Antibody conjugate

Abstract

The present disclosure provides a conjugate comprising i) an antigen binding protein, for example an antibody, comprising two antigen binding domains that each has the ability to bind to a peptide associated with an MHC class II molecule (pMHCll), wherein said peptide associated with an MHC class II molecule is present on the surface of a target cell, and ii) a silencing molecule that has the ability to target the expression of one or more of CD80, CD86 and CD40. Compositions comprising said conjugates and therapeutic methods are also provided.

Description

[0001] Antibody conjugate

[0002] This invention relates generally to the field of conjugates, in particular conjugates comprising antibodies that bind to, or specifically bind to, a peptide-Major Histocompatibility type II Complex (pMHCll), and deliver silencing molecules into target cells such as disease- associated antigen presenting cells (APCs), e.g. via conjugate internalisation. Such conjugates have therapeutic uses, such as in the suppression of an immune response to an antigen (e.g. immune tolerization to an antigen), and treatment of diseases, particularly autoimmune diseases e.g. celiac disease, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. Conjugate-based compositions, methods and kits are also provided.

[0003] Antibody-drug conjugates (ADCs) represent an exciting class of therapeutics which take advantage of the specificity of antibodies, in addition to their long half-life and low immunogenicity. For instance, ADCs have been widely researched as potential cancer therapeutics for delivering anti-cancer drugs to tumour cells, whereby the drug is conjugated to an antibody which binds to a receptor that is selectively expressed on tumour cells, or has higher expression on tumour cells compared to healthy cells.

[0004] To achieve further therapeutic selectivity and safety (e.g. prevent or minimise off-target effects of the drug) the antibody can be conjugated to a precision payload, including gene silencing molecules such as a siRNA (small interfering RNA), which can thus be selectively delivered to desired target cells.

[0005] Antigen presenting cells (APCs), such as dendritic cells, play an essential role in regulating the immune response to both foreign and host antigens. Antigen presentation by MHC (Major Histocompatibility Complex) on the cell membrane of the APC results in activation of lymphocytes (e.g. CD4+ T cells) expressing a cognate T cell receptor (TCR) as part of the immune response. Such T cell activation in response to disease associated peptide-MHC molecules presented on APCs is an important contributor to autoimmune diseases.

[0006] TCR-like antibodies are antibodies developed to bind to, or specifically bind to, peptide- MHC (pMHC) on the surface of APCs, as an alternative to using recombinant TCRs which are limited by low affinity for pMHC and poor stability. A current aim with such TCR-like antibodies in the treatment of autoimmune diseases, is for such antibodies to bind to disease-associated pMHC molecules on the surface of an APC and then hinder the binding of a cognate TCR to the pMHC complex, hence preventing T cell activation. Thus, these antibodies can be used to directly affect the T cells interacting with that specific target pMHC. However, a primary goal for treating autoimmune disease, which is yet to be achieved, is to selectively suppress the immune response to an autoimmune disease-associated antigen, while maintaining a normal immune response to foreign antigens (e.g. induction of immune tolerance to the autoimmune disease-associated antigen).

[0007] Further complicating the matter is the high epitope-spread phenotype observed in many autoimmune diseases where the immune system, initially targeting a specific epitope, begins to recognize additional epitopes on the same or different proteins. A single antigen presenting cell (APC) can present thousands of different pHLAs covering multiple epitopes. Thus, multiple disease epitopes are involved in the disease process, many of which are unknown, and which differ between patients and disease stages. As multiple epitopes / antigens are then involved in the disease process, this in turn results in the need to take multiple disease causing antigens into consideration in successful therapy, which is extremely complicated and difficult to do successfully (Nel HJ, et al., The Lancet Rheumatology. 2020;2(11), e712-e723; Koning F, et al., Nat Rev Rheumatol. 2015; 11 , 450- 461).

[0008] Thus, there is a need for alternative and preferably improved therapeutics that selectively target and suppress the immune activating ability of APCs presenting disease-associated antigens, particularly autoimmune disease-associated antigens, and their cognate T cells, while avoiding global immune suppression. Put another way, there is a need for therapeutic methods of inducing antigen-specific immune tolerance which target improperly activated T cells, but which don’t interfere with protective immunity to pathogens and cancer. In particular, there is a need for methods which can address the issues associated with the epitope spread observed in many autoimmune diseases that are associated with multiple antigens, and to provide a therapy that can target multiple types of T cells that are activated against multiple autoantigens presented by APCs.

[0009] The present invention addresses this unmet need by conjugating i) antibodies (TCR-like antibodies) binding to, or specifically binding to, pMHCs associated with autoimmune disease, and ii) silencing molecules that target the expression of one or more of the costimulatory molecules CD80, CD86 or CD40 on APCs. By using such conjugates, which are taken up into APCs, selective suppression of the immune response to multiple of the autoimmune-disease associated antigens presented on the targeted APC can be achieved. However, as explained in more detail elsewhere herein, advantageously this also results in immune tolerance by in effect reprogramming the APC to become generally antiinflammatory and to inhibit the activation of T cells recognising other MHC associated antigens (other pMHCs) presented on the targeted APC. Thus, the present invention provides a conjugate comprising (or consisting of) i) an antigen binding protein, for example an antibody, comprising two (or at least two) antigen binding domains that each has the ability to bind to a peptide associated with an MHC molecule (a pMHC molecule), preferably wherein the pMHC molecule is expressed on (or is present on) the surface of a target cell, and ii) a silencing molecule targeting (that has the ability to target) the expression of one or more of CD80, CD86 and CD40.

[0010] Thus, the present invention further provides a conjugate comprising (or consisting of) i) an antigen binding protein, for example an antibody, comprising two (or at least two) antigen binding domains that each has the ability to bind to a peptide associated with an MHC class II molecule (a pMHCll molecule), and ii) a silencing molecule targeting the expression of one or more of CD80, CD86 and CD40.

[0011] The present invention further provides a conjugate comprising (or consisting of) i) an antigen binding protein, for example an antibody, comprising two (or at least two) antigen binding domains that each has the ability to bind to a peptide associated with an MHC class II molecule (a pMHCll molecule), wherein said peptide associated with an MHC class II molecule is expressed on (or is present on) the surface of a target cell, and ii) a silencing molecule targeting the expression of one or more of CD80, CD86 and CD40.

[0012] Such conjugates of the invention can advantageously be used for the targeted delivery of (or selective delivery of or specific delivery of) silencing or inhibitory molecules to target cells (e.g. target APCs) presenting disease-associated antigens (preferably autoimmune disease- associated antigens) associated with an MHC molecule, e.g. an MHC class I or MHC class II molecule (or an MHC class I or MHC class II molecule loaded with an appropriate peptide). Internalisation of the conjugate of the invention, e.g. to the lysosomal compartment, leads to the delivery of such inhibitory molecules into the target cells and reduces the expression of, preferably the cell surface expression of, one or more of the costimulatory molecules CD80, CD86 and CD40, preferably CD80 or at least CD80, thus suppressing the immune response to (and / or inducing or promoting immune tolerization to) the autoimmune-disease associated antigens.

[0013] After binding of the antigen binding protein, for example an antibody, of part i) of the conjugate of the invention to the specific disease-associated pMHC molecules on the surface of the target cell (e.g. the target APC), the conjugate and the pMHC are then internalized. As a result of the binding of the conjugate to the pMHC, the interaction between the pMHC and its cognate T cell (also referred to herein as the “target T cell”) is prevented (for example due to the blocking of the interaction between the pMHC and the T cell due to conjugate binding, and / or due to the internalization of the pMHC), thereby preventing T cell activation in response to the target disease-associated pMHC. As a result of the conjugate also being internalized, the silencing molecules of part ii) of the conjugate act to reduce the expression of one or more of the costimulatory molecules CD80, CD86 and CD40 (preferably CD80 or at least CD80) on the surface of the target cell (e.g. the target APC), thereby further preventing the activation of the target T cell and preventing the activation of T cells recognising other types of pMHC (e.g. other disease-associated peptides coupled to an MHC molecule) (also referred to herein as “bystander T cells”) on the target cell surface (e.g. the target APC cell surface) (see the exemplary schematic shown in Figure 1).

[0014] Since a single APC can present thousands of different pMHCs covering multiple epitopes, the downregulation of the costimulatory molecules by the conjugates of the invention results in an inability of this APC to activate T cells to all pMHCs it presents. In this way, the immune response / T cell activation to multiple epitopes, e.g. disease-associated epitopes, or multiple pMHCs, is inhibited. This results in immune tolerance by reprogramming the target cell (e.g. the target APC) to become generally anti-inflammatory and to inhibit the activation of bystander T cells recognising other pMHC associated antigens (e.g. other disease-associated antigens coupled to an MHC molecule) presented on the target cell (e.g. the target APC), thus overcoming the well-recognised issues associated with epitope spread.

[0015] As used herein the term “target T cell” refers to T cells that specifically recognise and are activated by the same peptide (e.g. disease-associated peptide) associated with an MHC (pMHC) as the pMHC binding antibodies (TCR-like antibodies) used in the conjugates of the present invention (for example see “T1” in Figure 1).

[0016] As used herein the term “bystander T cell” refers to T cells that recognise and are activated by a different peptide (e.g. a different disease-associated peptide, e.g. a different peptide associated with the same disease) associated with an MHC (pMHC) than the pMHC binding antibodies (TCR-like antibodies) used in the conjugates of the present invention, wherein the different pMHC is present on the same target cell (e.g. APC) as the pMHC bound by the pMHC binding antibodies (TCR-like antibodies) used in the conjugates of the present invention (for example see “T2” and “T3” in Figure 1). Such different pMHC molecules recognised by the bystander T cells can be MHC / HLA class I (HLA-A, HLA-B or HLA-C) or class II (HLA-DR, HLA-DP or HLA-DQ).

[0017] As such, the specificity of the “target T cell” referred to herein depends on the peptide associated MHC (e.g. disease-associated peptide associated MHC) targeted with the pMHC binding antibodies (TCR-like antibodies) used in the conjugates of the present invention. Thus, the disease (e.g. autoimmune disease) treated according to certain aspects of the present invention will determine the target pMHC and thus determine the specificity of the “target T cell”. Viewed alternatively, the specificity of the “target T cell” is dependent on the pMHC binding antibodies (TCR-like antibodies) used in the conjugates of the present invention.

[0018] In some embodiments, reference to pMHC binding antibodies (TCR-like antibodies) in the above (and elsewhere herein) can also refer more generally to an antigen binding protein, e.g. as described elsewhere herein.

[0019] In the invention described herein, the MHC molecule, and hence the pMHC molecule can be a class I MHC (MHCI) or a class II MHC (MHCII) molecule. In preferred embodiments the MHC molecule, and hence the pMHC molecule is a class II MHC (MHCII) molecule. However, the embodiments described herein relating to MHCII and pMHCll molecules, can equally apply, mutatis mutandis, to MHCI and pMHCI molecules.

[0020] Thus, such conjugates can be conveniently and advantageously used for selective or targeted immune suppression, or antigen tolerization, and hence for the treatment of autoimmune diseases (as defined elsewhere herein). By targeting (or specifically targeting or selectively targeting) cells which express the disease-associated pMHC, e.g. pMHCll (or more than one disease-associated pMHC, e.g. peptide MHCII), treatment of said diseases can be achieved while preventing (or minimising or limiting) side effects associated with global (or non-selective or non-specific, therapies, e.g. therapies not targeting, or not specifically or selectively targeting, the disease-associated pMHC) suppression of the immune system. For example, by targeting peptides predominantly located in the small intestine (e.g. targeting gluten-derived peptides for the treatment of celiac disease), T cell activation should only be affected in the small intestine and should be unaltered in peripheral tissues due to the absence of the target peptide.

[0021] Thus, the conjugates of the present invention can bind to target cells which express pMHC, e.g. pMHCll molecules, in particular disease-associated pMHCll molecules, on their surface. Such binding is carried out through the antigen binding protein, e.g. antibody, part (part i)) of the conjugate.

[0022] To the inventors’ knowledge no other therapeutics (e.g. conjugates) have been disclosed to have the advantageous properties of targeting cells presenting autoimmune disease- associated pMHC, in particular pMHCll, and delivering silencing molecules inducing immune suppression (e.g. targeting CD80, CD86 and / or CD40) into these cells. As set out above, use of the conjugates of the invention not only allows the targeting of cells presenting a specific autoimmune disease-associated pMHC, and hence the inhibition of activation of T cells that recognise that pMHC on the target cell (target T cells), but the function of the conjugate advantageously allows the inhibition of activation of T cells that recognise other types of pMHC expressed by (or presented on) the target cell (bystander T cells). These combined properties are highly advantageous and in particular address the well-recognised problem of epitope spread associated with many such autoimmune diseases.

[0023] In some embodiments the conjugate is an antibody-drug conjugate (ADC). In such embodiments an antibody (e.g. as described elsewhere herein) that is capable of binding to a pMHC, e.g. a pMHCll molecule (e.g. as described elsewhere herein) is conjugated to a silencing molecule that targets (and preferably reduces) the expression (preferably cell surface expression) of one or more of CD80, CD86 and CD40.

[0024] A silencing molecule according to any aspect of the present invention may be defined as a molecule, construct or system that reduces (or inhibits or abolishes) the interaction between or signalling between one or more co-stimulatory molecules expressed on the surface of a target APC (e.g. CD80, CD86 and / or CD40, preferably CD80 or at least CD80) and one or more cognate ligand (e.g. cognate receptor) expressed on the surface of a T cell (e.g. CD28 and / or CD40L, as appropriate, preferably at least CD28), such that the activation of or stimulation of the one or more T cells is reduced (or inhibited or abolished) and preferably T cell anergy, T cell unresponsiveness and / or antigen tolerization is induced (or promoted or increased).

[0025] Preferred silencing molecules reduce (or inhibit or abolish) the expression of one or more of the target proteins CD80, CD86 and CD40, preferably CD80 or at least CD80, on the target APC.

[0026] Preferably, the silencing molecule used in the conjugates of the invention reduces (or inhibits or abolishes) the cell surface expression of one or more of CD80, CD86 and CD40, preferably CD80 or at least CD80, on the target APC.

[0027] In some embodiments, the target protein is CD80. In alternative embodiments, the target protein is CD86. In alternative embodiments, the target protein is CD40. In some such embodiments only one of CD80, CD86 or CD40 is targeted by the conjugate of the invention.

[0028] In another embodiment, the target protein is CD80 and CD86. In alternative embodiments, the target protein is CD80 and CD40. In alternative embodiments, the target protein is CD86 and CD40. In some such embodiments only two of CD80, CD86 or CD40 are targeted by the conjugate of the invention. In some embodiments, the target protein is CD80, CD86 and CD40. In such embodiments all three of CD80, CD86 or CD40 are targeted by the conjugate of the invention.

[0029] In a preferred embodiment, the target protein is CD80. In some such embodiments only CD80 is targeted by the conjugate of the invention.

[0030] Multiple target proteins (e.g. more than one of CD80, CD86 or CD40) can either be targeted with single conjugates of the invention that have one or more silencing molecules which target multiple target proteins, or can be targeted using multiple different conjugates of the invention which, for example, each have silencing molecules that target a single target protein. Such multiple conjugates can conveniently be used together in combination.

[0031] Thus, by reducing (or inhibiting or abolishing) the expression of (or levels of), preferably the cell surface expression of (or levels of), one or more of CD80, CD86 and CD40, the conjugates of the present invention advantageously reduce (or inhibit or abolish or limit) the ability of the target APC to provide sufficient co-stimulatory signalling to fully activate a cognate T cell after the T cell receptor (TCR) expressed on the T cell interacts with (or binds to) its cognate peptide MHC, e.g. peptide-MHCH (e.g. autoimmune disease-associated pMHCll) presented on the surface of the target APC. The conjugates of the invention can thus suppress the immune activation (i.e. suppress the immune response) induced by the target APC presenting the peptide-MHC, and suppress the immune activation (i.e. suppress the immune response) of the cognate T cells interacting thereto, including the target T cells (the T cells that specifically interact with the particular disease-associated peptide MHC on the target APC) and preferably also bystander T cells (T cells that interact with other pMHCs (e.g. other disease-associated pMHCs) on the same APC), and / or (preferably and) inducing or promoting immune tolerization (i.e T cell unresponsiveness or anergy) to the autoimmune disease-associated antigen and preferably to other pMHC disease-associated antigens presented on the target cells.

[0032] For a T cell to be fully activated, it requires two signals from an APC. First, it requires the interaction of the TCR expressed on the surface of a T cell with its cognate peptide- associated MHC (pMHC) (e.g. autoimmune-disease associated pMHC) on the surface of an APC. Second, full T cell activation also requires the interaction of one or more of the costimulatory molecules CD80 (B7.1), CD86 (B7.2) and CD40 (TNR5), expressed on the APC surface, with their cognate T cell ligands. This engagement of costimulatory molecules (or cofactor molecules) then triggers the secretion of cytokines which is regarded as the third signal necessary for complete activation of T cells. T cell activation can be measured and quantified using various methods known in the art, for example by detecting the expression of T cell activation markers such as CD69 and CD25, and / or by detection of the secretion of one or more of the cytokines associated with T cell activation, e.g. one or more of the cytokines: interleukin-2 (IL-2), Tumour Necrosis Factor alpha (TNF-a) and Interferon gamma (IFN-y).

[0033] CD80 and CD86 are members of the B7 family of transmembrane proteins and provide a co-stimulatory signal by interacting with CD28 expressed on T cells. CD40 is a member of the TNF receptor family transmembrane protein which interacts with CD40L expressed on T cells to promote activation of T cells.

[0034] DNA and mRNA sequences encoding human CD80, CD86 and CD40 proteins, and amino acid sequences of human CD80, CD86 and CD40 proteins are well known and described in the art and can be obtained from various sequence databases.

[0035] For example, Uniprot entry P33681 provides an exemplary amino acid sequence of human CD80 (SEQ ID NO:55). GenBank accession M27533.1 provides an exemplary mRNA sequence of human CD80 (SEQ ID NO:56). Ensembl transcript ID ENST00000264246.8 provides an exemplary DNA coding sequence encoding human CD80.

[0036] For example, Uniprot entry P42081 provides an exemplary amino acid sequence of human CD86 (SEQ ID NO:57). GenBank accession L25259.1 provides an exemplary mRNA sequence encoding human CD86 (SEQ ID NO:58). Ensembl transcript ID ENST00000330540.7 provides an exemplary DNA coding sequence encoding human CD86.

[0037] For example, Uniprot entry P25942 provides an exemplary amino acid sequence of human CD40 (SEQ ID NO:59). GenBank accession X60592.1 provides an exemplary mRNA sequence encoding human CD40 (SEQ ID NQ:60). Ensembl transcript ID ENST00000372285.8 provides an exemplary DNA coding sequence encoding human CD40.

[0038] The term “target protein” as used herein can also apply mutatis mutandis to precursors of the target protein, such as DNA and mRNA (also referred to as “target DNA” and “target mRNA” as defined elsewhere herein). As used herein, the term “target protein” refers to one or more of CD80, CD86 and CD40 proteins.

[0039] Preferred silencing molecules reduce (or inhibit or abolish) the expression of one or more target proteins (as defined elsewhere herein). In some preferred embodiments, the silencing molecules reduce (or inhibit or abolish) the expression of one or more target proteins on the cell surface, e.g. of a target cell, e.g. an APC. In different embodiments, the silencing molecule can target DNA, mRNA or protein, e.g. can target CD80, CD86 and / or CD40 DNA, mRNA or protein. Preferred silencing molecules in accordance with present invention target CD80, CD86 and / or CD40 mRNA (e.g. one preferred silencing molecule is siRNA), preferably targeting CD80 mRNA or at least CD80 mRNA.

[0040] Thus, in some embodiments the silencing molecule used in the conjugates of the invention may target the DNA encoding the target protein (i.e. “target DNA” as defined elsewhere herein), for example altering the DNA sequence encoding the target protein, which can for example reduce (or limit or abolish) the expression of (or level of) the target protein, preferably the cell surface expression (or level of) of the target protein. Such alterations in the DNA sequence would typically be detrimental alterations, for example resulting in a truncated protein being encoded which can for example be degraded, and / or encoding a mutant or otherwise non-functional target protein which for example has reduced (or limited or abolished) biological activity (e.g. reduced or limited or abolished functional expression as defined elsewhere herein). Alternatively, the silencing molecule can prevent transcription of the target DNA, for example resulting in reduced (or limited or abolished) target protein expression.

[0041] In alternative embodiments, the silencing molecule used in the conjugates of the invention may target the mRNA encoding the target protein (i.e. “target mRNA” as defined elsewhere herein), e.g. may reduce (or inhibit or abolish) the expression of (or levels of) mRNA encoding the target protein (i.e. “target mRNA” as defined elsewhere herein), which can for example reduce (or limit or abolish) the expression of (or levels of) the target protein, preferably the cell surface expression of the target protein. For example the silencing molecule may comprise or consist of one or more of siRNA (small interfering RNA), and shRNA (short hairpin RNA), for example an shRNA expression construct (e.g. a DNA sequence encoding an shRNA, such as a compact shRNA (cshRNA)), that may reduce (or inhibit or abolish) the expression of (or levels of) mRNA encoding the target protein (i.e. “target mRNA” as defined elsewhere herein). Alternatively, or additionally, the silencing molecule may reduce (or inhibit or abolish) the translation of the target mRNA by the ribosome. Preferred silencing molecules targeting mRNA are siRNA.

[0042] In alternative embodiments, the silencing molecule used in the conjugates of the invention may target the target protein directly, e.g. may reduce (or inhibit or abolish) the expression of (or levels of) the target protein, preferably reduce (or inhibit or abolish) the cell surface expression of (or levels of) the target protein. For example, in some embodiments the silencing molecule may conjugate with (or interact with or modify) the target protein to promote (or induce or increase) degradation of the target protein by the proteasome.

