-GFP 2063 pMP71-CD19BiTE-cCD3_scFv(L-H)-GFP 2064 pMP71-TP3BiTE-cCD3_scFv(L-H)-GFP Figure 6 visualizes a Western blot of the different constructs used in the present disclosure. Note that anti-canine CD3 scFv was arranged either as VL-linker-VH or VH-linker-VL.
Figure 7 visualizes Western blot of the production of BiTEs. Hek cells were transfected with the indicated constructs and supernatants were harvested after 2 days. Ten microliter of a 3 mL supernatant was separated on an SDS-PAGE and
transferred to a nitrocellulase membrane. The produced BiTE were detected with anti-His tag antibody. A unique single product migrating at the expected size is observed.
1 SW2061 (old)pMP71-CD19BiTE-cCD3_scFv(H-L)-GFP 2 SW1529 pMP71-CD19BiTE-hCD3-GFP 3 SW1850 human BiTE 4 SW1851 human BiTE 5 SW1971 pMP71-TP-3BiTE-hCD3-GFP 6 SW2061 pMP71-CD19BiTE-cCD3_scFv(H-L)-GFP (DogBiTE) 7 SW2062 pMP71-TP-3BiTE-cCD3_scFv(H-L)-GFP (DogBiTE) Figure 8a, 8b, 8c visualize the target binding capacity and selectivity of the BiTE produced in Figure7. The lymphoblast cell line Jeko (CD19+, a) and the
osteosarcoma cell line OSA (ALPL-1+, b) were incubated with 25 uL of the supernatant containing the indicated BiTE constructs (SW2061 or SW2062) or no BiTE (negative control) for 15 minutes at room temperature and washed. The bound BiTEs were detected with an anti-His Tag antibody combined with AF-647
fluorophore for 5 min at room temperature in the dark. After washing, the cells were analyzed on a flowcytometer and AF-647 positive cells were detected only when the BiTEs were harboring the TP3 scFv (osteosarcoma marker 2062in b and all
constructs in c), but not when anti-CD19FMC63 scFv was present (2061 in b and c) and not with mock supernatant. Thus, DogBiTE can generate potent scFv binding to their target.
Figure 9a and 9b show a “caninized”-CD3 (cCD3) and human (hCD3) Jurkat T-cell line created from the E6 clone expressing GFP under the control of NFAT in two different experiments with two different preparation of DogBiTEs. These cell lines and the non-modified Jurkat (Mock) were incubated with the indicated target cells
(BL41, CD19+; OSA, ALPL-1+) or no target cells. Fifty µL of the indicated BiTE
preparation from Fig. 7 or a new one were added and the mix was left for 12 hours at 37°C (hBiTE: CD19-BiTE and TP3-BiTE; dogBiTE-1 and 2: TP3 DogBiTE VH- linker-VL and VL-linker-VH, respectively). The presence of GFP (produced only upon TCR stimulation) was detected by flowcytometry. 9a % of positive Jurkat cells and 9b % of positive GFP-cells. Note that the human constructs work in all conditions due to the presence of endogenous human CD3 in these cells. Importantly DogBiTEs only stimulate caninized Jurkat cell line.
Figure 10a visualizes “Caninized” or human NK-TCR cells: NK-92 were transduced or not (NT) with caninized (c)CD3 chains (SW2069) or human (h)CD3 chains (SW652, from Mensali et al, 2019) and subsequently transduced with a human TCR (SW907). The presence of the TCR was detected by flowcytometryt with an anti- hTCRa/b-PE. As shown cCD3 is able to escorte human TCR molecules to the plasma membrane in NK cells. Figure 10b depicts a functional activity test where
NK-92, NK-92-hCD3-TCR, NK-92-cCD3-TCR were incubated with BL41 expressing GFP in the presence or not of the indicated BiTE construct for 12 hours. The mix was run on flowcytometer and the presence of target cells was monitored by the % of remining GFP. As shown, NK-92 alone cannot kill BL41 cells (confirming Walseng et al.2017) and neither can NK-92-CD3-TCR without BiTEs. DogBiTEs can lead to target cell killing, only when cCD3 is expressed in the
effector cells (triangles). Note that the human BiTE also activated the cCD3-NK- TCR cells (squares), probably due to the presence of hCD3e in these cells which is sent to the plasma membrane in complex with cCD3 proteins. Indeed, it was reported that some NK cells can express CD3 proteins (see Lanier et al J Immunol,
1992 Sep 15;149(6):1876-80 “Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes” PMID: 1387664). It is tempting to speculate that due to the conservation of protein sequences between human and dog CD3 proteins, complexes of heterogenous proteins are formed as it has been observed for humans and mice (PMID: 28368009; PMID: 16951205).
Figure 11 visualizes the BLI killing assay. OSA cells expressing luciferase were incubated with “caninized” or human NK-TCR and the indicated BiTE. % of lysis is depicted as % of a saturating control. Only TP3-Bite can lead to OSA killing, and dogBiTE (cTP3BiTE) can only induce target cell killing in “caninized” NK-TCR cells.
Figure 12 visualizes the BLI killing assay. BL-41 cells expressing luciferase were incubated with “caninized” or human NK-TCR and the indicated BiTE. % of lysis is depicted as % of a saturating control. Only CD19-BiTe can lead to BL-41 killing,
and dogBiTE (cCD19BiTE) can only induce target cell killing in “caninized” NK- TCR cells.
Figure 13 visualizes the cytotoxic activities of the DogBiTEs against cancer cells expressing the targeted antigens in a BLI killing assay. Jeko-1 (CD19+) cells expressing luciferase were incubated with canine primary T cells and the indicated BiTE. % of lysis is depicted as % of a saturating control. Only CD19-BiTE can lead to Jeko-1 killing, and dogBiTE (cCD19BiTE) can only induce target cell killing in canine primary T cells.
Panel A. Canine T-cells (CTC) were incubated or not with different amounts of the 2061 BiTE construct targeting the CD19 antigen on the target cells, and with the Jeko-1 B-cell lymphoma cell line. An irrelevant BiTE construct, 2062 targeting TP3, was used as a negative control to evaluate the basic killing of the BiTE
construct. A strong and almost complete cancer cell killing with the BiTE specifically targeting the CD19 antigen on the B-cell lymphoma target cell can be observed. When no BiTES were present, only the expected non-self-killing could be observed (CTC reacting on the human cancer cells). Panel B. CTC were incubated or not with different amounts of the 2062 BiTE construct targeting the ALPL-1 (TP-3 antigen) on the target cells (OSA-787). An irrelevant BiTE construct, 2061 targeting CD19, was used as a negative control to
evaluate the basic killing of the BiTE construct. We can observe a strong and almost complete cancer cell killing with the BiTE specifically targeting the TP3 antigen on the osteosarcoma target cell. When no BiTES were present, only the expected non-
self-killing could be observed (CTC reacting on the human cancer cells). Panel C and D. CTC from two donor dogs were incubated with BiTE targeting the TP3 antigen on the osteosarcoma cell line OSA 787. Several effector to target (E:T) ratios of CTC versus target cancer cells were used with TP3 BiTE. The BiTE construct is already demonstrating cell-killing efficiency at a ratio of one Dog T- cell for one target cancer cell. The 2061 BiTE construct targeting CD19 is an irrelevant control to evaluate the non-self-killing of dog T-cells toward the human cancer cell line. Panel E. CTC from 5 different donor dogs were incubated with BiTE targeting the TP3 antigen on the osteosarcoma cell line OSA787, using 10:1 E:T ratio.
Concentration dependent specific killing with the 2062 BiTE is clearly demonstrated in all the different donor dogs.
Figure 14 illustrates the killing ability of BiTEs, highlighting the impact of different BiTE designs, in some the size of the linker between the scFvs was changed and one had a Fc-based design with knobs-in-hole (KIH) structure.