[0043] The term “silencing molecule” as used herein can refer to a plurality of (or more than one, or at least two) silencing molecules, for example the term silencing RNA or siRNA can refer to a plurality of (or more than one, or at least two) siRNA molecules. Thus, in preferred embodiments the conjugate of the invention comprises one antigen binding protein, for example an antibody (as defined elsewhere herein), linked to or coupled to a plurality of (or more than one, or at least two) silencing molecules. This applies to all silencing molecule types or modalities as described herein (e.g. inhibitory RNA, CRISPR-Cas and targeted protein degradation (TPD) molecules).

[0044] In such embodiments the plurality of (or more than one, or at least two) silencing molecules are typically multiple copies of the same silencing molecule, e.g. multiple copies of the same siRNA molecules. However, as described in more detail elsewhere herein, the conjugates of the invention can also contain more than one different silencing molecule, e.g. which targets more than one different target sequence in the same target protein, or which can target different target proteins. Again, in such embodiments, a plurality of (or more than one, or at least two) silencing molecules of each different silencing molecule can be present.

[0045] In different embodiments, the silencing molecule used in the conjugates of the invention may reduce (or inhibit or abolish) the expression of, preferably the cell surface expression of, the target protein (e.g. CD80, CD86 or CD40) by targeting one of, or two of, or all three of: target DNA, target mRNA and target protein, as defined above, e.g. the silencing molecule may target one of, or two of, or all three of: CD80 DNA, CD80 mRNA and CD80 protein. In such embodiments, where two or all three of DNA, mRNA and protein are targeted, then appropriate different types (or modalities) of silencing molecule (e.g. as described elsewhere herein) are used. Again, in such embodiments, a plurality of (or more than one, or at least two) silencing molecules of each different silencing molecule can be present.

[0046] In one embodiment, the conjugates of the present invention include a silencing molecule comprising or consisting of an inhibitory RNA construct / molecule. The term inhibitory RNA construct as used herein may be defined as any RNA molecule or plurality of RNA molecules, or any construct or plurality of constructs (e.g. one or more DNA sequences) encoding an RNA molecule or plurality of RNA molecules, wherein the RNA molecule anneals to (or interacts with or complexes with), preferably specifically anneals to (or specifically interacts with or specifically complexes with), a target mRNA, i.e. an mRNA encoding the target protein, as defined elsewhere herein, and reduces (or inhibits or abolishes) the expression of (or level of) the target mRNA and / or reduces (or inhibits or abolishes) the translation of the target mRNA by the ribosome (e.g. inhibits, reduces or abolishes the expression of the target protein).

[0047] Thus, the term inhibitory RNA (sometimes referred to as RNAi) includes any RNA molecule, or any construct encoding an RNA molecule, that is capable of inducing gene silencing by targeting complementary mRNA for degradation.

[0048] For example, siRNA molecules typically comprise an RNA duplex of 19 to 25 base pairs, while shRNA molecules typically comprise 50 to 70 nucleotides forming a stem-loop secondary structure. After introduction into the cell, both siRNA and shRNA are processed by the enzyme Dicer, loaded on to multiprotein RISC complexes and separated into singlestranded RNA molecules which guide the complex to the target mRNA. Complementary base pairing between the RISC-associated single-stranded RNA molecules and the target mRNA sequence leads to cleavage of the target mRNA sequence by the RISC complex, preventing translation of the target mRNA sequence into a protein. siRNA is typically transfected into cells for targeting gene expression. shRNA is typically introduced into a cell by transfection or transduction of a DNA sequence (e.g. a plasmid or a gene expression cassette (i.e. a DNA sequence that includes a gene of interest and its regulatory DNA sequence)) encoding said shRNA, wherein the expression of the shRNA is typically driven by RNA polymerase III (RNAP III) type III promoters (e.g. U6 or H1 promoters). The transfection or transduction of an shRNA-encoding DNA sequence is thus associated with the long-term expression of the shRNA and the long-term suppression of target gene expression. Recent developments have also optimised the shRNA delivery system, whereby RNAP III type II promoters are employed to produce more compact shRNA (cshRNA) molecules (also referred to as an “cshRNA expression cassette” herein) (Burke, et al, Nucleic Acids Res. 2017;45(17):e154).

[0049] Thus, as used herein the term “shRNA” also includes compact shRNAs (cshRNAs).

[0050] Thus, the inhibitory RNA construct used in the conjugates of the present invention can be one or more inhibitory RNA molecules (for example siRNA molecules), e.g. one or more siRNA molecules linked to or coupled to the antigen binding protein according to the conjugates of present invention.

[0051] Alternatively, the inhibitory RNA construct used in the conjugates of the present invention can be one or more DNA sequences (e.g. a plasmid or a cshRNA expression cassette) that encode one or more inhibitory RNA molecules (e.g. shRNA or cshRNA), e.g. one or more DNA sequences linked to or coupled to the antigen binding protein according to the conjugates of present invention. The presence of the one or more DNA sequences in a cell (i.e. in a target cell after the internalisation of the antigen binding protein used in the conjugates of the present invention, as described elsewhere herein), results in the expression of one or more inhibitory RNA molecules (e.g. one or more shRNA molecules), which in turn target the expression of the target mRNA (as described elsewhere herein) in the cell.

[0052] Thus, the inhibitory RNA construct used in the conjugates of the present invention can be any RNA construct, or plurality of RNA constructs, that comprise or consist of or express (or encode) an inhibitory RNA molecule or a plurality of inhibitory RNA molecules, for example a construct that comprises or consists of one or more siRNA molecules, or a construct that comprises or consists of one or more DNA sequences that encodes or expresses one or more shRNA or cshRNA molecules.

[0053] Viewed alternatively, the inhibitory RNA construct can be any RNA molecule, or any construct encoding an RNA molecule, that reduces (or inhibits or abolishes) the expression of (or levels of) CD80, CD86 and / or CD40 mRNA or, preferably, any RNA construct that reduces (or inhibits or abolishes) the expression of, preferably the cell surface expression of, CD80, CD86 and / or CD40 protein, preferably CD80 protein or at least CD80 protein.

[0054] In different embodiments, the inhibitory RNA construct may comprise or consist of one or more of: siRNA and a shRNA (e.g. a DNA sequence encoding a shRNA). Thus, one or more different types of inhibitory RNA construct may be used in a single conjugate of the present invention. In addition, one, or preferably a plurality, of each individual inhibitory RNA molecule may be used in a single conjugate. In a particularly preferred embodiment, the inhibitory RNA may be one, or a plurality of, preferably a plurality of, siRNA molecules. The skilled person would be aware of other suitable inhibitory RNA construct formats that comprise or consist of or express or encode an inhibitory RNA molecule that is capable of annealing to (or interacting with or complexing with) a target mRNA and inhibiting (or reducing or abolishing) the expression of (or level of) the target mRNA and / or reducing (or inhibiting or abolishing) the translation of (or expression of) the target mRNA by the ribosome. Other aspects of the invention apply mutatis mutandis to these alternative inhibitory RNA molecule formats.

[0055] As mentioned elsewhere herein, the target mRNA can be one of CD80, CD86 or CD40 mRNA, or more than one of, or more than two of, including any combination of: CD80, CD86 or CD40 mRNA, i.e. the target mRNA can be any mRNA sequence which encodes an amino acid sequence of one of, or more than one of, or more than two of, including any combination of: CD80, CD86 or CD40 proteins, as appropriate. A particularly preferred target mRNA is CD80 mRNA, or at least CD80 mRNA, i.e. any mRNA sequence which encodes an amino acid sequence of CD80 protein, or at least an amino acid sequence of CD80 protein.

[0056] Viewed alternatively, the inhibitory RNA construct used in the conjugates of the present invention can be any RNA construct, or a plurality of RNA constructs, that comprise or consist of or express an inhibitory RNA molecule, or a plurality of inhibitory RNA molecules, that anneal to (or interact with or complex with), preferably specifically anneal to (or specifically interact with or specifically complex with), one or more of CD80, CD86 and CD40 mRNA, preferably at least CD80 mRNA, and for example reduce (or inhibit or abolish) expression of (or level of) one or more of CD80, CD86 and CD40 mRNA, preferably at least CD80 mRNA, and / or reduces (or inhibits or abolishes) the translation of one or more of CD80, CD86 and CD40 mRNA, preferably at least CD80 mRNA, by the ribosome.

[0057] The sequence of such inhibitory RNA molecules (e.g. siRNA and shRNA) is determined by the nucleotide sequence of the target mRNA. Thus, the nucleotide sequence of inhibitory RNA molecules are preferably complementary to (or significantly or sufficiently complementary to) the nucleotide sequence of the target mRNA molecule. Thus, preferably, the inhibitory RNA molecules anneal to (or interact with, or complex with), preferably specifically anneal to (or specifically interact with or specifically complex with), the target mRNA by complementary base-pairing.

[0058] Thus, the sequence of an inhibitory RNA construct that expresses or encodes the inhibitory RNA molecule (e.g. the DNA sequence of a plasmid or cshRNA expression cassette that expresses or encodes an shRNA or cshRNA molecule) is also determined by the nucleotide sequence of the target mRNA. Such DNA sequences typically comprise a promoter for expression of the inhibitory RNA molecule (e.g. shRNA), and optionally other regulatory elements, and can be modified (e.g. engineered or mutated) to comprise a DNA sequence which encodes the inhibitory RNA molecules (e.g. shRNA) that anneal to or interact with or complex with the target mRNA molecule, as described above. Such inhibitory RNA constructs (e.g. DNA sequences such as a plasmid or a cshRNA expression cassette), which can be modified to comprise the DNA sequence encoding the inhibitory RNA molecule (e.g. shRNA), as defined elsewhere herein, are known in the art and any such construct can be used (e.g. Burke, et al, Nucleic Acids Res. 2017;45(17):e154; and BLOCK-iT™ U6 RNAi Entry Vector Kit, Invitrogen).

[0059] Preferably, the inhibitory RNA molecule has at least 80%, at least 85%, at least 90%, at least 95%, or is the reverse complement (e.g. 100% complementarity) to the target mRNA. Such levels of complementarity are generally required in order to have sufficient complementarity for functionality (e.g. to target the mRNA for degradation by the RISC complex). However, any level of complementarity can be used provided there is annealing of (or interaction between), preferably specific annealing of (or interaction between), the inhibitory RNA molecule and the target mRNA, and such an annealing or interaction leads to cleavage of the target mRNA by the RISC complex. For example, the inhibitory RNA molecule may have at least 80%, at least 85%, at least 90%, at least 95%, or is the reverse complement (e.g. 100% complementarity) to an appropriate number of nucleotides, e.g. at least 10, 15 or 18 nucleotides, e.g. an 18 or 19 to 25 nucleotide sequence within an exemplary CD80, CD86 or CD40 mRNA sequence (SEQ ID NOs:56, 58 and 60).

[0060] Such inhibitory RNA molecules thus preferably comprise or consist of a nucleotide sequence, or a number of different nucleotide sequences, which are complementary to a nucleotide sequence, or a number of different nucleotide sequences, within the nucleotide sequence of CD80, CD86 and / or CD40 mRNA (e.g. SEQ ID NOs:56, 58 and 60), preferably at least CD80 mRNA.

[0061] Thus, any nucleotide sequence within the target mRNA (e.g. CD80, CD86 or CD40 mRNA) can be the target sequence for inhibitory RNA molecules as described herein, provided the annealing of (or interaction of or complexing of) the inhibitory RNA molecule to the target mRNA results in reduced (or inhibited or abolished) target protein expression (i.e. reduced, inhibited or abolished CD80, CD86 and / or CD40 expression), preferably reduced (or inhibited or abolished) cell surface expression, as defined elsewhere herein. Thus any nucleotide sequence which shares sufficient complementarity (e.g. as defined above) with the target mRNA sequence in order to produce such reductions, etc., can be appropriately used as an inhibitory RNA molecule. Such nucleotide sequences would typically be regarded as sequences that were significantly complementary to the nucleotide sequence of the target mRNA molecule. The skilled person would be aware of suitable assays to analyse the reduction of target mRNA expression and / or target protein expression and thus determine suitable inhibitory RNA molecule sequences, for example quantitative reverse transcription polymerase chain reaction (RT-qPCR), northern blot or western blot. Furthermore, the skilled person would be aware of publicly available tools to design inhibitory RNA molecules, for example siRNA, with sufficient complementarity to one or more target mRNA (e.g. siMAX siRNA design tool, Eurofins Genomics).

[0062] The skilled person would also be aware of suitable (and optimised) DNA sequences that encode such inhibitory RNA molecules (e.g. DNA sequences that encode suitable shRNA sequences), and would also be aware of publicly available tools to design DNA sequences that encode such inhibitory RNA molecules (e.g. Burke, et al, Nucleic Acids Res. 2017;45(17):e154, discloses a tool for designing DNA sequences for cshRNAs; and BLOCK- iT™ U6 RNAi Entry Vector Kit, Invitrogen, discloses a tool for designing DNA sequences that can be cloned into an expression vector for expression of a specific shRNA). Such DNA sequences can be combined with any suitable construct (e.g. expression construct) which promotes the expression of (i.e. transcription of) the DNA sequence to form for example an shRNA (as described elsewhere herein), and the skilled person would be aware of suitable techniques (e.g. molecular cloning) and commercially available kits (e.g. BLOCK-iT™ U6 RNAi Entry Vector Kit, Invitrogen) to combine such DNA sequences with said constructs thus forming certain embodiments of the inhibitory RNA constructs used in the conjugates of the present invention.

[0063] In some embodiments, the inhibitory RNA construct may comprise or express more than one type of inhibitory RNA molecule, for example comprise a siRNA and express an shRNA (e.g. a cshRNA).

[0064] In some embodiments, the inhibitory RNA construct may comprise or express more than one different inhibitory RNA molecule which are each complementary to a different (overlapping or non-overlapping) nucleotide sequence within the same target mRNA, e.g. CD80, CD86 or CD40 mRNA, preferably CD80 mRNA.

[0065] In other embodiments, the inhibitory RNA construct may comprise or express more than one different inhibitory RNA molecule, where each inhibitory RNA molecule is complementary to a nucleotide sequence within different target mRNAs. In other words, the inhibitory RNA construct may comprise or express at least two inhibitory RNA molecules which are complementary to a nucleotide sequence within at least two of: CD80, CD86 and CD40 mRNA. In some embodiments, the inhibitory RNA construct may comprise or express different inhibitory RNA molecules where each different inhibitory RNA molecule is complementary to a nucleotide sequence within the CD80, CD86 and CD40 mRNAs.

[0066] Alternatively, if it is desired to target more than one of CD80, CD86 and CD40, then multiple different conjugates of the invention can be used, each of which individually targets a single target protein (here CD80 or CD86 or CD40).

[0067] Thus, multiple target proteins (e.g. more than one of CD80, CD86 or CD40) can either be targeted with single conjugates of the invention that have one or more inhibitory RNA constructs (e.g. siRNA molecules or shRNA expressing constructs) which target multiple target proteins, or can be targeted using multiple different conjugates of the invention which, for example, each have inhibitory RNA constructs (e.g. siRNA molecules or shRNA expressing constructs) that target a single target protein (here CD80 or CD86 or CD40). Such multiple conjugates can conveniently be used together in combination. Again, in such embodiments, a plurality of (or more than one, or at least two) inhibitory RNA constructs (e.g. siRNA molecules or shRNA expressing constructs) of each different inhibitory RNA construct can be present in a single conjugate.

[0068] Thus, the inhibitory RNA construct used in the conjugates of the present invention may be any construct comprising or consisting of or expressing or encoding an RNA molecule that anneals to (or interacts with or complexes with) CD80, CD86 and / or CD40 mRNA, and reduces (or inhibits or abolishes) the expression (or level) of CD80, CD86 and / or CD40 mRNA and / or reduces (or inhibits or abolishes) the translation of CD80, CD86 and / or CD40 mRNA by the ribosome.

[0069] Exemplary silencing molecules of the present invention comprise siRNA targeting the mRNA of CD80, CD86 or CD40 and have been shown to reduce the relative mRNA levels of CD80, CD86 and CD40, while the particularly preferred siRNA targeting the mRNA of CD80 has further been shown to reduce the surface expression of CD80 protein.

[0070] In another embodiment, the conjugates of the present invention include a silencing molecule comprising or consisting of a CRISPR-Cas gene silencing construct. The term CRISPR-Cas gene silencing construct as used herein may be defined as a system comprising or consisting of a guide RNA (gRNA) and a Cas nuclease (or multiple different gRNAs and a Cas nuclease), or one or more constructs which encode a system comprising or consisting of a gRNA and a Cas nuclease (or multiple different gRNAs and a Cas nuclease), wherein the Cas nuclease cleaves a target site within a target DNA sequence annealing to (or interacting with or complexing with or targeted by) the gRNA, resulting in reduction of (or inhibition of or abolishment of) the expression of a target protein (as defined herein), or results in the expression of a mutant or otherwise non-functional target protein that has reduced (or limited or abolished) biological activity (i.e reduced functional expression as defined elsewhere herein).

[0071] Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas enables precision genome engineering, linking the targeting of a specific genomic (DNA) sequence by complementary base pairing to the DNA cleavage activity of a Cas nuclease, such that cleavage of DNA can be induced at a specific pre-determined genomic location. Cleavage of the DNA (e.g. the formation of a double-strand break) activates cellular DNA damage response pathways which repairs the cleaved DNA. Repair of the cleaved DNA by error- prone DNA repair pathways, such as Non-Homologous End Joining (NHEJ), may introduce mutations (e.g. deletions or additions or substitutions) into the repaired DNA product, which for example may lead to the expression of a truncated protein product which can be targeted for degradation by nonsense-mediated decay (NMD) or lead to the expression of a mutated protein which is inactive (or has reduced activity). Alternatively, repair of the cleaved DNA by DNA repair pathways which utilise a homologous template for repair (e.g. homology- directed repair (HDR) pathways) can be used to introduce specific sequence mutations in the DNA repair product by providing an exogenous, homologous template DNA sequence containing the mutation.

[0072] Any appropriate CRISPR-Cas gene silencing construct may be used in order to target the expression of a target protein (as defined herein). In different embodiments, the CRISPR-Cas gene silencing construct of the invention may comprise or consist of: DNA, RNA, or ribonucleoprotein (RNP).

[0073] Thus, in some embodiments, the CRISPR-Cas gene silencing construct may comprise or consist of DNA, such as a singular plasmid DNA encoding one or more Cas nuclease and one or more gRNA, or multiple plasmids encoding one or more Cas nuclease and / or one or more gRNA.

[0074] In alternative embodiments, the CRISPR-Cas gene silencing construct may comprise or consist of RNA, for example one or more gRNA and one or more RNA polynucleotides encoding at least one Cas nuclease.

[0075] In alternative embodiments, the CRISPR-Cas gene silencing construct may comprise or consist of ribonucleoprotein (RNP), for example one or more gRNA complexed with (or interacting with) one or more Cas proteins.

[0076] The target DNA can be more than one of, or more than two of, including any combination of, the CD80, CD86 and CD40 genes as described elsewhere herein. In a preferred embodiment, the target DNA is the CD80 gene, or at least the CD80 gene.

[0077] Thus, in some embodiments, the CRISPR-Cas gene silencing construct used in the conjugates of the present invention is any CRISPR-Cas gene silencing construct which comprises, or encodes, at least one gRNA molecule that anneals to (or interacts with or complexes with) at least one of the CD80, CD86 and CD40 genes (i.e. target DNA as defined herein), preferably at least the CD80 gene, resulting in cleavage of the at least one target DNA sequence by at least one Cas nuclease, and reduction of (or inhibition of or abolishment of) CD80, CD86 and / or CD40 protein expression, preferably at least CD80 protein expression. Preferably, the cell surface expression of CD80, CD86 and / or CD40 (preferably CD80 or at least CD80) is reduced (or inhibited or abolished) by the CRISPR- Cas gene silencing construct used in the conjugates of the invention.

[0078] Generally, the sequence of the gRNA molecule, or multiple different gRNA molecules (where more than one target DNA is targeted), is determined by the nucleotide sequence of the target DNA. Preferred gRNA sequences are 17 to 24 nucleotides and target (e.g. anneal to) coding DNA sequences (e.g. exons). Thus, the nucleotide sequence of gRNA molecules are preferably complementary to (or significantly or sufficiently complementary to) one or more nucleotide sequences within the target DNA. Thus, preferably, the gRNA molecules of the CRISPR-Cas gene silencing construct used in the conjugates of the present invention anneal to (or interact with or complex with), preferably specifically anneal to (or specifically interact with or specifically complex with), one or more nucleotide sequences within the target DNA, e.g. within the CD80, CD86 or CD40 genes, preferably within the CD80 gene, or at least the CD80 gene.

[0079] Preferably, the gRNA molecule has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or is the reverse complement (e.g. 100% complementarity) to the target DNA. Such levels of complementarity are generally required in order to have sufficient complementarity for functionality (e.g. for cleavage of the target site, as defined elsewhere herein, by the Cas nuclease). However, any level of complementarity can be used provided there is annealing of (or interaction between), preferably specific annealing of (or interaction between) the gRNA and the target DNA, and such an annealing or interaction leads to cleavage of the target site by the Cas nuclease. For example, the gRNA molecule may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or is the reverse complement (e.g. 100% complementarity) to an appropriate number of nucleotides, e.g. at least 10, 14 or 17 nucleotides, e.g. a 17 to 24 nucleotide sequence within an exemplary DNA (genomic) coding sequence of CD80, CD86 or CD40 (e.g. Ensembl transcript IDs ENST00000264246.8, ENST00000330540.7 or ENST00000372285.8). Publicly available tools are also available which can be used to find suitable gRNA sequences by entering the gene identifier (e.g. “CD80”, “CD86” or “CD40”) (Invitrogen™ TrueGuide™ Synthetic gRNA).

[0080] Such gRNA molecules thus preferably comprise or consist of a nucleotide sequence or a number of different nucleotide sequences, which are complementary to a nucleotide sequence or a number of different nucleotide sequences within the CD80, CD86 and / or CD40 genes, preferably within the CD80 gene, or at least the CD80 gene.

[0081] Thus, any nucleotide sequence within the target gene (e.g. within CD80, CD86 and / or CD40 genes) can be the target sequence, provided annealing of (or interaction with or complexing of) the gRNA to said sequence results in cleavage of the target site, as defined elsewhere herein, by a Cas nuclease and results in reduced (or inhibited or abolished) CD80, CD86 and / or CD40 protein expression, preferably reduced (or inhibited or abolished) CD80, CD86 and / or CD40 cell surface expression. Thus any nucleotide sequence that shares sufficient complementarity (e.g. as defined above) with nucleotide sequences within CD80, CD86 or CD40 genes in order to produce such reductions, etc., can be appropriately used as a gRNA. Such nucleotide sequences would typically be regarded as sequences that were significantly complementary to the nucleotide sequence of the target gene. The skilled person would be aware of suitable assays to determine reduction of protein expression and / or reduction of cell surface protein expression, in addition to suitable assays to determine the sequence of the target DNA and predicted translated protein sequence after CRISPR-Cas gene editing, and thus determine suitable gRNA sequences, and (optionally) DNA sequences encoding such gRNAs. For example, common DNA sequencing techniques e.g. Sanger sequencing, together with western blot or flow cytometry could be used. Furthermore, the skilled person would be aware of publicly available tools to design suitable gRNAs, and DNA encoding such gRNAs (e.g. Invitrogen™ TrueDesign™ Genome Editor) or commercially available pre-designed gRNAs (e.g. Invitrogen™ TrueGuide™ Synthetic gRNA).

[0082] In some embodiments, the CRISPR-Cas gene silencing construct may comprise more than one different gRNA molecule which are each complementary to a different (overlapping or non-overlapping) nucleotide sequence within the same target DNA, e.g. within the CD80, CD86 or CD40 genes.

[0083] In other embodiments, the CRISPR-Cas gene silencing construct may comprise more than one different gRNA molecule, where each gRNA molecule is complementary to a nucleotide sequence within different target DNAs. In other words, the CRISPR-Cas gene silencing construct may comprise at least two different gRNA molecules which are complementary to a nucleotide sequence within at least two of: CD80, CD86 and CD40 genes. In some embodiments, different gRNA molecules where each different gRNA molecule is complementary to a nucleotide sequence within the CD80, CD86 and CD40 genes may be used in the CRISPR-Cas gene silencing constructs.

[0084] Alternatively, if it is desired to target more than one of the CD80, CD86 and CD40 genes, then multiple different CRISPR-Cas gene silencing conjugates of the invention can be used, each of which individually targets a single target gene (here CD80 or CD86 or CD40).

[0085] Thus, multiple target proteins (e.g. more than one of CD80, CD86 or CD40) can either be targeted with single conjugates of the invention that have one or more CRISPR-Cas gene silencing constructs which target multiple target genes, or can be targeted using multiple different CRISPR-Cas gene silencing conjugates of the invention which, for example, each have CRISPR-Cas gene silencing constructs that target a single target gene (here CD80 or CD86 or CD40). Such multiple conjugates can conveniently be used together in combination. Again, in such embodiments, a plurality of (or more than one, or at least two) CRISPR- Cas gene silencing constructs for each different CRISPR-Cas gene silencing construct can be present in a single conjugate.

[0086] The CRISPR-Cas gene silencing construct used in the conjugates of the present invention is any CRISPR-Cas gene silencing construct which comprises a Cas nuclease, or variant thereof, that is able to cleave the target site as defined herein (e.g. within the CD80, CD86 and / or CD40 genes, preferably the CD80, or at least the CD80, gene). In preferred embodiments the Cas nuclease is a Cas9 nuclease, however the conjugates of the invention are not limited thereto and the skilled person would appreciate that alternative Cas nucleases can be used.

[0087] Thus, the CRISPR-Cas gene silencing construct used in the conjugates of the present invention may comprise any Cas nuclease, provided the Cas nuclease is capable of cleaving the target site after (preferably specifically after) the gRNA has annealed to (or interacted with or complexed to) the target DNA sequence as described elsewhere herein (e.g. one or more sequence within the CD80, CD86 and / or CD40 genes), provided said cleavage results in reduction of (or inhibition of or abolishment of) the expression of (or level of) CD80, CD86 and / or CD40 proteins, or results in the expression of a mutant or otherwise non-functional CD80, CD86 and / or CD40 protein that has reduced (or limited or abolished) biological activity (e.g. reduced functional expression as defined elsewhere herein). In preferred embodiments, the Cas nuclease is a Cas9 nuclease.

[0088] The skilled person would be aware of suitable Cas protein sequences, and variants thereof, and (optionally) DNA and / or RNA sequences encoding such Cas protein sequences.

[0089] Thus, in some embodiments, the CRISPR-Cas gene silencing construct used in the conjugates of the present invention may comprise one or more gRNA molecules, targeting one or more DNA target sequences within one or more of the CD80, CD86 and CD40 genes, as defined elsewhere herein, and one or more Cas nucleases as defined elsewhere herein.

[0090] Thus, in some embodiments, the CRISPR-Cas gene silencing construct used in the conjugates of the present invention comprises or consists of a) one or more gRNA molecules (or one or more DNA construct encoding the one or more gRNA molecules) as described elsewhere herein, which is capable of annealing to a target DNA sequence (as defined elsewhere herein, e.g. within the CD80, CD86 and / or CD40 genes, preferably the CD80 gene or at least the CD80 gene), and b) one or more Cas nuclease (or one or more DNA construct and / or one or more RNA polynucleotide encoding the one or more Cas nuclease) as defined elsewhere herein, which is capable of cleaving a nucleotide sequence (i.e. the target site as defined elsewhere herein) within the target DNA sequence (e.g. cleaving at least one nucleotide sequence within the CD80, CD86 and / or CD40 genes, preferably within the CD80 gene or at least the CD80 gene), leading to reduction of (or inhibition of or abolishment of) the expression of, preferably the cell surface expression of, at least one target protein (as defined herein, e.g. one or more of CD80, CD86 and CD40 proteins, preferably CD80 or at least CD80).

[0091] In some embodiments, the CRISPR-Cas gene silencing construct may comprise one or more gRNAs and one or more Cas nucleases, as described above, and further comprise one or more template DNA sequences. Such template DNA sequences may comprise or consist of a DNA sequence which shares sufficient homology with the target DNA sequence to be used as template for homology-directed repair (HDR), preferably wherein the template DNA sequence includes one or more mutations (e.g. substitutions, deletions or additions). As such, after site-directed DNA cleavage by the gRNA-Cas complex, the template DNA sequence may be used for repair of the cleaved DNA, resulting in a repaired DNA product including the one or more mutations. Such CRISPR-Cas gene silencing constructs can thus be used to generate mutant protein sequences which for example have reduced (or inhibited or abolished) functional expression (as defined elsewhere herein) and / or expression, preferably cell surface expression.

[0092] The skilled person would be aware of other forms of CRISPR-Cas gene silencing, e.g. CRISPR inhibition (CRISPRi), and other forms of gene editing including e.g. base editing or prime editing, that may be used to reduce the expression of, preferably the cell surface expression of, one or more of CD80, CD86 and CD40, preferably CD80 or at least CD80.

[0093] In another embodiment, the conjugates of the present invention include a silencing molecule comprising or consisting of a targeted protein degradation (TPD) molecule, or a plurality of (e.g. more than one or at least two) TPD molecules. The term TPD molecule as used herein may be defined as a molecule that has the ability to couple a target protein as defined elsewhere to a cellular protein degradation pathway e.g. a proteasomal or lysosomal degradation pathway. The TPD molecule may thus interact with (bind to or conjugate to) the target protein and in turn target the protein for degradation by the proteasomal or lysosomal pathways, without targeting or affecting the DNA or mRNA of the target protein. In other words such TPD molecules can allow the target protein to be directly targeted.

[0094] Targeted protein degradation (TPD) uses molecules, for example ligands or antibodies, to specifically target an endogenous protein (here CD80, CD86 or CD40) for degradation by the proteasomal or lysosomal pathways. TPD via the proteasome utilises the canonical ubiquitination pathway, whereby addition of ubiquitin chains to a target protein by an E3 ubiquitin ligase leads to degradation of the target protein by the proteasome. For example, PROTAC (proteolysis-targeting chimera) is a form of TPD that uses a molecule which concurrently binds a target protein (here CD80, CD86 or CD40) and an E3 ubiquitin ligase, increasing the proximity between the two proteins leading to ubiquitination of the target protein by the E3 ubiquitin ligase and thus degradation of the target protein by the proteasome. An alternative example of a TPD system is TrimAway, which takes advantage of the cellular system for targeted degradation of antibody-bound pathogens by the proteasome. For example, TRIM21 is an E3 ubiquitin ligase expressed in a range of tissues and cell types that interacts with the Fc domain of antibodies and, via ubiquitination, targets the antibody-bound target protein for degradation by the proteasome. Therefore an antigen binding protein comprising an Fc domain, for example an antibody, that binds to the target protein (here CD80, CD86 or CD40) can be used to induce TRIM21 -mediated protein degradation in the TrimAway system.

[0095] In preferred embodiments, the conjugates of the present invention include a silencing molecule comprising or consisting of a PROTAC molecule. The term PROTAC molecule as used herein may be defined as a molecule comprising or consisting of a) a ligand which binds to (or interacts with) a target protein as defined elsewhere herein, and b) a ligand which binds to (or interacts with) an E3 ubiquitin ligase, wherein the PROTAC molecule reduces (or limits or abolishes) expression of, preferably the cell surface expression of (or level of) the target protein (as defined elsewhere herein) by proteasomal degradation. Preferably, parts a) and b) of the PROTAC are linked by a linker moiety. In some embodiments, the target protein is one of CD80, CD86 and / or CD40 protein.

[0096] In some embodiments, the target protein can be more than one of, or more than two of, including any combination of: CD80, CD86 and CD40 protein.

[0097] In a preferred embodiment, the target protein is CD80 protein, or at least the CD80 protein.

[0098] Thus, a PROTAC molecule used in the conjugates of the present invention comprises a ligand which binds to (or interacts with), preferably specifically binds to or specifically interacts with, CD80, CD86 or CD40 proteins, preferably CD80 protein or at least the CD80 protein. The skilled person would be aware of appropriate methods to analyse binding or interaction of a ligand to a target protein to identify suitable ligands for CD80, CD86 and / or CD40 proteins, for example enzyme-linked immunosorbent assay (ELISA) or surface plasma resonance (SPR). For example, suitable ligands for CD80 include, but are not limited to, CD28 and CTLA-4; suitable ligands for CD86 include, but are not limited to, CD28 and CLTA-4; suitable ligands for CD40 include, but are not limited to, CD40L. Suitable ligands may also include antibodies to CD80, CD86 or CD40, or binding proteins comprising the antigen binding domains of such antibody sequences. For example, there are publicly available antibody sequences for antibodies binding to CD80, CD86 or CD40 and any of these can be used in the conjugates of the invention. In addition, antibodies binding to CD80, CD86 or CD40 are commercially available and such antibodies can also be used in the conjugates of the invention.

[0099] An appropriate PROTAC molecule used in the conjugates of the present invention further comprises a ligand which binds to (or interacts with) (preferably specifically binds to or specifically interacts with) an E3 ubiquitin ligase.

[0100] The term “E3 ubiquitin ligase” is used herein to refer to a protein, or protein complex, which is capable of assisting or catalysing the transfer of one or more ubiquitin molecules (or one or more ubiquitin chains) to a target protein (here CD80, CD86 or CD40), preferably the transfer of one or more ubiquitin chains that leads to degradation of the target protein by the proteasome.

[0101] Thus, a PROTAC molecule used in the conjugates of the present invention comprises a ligand which binds to (or interacts with) (preferably specifically binds to or specifically interacts with) any E3 ubiquitin ligase protein, provided said ligand binding or interaction does not inhibit or significantly reduce the biological activity of the E3 ubiquitin ligase, e.g. does not inhibit the ability of the E3 ubiquitin ligase to assist or catalyse the transfer of one or more ubiquitin molecules or (preferably) one or more ubiquitin chains to a target protein. The skilled person would be aware of appropriate methods to analyse binding or interaction of a ligand to an E3 ubiquitin ligase, and to analyse the subsequent biological activity of the E3 ubiquitin ligase, for example ELISA and western blot, to identify suitable ligands for the PROTAC molecules according to this aspect of the invention. Ligands which interact with or bind to E3 ubiquitin ligases are well known in the art and any of these may be used.

[0102] By way of non-limiting example, common E3 ubiquitin ligase ligands in the art include those which bind to or interact with the E3 ubiquitin ligases CRBN or VHL.

[0103] Appropriate linker molecules for use in such PROTAC molecules can be any moiety or molecule which is capable of covalently or non-covalently conjugating (preferably covalently conjugating) the target protein ligand and the E3 ubiquitin ligase ligand, as defined elsewhere herein. Linker moieties used in the PROTAC molecules in the conjugates of the present invention conjugate to the target protein ligand and the E3 ubiquitin ligase ligand concurrently. Any linker moiety can be used provided it is an appropriate length or size to allow sufficient proximity for the ubiquitination of the target protein by the E3 ubiquitin ligase, and does not otherwise inhibit, reduce or limit the biological activity of the E3 ubiquitin ligase. Appropriate linker moieties are well known in the art and any of these may be used. Thus, in some embodiments, the PROTAC molecule used in the conjugates of the present invention can be any molecule, that can concurrently bind to (or interact with), preferably specifically bind to (or specifically interact with), one or more of the CD80, CD86 and CD40 proteins, preferably the CD80 protein or at least the CD80 protein, and one or more E3 ubiquitin ligase, resulting in reduction of (or limitation of or abolishment of) the expression of (or level of), preferably the cell surface expression of (or level of), one or more of CD80, CD86 and CD40 proteins, preferably CD80 protein or at least CD80 protein, by proteasomal degradation.

[0104] In some embodiments, the conjugates of the invention may comprise a number of different TPD (e.g. PROTAC) molecules with different target protein ligands while comprising the same or different E3 ubiquitin ligase ligands. The different target protein ligands may be different ligands targeting the same target protein (e.g. CD80, CD86 or CD40),for example targeting different protein domains of the same target protein. Alternatively, the different target protein ligands may be ligands targeting different target proteins (e.g. two or more of CD80, CD86 and CD40).

[0105] Alternatively, if it is desired to target more than one of CD80, CD86 and CD40, then multiple different TPD, e.g. PROTAC, conjugates of the invention can be used, each of which individually targets a single target protein (here CD80 or CD86 or CD40).

[0106] Thus, multiple target proteins (e.g. more than one of CD80, CD86 or CD40) can either be targeted with single TPD, e.g. PROTAC, conjugates of the invention that have different target protein ligands which target multiple target proteins (here more than one of CD80 or CD86 or CD40), or can be targeted using multiple different TPD, e.g. PROTAC, conjugates of the invention which, for example, each have TPD or PROTAC molecules that target a single target protein (here CD80 or CD86 or CD40). Such multiple conjugates can conveniently be used together in combination.

[0107] Again, in such embodiments, a plurality of (or more than one, or at least two) TPD molecules (e.g. PROTAC molecules) of each different TPD molecule (e.g. PROTAC molecule) can be present in a single conjugate.

[0108] In some embodiments, the silencing molecule used in the conjugates of the invention may comprise or consist of one of, or two of, or all three of the silencing molecule modalities: inhibitory RNA, CRISPR-Cas and TPD (preferably PROTAC), as defined above. For example, a single antigen binding protein, for example an antibody, may be conjugated to a silencing molecule that comprises or consists of one of, or two of, or all three of: inhibitory RNA such as siRNA, CRISPR-Cas, and TPD such as PROTAC. In embodiments where the silencing molecule comprises or consists of at least two of the above silencing molecule modalities, at least siRNA and PROTAC are preferred.

[0109] In some embodiments, when the silencing molecule used in the conjugates of the invention comprises or consists of two or more of inhibitory RNA, CRISPR-Cas and TPD (preferably PROTAC), each silencing molecule modality may target the same gene or gene product (e.g. targeting two or more of: DNA, mRNA and protein of one gene selected from CD80, CD86 or CD40, preferably CD80). Alternatively, each silencing molecule modality may target a different gene or gene product (e.g. targeting two or more of: DNA, mRNA and protein, of two or more genes selected from: CD80, CD86 and CD40, preferably including CD80).

[0110] Alternatively, a single antigen binding protein, for example an antibody, may be conjugated separately to multiple different types / modalities of silencing molecules, e.g. two or more of inhibitory RNA (such as siRNA), CRISPR-Cas, and TPD (preferably PROTAC).

[0111] Again, in some such embodiments, where multiple different types / modalities of silencing molecules are conjugated separately to an antigen binding protein, for example an antibody, then each silencing molecule type / modality may target the same gene or gene product (e.g. targeting two or more of: DNA, mRNA and protein of one gene selected from CD80, CD86 or CD40, preferably CD80). Alternatively, each silencing molecule type / modality may target a different gene or gene product (e.g. targeting two or more of: DNA, mRNA and protein, of two or more genes selected from: CD80, CD86 and CD40, preferably including CD80).

[0112] Alternatively, a single antigen binding protein, for example an antibody, may be conjugated to a single type / modality of silencing molecule, e.g. inhibitory RNA (such as siRNA), CRISPR-Cas or TPD (preferably PROTAC). If there are multiple target proteins, e.g. more than one of CD80 or CD86 or CD40, then such multiple target proteins (e.g. more than one of CD80, CD86 or CD40) can either be targeted with a single conjugate of the invention that comprises appropriate different silencing molecules which target multiple target proteins (here more than one of CD80 or CD86 or CD40), or can be targeted using multiple different conjugates of the invention of a single type, e.g. a single type selected from inhibitory RNA (such as siRNA), CRISPR-Cas or TPD (preferably PROTAC) conjugates which, for example, each have silencing molecules that target a single target protein (here CD80 or CD86 or CD40). Such multiple conjugates can conveniently be used together in combination.

[0113] Again, in such embodiments, a plurality of (or more than one, or at least two) silencing molecules of each different silencing molecule can be present in a single conjugate. In preferred embodiments of the invention where all three of CD80, CD86 and CD40 are targeted, then multiple different conjugates of the invention are used, e.g. in combination, which each target a single target protein (here CD80 or CD86 or CD40). A preferred modality of silencing molecule is inhibitory RNA, more preferably siRNA. In other preferred embodiments of the invention only, or at least, CD80 is targeted. Again for such embodiments a preferred modality of silencing molecule is inhibitory RNA, more preferably siRNA.

[0114] In some embodiments, the antigen binding proteins, e.g. antibodies, used in the conjugates of the present invention and the silencing molecules used in the conjugates of the present invention are linked or coupled or conjugated using a linker. Thus, in such embodiments parts i) and ii) of the conjugates of the invention, as described herein, are coupled (or linked or conjugated) by a linker / linker molecule.

[0115] An appropriate linker for use in the conjugates of the present invention may be any molecule, or combination of molecules, that is able to concurrently bind to (or interact with or conjugate to) the antigen binding proteins, e.g. antibodies, used in the conjugates of the invention, and bind to (or interact with or conjugate to) one or more of the silencing molecules used in the conjugates of the present invention, as described elsewhere herein. Thus, the linkers used in the conjugates of the present invention link or couple or conjugate the antigen binding proteins, e.g. antibodies, and silencing molecules, thus forming the preferred conjugates of the present invention.

[0116] Appropriate linkers are known in the art, and any such linker may be used, provided it is capable of binding to (or interacting with or conjugating to) an antigen binding protein, for example an antibody, used in the conjugates of the present invention and binding to (or interacting with or conjugating to) one or more silencing molecules used in the conjugates of the present invention concurrently (i.e. at the same time), thereby joining, linking or conjugating parts i) and parts ii) of the conjugates of the invention together. It is further provided that such a linker does not affect or reduce (preferably does not significantly affect or significantly reduce) the biological activity of the antigen binding protein, e.g. antibody, or the one or more silencing molecules of the conjugates of the invention. In other words the linker used is such that the biological activity of parts i) and ii) of the conjugates of the invention are retained.

[0117] Alternatively viewed, any linker may be used provided it allows (or induces or promotes) the internalisation of the one or more silencing molecules (as defined elsewhere herein) alongside (or coupled to or simultaneously with) the antigen binding proteins (e.g. antibodies) in the conjugates according to the present invention, provided such a linker does not affect or reduce (or significantly affect or significantly reduce) the biological activity or the specificity of the antigen binding protein, e.g. antibody, or the biological activity of the one or more silencing molecules, as defined previously herein. Methods of assessing the internalisation of the antigen binding protein and the silencing molecule, the specificity of the antigen binding protein, and the biological activity of the silencing molecules are described elsewhere herein.

[0118] In some embodiments, a plurality of linkers (i.e. at least two) bind to (or interact with or conjugate to) the antigen binding protein, for example an antibody, used in the conjugates of the present invention, for example a plurality of linkers can bind to different locations (e.g. different cysteine or lysine residues) within the antigen binding protein, e.g. antibody, for example at least two cysteine or lysine residues (preferably at least two cysteine residues) in the immunoglobulin chains of the antibody. Conveniently for example, the linkers can be bound to appropriate residues, e.g. to cysteine or lysine residues (preferably to cysteine residues), in the Fc region (or other constant regions) of an antibody. Where residues in the Fc region are used then the linkers can for example be bound to the same residue(s) in each of the two chains of the Fc region in order for a plurality of linkers to be present.