NT = control (no BiTE). Here, the DogBiTE used is the TP-3DogBiTE targeted against ALPL-1. Fig. 14 A depicts the G4S linker position in the DogBiTE molecule. Fig. 14 B depicts the design of the expression vector of the KIH DogBiTE construct and the expected molecular product. A: BiTE with G4S-linker between the scFvs B: BiTE with (G4S)2-linker between the scFvs C: BiTE with (G4S)3-linker between the scFvs D: BiTE with (EAAAK)3-linker between the scFvs
E: BiTE with knob-in-hole Fc connecting the scFv’s Figure 14 C depicts an example of functional assay which was performed with NK92-cCD3-TCR cells as in Fig. 10b. Here the target cells, OSA, were co-cultured with NK92-cCD3-TCR in the presence of the different BiTE constructs at saturating concentration for 8 hours. The killing was quantified using BLI Figure 14 D shows a Western blot with DogBiTE TP3 constructs designed with different linkers or a Knobs in Hole design. The indicated constructs were produced in Hek cells and supernatants were harvested and 10 µL/2 mL were loaded on a SDS-PAGE and detected by Western blot using an anti-His tag antibody. Concentrations were determined by density using TP3BiTE (a human TP3 BiTE used as a standard). Figure 14 E These constructs were then used at equal amount to stain the ALPL-1 positive cell line OSA or the BL41 (ALPL-1 negative cell line and CD19 positive). As shown, all constructs efficiently bound the ALPL-1+ target but not the negative one (CD19 is used as a binding control for BL41). Notably, (G4S)2 linker seems to bind with a greater affinity than the other constructs.
Figure 15 A shows the TCR complex comprising a TCR and a CD3 complex. Figure 15B shows the constructs tested in the CD3 experiment. Figure 15C illustrates the cells in the CD3 experiment.
Figure 15 D shows a flow plot of NK-92 cells transduced with the indicated cCD3 constructs +/- TCR. As shown, only the full CD3 and the cCD3εδγ have the capacity to mix with the endogenous human CD3ε and be recognized by OKT-3 in the presence of a TCR transgene. Thus, the cCD3ζ subunit is not necessary to create a functional complex. DETAILED DESCRIPTION It is to be noted that the term “a” or “an” entity refers to one or more of that entity;
for example, “a cell”, is understood to represent one or more cells. As any skilled person will understand, a cell expressing a TCR will normally express numerous such TCRs. As such, the terms “a” (or “an”), “one or more”, and “at least one” can
be used interchangeably herein. Accordingly, a cell expressing at least two different canine CD3-chains in its cell membrane will normally express numerous CD3- chains of each type in its cell membrane. The concept of specific binding to an epitope under physiological conditions is well known. Specific binding to a target may be distinguished from non-specific binding.
Accordingly, the targeting units herein (e.g. Fv’s, in particular scFv’s) display a non-covalent and reversible binding to their particular epitopes under the relevant physiological conditions. They bind with a higher affinity to the target epitope than they bind non-related molecules. The bispecific proteins herein can thus function as a coupling agent between cells expressing T-cell complexes and target cells. The binding of a targeting unit to a target epitope can be measured by conventional methods, e.g. surface plasmon resonance (SPR). The affinity between the targeting unit and its epitope can be represented by a KD-value (the equilibrium dissociation constant) for example in the range of 10
-4M to 10
-12M, such as 10
-5M to 10
-11M or 10
-6M to 10
-10M. As used herein, when referring to “sequence identity” of proteins, an amino acid sequence having at least x% identity to a second amino acid sequence means that
x% represents the fraction of amino acid residues in the first sequence which are identical to their matched amino acid residues of the second sequence when both sequences are optimally aligned via a global alignment, relative to the total length of the second amino acid sequence. Both sequences are optimally aligned when the
sequence identity value is at its maximum by using the comparison matrix BLOSUM62 with gap costs: existence 11, extension 1.
The cells for testing immunotherapeutic compounds for dogs. The present disclosure concerns human cells as a starting material. Such cells may be isolated from a healthy subject by leukapheresis or other suitable methods. The
human cells may endogenously express a TCR. Suitable examples include cytotoxic T-cells and T-helper cells. Cells isolated from a healthy subject will be primary
cells. Alternatively, the human cells can be transduced or transfected with nucleic acids encoding a TCR and CD3-chains if needed. Culturing of primary cells can be challenging, but culturing of “immortalized” cells i.e. cell-lines, is generally easier for consistently obtaining larger cell populations. The human cells in the present disclosure can for example be cell-lines suitable for
clinical use like NK-92 or Jurkat cell-lines. Jurkat cells are “immortalized” human T-cells expressing an αβTCR. Numerous Jurkat cell-lines are commercially available from the American Type Culture Collection (ATCC). NK-92 can express TCRs as disclosed by Mensali et al 2019 eBioMedicine, 40: 106–11. Such human cells, already expressing TCRs, can be modified to also express canine CD3-chains. For example, the cells can be transduced or transfected with nucleic acids encoding canine CD3-chains for transient expression. Alternatively, nucleic acids encoding canine CD3-chains can be incorporated in the cell genome for stable expression by known methods. TCRs are well-known receptors. Their variable domains, either Vα and Vβ or Vγ
and Vδ, form a single antigen binding unit based on six complementarity determining regions, CDRs. The CDRs are flanked by framework sequences. The first framework sequence is N-terminal to the CDR1, the second framework sequence is located between CDR1 and CDR2, while the third framework sequence is located between CDR2 and CDR3. Accordingly, both a Vα and a Vβ can be roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-: N-FRAMEWORK1CDR1FRAMEWORK2CDR2FRAMEWORK3CDR3FRAMEWORK4
However, the detailed mechanism of action of TCRs is not completely understood. As visualized in Figure 2, they are believed to form a TCR-complex upon specific binding to its target. Such TCR-complex involves interaction between the TCR and several CD3-chains. It is found that human cells expressing a human TCR can form a hybrid TCR-
complex with canine CD3-chains. Notably, co-expression of canine CD3ε alone in
cells expressing a human TCR, failed to present the canine CD3ε epitope on the cell surface.
However, it was found that by co-expressing all four canine CD3-chains (canine CD3ζ, canine CD3γ, canine CD3δ and canine CD3ε) in cells expressing a human
TCR, successful presentation of the canine CD3ε epitope was achieved. Accordingly, such cells are believed to express a hybrid TCR-complex in the cell
membrane comprising a human αβTCR and at least two canine CD3-chains either upon binding of the TCR target or upon clustering of CD3-chains due to binding of the bispecific proteins disclosed herein. This TCR-complex is referred to as hybrid
because it contains both human and canine proteins. As demonstrated herein, these hybrid TCR-complexes can activate the cells. Accordingly, they are believed to form a functional TCR-complex in the cell membrane. These cells can be used during drug development of immunotherapeutic drugs intended for dogs. If the candidate compounds rely on binding to TCR-complexes,
the cells may be used as in vitro tools to validate immunotherapeutic candidate compounds for dogs. These human cells are modified to represent a reliable and convenient canine model simply by expression of canine CD3-chains.
This approach can thus reduce the need for in vivo testing in living dogs or the need to create fully canine in vitro system such as an equivalent Jurkat or NK-92 cell line.
For example, the present disclosure provides an in vitro method for testing immunotherapeutic candidate proteins for dogs, comprising the steps; a) providing a cell population comprising human cells expressing a TCR and canine CD3-chains, together forming a TCR-complex, in a suitable medium, b) incubating the human cell population in step a) with an immunotherapeutic bispecific candidate protein comprising a first Fv for specific binding to an epitope located on the extracellular part of a canine CD3-chain and a second Fv for binding to an epitope located on target cells, as well as incubating the human cell population in step a) with target cells, and then
c) measuring the target cell mortality, viability, or proliferation. Culturing of the cells disclosed herein can be done by well-known methods, and a detailed example is provided in the experimental part.
Nucleic acids encoding proteins are well known for example DNA and RNA. The proteins mentioned herein can be encoded by recombinant nucleic acids.