[0119] In the conjugates of the invention, each linker may itself interact with or conjugate to a plurality of silencing molecules as defined elsewhere herein. Thus, preferred conjugates of the invention comprise one antigen binding protein, for example an antibody, bound to (or interacting with or conjugating to) a plurality of linkers (i.e. at least two), wherein each linker may itself interact with or conjugate to a plurality of silencing molecules as defined elsewhere herein.

[0120] Preferred linkers (linker molecules) comprise or consist of a chemical crosslinker and optionally (but preferably) a carrier peptide.

[0121] Such chemical crosslinkers and optional carrier peptides are generally used to link parts i) and parts ii) of the conjugates of the invention in any appropriate way or in any appropriate order. Conveniently part i) of the conjugate is joined to (or conjugated to) a chemical crosslinker and this chemical crosslinker then joins to (or conjugates to) part ii) of the conjugate, optionally through a carrier peptide. Appropriate carrier peptides and chemical crosslinkers are well known and described in the art and any of these can be used in the conjugates of the invention.

[0122] In the exemplary arrangement described above an appropriate “chemical crosslinker” refers to any molecule, or combination of molecules, that are capable of conjugating to (or interacting with) the antigen binding protein, for example an antibody, (or part i)) of the conjugates of the present invention, and concurrently conjugating to (or interacting with) the silencing molecule (or part ii)) of the conjugates of the present invention, optionally via a carrier peptide, e.g. as described elsewhere herein. An appropriate “chemical crosslinker” will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the function of any components of the complete conjugate. Thus for example such a “chemical crosslinker” will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the biological activity or specificity (e.g. specificity for a pMHC) of the antigen binding protein, e.g. antibody, or the silencing molecules used in the conjugates of the invention, will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the interaction between any carrier peptide and the silencing molecules used in the conjugates of the invention, and will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the internalisation of the antigen binding protein and / or the one or more silencing molecules used in the conjugates of the present invention.

[0123] Appropriate chemical crosslinkers, e.g. chemical cross linkers that can be used to conjugate proteins such as antibodies to other molecular entities such as nucleic acids or other proteins, are known in the art and the skilled person would be aware of methods to assess whether the chemical crosslinkers inhibit or reduce the interaction between the carrier peptide and the silencing molecules (e.g. electrophoretic mobility shift assay (EMSA) or fluorescence microscopy using a fluorescently labelled siRNA), assess the biological activity or specificity (e.g. specificity for a pMHC) of the antigen binding proteins (e.g. antibodies) or silencing molecules, and assess the internalisation of the antigen binding protein or silencing molecules (e.g. as described elsewhere herein).

[0124] Preferred chemical crosslinkers for use in the conjugates of the invention are molecules which can conjugate to one or more residues, e.g. one or more cysteine residues and / or one or more lysine residues (preferably one or more cysteine residues) within the antigen binding protein component (part i) of the conjugate of the invention (e.g. one or more cysteine residues and / or lysine residues, preferably one or more cysteine residues, within the immunoglobulin chains of an antibody, e.g. within the Fc region), and concurrently conjugate to a carrier peptide (e.g. via an amino group). Preferred chemical crosslinkers comprise an N-hydroxysuccinimide (NHS) ester functional group and an alkene functional group, which are capable of reacting respectively with amine groups (e.g. present on amino acid side chains of the carrier peptide), thus forming a covalent amide bond with a carrier peptide, and with thiol functional groups (e.g. present on amino acid side chains of the immunoglobulin chains of an antibody), forming a covalent thioether bond with an antigen binding protein e.g. an antibody.

[0125] For example, particularly preferred chemical crosslinkers are sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate) molecules, which are heterobifunctional crosslinkers that contain a sulfo-NHS ester group (i.e. comprising an NHS ester functional group) and a maleimide group (i.e. comprising an alkene functional group), and are thus capable of concurrently forming an amide bond with the carrier peptide and forming a thioether bond with the antigen binding protein, e.g. antibody (and thus linking the antigen binding protein component of part i) to the silencing molecule component of part ii)). Other examples of chemical crosslinkers include, but are not limited to: / V-succinimidyl-4-(2- pyridyldithio)butyrate (SPDB), avidin-biotin, and hydrazinonicotinate acetone hydrazine (HyNic)-disulfide formyl benzoate (SS-FB).

[0126] Thus, in a particularly preferred embodiment, the chemical crosslinker used in the conjugates of the present invention comprises or consists of one or more sulfo-SMCC molecule.

[0127] In preferred embodiments, the thiol functional groups are present on amino acid side chains of the antigen binding protein used in the conjugates of the present invention, for example present on the amino acid side chains of the immunoglobulin chains of an antibody, wherein the thiol functional groups are provided by one or more (preferably at least two) cysteine residues within the amino acid sequence of the antigen binding protein.

[0128] Alternatively, the thiol functional groups present on amino acid side chains of the antigen binding protein used in the conjugates of the present invention, for example present on the amino acid side chains of the immunoglobulin chains of an antibody, may be introduced post-translationally, e.g. by using chemicals or molecules that target certain amino acid residues (non-cysteine residues) and converts a side chain on said amino acid residue to a thiol functional group. Such post-translationally modified amino acid residues (e.g. lysine residues) can thus conveniently react with sulfhydryl functional groups (e.g. a maleimide group provided by a sulfo-SMCC chemical crosslinker) to form a thioether bond and link the antigen binding protein (e.g. immunoglobulin chain of an antibody) to the chemical crosslinker for use in the conjugates of the present invention. Chemicals or molecules that can convert an amino acid residue side chain to a thiol functional group are known in the art, for example 2-iminothiolane (also known as Traut’s reagent) and N-succinimidyl-S- acetylthioacetate (SATA) can be used to convert a primary amine on an amino acid residue side chain (e.g. the primary amine on the side chain of a lysine residue) to a thiol functional group and thus provide the addition of a thiol functional group. Thus, in some embodiments the chemical crosslinkers for use in the conjugates of the invention are molecules which can conjugate to one or more lysine residues treated with a chemical or molecule that introduces a thiol group into the side chain of the lysine residue. Carrier peptides are also often used in the conjugates of the invention in order to link parts i) and parts ii) of the conjugates. In the exemplary arrangement described above an appropriate “carrier peptide” can refer to any peptide that is capable of interacting with (or conjugating to) the silencing molecule (or part ii)) of the conjugates of the present invention, and concurrently conjugating to (or interacting with) the chemical crosslinker used in the conjugates of the invention, which in turn is conjugated to (or interacts with) the antigen binding protein, for example an antibody, (or part i)) of the conjugates of the present invention, e.g. as described elsewhere herein. In other embodiments such carrier peptides can be conjugated via an alternative intermediate molecule or conjugated directly to the antigen binding protein, for example an antibody, (or part i)) of the conjugates of the present invention.

[0129] An appropriate “carrier peptide” will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the function of any components of the complete conjugate. Thus for example such a “carrier peptide” will not inhibit or reduce the biological activity or specificity of the antigen binding protein, e.g. antibody, (as described elsewhere herein) or the silencing molecules used in the conjugates of the invention and will not inhibit or reduce the conjugation or interaction between any chemical crosslinker and the antigen binding proteins, e.g. antibodies, used in the conjugates of the invention. Furthermore, such a carrier peptide will not inhibit or reduce (e.g. will not significantly inhibit or reduce) the internalisation of the antigen binding protein or silencing molecules of the conjugate of the present invention.

[0130] Appropriate carrier peptides are known in the art and the skilled person would be aware of methods to assess whether the carrier peptides inhibit or reduce the conjugation or interaction between any chemical crosslinker and the antigen binding proteins, e.g. antibodies, and assess the biological activity of the antigen binding proteins, e.g. antibodies, or the silencing molecules (as described elsewhere herein). Methods of assessing the internalisation of the antigen binding protein and the silencing molecule and the specificity of the antigen binding protein (e.g. for a specific pMHC) are described elsewhere herein.

[0131] Preferred carrier peptides are capable of interacting with (or conjugating to) more than one, preferably multiple or a plurality of (e.g. at least two), silencing molecules as described herein in order to form the conjugates of the present invention.

[0132] Preferred carrier peptides used in the conjugates of the invention are cationic (positively charged) peptides (cationic carrier peptides), e.g. protamine, which can electrostatically interact with anionic (negatively charged) silencing molecules, for example RNA (including siRNA, shRNA, cshRNA and gRNA) and / or DNA (including plasmid DNA which can encode for example an shRNA or a CRISPR-Cas system, or a cshRNA expression cassette which can encode a cshRNA as described elsewhere herein). Such carrier peptides can thus interact with (or conjugate to) more than one (or multiple, or a plurality of, e.g. at least two) silencing molecules.

[0133] In a particularly preferred embodiment, the carrier peptide used in the conjugates of the invention comprises or consists of one or more protamine molecules. Protamine is a low molecular weight protein rich in arginine and is thus highly positively charged, meaning this protein can electrostatically interact with negatively charged molecules such as nucleotides. In some embodiments, protamine can be covalently attached (for example via an amine group) to an NHS ester functional group on a chemical crosslinker, for example sulfo-SMCC, and as such can be linked via a sulfo-SMCC molecule to the antigen binding proteins (e.g. antibodies) used in the conjugates of the present invention. Any version or isoform of protamine can be used provided it is sufficiently positively charged to enable ionic interaction with nucleotides (e.g. using assays as described above) and does not adversely affect the function of the conjugate as described elsewhere herein.

[0134] Thus, in a preferred embodiment, the conjugate of the invention comprises a linker comprising or consisting of i) a chemical crosslinker which can conjugate to one or more cysteine residues and / or one or more lysine residues (preferably one or more cysteine residues) within the antigen binding protein of the conjugate (for example one or more cysteine residues and / or one or more lysine residues within the immunoglobulin chains, e.g. an Fc region, of an antibody), and / or (preferably and) ii) a cationic carrier peptide.

[0135] In a particularly preferred embodiment, the conjugate of the invention comprises a linker comprising or consisting of i) a chemical crosslinker which comprises or consists of one or more sulfo-SMCC molecules, and / or (preferably and) ii) a carrier peptide which comprises or consists of one or more protamine peptides.

[0136] Part i) (or component i)) of the conjugates of the invention comprises an antigen binding protein, e.g. an antibody, comprising two antigen binding domains that each has the ability to bind to (or specifically bind to) a peptide associated with an MHC molecule, e.g. an MHC class I or MHC class II molecule (or an MHC molecule, e.g. an MHC class I or MHC class II molecule loaded with peptide). Thus, the target antigen for the antigen binding domains of such antibodies is a peptide associated with an MHC molecule, e.g. an MHC class I or MHC class II molecule (or an MHC molecule, e.g. an MHC class I or MHC class II molecule loaded with peptide), also referred to herein as pMHC, pMHCI or pMHCll (which can equally be referred to as pHLA, pHLAI or pH LAI I). In nature, such pMHC antigens are recognised by T Cell Receptors (TCRs), for example TCRs expressed on T cells. Thus, antigen binding proteins or antibodies or antigen binding domains with this type of binding (specificity of binding) for pMHC complexes are sometimes referred to as TCR-like antibodies / antigen binding domains / antigen binding proteins.

[0137] Preferably the pMHC molecules to which these antigen binding proteins or antibodies or antigen binding domains bind are expressed on the surface of a cell, and such cells can thus be referred to herein as target cells. Any cells expressing pMHC, e.g. pMHCI or pMHCll molecules, can be targeted in this way. Exemplary cells would include antigen presenting cells (APCs), e.g. immune cells that present antigens (e.g. disease-associated antigens) associated with an MHC molecule (i.e. a pMHC molecule), for example dendritic cells, B cells, macrophages, monocytes and sub-types of these.

[0138] Preferred pMHC molecules that can be bound by the antibody component of the conjugates of the invention are pMHCI or pMHCll molecules associated with disease, in particular autoimmune diseases.

[0139] In preferred embodiments of the invention pMHCll molecules are targeted. However, embodiments described herein for pMHCll can also be used, mutatis mutandis, for pMHCI.

[0140] Thus, a pMHCll target antigen that is bound by an antigen binding domain of an antibody or antigen binding protein as referred to herein is made up of two components. The first component of the pMHCll target antigen is a peptide (p) that comprises a particular defined amino acid sequence, for example an amino acid sequence which comprises a T-cell epitope, for example a 9-mer core sequence of a T-cell epitope. Exemplary peptide sequences (and T-cell epitope sequences) of peptides that are associated with MHC class II molecules, for example disease-associated peptides that are associated with MHC class II molecules, are well known and described in the literature. The second component of the pMHCll target antigen is a particular MHC class II component, e.g. a particular, HLA-DP, HLA-DQ or H LA-DR molecule. The appropriate peptide (p) is associated with or bound to the MHC class II molecule, for example loaded into the peptide groove of the MHC class II molecule, and the antigen binding proteins, e.g. antibodies, or antigen binding domains, used in the conjugates of the invention have the ability to bind to peptide associated (or when associated) with the MHC class II molecule, in other words have the ability to bind to the pMHCll complex.

[0141] Preferred antigen binding proteins, e.g. antibodies, will thus bind to a chosen combination of peptide and MHC, e.g. peptide and MHC class II, e.g. a chosen pMHCll complex. More preferred antibodies will have a level of specificity in binding and will for example not bind (or not significantly bind) to target antigens other than the chosen pMHC, e.g. chosen pMHCll, target antigens. More preferred antibodies will specifically bind to the chosen combination of peptide and MHC, e.g. a chosen pMHCll complex. Such antibodies generally will not show binding or significant binding to other pMHC or pMHCll combinations.

[0142] As used herein, the term "specifically bind(s)” or “specifically recognise(s)” in the context of an antigen binding domain binding to a pMHCll molecule means that those binding proteins (e.g. antibodies or antigen binding domains of antibodies) are capable of binding to a chosen pMHCll molecule, e.g. a peptide loaded or presented on HLA-DQ, e.g. HLA-DQ2.5 or HLA-DQ2.2, and do not cross-react (or do not bind) or do not significantly cross-react (or do not significantly bind) with other antigens or other peptides loaded or presented on the same MHCII molecule, e.g. HLA-DQ2.5 or HLA-DQ2.2.

[0143] Such binding proteins (e.g. antibodies or antigen binding domains of antibodies) which bind (or specifically bind) to a pMHCll molecule in accordance with the invention generally do not bind to the peptides (p) when they are in a naked, isolated or uncomplexed form, but only bind when said peptide (p) is associated with an MHC class II molecule. In addition, such binding proteins (e.g. antibodies or antigen binding domains of antibodies) which bind (or specifically bind) to a pMHCll molecule in accordance with the invention generally do not bind to MHC class II molecules alone, e.g. MHCII molecules that are empty or unloaded with peptide.

[0144] The antigen binding proteins, e.g. antibodies, used in the conjugates of the invention comprise two (or at least two) antigen binding domains that each has the ability to bind to (or specifically bind to) a pMHC, e.g. a pMHCll, molecule. Thus, such antibodies can be bivalent (divalent) or multivalent for pMHCll molecules (or can bind bivalently or multivalently to pMHCll molecules), e.g. can comprise two antigen binding domains that can bind to pMHCll molecules (e.g. only two antigen binding domains that can bind to pMHCll molecules) or more than two antigen binding domains that can bind to pMHCll molecules. Such antigen binding proteins, e.g. antibodies, are hence at least bivalent (or at least divalent) for pMHCll. The pMHCll molecules bound by the two (or at least two) antigen binding domains can be the same or different, providing that the pMHCll molecules are located on the surface of the same target cell. As described elsewhere herein, in some preferred embodiments the pMHCll molecules bound by the antigen binding domains of the antigen binding proteins, e.g. antibodies, used in the conjugates of the present invention are pMHCll molecules loaded with the glia-a2 or glia- ala peptides, e.g. HLADQ2.5-glia-a2 or HLADQ2.5-glia-a1a.

[0145] Antibodies that bind to the same type of pMHC, e.g. pMHCll, molecule (e.g. that bind to the same peptide-MHCH complex), or the same epitope of the same type of pMHCll molecule (e.g. that bind to the same epitope in the same peptide-MHCH complex), can be referred to as monospecific, i.e. bind to one type of target antigen, or the same epitope on the same type of target antigen. In some embodiments, monospecific antigen binding proteins, e.g. antibodies, are preferred. However, such antigen binding proteins or antibodies are still required to have two or more antigen binding domains that bind to pMHCll molecules and are hence at least bivalent (or at least divalent) for pMHCH. In such embodiments, the two or more antigen binding domains that bind to the target pMHCll molecules can be the same or different, providing that the same specific pMHCll molecule is targeted (e.g. HLADQ2.5-glia-a2 or HLADQ2.5-glia-a1a). In some embodiments, monospecific antigen binding proteins, e.g. antibodies, in which the two, or at least two, antigen binding domains that bind to the target pMHC, e.g. pMHCll, molecules are the same, are preferred. In other words, two or more identical antigen binding domains are used. Thus, such antigen binding domains comprise the same CDRs and optionally, but preferably, the same FR regions, or the same VL and VH domains. Such identical antigen binding domains can conveniently be provided by using a full length antibody format, e.g. a conventional full length antibody, e.g. a full length IgG antibody.

[0146] Any TCR-like antibodies can be used to provide such CDR, FR, VL and VH sequences (and indeed provide the CDR, FR, VL and VH sequences for use in any of the antigen binding proteins, antibodies, or antigen binding domains, used in the conjugates of the invention), examples of which are known in the art. Preferred CDR, FR, VL and VH sequences for antigen binding proteins, antibodies, or antigen binding domains, used in the conjugates of the invention, such as bivalent and multivalent antigen binding proteins, antibodies, or antigen binding domains, are as provided elsewhere herein, see e.g. the exemplary TCR-like antibodies 3.C11, 4.7C and RF117 as shown in Tables A, B and C, respectively.

[0147] Thus, in some embodiments an exemplary antigen binding protein, e.g. antibody, or antigen binding domain, for use in the conjugates of the invention is an antigen binding protein, antibody, or antigen binding domain which binds to HLA-DQ2.5 presenting the a2 gliadin peptide or HLA-DQ2.5:DQ2.5 presenting the a2 gliadin peptide, and which comprises at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises: a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTVRSRVHA (SEQ ID NO:5) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:6) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDVQRMGMDV (SEQ ID NO:7) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of QDISNW (SEQ ID NO:8) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VL CDR2 that comprises the amino acid sequence of DSS (SEQ ID NO:9) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QQFNSYPLT (SEQ ID NO: 10) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative said sequence.

[0148] In other embodiments, antibodies (or other binding proteins) comprising antigen binding domains with VH and / or (preferably “and”) VL domains, that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the given amino acid sequence in Table A can be used in the conjugates of the invention, or antibodies (or other binding proteins) comprising antigen binding domains with a set of 6 CDR domains that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the set (combined set) of 6 CDRs of Table A, i.e. the set of CDRs taken as a whole, can be used in the conjugates of the invention. Such variant antibodies (or other binding proteins) should comprise antigen binding domains which retain the ability to bind to HLA-DQ2.5:DQ2.5 presenting the a2 gliadin peptide.

[0149] Antibodies with all 6 of the CDR sequences of Table A are also referred to herein as the 3.C11 antibody. Such antibodies (or other binding proteins or antigen binding domains) with all 6 of these CDR sequences, for example, the antibody as defined in Table A, for example, antibodies with the VH and VL domains as outlined in Table A, or one or more of the other sequences as outlined in Table A, are preferred for use in the conjugates of the present invention.

[0150] Thus, in some embodiments an exemplary antigen binding protein, e.g. antibody, or antigen binding domain, for use in the conjugates of the invention is an antigen binding protein, antibody, or antigen binding domain which binds to HLA-DQ2.5 presenting the ala gliadin peptide or HLA-DQ2.5:DQ2.5 presenting the a1 a gliadin peptide, and which comprises at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises: a variable heavy (VH) CDR1 that comprises the amino acid sequence of GDSVSSNSAA (SEQ ID NO:23) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of TYYRSKWYN (SEQ ID NO:24) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDRTTGWHPYGMDV (SEQ ID NO:25) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of HDISSY (SEQ ID NO:26) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:27) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QDLNSYPL (SEQ ID NO:28) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative said sequence.

[0151] In other embodiments, antibodies (or other binding proteins) comprising antigen binding domains with VH and / or (preferably “and”) VL domains, that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the given amino acid sequence in Table B can be used in the conjugates of the invention, or antibodies (or other binding proteins) comprising antigen binding domains with a set of 6 CDR domains that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the set (combined set) of 6 CDRs of Table B, i.e. the set of CDRs taken as a whole, can be used in the conjugates of the invention. Such variant antibodies (or other binding proteins) should comprise antigen binding domains which retain the ability to bind to HLA-DQ2.5:DQ2.5 presenting the ala gliadin peptide. Antibodies with all 6 of the CDR sequences of Table B are also referred to herein as the 4.7C antibody. Such antibodies (or other binding proteins or antigen binding domains) with all 6 of these CDR sequences, for example, the antibody as defined in Table B, for example, antibodies with the VH and VL domains as outlined in Table B, or one or more of the other sequences as outlined in Table B, are preferred for use in the conjugates of the present invention.

[0152] Thus, in some embodiments an exemplary antigen binding protein, e.g. antibody, or antigen binding domain, for use in the conjugates of the invention is an antigen binding protein, antibody, or antigen binding domain which binds to HLA-DQ2.5 presenting the ala gliadin peptide or HLA-DQ2.5:DQ2.5 presenting the a1 a gliadin peptide, and which comprises at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises: a variable heavy (VH) CDR1 that comprises the amino acid sequence of GDSVSSSSAA (SEQ ID NO:41) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of TYYRSKWYN (SEQ ID NO:42) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDRTTGWHPYGMDV (SEQ ID NO:43) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of HDISSY (SEQ ID NO:44) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:45) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QNLNSYPL (SEQ ID NO:46) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative said sequence.