Recombinant nucleic acids are non-naturally occurring nucleic acids. In particular,
the recombinant nucleic acid may encode canine CD3ε and at least one canine CD3- chain selected from the group consisting of CD3γ and CD3δ. The recombinant nucleic acid may encode canine CD3ε and canine CD3γ, or canine CD3ε and canine CD3δ, or canine CD3ε and canine CD3ζ, or canine CD3ε, canine CD3γ and canine
CD3δ. A recombinant nucleic acid may encode canine CD3ζ, canine CD3γ, canine CD3δ and canine CD3ε. Such recombinant nucleic acids may encode canine CD3- chains in any order. The recombinant nucleic acids may comprise promoters, enhancers and other well- known elements for providing expression of the CD3-chains in the intended cell.
Accordingly, the recombinant nucleic acid may encode and express canine CD3ε and at least one canine CD3-chain selected from the group consisting of canine
CD3γ and canine CD3δ in human cells. The recombinant nucleic acid may encode and express, in human cells, canine CD3ε and canine CD3γ, or canine CD3ε and canine CD3δ, or canine CD3ε and canine CD3ζ, or canine CD3ε, canine CD3γ and
canine CD3δ. A recombinant nucleic acid may encode and express, in human cells, canine CD3ζ, canine CD3γ, canine CD3δ and canine CD3ε. For expression in equimolar ratio, the CD3-chain can be produced as a polyprotein wherein ribosomal skipping sequences separate the CD3-chains. Ribosomal skipping sequences are well-known. NIH defines them as: a 2A is an oligopeptide sequence mediating a ribosome 'skipping' effect, producing an apparent 'cleavage' of polyproteins. First identified and characterized in picornaviruses, '2A-like' sequences are found in other mammalian viruses and a wide range of insect viruses. They are commonly used in biotechnology to produce two or more proteins in equimolar ratio, in a polycistronic design. The recombinant nucleic acid may
encode at least one ribosomal skipping sequence between two CD3-chains. The recombinant nucleic acid may encode a ribosomal skipping sequence between each of the CD3-chains.
SEQ ID NO: 1 shows one example of a polyprotein comprising four canine CD3- chains (cCD3ζ-F2A-cCD3ε-T2A-cCD3δ-E2A-cCD3γ) with leader peptides underlined and three different ribosomal skipping sequences in italic. The Ribosomal skipping sequence F2A: VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 2)
The Ribosomal skipping sequence T2A: EGRGSLLTCGDVEENPGP (SEQ ID NO: 3) The Ribosomal skipping sequence E2A: QCTNYALLKLAGDVESNPGP (SEQ ID NO: 4)
Accordingly, the recombinant nucleic acids herein can be SEQ ID NO: 1 or nucleic acids with more than 75% sequence identity thereto, such as more than 80%, 90% or
95% sequence identity thereto. The leader peptide is believed to be trimmed off and will likely not be present in the
protein in the cell membrane. In one embodiment, the present disclosure provides a human cell immune cell, such as a T-cell or NK-cell, expressing a hybrid TCR-complex in the cell membrane comprising a human αβTCR and at least two, such as three or four different canine CD3-chains including canine CD3ε. The bispecific proteins In contrast to TCRs, most naturally occurring antibodies comprise two identical
Fv’s (see Figure 2). Because both Fv’s are able to bind the same target epitope, such antibodies are monospecific and their binding to the target epitope is bivalent. An Fv is a protein moiety able to bind a target epitope under physiological conditions. The Fv comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH). Such variable domains are well-known for skilled persons.
Provided herein are proteins comprising a first targeting unit for binding to a first target epitope and a second targeting unit for binding to a second target epitope. Such proteins are bispecific. The first and second targeting unit may have any format as long as they provide
specific binding, under physiological conditions, to their target epitopes. Such binding units may be obtained by phage display libraries by conventional screening. They can also be obtained from TCRs. However, more conventionally, such targeting units may be obtained from antibodies of any type including single- domain antibodies.
In one embodiment, the targeting units can provide specific binding to a conformational epitope (i.e. a 3Dimensional epitope) rather than conventional
epitopes which often are 2Dimensional or linear. This may allow targeting of proteins that are also expressed on normal tissue, but with a different conformation in diseased tissue. Accordingly, such target epitopes can be used to distinguish
healthy cells from cancer cells.
In particular, provided herein are proteins comprising a first and a second targeting unit for binding to a first and a second target epitope, wherein the first targeting unit specifically binds, under physiological conditions, to an epitope located on the extracellular part of a canine CD3-chain, and wherein the
second targeting unit specifically binds, under physiological conditions, to an epitope located on canine or human target cells.
In one embodiment, the bispecific proteins comprise a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope. In particular, provided herein are proteins comprising a first and a second Fv for binding to a first and a second target epitope, wherein each Fv comprises a VL and a VH, and the VL comprises three CDRs flanked by framework sequences, and the VH comprises three CDRs flanked by framework sequences, wherein the first Fv specifically binds, under physiological conditions, to an epitope located on the extracellular part of a canine CD3-chain, and wherein the second Fv specifically binds, under physiological conditions, to an epitope located on canine
or human target cells. Physiological conditions, as used herein, means the environment encountered or
simulated, in a living human or canine patient, where the disclosed bispecific proteins are intended to bind an epitope located on canine or human target cells. In general, the physiological conditions in most human extracellular fluids are normally ca. 37°C, pH in the range of 6.0 to 7.5. The physiological conditions in canine extracellular fluids are normally 38-39°C, pH in the range of 6.0 to 7.5. If the target cells are cancer cells in a solid tumor, the physiological conditions are the
ones found in the solid tumor, i.e. the tumor microenvironment (TME). The TME is often hypoxic and acidic. Each VL and VH herein comprises three complementarity determining regions (CDRs) flanked by framework sequences. Framework sequences are structurally conserved regions that normally tend to form a β-sheet structure positioning the
CDRs for specific binding to the target epitope under physiological conditions. The CDR sequences herein have been determined using the well-known Kabat system. The first framework sequence is N-terminal to the CDR1, the second framework
sequence is located between CDR1 and CDR2, while the third framework sequence is located between CDR2 and CDR3. Accordingly, both a VL and VH can be
roughly visualized as follows, with the CDRs boxed and the N-terminal indicated as N-: N-FRAMEWORK1CDR1FRAMEWORK2CDR2FRAMEWORK3CDR3FRAMEWORK4
Antibodies obtained from dogs will have canine framework sequences. Antibodies obtained from mice will have murine framework sequences. Antibodies obtained from humans will have human framework sequences. However, it is well known that murine framework sequences can be completely or partially replaced by human
framework sequences. This process is called humanization, and the resulting Fv is thus humanized. Accordingly, if murine or human framework sequences in the Fv are completely or partially replaced by canine framework sequences, the resulting Fv is caninized. In one embodiment of the bispecific proteins herein, each of the framework sequences in the first Fv may independently be human, murine or canine. In one embodiment of the bispecific proteins herein, each the framework sequences in the second Fv may independently be human, murine or canine. In one embodiment of the bispecific proteins herein, all the framework sequences in the first Fv may be human, murine or canine. In one embodiment of the bispecific proteins herein, all the framework sequences in the second Fv may be human, murine or canine. A framework sequence is human if it is present in a human antibody, and it is flanking a CDR in a human VL or human VH, or it is encoded by a human genomic sequence flanking a CDR in a human VL-sequence or human VH-sequence. A framework sequence is canine if it is present in a canine antibody and it is flanking a CDR in a canine VL or canine VH, or it is encoded by a canine genomic sequence flanking a CDR in a canine VL-sequence or canine VH-sequence. A framework sequence is murine if it is present in a murine antibody and it is flanking a CDR in a murine VL or murine VH, or it is encoded by a murine genomic sequence flanking a CDR in a murine VL-sequence or murine VH- sequence.
The framework sequences may also comprise amino acid substitutions. Each of the canine framework sequences may optionally comprise 1 to 5 amino acid
substitutions if needed based on discovered or predicted immunogenicity by known methods. An amino acid substitution is a sequence wherein an amino acid residue in a specific position is substituted for a different amino acid residue at the
corresponding position, apparent when the sequences are aligned. The substitutions may be conservative substitutions. Even if such framework sequences are not
necessarily previously known from canine antibodies, they may provide lower immunogenic risk in dogs compared to a murine framework sequence. In one embodiment, the canine framework sequences are mature canine framework sequences available from known canine antibodies. Without being bound by theory, such framework sequences may convey very low risk of triggering unwanted
immunogenic responses against the Fv in dogs, and at the same time increase the likelihood of obtaining stable binding units which are expressed well in cellular systems.