[0153] In other embodiments, antibodies (or other binding proteins) comprising antigen binding domains with VH and / or (preferably “and”) VL domains, that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the given amino acid sequence in Table C can be used in the conjugates of the invention, or antibodies (or other binding proteins) comprising antigen binding domains with a set of 6 CDR domains that have at least 60%, 70%, 80%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the set (combined set) of 6 CDRs of Table C, i.e. the set of CDRs taken as a whole, can be used in the conjugates of the invention. Such variant antibodies (or other binding proteins) should comprise antigen binding domains which retain the ability to bind to HLA-DQ2.5:DQ2.5 presenting the ala gliadin peptide.

[0154] Antibodies with all 6 of the CDR sequences of Table C are also referred to herein as the RF117 antibody. Such antibodies (or other binding proteins or antigen binding domains) with all 6 of these CDR sequences, for example, the antibody as defined in Table C, for example, antibodies with the VH and VL domains as outlined in Table C, or one or more of the other sequences as outlined in Table C, are preferred for use in the conjugates of the present invention.

[0155] In addition, for all the above embodiments, at the amino acid level, other preferred CDR sequences contain up to 6, e.g. only 1 , 2, 3, 4, 5 or 6, for example 1 , 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1 , 2 or 3, more preferably 1 or 2, altered amino acids, in the provided sequence. Said alterations can be with conservative or non-conservative amino acids, or a mixture thereof. Preferably said alterations are substitutions, preferably conservative amino acid substitutions.

[0156] In certain embodiments, if a given starting CDR sequence is relatively short (e.g. three or six amino acids in length), then fewer amino acid substitutions may be present in such CDR sequences as compared with the number of amino acid substitutions that might optionally be made in a longer starting CDR sequence. For example, in certain embodiments, a starting CDR sequence which may be six amino acid residues in length, preferably has 1 or 2 (more preferably 1) altered amino acids in comparison with the starting sequence. In addition, in certain embodiments, a starting CDR sequence which may be three amino acid residues in length, preferably has 1 or 2 (more preferably 1 , and sometimes none) altered amino acids in comparison with the starting sequence. Accordingly, in some embodiments the number of altered amino acids in CDR sequences can be tailored to the length of a given starting CDR sequence. For example, different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence such as to achieve a particular % sequence identity in the CDRs, for example a sequence identity of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 93%, 95%, 96%, 97%, 98% or 99% in each CDR. Said alterations can be with conservative or non-conservative amino acids, or a mixture thereof. Preferably said alterations are substitutions, preferably conservative amino acid substitutions.

[0157] For the variant antibodies described above, e.g. antibodies (or other binding proteins) having a sequence containing 1, 2 or 3 (preferably 1 or 2, or 1) amino acid substitutions, additions or deletions relative to the exemplary TCR-like antibody CDR sequences as set out in Tables A, B and C, preferred variant antibodies (or other binding proteins) maintain the biological activity and / or the specificity and / or the ability to conjugate to one or more silencing molecules as the exemplary TCR-like antibodies. For example, the variant antibodies (or other binding proteins) maintain the specificity for binding to the same peptide MHC (pMHC) molecule on the surface of the target cell (i.e. binding to the same pMHC), and / or (preferably “and”) maintain the ability to conjugate to the one or more silencing molecules as described elsewhere herein (e.g. via sulfo-SMCC and protamine linkage), and / or (preferably “and”) maintain the ability to be internalized along with the one or more silencing molecules into the target cell. Methods of assessing the internalization of the antigen binding protein and the silencing molecule, the specificity of the antigen binding protein, and the biological activity of the silencing molecules are described elsewhere herein.

[0158] Antigen binding proteins, e.g. antibodies, that bind to two (or at least two) different types of pMHCll molecules can be referred to as bispecific (e.g. that bind to HLADQ2.5-glia-a2 and HLADQ2.5-glia-a1a), trispecific or multispecific, i.e. bind to more than one type of target antigen. However, for use in the conjugates of the invention, such antigen binding proteins, e.g. antibodies, are still required to have two or more antigen binding domains that bind to pMHCll molecules and are hence at least bivalent (or divalent) for pMHCll.

[0159] Exemplary and preferred antibodies have two, or only two, antigen binding domains, or 12, or only 12, CDRs (e.g. two sets of 6 CDRs) that bind to pMHCll molecules.

[0160] The term “bivalent” as used herein refers to a binding protein (or antibody) with 2 antigen binding domains. The term “bivalent for pMHCll”, or equivalent terms, refers to a binding protein (or antibody) with 2 antigen binding domains which can hence bind to 2 molecules of pMHCll. The term “divalent” can be used as an alternative to bivalent. The term “multivalent” as used herein refers to a binding protein (or antibody) with more than 2 antigen binding domains. The term “multivalent for pMHCll”, or equivalent terms, refers to a binding protein (or antibody) with more than 2 antigen binding domains which can hence bind to more than 2 molecules of the pMHCll. Trivalent (with three antigen binding domains) and tetravalent (with four antigen binding domains) binding proteins or antibodies are therefore provided. Such binding proteins or antibodies may be “bivalent for pMHCll” or “multivalent for pMHCll”, but may also include additional antigen binding domains that bind to target antigens other than pMHCll. Thus, the antibodies used in the conjugates of the invention as described above and elsewhere herein are at least bivalent, e.g. bivalent or multivalent, for pMHCll, and preferably for a single particular pMHCll. Put another way, they can bind bivalently or multivalently to pMHCll, and preferably a single particular pMHCll.

[0161] Exemplary at least bivalent formats would be well known in the art but include antibodies (or binding proteins) comprising all or a portion of antibody constant regions (e.g. all or a portion of a heavy chain and / or a light chain constant region), and in particular human antibody constant regions, e.g. full length (whole) antibody formats such as full length IgG antibodies, e.g. lgG1, lgG2, lgG3, lgG4, antibodies, or other full length antibodies such as lgA1, lgA2, IgE, IgM, or IgD antibodies.

[0162] Other antibody (or binding protein) bivalent or multivalent formats comprising antibody constant regions, and in particular human antibody constant regions, such as antibodies comprising an Fc region, e.g. Fc fusions, are also provided. Thus, preferred antibodies (or binding proteins) for use in the conjugates of the invention have an Fc region of an antibody. Such Fc regions can provide a convenient way of obtaining an antibody which is bivalent for pMHCll as an antigen binding domain can be attached to each chain of the Fc region (Fc dimer). Exemplary Fc regions comprise (or consist of, or consist essentially of) CH2 and CH3 domains, and optionally also a hinge domain. An exemplary such format might be a bivalent scFv-Fc fusion protein format. Alternative formats which are bivalent or multivalent for pMHCll are also envisaged, e.g. F(ab’)2 formats, Fab3, diabody, triabody, minibody, 2xscFv linked, and bivalent nanobodies.

[0163] Preferred antigen binding proteins, e.g. antibodies, for use in the conjugates of the present invention are antibodies that are internalized into the target cell, preferably to the lysosomal compartment of the target cell, once they have bound to the pMHC, e.g. pMHCll, molecules on the cell surface. Such internalization of the antibody also allows internalization of the conjugate of the invention into the target cells. This, in turn, allows internalization of and delivery of the silencing molecule into the target cell, which can then act to target the expression of (and ultimately the cell surface expression of, preferably to reduce the cell surface expression of) one or more of the CD80, CD86 or CD40 target proteins as appropriate, depending on the nature of the silencing molecule in the conjugate. The nature of the silencing molecule in the conjugate will not only determine which of CD80, CD86 or CD40 is targeted, but also the mode of action of the targeting, e.g. whether the expression of CD80, CD86 or CD40 is targeted (and also whether more than one of CD80, CD86 or CD40 is targeted) at the level of DNA, RNA and / or protein as described elsewhere herein.

[0164] Not all antigen binding proteins or antibodies have the ability to be internalized, e.g. to the lysosomal compartment. This depends on the nature of the target antigen and also on the particular antigen binding protein or antibody that is used. Thus selection of antigen binding proteins / antibodies which have the ability to be internalized, e.g. to the lysosomal compartment, is an important part of the selection process when choosing the components of the conjugates of the invention. Assays to determine this would be well known to a person skilled in the art, and an exemplary assay is outlined in the Examples. Alternatively, non-internalizing antigen binding proteins or antibodies can be engineered so that they have the ability to be internalized, e.g. to the lysosomal compartment.

[0165] The bivalent (or at least bivalent) nature of the antigen binding proteins, e.g. antibodies, used in the conjugates is believed to also be important to this process as the fact that such antigen binding proteins, e.g. antibodies, can interact with two pMHC, e.g. pMHCll, target molecules on the surface of the same target cell (by way of the two antigen binding domains) is believed to allow the cross-linking or cross-coupling of pMHC / pMHCll target antigens and the subsequent internalization of the target antigens (pMHCll molecules), e.g. to the lysosomal compartment, together with the bound antigen binding protein / antibody (and any attached silencing molecules). Not only does this enable the delivery of the silencing molecules to the target cells (and the subsequent targeting, and preferably reduction, in expression of CD80, CD86 and / or CD40), but also prohibits or reduces ADCC of the target cells as the antibody is no longer associated with the cell surface.

[0166] The term "antibody" as used herein, refers broadly to any immunological binding agent that comprises one or more antigen binding domains made up of CDRs and FR regions derived from, or based on such regions derived from, an antibody molecule, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred. However, antigen binding proteins and antibodies used in the conjugates of the invention have a structure or format such that they bind bivalently or multivalently to pMHC, e.g. pMHCll, e.g. can comprise antibodies or antibody fragments that bind bivalently or multivalently to pMHC, e.g. pMHCll. Such antigen binding proteins or antibodies used in the conjugates of the invention thus comprise two, or at least two, antigen binding domains that each has the ability to bind to a pMHC, e.g. pMHCll, molecule.

[0167] Depending on the type of constant domain in the heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM and the antigen binding proteins or antibodies used in the conjugates of the invention may be in any one of these classes. Several of these are further divided into subclasses or isotypes, such as I gG 1 , I gG2, I gG3, I gG4, and the like. The heavy-chain constant domains that correspond to the difference classes of immunoglobulins are termed a, 5, s, y and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Generally, where whole antibodies are used in the invention, IgG are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.

[0168] The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types: kappa (K) and lambda ( ), based on the amino acid sequences of their constant domains and some amino acids in the framework regions of their variable domains.

[0169] The term "heavy chain complementarity determining region" ("heavy chain CDR") as used herein refers to regions of hypervariability within the heavy chain variable region (VH domain) of an antibody molecule. The heavy chain variable region has three CDRs termed heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 from the amino terminus to carboxy terminus. The heavy chain variable region also has four framework regions (FR1, FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

[0170] The term "heavy chain variable region" (VH domain) as used herein refers to the variable region of a heavy chain of an antibody molecule.

[0171] The term "light chain complementarity determining region" ("light chain CDR") as used herein refers to regions of hypervariability within the light chain variable region (VL domain) of an antibody molecule. Light chain variable regions have three CDRs termed light chain CDR1 , light chain CDR2 and light chain CDR3 from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions (FR1 , FR2, FR3 and FR4 from the amino terminus to carboxy terminus). These framework regions separate the CDRs.

[0172] The term "light chain variable region" ( L domain) as used herein refers to the variable region of a light chain of an antibody molecule.

[0173] As described elsewhere herein some of the antigen binding proteins or antibodies for use in the conjugates of the invention comprise constant regions of heavy and / or light chains. All or part of such constant regions may be produced naturally or may be wholly or partially synthetic. Appropriate sequences for such constant regions are well known and documented in the art. When a full complement of constant regions from the heavy and light chains are included in the antibodies used in the invention, such antibodies are typically referred to herein as "full length" antibodies or "whole" antibodies. In some embodiments such full length or whole antibodies are preferred.

[0174] The antibodies or binding proteins for use in the conjugates of the invention can be produced naturally or can be wholly or partially synthetically produced. The antigen binding domains of the antibodies or binding proteins for use in the conjugates of the invention generally comprise an antibody light chain variable region (VL) that comprises three CDR domains and an antibody heavy chain variable region (VH) that comprises three CDR domains.

[0175] As described above and elsewhere herein, preferred antigen binding proteins for use in the conjugates of the invention are, or comprise, antibodies. However, embodiments as described herein which relate to antibodies, apply equally, mutatis mutandis, to other types of antigen binding proteins, or vice versa.

[0176] Preferred antigen binding proteins as referred to herein are polypeptides which can bind (e.g. specifically bind) to pMHC molecules as described herein. Appropriate types of antigen binding protein which could be used in the conjugates of the invention are known in the art. For example, in some embodiments immunoglobulin based polypeptides are used, which generally comprise CDR regions (and optionally FR regions or an immunoglobulin based scaffold), such that the CDR regions (and optionally FR regions) of appropriate antibodies, e.g. TCR-like antibodies, can be grafted onto an appropriate scaffold or framework, e.g. an immunoglobulin scaffold.

[0177] However, in other embodiments, non-immunoglobulin based antigen binding proteins / scaffold proteins can be used which can be selected for the ability to bind (e.g. specifically bind) to a particular target antigen (e.g. pMHC molecules as described herein) in their own right. Such molecules are also referred to as antibody mimics (or antibody mimetics). Examples of appropriate non-immunoglobulin based antigen binding proteins are known and described in the art and include fibronectins (or fibronectin-based molecules), for example based on the tenth module of the fibronectin type III domain, such as Adnectins (e.g. from Compound Therapeutics, Inc., Waltham, MA); affimers (e.g. from Avacta); ankyrin repeat proteins or DARPins (e.g. from Molecular Partners AG, Zurich, Switzerland); lipocalins, e.g. anticalins (e.g. from Pieris Proteolab AG, Freising, Germany); human A- domains (e.g. Avimers); staphylococcal Protein A (e.g. from Affibody AG, Sweden); thioredoxins; and gamma-B-crystallin or ubiquitin based molecules, e.g. affilins (e.g. from Scil Proteins GmbH, Halle, Germany). Such molecules can also be used as scaffolds onto which appropriate CDRs which mediate target antigen binding can be grafted. For example, the CDR regions (and optionally FR regions) of TCR-like antibodies as described herein can be grafted onto an appropriate non-immunoglobulin scaffold. Nucleic acid-based molecules such as aptamers can also be used.

[0178] Types of autoimmune disease to be treated in accordance with the present invention include celiac disease (Sollid LM, et al. Immunogenetics. 2020;72(1-2):85-88), rheumatoid arthritis (Nel HJ, et al. The Lancet Rheumatology. 2020;2(11), e712-e723), type I diabetes (Nakayama M, Michels AW. Front Immunol. 2019;10:365), multiple sclerosis (Lutterotti A, et al. Front Immunol. 2021; 12:640935), and Sjogren's syndrome (Abe S, et al. JCI Insight. 2020;5(15):e135982), but are not limited thereto. Examples of pMHCll molecules (e.g. disease-associated or disease- specific pMHCll molecules) that are appropriate target antigens for the treatment or prevention of these diseases using the conjugates of the invention would thus be readily identified from the art.

[0179] The use of disease-associated or disease-specific pMHCll molecules as targets should ensure that the silencing molecules are targeted to the appropriate target tissue. In addition, various TCR-like antibodies are known in the art to target appropriate pMHCll molecules associated with various diseases and any of these may be used in the conjugates of the invention. Alternatively, appropriate TCR-like antibodies to chosen pMHCll molecules, e.g. disease-associated or disease-specific pMHCll molecules, could be generated and used in the conjugates of the invention. As described elsewhere herein, it is preferred that such antibodies have the ability to be internalized, preferably to the lysosomal compartment, once they have bound to the pMHCll target antigen.

[0180] As a preferred disease to be treated or prevented using the conjugates of the invention is celiac disease, preferred antigen binding proteins, e.g. antibodies, for use in the conjugates of the invention are antigen binding proteins or antibodies that target pMHCll molecules associated with or specific for CeD. The peptides in such pMHCll molecules will generally be gluten-derived peptides, e.g. a gliadin peptide. Such peptides will generally be associated with MHCII in the context of the HLA-DQ, e.g. HLA-DQ2 (especially HLA-DQ2.5), but also HLA-DQ2.2 or HLA-DQ8 molecules. There are a number of characterized HLA- DQ2.5 restricted epitopes (and therefore examples of peptides, p) associated with celiac disease. This number was 27 as of 2020 (Sollid et al., Immunogenetics 72(1-2):85-88) but it is widely accepted that many epitopes are still unknown. Any of these pHLA-DQ, e.g. pHLA- DQ2.5, complexes represent a target antigen for the antigen binding protein / antibody component of the conjugates of the invention, in particular through the use of TCR-like antibodies.

[0181] Thus, preferred conjugates of the invention may comprise antigen binding proteins, e.g. antibodies, comprising antigen binding domains which bind to, or bind specifically to, a pHLA-DQ or pHLA-DQ2 molecule presenting a gluten-derived peptide, e.g. a gliadin peptide, in particular a pMHC HLA-DQ2.5 or HLA-DQ2.2 or HLA-DQ8 presenting a gluten-derived peptide, e.g. a gliadin peptide, e.g. a gliadin alpha or omega peptide. More particularly, preferred conjugates of the invention may comprise antigen binding proteins, e.g. antibodies, comprising antigen binding domains which bind to, or bind specifically to, HLA- DQ2.5:DQ2.5-glia-a1a, or which bind to, or bind specifically to, HLA-DQ2.5:DQ2.5-glia-a2. Most preferably, preferred conjugates of the invention may comprise antigen binding proteins, e.g. antibodies, comprising antigen binding domains which bind to, or bind specifically to, HLA-DQ2.5:DQ2.5-glia-a2.

[0182] The glia-a1a peptide comprises the sequence: PFPQPELPY (SEQ ID NO:61). The glia- 02 peptide comprises the sequence: PQPELPYPQ (SEQ ID NO:62). HLA-DQ2.5:DQ2.5- glia-o1a means an HLA-DQ2.5 molecule that is presenting (or “loaded” with) a DQ2.5-glia- o1a epitope (PFPQPELPY (SEQ ID NO:61)). Put another way, HLA-DQ2.5:DQ2.5-glia-o1a means an HLA-DQ2.5-peptide complex (pMHCll) in which the DQ2.5-glia-o1a epitope is presented in the antigen binding groove (or accommodated in the antigen binding groove). HLA-DQ2.5:DQ2.5-glia-o2 means an HLA-DQ2.5 molecule that is presenting (or “loaded” with) a DQ2.5-glia-o2 epitope (PQPELPYPQ (SEQ ID NO:62)). Put another way, HLA- DQ2.5:DQ2.5-glia-o2 means an HLA-DQ2.5-peptide complex (pMHCll) in which the DQ2.5- glia-o2 epitope is presented in the antigen binding groove (or accommodated in the antigen binding groove).

[0183] Preferred antigen binding proteins, e.g. antibodies, for use in the conjugates of the invention, recognize peptides that are DQ2.5 restricted. In other words, they bind to peptides or epitopes which can be bound to or associated with or presented by the MHC class ll / HLA molecule HLA-DQ2.5. Alternatively, or additionally, preferred antibodies for use in the conjugates of the invention can bind to peptides or epitopes which are DQ2.2 restricted. In other words, they bind to peptides or epitopes which can be bound to or associated with or presented by the MHC class ll / HLA molecule HLA-DQ2.2. Thus preferred antibodies for use in the conjugates of the invention are capable of binding to peptides associated with the MHC class ll / HLA molecule HLA-DQ2.5 or HLA-DQ2.2, preferably HLA- DQ2.5.

[0184] HLA-DQ2.5 (encoded by DQA1*05 and DQB1*02) is a specific type of MHC Class II molecule that has a strong association with CeD. HLA-DQ2.2 (encoded by DQA1*02:01- DQB1*02) is another specific type of MHC Class II molecule that has an association with CeD.

[0185] HLA-DQ2.5 comprises an a-chain (typically having an ai domain and an 02 domain and typically encoded by DQA1*05) and a p-chain (typically having a Pi domain and a 2 domain and typically encoded by DQB1*02). Amino acid sequences of the a- and - chains of HLA- DQ2.5 are known in the art. HLA-DQ2.5 (or HLA-DQ2.2) can present gliadin peptides or epitopes, for example, peptides or epitopes of a-gliadin or co-gliadin, to T cells (e.g.CD4+ T cells). Such pMHCll complexes are preferred target antigens for the antigen binding protein / antibody parts of the conjugates of the present invention as described herein.

[0186] As used herein, the term “target cell” refers to any cell that expresses one or more MHC molecules, e.g. MHC class II molecules, bound to or associated with or presenting or loaded with a disease-associated antigen, preferably an autoimmune disease-associated antigen. Preferred target cells include, but are not limited to, antigen presenting cells (APCs), for example dendritic cells, B cells, macrophages, monocytes and sub-types of these.

[0187] Viewed alternatively, the “target cell” may be any APC that presents the correct pMHC / pMHCll on the cell surface (in this case a disease-associated pMHC / pMHCll which has the ability to interact with the antigen binding protein, for example an antibody, used in the conjugate of the present invention).

[0188] As used herein a “disease-associated antigen” or an “autoimmune disease-associated antigen” is an antigen that is linked to, correlated with, a causative factor in, or a biomarker for a disease, preferably an autoimmune disease, when it is bound to or associated with or presented by one or more MHC or MHC class II molecules on a cell membrane, e.g. an APC. Such cells can be target cells as defined herein. Such pMHC class II complexes (pMHCll) can also be referred to herein as a “disease-associated pMHCll” or an “autoimmune-disease associated pMHCll”). The term “disease-specific antigen” may also be used.