In one embodiment, the human framework sequences are mature human framework sequences available from known human antibodies. Without being bound by theory, such framework sequences may convey very low risk of triggering unwanted immunogenic responses against the Fv in humans, and at the same time increase the likelihood of obtaining stable binding units which are expressed well in cellular systems.
In one embodiment, the Fv comprises or consists of VL-linker-VH. In another embodiment, the Fv comprises or consists of VH-linker-VL. Such antigen binding domains are often referred to as single chain Fv (scFv’s). The linker between VL and VH in a scFv generally needs a certain length in order to allow the VH and VL to form a functional antigen binding domain. In one
embodiment, the linker comprises 10 to 50 amino acid residues, or 15 to 30 amino acid residues. In one embodiment, the linker comprises 10 to 50 glycine and/or serine residues. One such example is provided by SEQ ID NO: 13. The VH and VL may be connected by at least one disulphide bridge or alternative linkers. The VL and VH may be embedded in a Fab-fragment of an antibody or an antibody as such. Bispecific proteins for binding and recruiting T-cells to target cells can have a
variety of formats. Examples of such T-cell-engagers are BiTEs. For example, Blinatumomab is a bispecific CD19-directed CD3 T-cell engager that binds to
CD19 expressed on the surface of cells of B-lineage origin and CD3 expressed on the surface of T-cells. It activates endogenous T-cells by connecting CD3 in the T-
cell receptor (TCR) complex with CD19 on benign and malignant B cells. Blinatumomab mediates the formation of a synapse between the T-cell and the
tumor cell, upregulation of cell adhesion molecules, production of cytolytic proteins, release of inflammatory cytokines, and proliferation of T-cells, which result in redirected lysis of CD19+ cells. Many types of bispecific T-cell engagers are known. Some examples are described by Goebeler and Bargou (Nat. Rev. Clin. Oncol.17(7):418-434 (2020); doi:
10.1038/s41571-020- 0347-5), or by Zhou et al. (Biomarker Research 9(38) (2021)). The BiTEs disclosed in the examples comprise a well-known C-terminal oligopeptide comprising multiple histidine residues, also known as a “His-tag”, for purification and detection during research and early development. Such “His-tag” will not necessarily be present in a protein drug. The first targeting unit
The first targeting unit for specific binding to a first target epitope located on the extracellular part of canine CD3-chain will provide binding to cells expressing a TCR-complex comprising the canine CD3-chain. The TCR does not signal and associates at the ER with CD3 proteins which will serve of signaling units.
The first targeting unit may be a Fv for specific binding to a first target epitope located on the extracellular part of canine CD3-chain. It will provide binding to cells expressing a TCR-complex comprising the canine CD3-chain. Accordingly, a
first Fv for specific binding to a first target epitope located on the extracellular part of canine CD3ε-chain will provide binding to cells expressing a TCR-complex comprising a canine CD3ε-chain. However, as demonstrated herein, the first Fv may also provide specific binding to cells expressing a TCR-complex comprising a
human CD3ε-chain. Such cross-reactivity is beneficial because the same protein can thus bind both canine and human T-cells. One such example of first Fv with cross-reactivity for both canine and human
CD3ε-chains is represented by the scFv with the SEQ ID NO: 5. Accordingly, the present disclosure provides an Fv comprising a VL with the following three CDRs; CDR1: QSIFKN CDR2: YAS CDR3: LQAYSTPWT and a VH with the following three CDRs;
CDR1: GYTFSDYNM
CDR2: IYPYNGGT CDR3: ARLVYFDY. Another example is the scFv with the SEQ ID NO: 6. It comprises the same CDRs as the scFv with SEQ ID NO: 5. The present disclosure also provides scFv’s wherein the sequences have more than 70%, such as 75% to 99% sequence identity to SEQ ID NO: 5. The present disclosure also provides scFv’s wherein the sequences have more than 70%, such as 75% to 99% sequence identity to SEQ ID NO: 6. The present disclosure also provides scFv’s wherein the sequences have more than 70%, such as 75% to 99%, such as 85% to 99% sequence identity to SEQ ID NO: 5, provided the CDRs are identical to the ones in SEQ ID NO: 5. Such scFv’s may for example be humanized versions of the scFv. The present disclosure also provides scFv’s wherein the sequences have more than 70%, such as 75% to 99%, such as 85% to 99% sequence identity to SEQ ID NO: 6, provided the CDRs are identical to the ones in SEQ ID NO: 6. Such scFv’s may for example be humanized versions of the scFv. However, it was found that in the BiTEs comprising the scFv represented by SEQ ID NO: 5 performed better than BiTEs comprising the scFv represented by SEQ ID NO: 6. Accordingly, for this Fv, VH-linker-VL may be the preferred orientation in scFv’s.
It was found that this Fv activated the canine T-cells upon binding. Because the affinity and the position of the epitope may affect the efficacy of a synapse formation on the T-cell, any anti-CD3 Fv should be tested in a scFv-format for the ability to stimulate T-cells through TCR clustering. Indeed, the binding of an scFv to CD3 is not sufficient to make a potent BiTE. scFv must be tested experimentally
and the epitope location must be extracellular (see Zhou et al, Biomarker Research volume 9, Article number: 38 (2021) “The landscape of bispecific T cell engager in
cancer treatment” and Tian et al. Journal of Hematology & Oncology volume 14, Article number: 75 (2021) “Bispecific T cell engagers: an emerging therapy for the management of hematologic malignancies” https://biomarkerres.biomedcentral.com/articles/10.1186/s40364-021-00294-
9#Sec18; https://doi.org/10.1186/s13045-021-01084-4). The testing can be run using primary T cells, which are cocultured with target cells (expressing the epitope recognized by the second scFv). Activation of T cells is routinely performed and
can be the detection of activation marker (CD69), degranulation markers (CD107a) or killing (bioluminescence-based assays, BLI or Chromium release).
The BiTEs herein are believed to involve a mechanism for T-cell activation: 1) The tumor cell plays an indispensable role in the T cell activation induced by the BiTE. In other words, the BiTE cannot activate the T cells alone, but needs to be clustered by another mean (tumour antigen) to trigger T-cell activation. This T-cell activation is TCR dependent, meaning that it is the result of TCR clustering that will trigger the necessary signalling components to enable the cellular changes related to activation. 2) T-cells can be activated without costimulatory signals such as CD28 and interleukin (IL)-2 where this feature can be attributed to memory T-cells which play
an important role in the reaction to BiTEs (Godbersen C, Coupet TA, Huehls AM, Zhang T, Battles MB, Fisher JL, et al. NKG2D ligand-targeted bispecific T-Cell engagers lead to robust antitumor activity against diverse human tumors. Mol Cancer Ther.2017;16(7):1335–46.) 3) The immunological synapses between T-cells and tumor cells can assemble TCRs and amplify the first signals of activation because the Immunological synapses
between T-cells and tumor cells are also crucial to the BiTE-mediated tumor lysis by perforin (Huehls AM, Coupet TA, Sentman CL. Bispecific T-cell engagers for cancer immunotherapy. Immunol Cell Biol.2015;93(3):290–6.).
Accordingly, provided herein are proteins comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the first Fv is cross-reactive with both human and canine homologs of the target
protein. Especially suitable as such target proteins are CD3-chains such as CD3γ, CD3δ and CD3ε.
Based on the present disclosure, skilled persons can obtain such Fv’s by employing conventional methods. For example, the desired human epitope can be used as an antigen in mice or rabbits for obtaining antibodies with specific binding to it, and
then screen for cross-reactivity to the canine variant. For example, the desired canine epitope can be used as an antigen in mice or rabbits for obtaining antibodies with specific binding to it, and then screen for cross-reactivity to the human variant. There are several techniques to generate a single-chain variable fragment (scFv) for antigen recognition: 1) By amplification and cloning of VL and VH antibody sequences: scFv sequences have traditionally been cloned by amplifying the variable light (VL) and variable heavy (VH) antibody sequences of hybridomas.