[0189] As used herein an “autoimmune disease” is any disease which is caused by an undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive immune response to an antigen (e.g. an autoimmune disease-associated antigen as defined herein) which induces or promotes or increases the immune system mistakenly attacking healthy cells, tissues and organs in the body of a subject.

[0190] As used herein, the term “cognate T cell” or “cognate T cell receptor (TCR)” refers to the TCR, and the T cell (e.g. a target T cell or a bystander T cell) that expresses said TCR, that interacts with or binds to (preferably specifically interacts with or binds to) the pMHC, e.g. pMHCll, (preferably the disease-associated pMHCll) presented on the surface of the target APC. The term “cognate pMHCll” as used herein refers to the pMHCll (preferably the disease-associated pMHCll) presented on the surface of the target APC that interacts with or binds to (preferably specifically interacts with or binds to) the T cell receptor of the T cell (e.g. a target T cell or a bystander T cell). As used herein, the term “target protein” refers to one or more of CD80, CD86 and CD40 proteins.

[0191] The term “target mRNA” used herein refers to one or more of CD80, CD86 and CD40 mRNA.

[0192] The term “target DNA” is used herein to for example refer to the genomic DNA sequence that encodes the target mRNA and subsequently the target protein as defined elsewhere herein. Thus, in different embodiments, the target DNA can comprise or consist of one or more of the CD80, CD86 and CD40 genes or a relevant portion thereof (e.g. one or more of the CD80, CD86 and CD40 genomic DNA sequences or a relevant portion thereof).

[0193] As used herein, the term “target sequence” for example refers to the nucleotide or amino acid sequence which interacts with or binds to or anneals to the silencing molecules according to the present invention. For example, the inhibitory RNA and gRNA as defined elsewhere herein can interact with or bind to or anneal to an mRNA target sequence and DNA target sequence respectively. For example, the targeted protein degradation (TPD) molecules, e.g. the PROTAC molecules as defined elsewhere herein can interact with or conjugate to an amino acid target sequence, or a plurality of interrupted amino acid target sequences which form one or more protein domains, within the target protein.

[0194] The term “target site” is used herein for example to refer to the DNA sequence that is cleaved by the Cas nuclease as used in the present invention (i.e. the CRISPR-Cas cleavage site), and is used to differentiate this sequence from the target DNA and target sequence (which anneal to or interact with the gRNA) as defined elsewhere herein.

[0195] As used herein, the term “biological activity” in the context of proteins, e.g. target proteins, according to the invention refers to the ability of the protein to perform or maintain its normal or usual functions within the cell or on the cell membrane (e.g. catalysing or performing chemical reactions, interacting with ligands and other proteins, and / or transducing signalling cascades (e.g. costimulatory molecules)).

[0196] As used throughout the entire application, the term “expression of” (or equivalent terms) refer to the measurable amount of or the level of for example the DNA, mRNA or protein, which is dependent on the type of measurement. For example, these terms can refer to the number of copies of DNA or RNA, the units of protein, or the concentration of the DNA, RNA or protein, e.g. as compared to a control. Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no conjugate, or where a conjugate comprising a control antigen binding protein (e.g. a control antibody) that does not bind the target antigen, or where a conjugate comprising a control silencing molecule that does not reduce or abolish expression of the target protein, is used.

[0197] The term “cell surface expression of” as used herein refers to the measurable amount of protein on the cell surface (cell membrane). For example, this can refer to the units of protein or the concentration of the protein on the cell surface (cell membrane), e.g. as compared to a control. Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no conjugate, or where a conjugate comprising a control antigen binding protein (e.g. a control antibody) that does not bind the target antigen, or where a conjugate comprising a control silencing molecule that does not reduce or abolish expression of the target protein, is used.

[0198] The term “expression of” or “cell surface expression of” as used herein also includes the “functional expression of” or the “cell surface functional expression of” respectively, i.e. the amount of or level of a protein that has the ability to perform or maintain its normal or usual functions within the cell or on the cell surface (e.g. catalysing or performing chemical reactions, interacting with ligands and other proteins, and / or inducing or transducing signalling cascades (e.g. costimulatory molecules)). For example, a mutated protein sequence (e.g. generated by CRISPR-Cas) may have the same or similar protein expression level (i.e. the same or similar amount of protein) as the wild-type protein (or similar cell surface protein expression level), while displaying a reduced biological function activity compared to the wild-type protein, and thus has a reduced (or inhibited or abolished) functional expression or cell surface functional expression.

[0199] The term “expression of’ may also refer to the localisation of or translocation of the protein to the cell surface, or the localisation of the protein on the cell surface (e.g. reduced or inhibited or abolished localisation or translocation to the cell surface, or perturbed or modified or inhibited localisation on the cell surface).

[0200] As used herein, the term “biological activity” in the context of antigen binding proteins or antibodies, e.g. antigen binding proteins and antibodies for use in the conjugates of the invention refers to the ability of the antigen binding protein or antibody to, for example, bind to or interact with its target antigen, the specificity of the antigen binding protein or antibody for its target antigen, or the affinity of the interaction with the target antigen. Thus, as described elsewhere herein, appropriate antigen binding proteins or antibodies for use in the conjugates of the invention have pMHCll as a target antigen (e.g. can be referred to as TCR- like antibodies), in particular disease-associated or autoimmune disease-associated pMHCll as a target antigen. The ability of the antigen binding protein or antibody to cross-link cell membrane receptors after target antigen binding, or the ability of the antigen binding protein or antibody to be internalised, e.g. to the lysosomal compartment, after binding to its target antigen, are additional preferred biological activities of such pMHCll targeting antigen binding proteins or antibodies used in the conjugates of the invention.

[0201] As used herein, the term “biological activity” in the context of silencing molecules, e.g. silencing molecules for use in the conjugates of the invention refers to the ability of the silencing molecule to perform or induce or promote its inhibitory activity, e.g. the biological mechanism by which it reduces the expression of (or level of) target mRNA and target protein (as defined herein), for example its ability to target and cleave target DNA (e.g. CRISPR-Cas), or anneal to or interact with and / or induce or promote the degradation of target mRNA (e.g. inhibitory RNA) or target protein (e.g. TPD, preferably PROTAC).

[0202] By “complementary” and “complementarity” are meant for example that a nucleic acid molecule can form hydrogen bond(s) with a second nucleic acid molecule for example by Watson-Crick base pairing. A nucleic acid molecule which can form hydrogen bond(s) with another nucleic acid molecule through non-Watson-Crick base pairing also falls within the definition of having complementarity. A percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively).

[0203] As used herein, the terms “anneal” and “annealing” refer for example to the hydrogen bonding between complementary nucleotides of two nucleic acid molecules by Watson-Crick base pairing.

[0204] The term “mutant” in the context of a protein is used herein for example to refer to a protein comprising one or more mutations (e.g. substitutions, deletions, insertions) in the amino acid sequence of the protein. Such mutations are defined as compared to an appropriate original protein sequence, typically a wild-type, parent or native protein sequence.

[0205] Another aspect of the invention provides nucleic acid molecules (e.g. one or more nucleic acid molecules), comprising nucleic acid sequences which encode the amino acid sequences (or amino acid portions or segments), or fragments thereof, of the conjugates of the invention, as defined herein.

[0206] Compositions comprising at least a first conjugate of the invention, or at least a first nucleic acid molecule of the invention, constitute further aspects of the present invention. Formulations or compositions comprising one or more conjugates, etc., of the invention, optionally in admixture with other components, such as a suitable diluent, carrier or excipient constitute a further embodiment of the present invention. Such formulations or compositions may be for pharmaceutical use, and thus formulations or compositions of the invention are preferably pharmaceutically acceptable or otherwise acceptable for administration to human or non-human mammals, but in particular humans. Suitable diluents, excipients and carriers are known to the skilled person.

[0207] In some embodiments, compositions may comprise more than one type of conjugate of the invention, e.g. comprise at least a second conjugate of the invention. Such compositions may thus comprise more than one different conjugate of the invention, for example each comprising a different antigen binding protein, for example a different antibody, and / or a different silencing molecule.

[0208] In this context a “different antigen binding protein” or a “different antibody” may either bind to the same epitope on the target antigen as the antigen binding protein or antibody used in the first conjugate of the invention, bind to a different epitope on the same target antigen, or bind to a different target antigen, e.g. to a different epitope on a different target antigen. Appropriate target antigens are pMHC molecules, e.g. pMHCll molecules, in particular cell-associated pMHCll molecules (or pMHCll molecules expressed on a cell surface), examples of which are described elsewhere herein.

[0209] In this context, a “different silencing molecule” may either target a different nucleotide or amino acid sequence on the same target sequence (e.g. the different silencing molecules target distinct sequences of the same DNA, RNA or amino acid sequences of the target sequence), or target a different sequence format of the same target protein (e.g. different formats of silencing molecules may be used that can target one of the other (e.g. DNA, RNA or amino acid) of the same protein than that targeted by the silencing molecule used in the first conjugate of the invention), or target the DNA and / or RNA encoding, and / or amino acid sequence of, a different target protein (e.g. if the first conjugate of the invention targets CD80, then different conjugates may be included which target CD86 and / or CD40).

[0210] In such embodiments different linker molecules, e.g. different linkers between parts i) and parts ii) of the conjugates of the present invention may be used if appropriate.

[0211] Thus, some embodiments of the invention provide a composition comprising two, or at least two, e.g. three, conjugates of the invention, or two, or at least two, e.g. three, nucleic acid molecules of the invention. Viewed alternatively such compositions comprise at least a first and at least a second conjugate of the invention, and optionally at least a third conjugate of the invention. In such compositions, the multiple (two or more) conjugates of the inventionwhich are included (i.e. the first, second, third, etc., conjugates of the invention) are different from each other as described elsewhere herein.

[0212] Any appropriate mode of administration can be used. The compositions or conjugates according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular), topical or rectal administration, or for mucosal delivery, and any of these modes of administration, or indeed any other appropriate mode of administration, can be used. In a preferred embodiment, compositions according to the invention are presented in a form suitable for intravenous administration. In some embodiments, compositions according to the invention are presented in a form suitable for intraperitoneal (i.p.) administration.

[0213] The active compounds (e.g. the conjugates or compositions of the invention) as defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, exosomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.

[0214] Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions may then be filled into injection vials or ampoules.

[0215] The pharmaceutical compositions (formulations) of the present invention are preferably administered parenterally. Intravenous administration is preferred. In some embodiments, administration is intraperitoneal (i.p.) administration. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal or intravenous injection by means of a syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the conjugate in the form of a nasal or pulmonal spray. As a still further option, the conjugates or compositions of the invention can also be administered transdermally, e.g. from a patch, optionally an iontophoretic patch, or transmucosally, e.g. bucally.

[0216] Suitable dosage units can be determined by a person skilled in the art.

[0217] A further aspect of the present invention provides the conjugates or compositions of the invention for use in therapy, in particular for use in preventing or treating a disease or disorder, for example an autoimmune disease or disorder. In some embodiments, the disease or disorder is linked to, caused by, characterised by or associated with, undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive immune activation in response to a disease-associated antigen (preferably an autoimmune disease-associated antigen), preferably undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive CD4+ or CD8+ T cell activation in response to a disease-associated antigen (preferably an autoimmune disease-associated antigen).

[0218] Alternatively viewed, the disease or disorder to be treated in accordance with the present invention is linked to, caused by, characterised by or associated with insufficient, a lack of, a decrease in, or reduction in, immune tolerance to a disease-associated antigen (preferably an autoimmune disease-associated antigen).

[0219] As the preferred conjugates of the invention have pMHCll molecules as a target antigen, appropriate diseases and disorders can be any that are linked to, caused by, characterised by or associated with such pMHCll molecules (disease-associated or disease-specific pMHCll molecules).

[0220] Preferred diseases are autoimmune diseases. Other preferred diseases are those which are confined to specific tissues. In this regard, it is believed that the treatment of such diseases will allow a tissue compartmentalised, or localised, inactivation of T cells and hence not induce a general immune deactivation or suppression. Types of autoimmune disease to be treated in accordance with the present invention include celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis and Sjogren's syndrome, but are not limited thereto. Thus, the autoimmune-disease associated antigens in accordance with the present invention include, but are not limited to, antigens associated with, antigens specific for, antigens linked to, antigens causing, or antigen biomarkers for, celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis, and Sjogren's syndrome, but are not limited thereto. Such diseases are known to be associated with pMHCll or have disease- associated or disease-specific pMHCll molecules. In some embodiments a preferred disease or disorder is celiac disease. Examples of pMHCll molecules that are appropriate target antigens for the treatment or prevention of these diseases using the conjugates of the invention are described in the art and some examples are described elsewhere herein.

[0221] Preferred diseases to be treated in accordance with the present invention include celiac disease (discussed elsewhere herein), rheumatoid arthritis (which is e.g. associated with DRB1*04:01, HLA-DRA*01 :01 / HLA-DRB1*04:01), type I diabetes (which is e.g. associated with HLA DR3 (DRB1*0301) or HLA DQ8 (DQB1*0302) or HLA DQ2 (DQB1*0201) or HLA DR4 (DRB1*0401) and multiple sclerosis (which is e.g. associated with HLA DRB1*1501 or HLA DRB5*0101). As other conjugates of the invention have pMHCI molecules as a target antigen, further diseases which may be treated in accordance with the present invention include diseases or disorders (preferably autoimmune diseases) linked to, caused by, characterised by or associated with disease-associated or disease-specific pMHCI molecules, for example psoriasis vulgaris (which is e.g. associated with HLA-Cw6) and ankylosing spondylitis (which is e.g. associated with HLA-B27).

[0222] Alternatively viewed, the conjugates or compositions of the invention can be used to suppress, reduce, inhibit or limit the immune response to an autoimmune disease- associated antigen in vivo, and / or induce or increase tolerance to an autoimmune disease- associated antigen (i.e. induce or increase immune tolerance) in vivo. Such therapies can also be referred to as tolerising therapies.

[0223] The conjugates or compositions of the invention also provide a means of silencing immune cells, in particular autoreactive immune cells such as autoreactive T cells by means of modifying the APC responsible for activating these autoreactive T cells. This silencing thus advantageously provides a means for using disease-specific tools, here diseasespecific antibodies (e.g. TCR-like antibodies) which bind to particular pMHCI or pMHCll molecules to then achieve general silencing or broad suppression of any immune cells which present such disease specific peptides and hence re-establish homeostasis without compromising systemic immunity.

[0224] Thus, a yet further aspect of the present invention provides the conjugates or compositions of the invention for use in reducing or suppressing or limiting an immune response (or autoimmune response) in vivo, and / or inducing or increasing immune tolerance (or autoimmune tolerance) in vivo, for example in tolerising therapy.

[0225] The conjugates or compositions of the invention can advantageously be used to induce or promote local, tissue-compartmentalised silencing (or suppression) of immune cells, in particular autoreactive immune cells such as autoreactive T cells, thus preventing or minimising global immune silencing. Thus, as explained elsewhere herein, preferred diseases to be treated in accordance with the present invention are tissue compartmentalised autoimmune diseases.

[0226] In other embodiments, the nucleic acid molecules of the invention (or compositions comprising said nucleic acid molecules) can also be used in the therapeutic methods described herein.

[0227] The administration of the conjugates or compositions in the therapeutic methods and uses of the invention is carried out in pharmaceutically, therapeutically, or physiologically effective amounts, to subjects (e.g. mammals, preferably humans) in need of treatment. Thus, said methods and uses may involve the additional step of identifying a subject in need of treatment. Appropriate and effective concentrations / doses to be administered can readily be determined by a person skilled in the art.

[0228] Treatment of diseases or conditions in accordance with the present invention (for example treatment of pre-existing disease) includes cure of said disease or condition, or any reduction or alleviation of disease, e.g. reduction in disease severity, or symptoms of disease.

[0229] In rheumatoid arthritis, the detection of autoantibodies (specifically anti-citrullinated protein antibodies (ACPA)) precedes the clinical manifestation of the disease by years, meaning there is activation of autoreactive T cells well before the onset of disease (de Brito Rocha S, et al. Adv Rheumatol. 2019;59(2)). A similar manifestation is also thought to occur in type 1 diabetes (Herold KC, et al. N Engl J Med. 2019;381(7):603-613).

[0230] Thus, the therapeutic methods and uses of the present invention are suitable for prevention of diseases as well as active treatment of diseases (for example treatment of preexisting disease). Thus, prophylactic treatment is also encompassed by the invention. For this reason in the methods and uses of the present invention, treatment also includes prophylaxis, or prevention where appropriate.

[0231] Such preventative (or protective) aspects can conveniently be carried out on healthy or normal or at risk subjects, e.g. those with relevant autoantibodies as described above, and can include both complete prevention and significant prevention. Similarly, significant prevention can include the scenario where severity of disease or symptoms of disease is reduced (e.g. measurably or significantly reduced) compared to the severity or symptoms which would be expected if no treatment is given.

[0232] Suitable subjects for treatment in accordance with the present invention are thus subjects, e.g. mammalian, preferably human subjects, capable of suffering from, suffering from, or at risk of suffering from, any of the diseases referred to above, including immune diseases, preferably autoimmune diseases, and more specifically autoimmune diseases associated with or caused by or characterised by undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive immune activation in response to an autoimmune disease-associated antigen.

[0233] Thus, the in vivo methods and uses as described herein are generally carried out in humans.

[0234] Thus, the term "patient" or “subject” as used herein preferably refers to humans. In another embodiment, the subject is a subject having, or suspected of having (or developing), or potentially having (or developing) the disease or condition in question as described above.

[0235] In an alternative embodiment, the invention provides a method for treating or preventing a disease or disorder comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition of the present invention. Exemplary diseases or disorders are described elsewhere herein. Preferably the disease or disorder is an autoimmune disease, preferably comprising one or more of: celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis and Sjogren's syndrome. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0236] For example, in an alternative embodiment, the invention provides a method for treating or preventing a disease or disorder that is linked to, caused by, characterised by or associated with, undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive immune activation in response to a disease-associated antigen or diseasespecific antigen (preferably an autoimmune disease-associated or disease-specific antigen), preferably undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive CD4+ or CD8+ T cell activation in response to a disease-associated antigen or disease-specific antigen (preferably an autoimmune disease-associated or disease-specific antigen), said method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition of the present invention.

[0237] Viewed alternatively, the invention provides a method for treating or preventing a disease or disorder that is linked to, caused by, characterised by or associated with insufficient, a lack of, a decrease in, or reduction in, immune tolerance to a disease-associated antigen or disease-specific antigen (preferably an autoimmune disease-associated or disease-specific antigen), said method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition of the present invention. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0238] In an alternative embodiment, the invention provides a method of suppressing an immune response and / or inducing immune tolerance in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition of the present invention. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention. By “therapeutically effective amount” is meant an amount sufficient to show benefit to the condition of the subject. Whether an amount is sufficient to show benefit to the condition of the subject may be determined by the subject him / herself or a physician; preferably it is determined by clinical assessment and can be readily monitored.

[0239] In an alternative embodiment, the invention provides the use of a conjugate or composition of the invention in the manufacture of a medicament for use in therapy, e.g. for use in the treatment or prevention of disease, preferably the treatment or prevention of an autoimmune disease. Exemplary diseases or disorders are described elsewhere herein. Preferably said therapy is the treatment or prevention of one or more of: celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis and Sjogren's syndrome. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0240] For example, in an alternative embodiment, the invention provides the use of conjugates or compositions of the invention in the manufacture of a medicament for use in treating or preventing a disease or disorder that is linked to, caused by, characterised by or associated with, undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive immune activation in response to a disease-associated antigen or disease-specific antigen (preferably an autoimmune disease-associated or disease-specific antigen), preferably undesired, inappropriate, aberrant, pathological, pathogenic, increased or excessive CD4+ or CD8+ T cell activation in response to a disease-associated antigen or disease-specific antigen (preferably an autoimmune disease-associated or disease-specific antigen).

[0241] Viewed alternatively, the invention provides the use of conjugates or compositions of the invention in the manufacture of a medicament for use in treating or preventing a disease or disorder that is linked to, caused by, characterised by or associated with insufficient, a lack of, a decrease in, or reduction in, immune tolerance to a disease-associated antigen or disease-specific antigen (preferably an autoimmune disease-associated or disease-specific antigen). Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0242] Further alternatively viewed, the present invention provides the use of a conjugate or composition of the invention as defined herein for the treatment or prevention of one or more of the diseases as outlined above. Embodiments of the therapeutic uses of the invention described herein apply, mutatis mutandis, to this aspect of the invention.

[0243] In an alternative embodiment, the conjugate of the invention may comprise (or consist of) i) an entity comprising two (or at least two) antigen binding domains that each has the ability to bind to (or specifically bind to) a peptide associated with an MHC molecule (a pMHC molecule), preferably a pMHCll molecule, preferably wherein the pMHC molecule is expressed on (or is present on) the surface of a target cell, and ii) a silencing molecule targeting (that has the ability to target) the expression of one or more of CD80, CD86 and CD40.

[0244] In some such embodiments, the entity may comprise or consist of a nanoparticle or a liposome. As is known to the skilled person, a liposome is a spherical vesicle formed from a lipid bilayer surrounding an aqueous core. In such embodiments, generally a plurality of (or multiple) antigen binding domains that target pMHC, e.g. pMHCll, molecules will be attached to the surface of the nanoparticle or liposome, and the silencing molecules will be encapsulated within the nanoparticles or liposomes. Thus, in such embodiments the entities according to this aspect of the invention, for example nanoparticles or liposomes, can be at least divalent or preferably multivalent for pMHCll molecules (or can bind at least divalently or multivalently to pMHCll molecules), e.g. can comprise at least two antigen binding domains that can bind to pMHCll molecules or more than two antigen binding domains that can bind to pMHCll molecules. The pMHCll molecules bound by the two (or at least two) antigen binding domains can be the same or different, providing that the pMHCll molecules are located on the surface of the same target cell. Generally however the use of entities such as nanoparticles or liposomes conveniently allows many or multiple antigen binding proteins, antibodies, or antigen binding domains, to be attached to the surface of the nanoparticles or liposomes, as such antigen binding proteins, antibodies, or antigen binding domains can be distributed across the whole surface of the nanoparticles or liposomes. Thus, a multivalent or polyvalent interaction between the antigen binding domains and the target pMHC / pMHCll molecules can take place.

[0245] Such entities can be loaded in the interior or core with one or more silencing molecules of the invention, for example, with RNAi such as siRNA, CRISPR-Cas or TPD such as PROTAC, and any combination thereof as described elsewhere herein. Thus in such embodiments the delivery of silencing molecules into the cell is linked to the antigen binding domains of such entities, forming the alternative conjugates of the invention.

[0246] Binding of the antigen binding domains of the entity, e.g. the nanoparticle or liposome, to pMHCll on the target cell surface can then result in fusion of the surface of the entity (e.g. fusion of the nanoparticle membrane or liposome membrane) with the surface of the target cell (e.g. the cell membrane of the target cell). This, in turn, allows internalization of and delivery of the silencing molecule (e.g. RNAi such as siRNA, CRISPR-Cas, or TPD such as PROTAC, or a combination thereof) into the target cell, which can then act to target the expression of (and ultimately the cell surface expression of, preferably to reduce the cell surface expression of) one or more of the CD80, CD86 or CD40 target proteins as appropriate, depending on the nature of the silencing molecule in the conjugate. Details of the targeting of CD80, CD86 or CD40 are described elsewhere herein.

[0247] The antigen binding domain may comprise or consist of an antibody (e.g. a full-length antibody) or an antibody fragment (preferably a Fab fragment or an scFv fragment), e.g. an appropriate Fab format or an scFv format of a TCR-like antibody (as described herein) which retains the ability to bind to a pMHCll (e.g. disease-associated pMHCll as described elsewhere herein) on the surface of a target cell.

[0248] Liposomes may be synthesised using any method known in the art. Suitable methods for liposome synthesis and drug loading are described in e.g. Akbarzadeh et al., Nanoscale Res Lett 8(1): 102, 2013. Furthermore, it is known in the art that C-terminal PEGylated Fab fragments can spontaneously insert into a liposome membrane, allowing a plurality of Fab fragments to be inserted into the membrane e.g. and thus providing multivalency.

[0249] Other features and properties of other aspects and embodiments of the invention apply, mutatis mutandis, to this aspect of the invention. For example pMHCI molecules can also be targeted by these entities such as nanoparticles or liposomes.

[0250] The invention further includes kits comprising one or more of the conjugates or compositions of the invention, or one or more of the nucleic acid molecules of the invention. Preferably said kits are for use in the methods and uses as described herein, e.g. the therapeutic methods or in vivo methods / assays as described herein. Preferably said kits comprise instructions for use of the kit components. Preferably said kits are for treating or preventing diseases or conditions as described elsewhere herein, and optionally comprise instructions for use of the kit components to treat or prevent such diseases or conditions.

[0251] As used throughout the entire application, the terms "a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, except in instances wherein an upper limit is thereafter specifically stated.

[0252] In addition, where the terms “comprise”, “comprises”, “has” or “having”, or other equivalent terms are used herein, then in some more specific embodiments, these terms include the term “consists of” or “consists essentially of’, or other equivalent terms.

[0253] The term "decrease" or "reduce" or “inhibit” or “limit” or “prevent” (or equivalent terms) as described herein includes any measurable decrease or reduction when compared with an appropriate control. Such terms would thus include partial or complete decreases or reductions or inhibitions, etc., e.g. when compared with an appropriate control. The term “abolish” as described herein refers more specifically to complete inhibition or silencing, or essentially complete inhibition or silencing, e.g. such that for example the entity being measured is no longer detectable, or no longer significantly detectable, e.g. when compared with an appropriate control. Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no conjugate, or where a conjugate comprising a control antigen binding protein, for example a control antibody, that does not bind the target antigen, or where a conjugate comprising a control silencing molecule that does not reduce or abolish expression of the target protein, is used.

[0254] The term "increase" or “promote” or “induce” (or equivalent terms) as described herein includes any measurable increase or elevation when compared with an appropriate control. Appropriate controls would readily be identified by a person skilled in the art and might include non-treated or placebo treated subjects or healthy subjects, or samples or assays where no conjugate, or where a conjugate comprising a control antigen binding protein, for example a control antibody, that does not bind the target antigen, or where a conjugate comprising a control silencing molecule that does not target the expression of the target protein, is used.

[0255] Preferably such increases (and indeed other increases, improvements or positive effects as mentioned elsewhere herein) or such decreases (and indeed other decreases, reductions, inhibitions or negative effects as mentioned elsewhere herein) are measurable increases, decreases, etc., (as appropriate), more preferably they are significant increases, decreases, etc., preferably clinically significant or statistically significant increases, for example with a probability value of <0.05 or <0.05, when compared to an appropriate control level or value (e.g. compared to an untreated or placebo treated subject or compared to a healthy or normal subject, or the same subject before treatment, or compared to samples or assays where no conjugate is present, or where a control conjugate that does not bind the target antigen or target the expression of the target protein, is present).

[0256] Methods of determining the statistical significance of differences between test groups of subjects or differences in levels of a particular parameter are well known and documented in the art. For example herein a decrease or increase in level of a particular parameter or a difference between test groups of subjects is generally regarded as statistically significant if a statistical comparison using a significance test such as a Student t-test, Mann-Whitney II Rank-Sum test, chi-square test or Fisher's exact test, one-way ANOVA or two-way ANOVA tests as appropriate, shows a probability value of <0.05 or <0.05. In all embodiments, reference to significant or non-significant differences is preferably a reference to statistically significant or non-significant differences.

[0257] Sequence tables

[0258]

[0259] Amino acid sequence of human CD80 (Uniprot entry P33681) (SEQ ID NO:55):

[0260] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQ

[0261] TRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKD AFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTT

[0262] VSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAI

[0263] TLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV mRNA sequence of human CD80 (GenBank accession M27533.1) (SEQ ID NO:56):

[0264] CCAAAGAAAAAGTGATTTGTCATTGCTTTATAGACTGTAAGAAGAGAACATCTCAGAAGT GGAGTCTTACCCTGAAATCAAAGGATTTAAAGAAAAAGTGGAATTTTTCTTCAGCAAGCT

[0265] GTGAAACTAAATCCACAACCTTTGGAGACCCAGGAACACCCTCCAATCTCTGTGTGTTT

[0266] TGTAAACATCACTGGAGGGTCTTCTACGTGAGCAATTGGATTGTCATCAGCCCTGCCTG

[0267] TTTTGCACCTGGGAAGTGCCCTGGTCTTACTTGGGTCCAAATTGTTGGCTTTCACTTTT

[0268] GACCCTAAGCATCTGAAGCCATGGGCCACACACGGAGGCAGGGAACATCACCATCCAA GTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTC

[0269] AGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCAC AATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAAT

[0270] GGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACC

[0271] ATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGA

[0272] GGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACAC

[0273] CTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTT

[0274] GAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAG

[0275] CCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTC

[0276] CCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAA

[0277] CCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTC

[0278] AACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCAT

[0279] TACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCC

[0280] AAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA

[0281] TAACAGTGTCCGCAGAAGCAAGGGGCTGAAAAGATCTGAAGGTAGCCTCCGTCATCTC

[0282] TTCTGGGATACATGGATCGTGGGGATCATGAGGCATTCTTCCCTTAACAAATTTAAGCT

[0283] GTTTTACCCACTACCTCACCTTCTTAAAAACCTCTTTCAGATTAAGCTGAACAGTTACAA

[0284] GATGGCTGGCATCCCTCTCCTTTCTCCCCATATGCAATTTGCTTAATGTAACCTCTTCTT

[0285] TTGCCATGTTTCCATTCTGCCATCTTGAATTGTCTTGTCAGCCAATTCATTATCTATTAAA CACTAATTTGAG

[0286] Amino acid sequence of human CD86 (Uniprot entry P42081) (SEQ ID NO:57):

[0287] MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELWFWQDQE

[0288] NLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRI

[0289] HQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVM

[0290] QKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWIT

[0291] AVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQR VFKSSKTSSCDKSDTCF mRNA sequence of human CD86 (GenBank accession L25259.1) (SEQ ID NO:58):

[0292] CACAGGGTGAAAGCTTTGCTTCTCTGCTGCTGTAACAGGGACTAGCACAGACACACGG

[0293] ATGAGTGGGGTCATTTCCAGATATTAGGTCACAGCAGAAGCAGCCAAAATGGATCCCC

[0294] AGTGCACTATGGGACTGAGTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGGTGCT

[0295] GCTCCTCTGAAGATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGC

[0296] AAACTCTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAAAACT

[0297] TGGTTCTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTCATTCCAAGTATA

[0298] TGGGCCGCACAAGTTTTGATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATC

[0299] AAGGACAAGGGCTTGTATCAATGTATCATCCATCACAAAAAGCCCACAGGAATGATTCG CATCCACCAGATGAATTCTGAACTGTCAGTGCTTGCTAACTTCAGTCAACCTGAAATAGT

[0300] ACCAATTTCTAATATAACAGAAAATGTGTACATAAATTTGACCTGCTCATCTATACACGG

[0301] TTACCCAGAACCTAAGAAGATGAGTGTTTTGCTAAGAACCAAGAATTCAACTATCGAGTA

[0302] TGATGGTATTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACGACGTTTCCATCAG

[0303] CTTGTCTGTTTCATTCCCTGATGTTACGAGCAATATGACCATCTTCTGTATTCTGGAAA

[0304] CTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAGCTTGAGGACCCTCAGCCT

[0305] CCCCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTATTATATGTGTGATG

[0306] GTTTTCTGTCTAATTCTATGGAAATGGAAGAAGAAGAAGCGGCCTCGCAACTCTTATAA

[0307] ATGTGGAACCAACACAATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAGAAAAA

[0308] ATCCATATACCTGAAAGATCTGATGAAGCCCAGCGTGTTTTTAAAAGTTCGAAGACATCT

[0309] TCATGCGACAAAAGTGATACATGTTTTTAATTAAAGAGTAAAGCCC

[0310] Amino acid sequence of human CD40 (Uniprot entry P25942) (SEQ ID NO:59):

[0311] MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLP

[0312] CGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCV LHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNK TDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSN

[0313] TAAPVQETLHGCQPVTQEDGKESRISVQERQ mRNA sequence of human CD40 (GenBank accession X60592.1) (SEQ ID NQ:60):

[0314] GCCTCGCTCGGGCGCCCAGTGGTCCTGCCGCCTGGTCTCACCTCGCCATGGTTCGTC

[0315] TGCCTCTGCAGTGCGTCCTCTGGGGCTGCTTGCTGACCGCTGTCCATCCAGAACCACC

[0316] CACTGCATGCAGAGAAAAACAGTACCTAATAAACAGTCAGTGCTGTTCTTTGTGCCAGC

[0317] CAGGACAGAAACTGGTGAGTGACTGCACAGAGTTCACTGAAACGGAATGCCTTCCTTG

[0318] CGGTGAAAGCGAATTCCTAGACACCTGGAACAGAGAGACACACTGCCACCAGCACAAA

[0319] TACTGCGACCCCAACCTAGGGCTTCGGGTCCAGCAGAAGGGCACCTCAGAAACAGACA

[0320] CCATCTGCACCTGTGAAGAAGGCTGGCACTGTACGAGTGAGGCCTGTGAGAGCTGTGT

[0321] CCTGCACCGCTCATGCTCGCCCGGCTTTGGGGTCAAGCAGATTGCTACAGGGGTTTCT

[0322] GATACCATCTGCGAGCCCTGCCCAGTCGGCTTCTTCTCCAATGTGTCATCTGCTTTCGA

[0323] AAAATGTCACCCTTGGACAAGCTGTGAGACCAAAGACCTGGTTGTGCAACAGGCAGGC

[0324] ACAAACAAGACTGATGTTGTCTGTGGTCCCCAGGATCGGCTGAGAGCCCTGGTGGTGA

[0325] TCCCCATCATCTTCGGGATCCTGTTTGCCATCCTCTTGGTGCTGGTCTTTATCAAAAAG

[0326] GTGGCCAAGAAGCCAACCAATAAGGCCCCCCACCCCAAGCAGGAACCCCAGGAGATC

[0327] AATTTTCCCGACGATCTTCCTGGCTCCAACACTGCTGCTCCAGTGCAGGAGACTTTACA

[0328] TGGATGCCAACCGGTCACCCAGGAGGATGGCAAAGAGAGTCGCATCTCAGTGCAGGA

[0329] GAGACAGTGAGGCTGCACCCACCCAGGAGTGTGGCCACGTGGGCAAACAGGCAGTTG GCCAGAGAGCCTGGTGCTGCTGCTGCAGGGGTGCAGGCAGAAGCGGGGAGCTATGC CCAGTCAGTGCCAGCCCCTC

[0330] The invention will now be further described in the following non-limiting Examples with reference to the following drawings in which:

[0331] Figure 1. Proposed mechanism of selectively suppressing the immune response to an autoimmune disease-associated antigen (i.e. inducing immune tolerance) using an antibodydrug conjugate (ADC). (A) Illustration of human anatomy showcasing the small intestine (labelled), the broader digestive system (depicted in grey), and the peripheral tissues (labelled). (B) The activation of T cells requires three signals: (1) Interaction of the TCR with a target peptide presented by the appropriate pH LAI I on the surface of an antigen presenting cell (APC), (2) The engagement of necessary cofactors on the APC with the T cell to trigger (3) the secretion of cytokines from the T cell. The presence of all three signals is imperative for the complete activation of T cells. The illustration also shows for example T cell activation occurring within the small intestine and peripheral regions. The mechanism is illustrated with the 3.C11 antibody which has specificity for a gluten-derived peptide, predominantly located in the small intestine, which suggests that T cell activation should be unaltered in peripheral tissues due to the absence of the target peptide. (C) The upper panel of the diagram details the interaction of the unmodified exemplary 3.C11 antibody with pH LAI I , which hinders the T cell's (referred to as the “target T cell” herein) (illustrated by T1) peptide-specific binding, thereby blocking signal 1 and averting T cell activation. However, T cells of other specificities (sometimes referred to as “bystander T cells” herein, i.e. T cells that interact with different target pH LAI I molecules) (illustrated by T2 and T3) retain the ability to bind with the APC, leading to their activation. In contrast, the exemplary 3.C11 antibody-drug conjugate (ADC), lower panel, attaches to the specific pH LAN and is internalized, eliminating signal 1 (i.e. preventing the interaction of the target T cell with its peptide-specific HLA molecule). Once internalized, the ADC's payload (i.e. silencing molecule) diminishes the expression of relevant cofactors on the APC surface, thereby obstructing signal 2 and preventing the activation of other specificity T cells (bystander T cells) (illustrated by T2 and T3), including preventing signal (3) and the secretion of cytokines from the T cells (illustrated by T2 and T3). As such, the activation of both the target T cell and bystander T cells is prevented. This proposed mechanism results in immune tolerance by reprogramming the APC to become generally anti-inflammatory and to inhibit the activation of bystander T cells recognising other HLA associated antigens (other pH LAs) presented on the target APC, thus overcoming the issues associated with epitope spread. Figure 2. Analysis of internalisation of the 3.C11 antibody in Raji cells. Raji cells were incubated with (A) 3.C11 antibody and NC peptide, (B) 3.C11 and index peptide (modified 33-mer), or (C) SPLV3 antibody and NC peptide. The ratio of membrane-bound to intracellular antibody, and colocalization of the intracellular antibody with the lysosome, from the images in (A) and (B) are quantified in (D), and the image in (C) is quantified in (E).

[0332] Figure 3. Analysis of internalisation of the 3.C11 antibody in K562-CIITA cells. K562- CIITA cells were incubated with (A) 3.C11 antibody and NC peptide, (B) 3.C11 and index peptide (modified 33-mer), or (C) SPLV3 antibody and NC peptide. The ratio of membranebound to intracellular antibody, and colocalization of the antibody with the lysosome, from the images in (A) and (B) are quantified in (D), and the image in (C) is quantified in (E).

[0333] Figure 4. ADCC assay on Raji cells pulsed with 33-mer. (A) Mechanism illustrating the ADCC assay. (B) ADCC assay on peptide-pulsed Raji cells incubated with Rituximab. (C) ADCC assay on Raji cells pulsed with 33-mer_E, 33-mer_Q or no peptide, in combination with the 3.C11 antibody or isotype control antibody.

[0334] Figure 5. Conjugation of sulfo-SMCC, protamine and siRNA to 3.C11. (A) Graphic summary of the chemical coupling steps (B) SDS-PAGE showing the efficient binding of the 3.C11 antibody to protamine via the sulfo-SMCC linker (SPC). Note the molecular weight increase in the 3.C11-SPC lanes. (C) Specific siRNA payload estimation of 3.C11-SP- siRNA complexes. Different molar ratios of CSP (3. C11-SPC) complexes to Alexa Fluor 488-siRNA were incubated for 2h at 1100rpm and loaded on a 1.5% (wt / vol) agarose gel, and the band shift was then documented. At a molecular excess of 1 : 1-1:2, siRNA duplexes are almost completely retarded by CSP. At a molecular excess of 1:4, CSP is saturated and unbound siRNA duplexes start to appear. The left lane depicts un-complexed siRNA without CSP addition.

[0335] Figure 6. Surface expression of the siRNA target proteins on Raji cells. 1x104cells / well were incubated with the indicated antibodies for 30min at 4°C. CD40, CD80 and CD86 are primarily expressed on dendritic cells, B cells, and macrophages. Samples were analysed on the Novocyte Advanteon flow cytometer and visualized using FlowJo.

[0336] Figure 7. Cell surface staining of Raji cells using 3.C11. 1x105Raji cells were incubated with 10pM of the indicated peptides for 24 hours. Cells were stained using the 3.C11 antibody with (3.C11_SMCC / protamine) and without SMCC linker conjugation (3.C11). Detection of the bound 3.C11 was done using anti-hlgG-biotin and Streptavidin-PE beads. Samples were analysed on the Novocyte Advanteon flow cytometer and processed using FlowJo. Figure 8. Cell surface staining of Raji cells using 3.C11. 1x105Raji cells were pulsed with 10pM of the indicated peptides for 24 hours. Cells were stained using the native 3.C11 antibody (3.C11 mAb) or the fully conjugated antibody including SMCC-protamine-siRNA (3.C11 mAb_SP batch#1 and #2). Detection of the bound 3.C11 was done using anti-hlgG- biotin and Streptavidin-PE beads and the samples analysed on the Novocyte flow cytometer. (A) Distribution of fluorescence intensities within the cell populations showing preferential binding of the ADC (3.C11 mAb_SP) to the 33mer (B). Quantification of the geometric mean of the 33mer and glia-a1a samples analysed in (A).

[0337] Figure 9. Knockdown efficiency of siRNA against CD40, CD80 and CD86. (A) qPCR was performed on cDNA from Raji cells treated with siRNA against CD40, CD80 or CD86 after 24 hours. (B) Surface expression of CD80 in Raji cells. The cells were stained with an anti-CD80 antibody 48 hours after transfection. Mock transfected controls were used.

[0338] Figure 10. ADC mediated downregulation of CD80 mRNA in Raji APC cells. The ADC was freshly prepared prior to the experiment. Raji cells were incubated with the indicated concentrations of ADC-siCD80 (3.C11 conjugated to CD80 siRNA), non-targeting control ADC (ADC-siNT) (3.C11 conjugated to a non-targeting control siRNA) or naked ADC (3.C11 not conjugated to a siRNA molecule) for 24 hours before cell pellets were harvested. RNA extraction and RT-PCR was performed to generate cDNA. qPCR was performed with PLIM1 and SNW1 as housekeeping genes. The data is presented as relative values compared to the naked ADC sample

[0339] Figure 11. Payload alternatives with different modalities. Payload estimation of 3.C11- SP-payload complexes using plasmid DNA encoding shRNA against CD80 (A) or a 100nts expression construct encoding cshRNA against CD80 (B). Different molar ratios of CSP (3.C11-SPC) complexes to the payload were incubated for 2h at 1100rpm and loaded on a 1.5% (wt / vol) agarose gel, and the band shift was then documented. Depending on the payload, molecular excess of 1 :0.5-1:8 saturated the 3. C11-SPC complex and unbound payload start to appear. The left lane depicts free payload without CSP addition. (C) Flow analysis of surface CD80 expression on Raji cells 48 hours after transfection of a cshRNA expression cassette targeting CD80 mRNA or a non-targeting control siRNA. Detection of surface CD80 was done using a directly labelled APC anti-human CD80 Antibody. Samples were recorded on the Novocyte Advanteon flow cytometer and analyzed using FlowJo. Data presentation was done with GraphPad Prism.

[0340] Figure 12. Peptide specific activation of Jurkat TIB152 T cells specific for the index peptide (33mer, Jurkat TIB152 #364) and the gliadin-a1a peptide (Jurkat TIB152 #380). (A, C) Jurkat TIB152 #364 T cells (A) or Jurkat TIB152 #380 T cells (B) were activated using Raji cells or K562-CIITA cells pulsed with 50pM of 33mer peptide, gliadin-a1a peptide, no peptide (NP, negative control) or a positive T cell stimulation control (PMA). To assess the activation status of the T cells, surface staining for CD69 and CD25 was performed. (B, D) IL-2 production by Jurkat TIB152 T cells upon exposure to K562-CIITA cells presenting the corresponding target peptides for Jurkat TIB152 T cells #364 and #380. Jurkat TIB152 #364 T cells exposed to K562-CIITA cells presenting the 33mer peptide, and the effect of addition of the naked 3.C11 antibody, is shown in (B); Jurkat TIB152 #380 T cells exposed to K562- CIITA cells presenting the gliadin-a1a peptide is shown in (D). K562-CIITA were pulsed with the indicated amount of peptide before addition of the corresponding T cells. After 20 hours, the supernatant was harvested, and an anti-human IL-2 ELISA was performed. Data processing and presentation was done using GraphPad Prism representing a minimum of n=2.