2) By a Phage Display approach: For phage display, a pooled library of scFv’s is displayed on the coats of bacteriophages and screening for antigen binding. 3) Recombinant scFv format (mouse, Rabbit): This involves generating a recombinant scFv format for a specific monoclonal antibody (mAb). However, the first targeting unit does not necessarily have to comprise an Fv. One example of a targeting unit in the form of a peptide ligand for human CD3ε is
disclosed by Ahmadi et al J Mater Chem B, 2021 Feb 14;9(6):1661-1675. doi: 10.1039/d0tb02235g. Epub 2021 Jan 22. Such peptide ligands which specifically binds, under physiological conditions, to an epitope located on the extracellular part
of a canine CD3-chain can also be obtained by conventional methods. Non-scFv based targeting units can be implemented in BiTEs. For example: 1) Aptamer based BitEs TLS11a Aptamer/CD3 Antibody Anti-Tumor System for Liver Cancer. DOI: https://doi.org/10.1166/jbn.2018.2619 2) Single domain antibody-based BiTes ang, Xm., Lin, Xd., Shi, W. et al. Nanobody-based bispecific T-cell engager (Nb-BiTE): a new platform for enhanced T-cell immunotherapy. Sig Transduct Target Ther 8, 328 (2023). https://doi.org/10.1038/s41392-023-01523-3 3) HLE Bite (to increase half-life of Bites by adding an Fc domain) 4) dAbs VH domain antibody (dAb) Development of single domain agents including: VHH domains, v-NARs, FN3-based binders, human dAbs. 5) CD3 is linked to a receptor, like NKG2D. 6) CD3 is linked to a 14FN3 Based Single Domain Proteins (doi.org/10.3390/biomedicines10123184) The second targeting unit
The second targeting unit for specific binding to a second target epitope located on the target cells will provide binding to said target cells. The second targeting unit may be a Fv for specific binding to a second target epitope located on the target cells providing binding to said target cells. The target cells may be any kind of cell where close localization of T-cells are desired, e.g. for cytotoxic activity. The target cells may be cancer cells, but the target does not have to be cancer specific. For example, CD19 is used in cancer treatment even though CD19 is also expressed
on healthy cells which are expendable. However, choosing a cancer associated antigen can provide better therapeutic specificity. A true cancer-restricted antigen would of course provide the best therapeutic specificity.
Many target epitopes located on the cancer cells are being explored. For example,
human epidermal growth factor receptor 2 (HER2), PCMA, CD22, CD19. CD19 and CD20 are targets for treatment of NHL. CD19 is the target for the treatment of acute lymphocytic leukemia. CD123, CD33, CLEC12A, WT1, and FLT3 are targets for treatment of acute myeloid leukemia. BCMA, CD38, and GPRC5D are targets for the treatment of multiple myeloma. EpCAM for solid
malignancies of epithelial origin, CEA for gastrointestinal tract, sweat gland and prostate epithelium, PSMA for prostate cancer. For example, the second Fv may specifically bind, under physiological conditions,
to an epitope on ALPL-1 on an osteosarcoma cell, both in human and dogs. Double targeting has been exploited (combi or dual), which can also be exploited in BiTE format.
The target epitope can be a secondary modification of a protein such as sugar or phosphorylation. One specific example of a suitable second Fv is represented by the scFv with the SEQ ID NO: 14 derived from the known antibody TP3. Said antibody specifically binds to osteosarcoma cells.
Another example is the scFv with the SEQ ID NO: 15 derived from the known antibody FMC63. The second Fv may have a TCR-like specificity, i.e. it may specifically bind an MHC presenting a specific peptide, in particular an HLA presenting a specific peptide. Methods for obtaining such Fv’s are well known and one example is provided in Yarmarkovich, M., Marshall, Q.F., Warrington, J.M. et al. Targeting of intracellular oncoproteins with peptide-centric CARs. Nature 623, 820–827 (2023). https://doi.org/10.1038/s41586-023-06706-0. Based on the present disclosure, skilled persons can obtain such Fv’s by employing
conventional methods. For example, the desired cancer epitope can be used as an antigen in mice or rabbits for obtaining antibodies with specific binding to it. The purified protein or an epitope in this protein (peptide) can be used to pan a phage library and isolate binding sequences. These sequences can be peptidic or scFv, they
will then be used as targeting unit of the BiTE.
The bispecific proteins herein can also contain Fv’s for specific binding to other
non-cancerous targets like CD19. Accordingly, provided herein are proteins comprising a first Fv for binding to a first target epitope and a second Fv for binding to a second target epitope, wherein the second Fv is cross-reactive with both human and canine homologs of the target protein. Pharmaceutical compositions may comprise the bispecific proteins or nucleic acids disclosed herein. The pharmaceutical compositions can be a composition suitable
for administration of therapeutic proteins or nucleic acids to a patient. The most common administration route for therapeutic proteins is parenteral administration. Accordingly, said pharmaceutical compositions may for example be sterile aqueous solutions with a neutral pH. Accordingly, said pharmaceutical compositions may for
example be sterile and isotonic aqueous solutions with a physiological pH. SEQUENCES
SEQ ID NO: 1 represents a polyprotein comprising canine CD3-chains (cCD3ζ- F2A-cCD3ε-T2A-cCD3δ-E2A-cCD3γ) with leader peptides underlined and the ribosomal skipping sequences in italic: MWKMLVITALLQAQLPVTGAQSLGLLDPKLCYLLDGVLFIYGVIITALFLRAKFGRSAA APEHQQGPNQLYNELNLRGREEYEVLDKRRGLDPEMGGKQRKRNPQEVVYNALQKDKMA EAYSEIGIKSENQRRRGKGHDGLYQGLSTATKDTYDALHMQALPPRVKQTLNFDLLKLA GDVESNPGPMQSRNLWRILGLCLLSVGAWGQDEDFKASDDLTSISPEKRFKVSISGTEV VVTCPDVFGYDNIKWEKNDNLVEGASNRELSQKEFSEVDDSGYYACYADSIKEKSYLYL RARVCANCIEVNLMAVVTIIVADICLTLGLLLMVYYWSKTRKANAKPVMRGTGAGSRPR GQNKEKPPPVPNPDYEPIRKGQQDLYSGLNQRGIEGRGSLLTCGDVEENPGPMEHCRFL AGLILAVLLSRVSPFKVSVEELEDRVFLSCNTSVLRIEGTMGIQLPHSRTLDLGKRILD PRGIYRCNATEEQSDKAPYMQVYYRMCQNCVELNSATLAGIVIADIIATLLLALGVYCF AGHETGRSSKSADTQILLENDQLYQPLRDRNDAQYSHLGENWPRKKQCTNYALLKLAGD VESNPGPMELGKHLAGLILAVTLLQGATAQKEQGFPMIKVDGNREDGSVLLICDSQSKD IKWFEDGKERTLPKKDKKTLDLGSSMKDPQGIYQCQVAKNISKPLQVYYRMCQNCIELN AGTIIGFVFAEIISIFFLAVGVYFIAGQDGVRQSRASDKQTLLSNDQLYQPLRDRENDQ YGHLQGKRLRKN SEQ ID NO: 2 (The Ribosomal skipping sequence F2A): VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 3 (The Ribosomal skipping sequence T2A): EGRGSLLTCGDVEENPGP
SEQ ID NO: 4 (The Ribosomal skipping sequence E2A): QCTNYALLKLAGDVESNPGP
SEQ ID NO: 5 (cCD3_scFv (VH-Linker-VL)) wherein the CDRs are boxed and the linker is underlined: EVQLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYNGGTY YNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALTVSSVEG GSGGSGGSGGSGGVDDILLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSP RLLIKYASDSISGIPSRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTK LEIK
SEQ ID NO: 6 (cCD3_scFv(VL-linker-VH)) wherein the CDRs are boxed and the linker is underlined:
SEQ ID NO: 7 (VH CDR1): GYTFSDYNM
SEQ ID NO: 8 (VH CDR2): IYPYNGGT
SEQ ID NO: 9 (VH CDR3): ARLVYFDY
SEQ ID NO 10 (VL CDR1): QSIFKN
SEQ ID NO 11 (VL CDR2): YAS
SEQ ID NO 12 (VL CDR3): LQAYSTPWT
SEQ ID NO: 13 Linker: VEGGSGGSGGSGGSGGVD
SEQ ID NO: 14 TP3 scFv (VL-linker-VH) wherein the CDRs are boxed and the leader peptide is underlined, and the linker is in italic: METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSD
SEQ ID NO: 15 CD19-fmc63 scFv wherein the CDRs are boxed and the leader peptide is underlined, and the linker is in italic: METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC
SEQ ID NO: 16 BiTE 2061 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPGGGGSEVQLQQSGP ELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYNGGTYYNQKFKS KATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALTVSSVEGGSGGS GGSGGSGGVDDILLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLL IKYASDSISGIPSRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKL EIKHHHHHH
SEQ ID NO: 17 BiTE 2062 with N-terminal leader peptide and C-terminal His-tag
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSDPGGGGSEV QLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYNGGTY YNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALTVSSV EGGSGGSGGSGGSGGVDDILLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKS HRSPRLLIKYASDSISGIPSRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWT FGGGTKLEIKHHHHHH
SEQ ID NO: 18 BiTE 2063 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPGGGGSDILLTQSPA TLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLLIKYASDSISGIPSRFTGSG SGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKLEIKVEGGSGGSGGSGGSGG VDEVQLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYN GGTYYNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALT VSSHHHHHH
SEQ ID NO: 19 BiTE 2064 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSDPGGGGSDI LLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLLIKYASDSISGIP SRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKLEIKVEGGSGGSG GSGGSGGVDEVQLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWI GYIYPYNGGTYYNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYW GQGTALTVSSHHHHHH
EXAMPLES Material and methods
Production of bispecificT-cell engager (BiTEs). HEK293T cells (ATCC) were seeded at 1.2x10
6 cells per 60 mm plate in 3 mL
DMEM (D5671, Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1% L-Glut (Sigma-Aldrich). After 24 hrs., cells were added a transfection mix. The transfection mix was prepared by mixing 300 ^L OptiMEM (Gibco, ThermoFisher Scientific), 9 ^L X-tremeGENE9 DNA Transfection Reagent (Roche, Merck) and 3.0 ^g expression vector (SW2061 or SW2062) and incubating it for 15 min. The HEK293T cells were incubated O/N at 37^C and 5% CO2 before the medium was changed to fresh IMDM (Cytiva) supplemented with 1% L-glut and 1% HI HyClone FBS (Cytiva). The cells were further incubated for 48 hrs at 32 ^C 5% CO2 before
the supernatant containing BiTEs was harvested by centrifuging at 500 X g for 5min to remove packaging cells. Supernatant form untransformed HEK293T cells treated the same way as the transformed HEK293T cells was collected as a control, termed
“noBiTE”. The supernatants were stored at 4 ^C until use (within 2 weeks). Verification of BiTE production by flow cytometry.
BiTE production was verified by flow cytometry. Target cells were washed in Phosphate buffer saline (PBS) and centrifuged at 300 X g for 5min. 1x 106 target cells were resuspended with 60 ^L BiTE supernatant, noBiTE supernatant or flow
buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide) (unstained control), transferred to FACS tubes and then incubated for 30 min at RT. Five mL flow buffer was added to all samples, and the cells were spun down at 300 X g for 5 min. The cells were then resuspended in 90 ^L flow buffer, added 10 ^L hFc Block (Miltenyi Biotec) and incubated for 10 min at RT. 0.5 ^L anti-His-Tag Ab-Alexa 647 (R&D systems, clone#AD1.1.10R) or flow buffer (unstained control) were directly added to the cells. After 30 min incubation at RT, protected from light, the cells were washed twice with flow buffer and centrifuged at 300 X g for 5 min. The
cells were resuspended in 300 ^L PBS and acquired on Accuri C6 (BD Biosciences).
CanineT-cell (cTc) isolation and activation. The canine blood collection received ethical approval by the Norwegian Food Safety Authority (FOTS 20944), according to the national regulation for use of
animals in research. Peripheral blood mononuclear cell (PBMC) was collected from healthy dogs in 6ml Heparin tubes and processed as soon as possible. The blood was kept at room temperature and under constant agitation in the meantime. Canine
PBMC´s were isolated by gradient centrifugation using Lymphoprep ™ (Alere Technologies AS). The PBMC´s were washed twice in PBS and resuspended in T- cell medium (TCM), consisting of HyClone IMDM supplemented with 25 mM
HEPES (Cytiva), 10% HI HyClone FBS (Cytiva), 1% Glutamax (ThermoFisher), 1% MEM non-essential amino acid solution (Sigma), 1% Sodium Pyruvate (Sigma Aldrich) and 1% Pen/Strep. The PBMC´s were counted, then centrifuged at 500 X g for 10 min, and the amount of TCM, antibodies and FcR blocking reagent needed
for the negativeT-cell selection were calculated (listed in table 1, below). Table 1: Antibodies and reagent volumes for canine T-cell isolation by MACS negative selection P
rimary antibodies (mAb) Amount Mouse anti-dog CD11b, clone CA16.3E10 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-dog CD11c, clone CA11.6A1 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-dog CD21, clone CA2.1D6 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-human CD14, clone TuK4 (Invitrogen) 1 µg/ 1x 107 cells Dog gamma globulins (Jackson Immunology) 10 µL/ 1x 107 cells TCM (90 µL/ 1x 107 cells) – mAb volume Total volume 100 µL/ 1x 107 cells From this step, the work was done on ice and the solutions kept cold. The PBMC´s were resuspended in TCM and the required amount of dog gamma-globulins were added. The cells were mixed gently and incubated for 10-15 min at RT. Then the primary antibodies were added, the cells gently mixed and incubated for 30 min at 4 ^C. The cells were then washed and centrifuged at 500 X g for 10 min, twice. The cell pellet was resuspended in 80 ^L TCM per 10
7 total cells and 20 ^L of anti- Mouse IgG MicroBeads (Miltenyi Biotec) was added per 10
7 cells. The cells were
mixed well and incubated for 15 min at 4 ^C before washing them twice in TCM
using up to 50 mL. The cell pellet was resuspended in TCM at 1x10
8 cells/500 mL and then applied to a LS Column (Miltenyi Biotec) according to the manufacturers protocol. A pre-selection sample and the antibody-bound fraction were also
collected to assess theT-cell purity by flow cytometry. Verification ofT-cell purity by flow cytometry. Cells were counted and centrifuged at 300 X g for 5 minutes. The samples were resuspended in staining buffer (FACS), before adding 10uL canine FC block (Invitrogen), and incubating for 10 mins at RT. The antibody master-mix was added as specified in table 2 below, and samples were incubated for 30min at 4°C. Cells were washed twice
with the staining buffer and centrifuged at 300 X g for 5 minutes, then re-suspended in PBS and acquired on the BD LSR Fortessa cytometer. Table 2: Antibodies used for flow cytometry evaluation of T-cell purity. mAb (clone) Channel Concentration Volume uL BFortessa Pr. Total sample C
D3e-FITC (CA17.2A12) 488 laser, filter 530/30 1:10 10 120 CD5-PE (YKIX322.3) * 561 laser, filter 582/15 1:50 2 24 CD4-SB645 (tandem, 407) 407 laser, filter 660/20 1:10 10 120 CD8-APC * 640 laser, filter 670/14 1:25 4 48 CD25-SB600 (tandem, 407) 407 laser, filter 610/20 1:50 2 24 Total 40uL pr sample x/12 480uL / 144 PBS Activation of T cells. The T-cells were activated by adding Dynabeads (M-450, Invitrogen) coated with canine anti-CD3, clone CA17.2A12 and canine anti-CD28, clone 1C6 (manufactured as described in Rotolo et al, doi.org/10.1016/j.xpro.2021.100905), at a ratio of 1:2. In addition, 10uL of canine specific IL-2 (100mg/uL, Cys147Ser, BioTechne) was added. The T-cells were seeded at a density of 1–2 x 10
6 cell/mL in 6-well plates (ThermoScientific), and expanded twice, before being frozen as described below.T-cells were counted, centrifuged at 300 X g, 5min and then resuspended in freeze medium (FBS (Sigma-Aldrich) with 10% DMSO (Sigma- Aldrich) or Cryostor-CS10 (Sigma-Aldrich), to a final concentration of 5x10
6 cells per 1 mL. Cryovials were placed in a freezing device (Naglene, Sigma-Aldrich) O/N
at -80 ^C. For short-term storage, the canineT-cells (cTcs) were kept at -80 ^C.
Bioluminescence killing assay. The osteosarcoma cell line OSA 787
luc+ was seeded in triplicate at 8000 cells/well
in a black walled 96 well plate (Corning Incorporated). The mantle cell lymphoma cell line JeKo-1
luc+ was also seeded in triplicate at 30,000 cells/well in a round bottom 96 well plate (Greiner).100 ^L RPMI 1640 (Sigma-Aldrich) supplemented
with 10% FBS (Sigma-Aldrich) and 1% L-Glut (Sigma-Aldrich) was added to each well. OSA 787luc+ was seeded 24 hrs before adding cTcs, whilst JeKo-1luc+ was seeded 2 hrs prior to adding of cTc. The cTcs were thawed by gently warming the cryotube for 1 min in a 37 ^C water bath, then the content was resuspended in 10
mL tissue culture medium (TCM) and centrifuged at 300 X g for 5 min to remove DMSO. The cTcs were counted and allowed to recover at a concentration of 0.5x106 cells/mL in TCM for 2 hrs at 37 ^C prior to adding them to target cells. The cTcs were added in 90 ^L TCM per well. In wells with no cTc, 90 ^L TCM was added. For the wells containing only cTcs, 100 ^L supplemented RPMI was added.
Treatment was added, in triplicate, with BiTE supernatant, noBiTE supernatant, 1% Triton X-100 (CAS-No: 9036-19-5, Sigma-Aldrich) or supplemented RPMI,
directly after the adding of cTcs. All wells, where needed, were supplemented with RPMI to reach a final volume of 200 ^L. The target cells mixed with cTcs and treatment, was then incubated for 24 hrs. After incubation, luciferin was added at a concentration of 2.5 mg/mL per well, incubated for 10 min protected from light, and then imaged using the IVIS Spectrum Series Imager (PerkinElmer Inc, Waltham,
MA, USA). Quantification using photons per second (p/s) was performed with the Live Image® Software (PerkinElmer Inc), and the percentage specific killing was calculated using the following equation: ^
^^^^^^^^^^ ^^^^ ^^^^ℎ − ^^^^^^ % ^^^^^^^^ ^^^^^^^ = 100 × ^^^^^^^^^^^ ^^^^ ^^^^ℎ − ^^^^^^^ ^^^^^^^ Spontaneous cell death is p/s from target cells only, sample is p/s from the sample where the specific killing is unknown, maximal killing is the p/s from the 1% Triton X-100 treated sample.
CD3 experiment We cloned different units of the canine CD3 (cCD3) isoforms to test the minimal
cCD3 requirement to accommodate TCRs. We monitored human CD3ε expression at the surface of NK-92 cells.
We compared three constructs: cCD3 full which comprises of all the CD3 units (ε,γ,δ, ζ,), cCD3 ε,γ,δ (with 3 CD3 subunits) and CD3ε with the ε subunit only. The results are presented in Figure 15D.
SEQ ID NO: 7 (VH CDR1): GYTFSDYNM SEQ ID NO: 8 (VH CDR2): IYPYNGGT SEQ ID NO: 9 (VH CDR3): ARLVYFDY SEQ ID NO 10 (VL CDR1): QSIFKN SEQ ID NO 11 (VL CDR2): YAS
SEQ ID NO 12 (VL CDR3): LQAYSTPWT SEQ ID NO: 13 Linker: VEGGSGGSGGSGGSGGVD SEQ ID NO: 14 TP3 scFv (VL-linker-VH) wherein the CDRs are boxed and the leader peptide is underlined, and the linker is in italic: METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSD SEQ ID NO: 15 CD19-fmc63 scFv wherein the CDRs are boxed and the leader peptide is underlined, and the linker is in italic: METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC
SEQ ID NO: 16 BiTE 2061 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPGGGGSEVQLQQSGP ELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYNGGTYYNQKFKS KATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALTVSSVEGGSGGS GGSGGSGGVDDILLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLL IKYASDSISGIPSRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKL EIKHHHHHH SEQ ID NO: 17 BiTE 2062 with N-terminal leader peptide and C-terminal His-tag
METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSDPGGGGSEV QLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYNGGTY YNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALTVSSV EGGSGGSGGSGGSGGVDDILLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKS HRSPRLLIKYASDSISGIPSRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWT FGGGTKLEIKHHHHHH SEQ ID NO: 18 BiTE 2063 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTL PYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVF LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPGGGGSDILLTQSPA TLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLLIKYASDSISGIPSRFTGSG SGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKLEIKVEGGSGGSGGSGGSGG VDEVQLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWIGYIYPYN GGTYYNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYWGQGTALT VSSHHHHHH SEQ ID NO: 19 BiTE 2064 with N-terminal leader peptide and C-terminal His-tag METDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASKSVSTGYSYLH WYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHS RELPLTFGAGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGAELVKPGASVKI SCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYDSTRYNQKFKGKATLTVDKSSS TAYMELRSLTSEDTAVYYCARGDYYVSSYGHDYAMDYWGQGTSVTVSSDPGGGGSDI LLTQSPATLSVTPGETVSLSCRASQSIFKNLHWYQQKSHRSPRLLIKYASDSISGIP SRFTGSGSGTDYTLSINSVKPEDEGVYYCLQAYSTPWTFGGGTKLEIKVEGGSGGSG GSGGSGGVDEVQLQQSGPELVKPGASVKISCKASGYTFSDYNMHWVKQSHGESLEWI GYIYPYNGGTYYNQKFKSKATLTVDNSSSTAYMEFRSLTSEDSAVYYCARLVYFDYW GQGTALTVSSHHHHHH
EXAMPLES Material and methods Production of bispecificT-cell engager (BiTEs). HEK293T cells (ATCC) were seeded at 1.2x106 cells per 60 mm plate in 3 mL DMEM (D5671, Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1% L-Glut (Sigma-Aldrich). After 24 hrs., cells were added a transfection mix. The transfection mix was prepared by mixing 300 ^L OptiMEM (Gibco, ThermoFisher Scientific), 9 ^L X-tremeGENE9 DNA Transfection Reagent (Roche, Merck) and 3.0 ^g expression vector (SW2061 or SW2062) and incubating it for 15 min. The HEK293T cells were incubated O/N at 37^C and 5% CO2 before the medium was changed to fresh IMDM (Cytiva) supplemented with 1% L-glut and 1% HI HyClone FBS (Cytiva). The cells were further incubated for 48 hrs at 32 ^C 5% CO2 before the supernatant containing BiTEs was harvested by centrifuging at 500 X g for 5min to remove packaging cells. Supernatant form untransformed HEK293T cells treated the same way as the transformed HEK293T cells was collected as a control, termed “noBiTE”. The supernatants were stored at 4 ^C until use (within 2 weeks). Verification of BiTE production by flow cytometry. BiTE production was verified by flow cytometry. Target cells were washed in Phosphate buffer saline (PBS) and centrifuged at 300 X g for 5min. 1x 106 target cells were resuspended with 60 ^L BiTE supernatant, noBiTE supernatant or flow buffer (PBS supplemented with 0.5% BSA and 0.1% sodium azide) (unstained control), transferred to FACS tubes and then incubated for 30 min at RT. Five mL flow buffer was added to all samples, and the cells were spun down at 300 X g for 5 min. The cells were then resuspended in 90 ^L flow buffer, added 10 ^L hFc Block (Miltenyi Biotec) and incubated for 10 min at RT. 0.5 ^L anti-His-Tag Ab-Alexa 647 (R&D systems, clone#AD1.1.10R) or flow buffer (unstained control) were directly added to the cells. After 30 min incubation at RT, protected from light, the cells were washed twice with flow buffer and centrifuged at 300 X g for 5 min. The
cells were resuspended in 300 ^L PBS and acquired on Accuri C6 (BD Biosciences). CanineT-cell (cTc) isolation and activation. The canine blood collection received ethical approval by the Norwegian Food Safety Authority (FOTS 20944), according to the national regulation for use of animals in research. Peripheral blood mononuclear cell (PBMC) was collected from healthy dogs in 6ml Heparin tubes and processed as soon as possible. The blood was kept at room temperature and under constant agitation in the meantime. Canine PBMC´s were isolated by gradient centrifugation using Lymphoprep ™ (Alere Technologies AS). The PBMC´s were washed twice in PBS and resuspended in T- cell medium (TCM), consisting of HyClone IMDM supplemented with 25 mM HEPES (Cytiva), 10% HI HyClone FBS (Cytiva), 1% Glutamax (ThermoFisher), 1% MEM non-essential amino acid solution (Sigma), 1% Sodium Pyruvate (Sigma Aldrich) and 1% Pen/Strep. The PBMC´s were counted, then centrifuged at 500 X g for 10 min, and the amount of TCM, antibodies and FcR blocking reagent needed for the negativeT-cell selection were calculated (listed in table 1, below). Table 1: Antibodies and reagent volumes for canine T-cell isolation by MACS negative selection Primary antibodies (mAb) Amount Mouse anti-dog CD11b, clone CA16.3E10 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-dog CD11c, clone CA11.6A1 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-dog CD21, clone CA2.1D6 (Invitrogen) 1 µg/ 1x 107 cells Mouse anti-human CD14, clone TuK4 (Invitrogen) 1 µg/ 1x 107 cells Dog gamma globulins (Jackson Immunology) 10 µL/ 1x 107 cells TCM (90 µL/ 1x 107 cells) – mAb volume Total volume 100 µL/ 1x 107 cells From this step, the work was done on ice and the solutions kept cold. The PBMC´s were resuspended in TCM and the required amount of dog gamma-globulins were added. The cells were mixed gently and incubated for 10-15 min at RT. Then the primary antibodies were added, the cells gently mixed and incubated for 30 min at 4 ^C. The cells were then washed and centrifuged at 500 X g for 10 min, twice. The cell pellet was resuspended in 80 ^L TCM per 107 total cells and 20 ^L of anti- Mouse IgG MicroBeads (Miltenyi Biotec) was added per 107 cells. The cells were mixed well and incubated for 15 min at 4 ^C before washing them twice in TCM
using up to 50 mL. The cell pellet was resuspended in TCM at 1x108 cells/500 mL and then applied to a LS Column (Miltenyi Biotec) according to the manufacturers protocol. A pre-selection sample and the antibody-bound fraction were also collected to assess theT-cell purity by flow cytometry. Verification ofT-cell purity by flow cytometry. Cells were counted and centrifuged at 300 X g for 5 minutes. The samples were resuspended in staining buffer (FACS), before adding 10uL canine FC block (Invitrogen), and incubating for 10 mins at RT. The antibody master-mix was added as specified in table 2 below, and samples were incubated for 30min at 4°C. Cells were washed twice with the staining buffer and centrifuged at 300 X g for 5 minutes, then re-suspended in PBS and acquired on the BD LSR Fortessa cytometer. Table 2: Antibodies used for flow cytometry evaluation of T-cell purity. mAb (clone) Channel Concentration Volume uL BFortessa Pr. Total sample CD3e-FITC (CA17.2A12) 488 laser, filter 530/30 1:10 10 120 CD5-PE (YKIX322.3) * 561 laser, filter 582/15 1:50 2 24 CD4-SB645 (tandem, 407) 407 laser, filter 660/20 1:10 10 120 CD8-APC * 640 laser, filter 670/14 1:25 4 48 CD25-SB600 (tandem, 407) 407 laser, filter 610/20 1:50 2 24 Total 40uL pr sample x/12 480uL / 144 PBS Activation of T cells. The T-cells were activated by adding Dynabeads (M-450, Invitrogen) coated with canine anti-CD3, clone CA17.2A12 and canine anti-CD28, clone 1C6 (manufactured as described in Rotolo et al, doi.org/10.1016/j.xpro.2021.100905), at a ratio of 1:2. In addition, 10uL of canine specific IL-2 (100mg/uL, Cys147Ser, BioTechne) was added. The T-cells were seeded at a density of 1–2 x 106 cell/mL in 6-well plates (ThermoScientific), and expanded twice, before being frozen as described below.T-cells were counted, centrifuged at 300 X g, 5min and then resuspended in freeze medium (FBS (Sigma-Aldrich) with 10% DMSO (Sigma- Aldrich) or Cryostor-CS10 (Sigma-Aldrich), to a final concentration of 5x106 cells per 1 mL. Cryovials were placed in a freezing device (Naglene, Sigma-Aldrich) O/N at -80 ^C. For short-term storage, the canineT-cells (cTcs) were kept at -80 ^C.
Bioluminescence killing assay. The osteosarcoma cell line OSA 787luc+ was seeded in triplicate at 8000 cells/well in a black walled 96 well plate (Corning Incorporated). The mantle cell lymphoma cell line JeKo-1luc+ was also seeded in triplicate at 30,000 cells/well in a round bottom 96 well plate (Greiner).100 ^L RPMI 1640 (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 1% L-Glut (Sigma-Aldrich) was added to each well. OSA 787luc+ was seeded 24 hrs before adding cTcs, whilst JeKo-1luc+ was seeded 2 hrs prior to adding of cTc. The cTcs were thawed by gently warming the cryotube for 1 min in a 37 ^C water bath, then the content was resuspended in 10 mL tissue culture medium (TCM) and centrifuged at 300 X g for 5 min to remove DMSO. The cTcs were counted and allowed to recover at a concentration of 0.5x106 cells/mL in TCM for 2 hrs at 37 ^C prior to adding them to target cells. The cTcs were added in 90 ^L TCM per well. In wells with no cTc, 90 ^L TCM was added. For the wells containing only cTcs, 100 ^L supplemented RPMI was added. Treatment was added, in triplicate, with BiTE supernatant, noBiTE supernatant, 1% Triton X-100 (CAS-No: 9036-19-5, Sigma-Aldrich) or supplemented RPMI, directly after the adding of cTcs. All wells, where needed, were supplemented with RPMI to reach a final volume of 200 ^L. The target cells mixed with cTcs and treatment, was then incubated for 24 hrs. After incubation, luciferin was added at a concentration of 2.5 mg/mL per well, incubated for 10 min protected from light, and then imaged using the IVIS Spectrum Series Imager (PerkinElmer Inc, Waltham, MA, USA). Quantification using photons per second (p/s) was performed with the Live Image® Software (PerkinElmer Inc), and the percentage specific killing was calculated using the following equation: ^^^^^^^^^^^ ^^^^ ^^^^ℎ − ^^^^^^ % ^^^^^^^^ ^^^^^^^ = 100 × ^^^^^^^^^^^ ^^^^ ^^^^ℎ − ^^^^^^^ ^^^^^^^ Spontaneous cell death is p/s from target cells only, sample is p/s from the sample where the specific killing is unknown, maximal killing is the p/s from the 1% Triton X-100 treated sample.
CD3 experiment We cloned different units of the canine CD3 (cCD3) isoforms to test the minimal cCD3 requirement to accommodate TCRs. We monitored human CD3ε expression at the surface of NK-92 cells. We compared three constructs: cCD3 full which comprises of all the CD3 units (ε,γ,δ, ζ,), cCD3 ε,γ,δ (with 3 CD3 subunits) and CD3ε with the ε subunit only. The results are presented in Figure 15D.