[0341] Figure 13. IL-2 secretion by Jurkat TIB152 T cells is CD80 dependent. Raji cells were incubated with the EC50 peptide concentration of peptide for the corresponding T cell (i.e. 33mer for Jurkat TIB152 #364 T cells and gliadin-a1a for Jurkat TIB152 #380 T cells), followed by the addition of the corresponding T cells. The cells were co-cultured in the presence of a CD80 blocking antibody at the indicated concentration (pg / ml) for 18 hours. The supernatant was harvested, and an anti-human IL-2 ELISA was performed. The data was normalized to show relative IL-2 secretion compared to the highest measured value within one dataset. Data processing and presentation was done using GraphPad Prism.

[0342] Figure 14. Graphical summary of the in vitro assay used for validation of the ADC in T cell activation. (1) human APC lines / primary APCs were pulsed (“first pulse”) with 33mer, gliadin-a1a or a non-pulsed control for 24 hours (2). Cells were incubated with the anti CD80-ADC or a non-targeting (NT) control ADC (3) for 24 hours. Cells were washed 3x to remove excess ADC and incubated for an additional 18 hours before the cells were pulsed (“second pulse”) with the 33mer or gliadin-a1a peptide for 6 hours (4). The corresponding T cells (Jurkat TIB152 #364 for 33mer, or Jurkat TIB152 #380 for gliadin-a1 a) were added to the cells (5). After 20 hours the cell culture supernatant was harvested and IL-2 secretion was assessed (6).

[0343] Figure 15. ADC mediated downregulation of CD80 expression in Raji cells reduces activation of both target T cells and bystander T cells. The assay was performed as described in Figure 14 using Raji cells as the APC. IL-2 secretion was measured using an ELISA assay and the data was normalized to the highest measured ELISA signal. Data processing and presentation was done with GraphPad Prism. (A-E) measures the activation of the target T cells. The test sample, wherein Raji cells were treated with a first pulse of 33mer peptide, treated with the CD80-ADC, treated with a second pulse of 33mer peptide and incubated with Jurkat TIB152 #364 T cells is presented as a comparative sample in each panel (the “CD80-33mer” sample). Each panel includes the comparison of the “CD80- 33mer” test sample to a relevant control sample. All control samples were also incubated with a second pulse of 33mer peptide and incubated with TIB152 #364 T cells for specific activation of this T cell line. However, the control samples were treated with a different combination of first pulse peptide and ADC, as follows: (A) Raji cells were treated with a first pulse of gliadin-a1a peptide and treated with the CD80-ADC before addition of second pulse peptide / T cells (“CD80-a1a”); (B) Raji cells were treated with a first pulse of a no peptide control and treated with the CD80-ADC before addition of second pulse peptide / T cells (“CD80-NP”); (C) Raji cells were treated with a first pulse of 33mer peptide and treated with a non-targeting control ADC before addition of second pulse peptide / T cells (“NT-33mer”); (D) Raji cells were treated with a first pulse of gliadin-a1a peptide and treated with a nontargeting control ADC before addition of second pulse peptide / T cells (“NT-a1a”); or (E) Raji cells were treated with a first pulse of a no peptide control and treated with a non-targeting control ADC before addition of second pulse peptide / T cells (“NT-NP”). (F-J) measures the activation of bystander T cells. The test sample, wherein Raji cells were treated with a first pulse of 33mer peptide, treated with the CD80-ADC, treated with a second pulse of gliadinala peptide and incubated with Jurkat TIB152 #380 T cells is presented as a comparative sample in each panel (the “CD80-33mer” sample). Each panel includes the comparison of the “CD80-33mer” test sample to a relevant control sample. All control samples were also incubated with a second pulse of gliadin-a1a peptide and incubated with TIB152 #380 T cells for specific activation of this T cell line. However, the control samples were treated with a different combination of first pulse peptide and ADC, exactly as described for A-E above.

[0344] Figure 16. ADC mediated downregulation of CD80 expression in primary APCs reduces activation of both target T cells and bystander T cells. The assay was performed as described in Figure 14 using CD14 / 19+ cells (primary APCs) from a DQ2.5 positive donor. IL-2 secretion was measured using an ELISA assay and the data was normalized to the highest measured ELISA signal. Data processing and presentation was done with GraphPad Prism. The data is presented in A-E and F-J exactly as described in the Figure 15 legend above, the only difference being primary APCs were used in these assays.

[0345] Figure 17. Heatmap showing the relative T cell activation of Jurkat TIB152 #364 and Jurkat TIB152 #380 T cell lines (i.e. target T cells and bystander T cells respectively) by Raji cells with HLA-DQ2.5 loaded with gluten peptides (33mer, glia-a1a, glia-a2). The peptides recited on the left of the heat map (33mer, glia-a1a, glia-a2, or glia-a1a + a2) represent the “first pulse” peptide. The “second pulse” peptide is specific for the T cell line used (i.e. 33mer for the Jurkat TIB152 #364 cells and glia-a1a for the Jurkat TIB152 #380 cells). The presence of the naked 3.C11 antibody, the CD80-ADC (“3.C11_ADC”) and the non-targeting control ADC (i.e. 3.C11 conjugated to a control siRNA, “CTRL_ADC”) is indicated. Signals were normalized to peptide-specific activation in the absence of antibodies or ADCs, and the heatmap represents the mean of at least n=2. The key comparative data (reduction of Jurkat TIB152 #380 T cell (bystander T cell) activation by the CD80-ADC (“3.C11_ADC”) as compared to the naked 3.C11 antibody) is highlighted with a “1” in the Figure.

[0346] EXAMPLES

[0347] Example 1 - Internalisation of a TCR-like antibody binding to a disease-associated peptide-MHC (pMHC)

[0348] Materials and Methods

[0349] Imaging

[0350] Raji cells (B lymphoblastoid cell line, ATCC-CCL-86) and K562-CIITA (lymphoblast cell line, ATCC-CCL-243) were cultured in appropriate media (for Raji: RPMI / 10 % FBS / 1% P / S, for K562 CIITA: IM DM / 10 % FBS / 1% P / S) and transferred to imaging-compatible dishes.

[0351] The following peptides were used:

[0352] • NC (omega2): PEQPYPQQEQPYGSS (SEQ ID NO:63)

[0353] • Index peptide; modified 33-mer (also referred to as “33mer” herein): LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF (SEQ ID NO:64) (comprising the glia-a2 epitope PQPELPYPQ (SEQ ID NO:62)).

[0354] The following antibodies were used:

[0355] • 3.C11 (binds to DQ2.5-glia-a2. Seguences provided in Table A).

[0356] • SPVL3 (anti-DQ) (binds HLA-DQ regardless of associated peptide).

[0357] • H11_3 (isotype control) (specificity irrelevant isotype-matched).

[0358] Fluorescent labeling for detection of the above antibodies was achieved by the Alexa Fluor 647 Antibody Labeling Kit from Thermo Fisher, following the manufacturer's protocols.

[0359] Imaging was conducted using an Olympus SpinSR super resolution imaging system. The light source was optimized to balance image guality and minimize phototoxicity. Environmental conditions, including temperature and CO2 levels, were meticulously controlled. 50|JM of peptide is incubated with the cells (pulsing) overnight. The antibodies are added at the start of the experiment at a standard concentration of 5pg / mL, so the indicated timepoints are reflecting the time after adding the antibody to the samples.

[0360] Kinetics analysis

[0361] Post-acquisition, images were processed using specialized software for analysis of the internalization kinetics.

[0362] Representative cells were selected and analyzed at the indicated time-points based on the ratio between the fluorescence intensity of membrane bound 3.C11 antibody and intracellular (cytosol) 3.C11 antibody, as well as the % of 3.C11 antibody co-localized with the Lysotracker as a direct measurement of internalization into the Lysosome compartment.

[0363] ADCC assay

[0364] Jurkat-ADCC cells (ADCC Bioassay Effector Cell V variant (High Affinity) BPS biosciences #60541) were centrifuged (1500 rpm, 5 min, room temperature), and the pellet was resuspended in 50 pl OptiMEM and incubated (37°C, 5% CO2, 2 h). After a second centrifugation (1200 rpm, 5 min), cells were resuspended in 4 ml OptiMEM, incubated (2 h), and adjusted to 1 x 106cells / ml with OptiMEM. Antibodies (Rituximab (anti-CD40 monoclonal antibody, INVIVOGEN , #hcd20-mab1), 3.C11 , and H11_3 (isotype control)) (100 pl, 2pg / mL) and Jurkat-ADCC cells (50 pl, 50,000 cells) were added to 96-well plates and incubated (37°C, 5% CO2, 5 h). Cells were pelleted (1500 rpm, 5 min), resuspended in 100 pl Bio-Gio Luciferase Assay substrate Promega, #G7940, transferred to a white 96-well plate, and luminescence was measured (Varioscan, 1000 ms). Luminescence data were analyzed using statistical software (Prism GraphPad), with significance set at p < 0.05 and results expressed as mean ± SD.

[0365] Results

[0366] Internalisation of 3.C11 antibody

[0367] Live cell imaging was used to assess the 3.C11 and control antibody behaviour upon binding to target positive versus target negative cells. Two different human HLA-DQ2.5 positive target cells were employed in these studies, namely Raji (Figure 2) and K562-CIITA (Figure 3). Consistent HLA-DQ2.5 expression was first confirmed in flow prior to the imaging (inserted histograms). The antibodies employed were directly labelled with a fluorophore (green), whereas the intracellular lysosomal compartment of the target cells were labelled with LysoTracker (red). In the absence of specific peptide (NC), no 3.C11 cell staining was seen with either target cell line, whereas the plasma membrane was immediately visible on the same cells when loaded with index peptide. In contrast, the plasma membrane was readily visible on both cells independent of loaded peptide with the pan-DQ Ab. No staining was seen with the isotype control in either case (not shown). At all times, the LysoTracker visualized the lysosomal compartment on both cells.

[0368] The results also clearly showed a time-dependent and antibody-dependent internalization of 3.C11 and the pan-DQ antibody where the plasma membrane staining was gradually decreasing, and intracellular accumulation of the antibodies emerged. In the case of 3.C11 this was completely index-peptide restricted, whereas in the case of the pan-DQ antibody, the effect was peptide independent. Overlying the images of the antibody staining and the LysoTracker revealed a clear colocalization indicating a time dependent antibody internalization with lysosomal targeting. Upon quantifying the time course of both plasma membrane to lysosomal trafficking, as well as the corresponding increase in co-localization of antibody and LysoTracker, it was apparent that both antibodies internalized and accumulated in the lysosomes at a similar rate (kinetics graph inserts). Thus, these data show the internalisation of the 3.C11 antibody in a peptide- and time-dependent manner, which is not seen with the specificity irrelevant isotype-matched H11-3. In contrast, the pan- DQ SPVL3 exhibits similar internalization, but in a peptide-independent manner.

[0369] Results of the ADCC assay are shown in Figure 4. Antibody dependent cell cytotoxicity (ADCC) mediated by cross-coupling of target cell surface bound antibody to FcgR on immune effector cells is a major endogenous effector mechanisms of antibodies in humoral immunity. For ADCC to be operational, a certain cell surface density of target bound antibody is required. Here, we used a reporter cell-based ADCC proxy assay based on Jurkat T cells engineered to express the high-affinity FcgRIIIa and a NFAT-NLuc response element (BPS Bioscience / cat no 60541). We used the anti-CD20 specific Rituximab as a positive control (PC) that is well documented to induce target B cell destruction through ADCC. The results showed sensitive and specific ADCC activity with the PC independent of gliadin peptide loaded on HLA-DQ2.5 (only with peptide shown). In contrast, using the 3.C11 antibody in the same setup showed no ADCC activity independent of index or NC peptide, as with the target irrelevant isotype control. Thus, despite peptide dependent target cell binding of 3.C11, the data strongly indicate that the antibody target cell surface density is below threshold to trigger ADCC activity even prior to antibody internalization, and as internalization occurs this effector mechanism will anyway not be expected to be operational. Example 2 - Production and validation of antibody-silencing molecule conjugate (ADC)

[0370] Materials and Methods

[0371] Antibody-payload conjugation

[0372] The production of the antibody-payload conjugate is based on the protocol disclosed in Baeumer et al. 2016., Nat Protoc 11, 22-36. The 3.C11 antibody was chemically coupled to the complex of the carrier protein protamine and the bispecific cross-linker sulfo- (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate) (sulfo-SMCC). First, protamine was conjugated amino-terminally to sulfo-SMCC, and then this conjugate was coupled via cysteine residues to the IgG backbone. The payload (siRNA, plasmid DNA or cshRNA expression cassette) is added prior to use, resulting in the fully formed 3.C11- SMCC-protamine-payload (e.g. siRNA, plasmid DNA or cshRNA expression cassette) conjugate. Specifically, different molar ratios of the 3.C11-SPC complexes (3.C11 linked to the protamine-SMCC linker) to the payload were incubated for 2h at 1100rpm.

[0373] For quality control after functionalisation (i.e. after the conjugation of sulfo-SMCC and protamine to 3.C11, also referred to as 3.C11-SPC), all samples were heated at 70°C / 10 min and loaded onto a Bolt 4-12% Tris Plus SDS gel. The bands were separated for 25min / 180V. The gel was washed in water 3x 5mL and stained with Instant Blue for 1h and then destained in water.

[0374] For quality control after conjugation (i.e. after the addition of siRNA to the 3.C11-SPC conjugate), different equimolar ratios of the functionalized 3. C11-SPC and Alexa Fluor 488- siRNA were incubated for 2h, 1100rpm in a Thermomixer at RT and loaded on a 1.5% (wt / vol) agarose gel.

[0375] Quantification of CD40, CD80 and CD86 protein surface expression on target cells

[0376] 50,000 cells / well (Jurkat (T cell line), SKW-3 (T cell line), K562-CIITA (lymphoblast cell line), Raji (B lymphoblastoid cell line) and T2 cells (T / B lymphoblast cell line) were centrifuged at 1300rpm for 5 min. The pellet was re-suspended in 50|JL PBS+2%FBS containing the detection antibody and incubated for 30min at 4 degrees Celsius. Cells were then centrifuged at 1300rpm for 5min and washed with 200|JL PBS+2%FBS (3X), followed by analysis on the Novocyte Advanteon flow cytometer.

[0377] Quantification of cell surface interaction of 3.C11 after conjugation with SMCC-protamine and SMCC-protamine-siRNA 1x105Raji cells were incubated with 10 pM of the indicated peptides for 24 hours. Cells were stained (using 5pg / mL antibody and incubating for 30min on ice) using the native 3.C11 antibody (3.C11 / 3.C11 mAb), 3.C11 conjugated to SMCC linker and protamine (3.C11_SMCC / protamine), and 3.C11 conjugated to SMCC linker, protamine and siRNA (3.C11 mAb_SP batch#1 and #2).

[0378] Detection of the bound 3.C11 (in native or conjugated form) was done using and anti- h IgG-biotin and Streptavidin-PE beads. Samples were analysed on the Novocyte Advanteon flow cytometer and processed using FlowJo. The protocol for this was to wash cells twice with 200 pl PBS / 2% FBS, then to add 50 pl anti-hlgG-Biotin (Goat F(ab')2 Anti-Human IgG, Southern Biotech, Cat. No.: 2043-08), and incubate on ice for 30 min. The cells were then washed twice with 200 pl PBS / 2% FBS, after which 50 pl of Streptavidin-PE (Biotech, #405204) was added and incubated on ice for 30 min protected from light. The cells were then washed twice with 200 pl PBS / 2% FBS. The cells were resuspended with 200 pl autoMACS rinsing solution containing 2% FBS and the data recorded on the Novocyte flow cytometer.

[0379] Quantification of CD40, CD80 and CD86 mRNA after siRNA transfection or incubation with ADC

[0380] For transfection of CD40, CD80 and CD86 siRNA, Raji cells were cultured in a suitable medium, seeding 1 million cells per dish. siRNA targeting CD40, CD80 or CD86 mRNA (Silencer Select siRNA from ThermoFisher ScientificCD40, CD40: #4392420, assaylD S2676; CD80: #4392420, assaylD s2624; CD86: #4392420, assaylD s2629), 7.5 nM, 20 nM and 6.5 nM, respectively, and Lipofectamine ™ RNAiMAX from Invitrogen, were diluted in serum-free medium as per manufacturer’s instructions. Next, diluted siRNA and Lipofectamine were mixed and incubated for 5-15 minutes and added to cells. Cells were then incubated for 24h under normal conditions.

[0381] For incubation with ADC, 1x106Raji cells were incubated with 110 nM non-targeting control ADC (i.e. 3.C11 conjugated to a non-targeting control siRNA), naked ADC (i.e. 3.C11 not conjugated to a siRNA molecule) or increasing concentrations of CD80-ADC (i.e. 3.C11 conjugated to the above-mentioned CD80 siRNA) for 24 hours before cell pellets were harvested.

[0382] To analyse CD40, CD80 and CD86 mRNA, cellular RNA was subsequently extracted from treated cells using commercially available kits (RNeasy minikit from Qiagen (Cat. No. I ID: 74104)) according to manufacturer’s instructions. The RNA concentration and quality were measured before proceeding. Next, cDNA was produced from the isolated RNA using commercial kits (QuantiTect Reverse Transcription Kit (Cat No. / ID: 205311)) according to manufacturer’s instructions. qPCR was then used to quantify the cDNA of CD40, CD80 and CD86, using the commercially available SYBR green kit (Powerllp™ SYBR™ Green Master Mix for qPCR (ThermoFisher Scientific, A25741)) according to manufacturer’s instructions and commercially available primers (Integrated DNA Technologies, PrimeTime qPCR Primers) against CD40, CD80 and CD86 cDNA, with PUM1 (fwd: TGCGGGAGATTGCTGGACAT (SEQ ID NO:65), rev: GTGTGGCACGCTCCAGTTTC (SEQ ID NO:66)) and SNW1 (fwd: GCAGCTCCTGATAAGAGGTCG (SEQ ID NO:67), rev: CCGAGGATTAGGAACACCGAG (SEQ ID NO:68)) included as housekeeping (i.e. control) genes. All samples were run on the Quantstudio 5 real-time PCR machine and analyzed using the Design and Analysis software (DA2.7.02).

[0383] Quantification of CD80 cell surface protein expression

[0384] Raji cells were cultured in a suitable medium, seeding 1 million cells per dish. siRNA (20 nM) or a cshRNA expression cassette (100 ng) (both targeting CD80 mRNA) and Lipofectamine were diluted in serum-free medium as per manufacturer’s instructions. Next, diluted siRNA / schRNA expression cassette and Lipofectamine were mixed and incubated for 5-15 minutes and added to cells. Cells were then incubated for 48h under normal conditions.

[0385] The sequences of the cshRNA are shown below. cshRNA2:TTCACGCACCCTCACCAGTGACTAGGGAAGTGGCTTAGTGGAATAGTCAC TTCCTTGGTCACGTGGTCTTCTTGCTGCCAGCACCGAGTTCGAACACAGC (SEQ ID NO:69). cshRNA3:TTCACGCACCCTCATGTTCTGTTGGAGAGCTGGCTTAGTGGAATAGCCAG CTCTTCAACAGAAACATCTTCTTGCTGCCAGCACCGAGTTCGAACACAGC (SEQ ID NQ:70).

[0386] For detection of CD80, 50,000 cells / well were centrifuged at 1300rpm for 5 min. The pellet was re-suspended in 50pL PBS+2%FBS containing a dilution of the detection antibody (BD Pharmingen™ PE Mouse Anti-Human CD80, #560925, 1 :20 dilution (650ng / mL)). Cells were incubated for 30min at 4 degrees Celsius. Cells were then centrifuged at 1300rpm for 5min and wash with 200pL PBS+2%FBS (3x) and analysed on the Novocyte Advanteon flow cytometer.

[0387] Results Antibody-siRNA conjugation

[0388] The chemical coupling of the SMCC-protamine linker to the 3.C11 antibody (performed as described in Baeumer et al, 2016, Figure 5A) has no negative impact on the integrity of the antibody. This was confirmed by SDS-PAGE, comparing 3.C11 before and after the chemical coupling. The electrophoresis analysis (Figure 5B) showed one specific band corresponding to either the native antibody (lane 1) or the functionalized versions (lane 2 and 3).

[0389] At molecular excess of 1 :1 - 1:2 (Figure 50), siRNA duplexes are almost completely retarded by both batches of the 3. C11-CSP conjugates. At a molecular excess of 1:4, 3.011-CSP is saturated and unbound siRNA duplexes begin to appear.

[0390] Analysis of CD40, CD80 and CD86 protein surface expression on target cells

[0391] Expression of CD40, CD80 and CD86 could be detected on the surface of different target cell lines, including K562-CIITA (lymphoblast), Raji (B lymphoblastoid) and T2 (T / B lymphoblast) cells (Figure 6). Based on the fluorescent intensities observed, T2 cells have the highest surface expression for all markers of all tested cell lines, with Raji cells being a medium and K562-CIITA a low presenting cell line. Minimal to no expression of CD40, CD80 or CD86 could be detected on the surface of T cell lines (Jurkat and SKW-3).

[0392] Analysis of pHLA specificity of 3.C11 after conjugation with SMCC-protamine and SMCC-protamine-siRNA

[0393] Cell surface staining of Raji cells pulsed with either relevant, irrelevant or no peptides were stained with the native 3.C11 antibody (3.C11, Figure 7), conjugated (3.C11_SMCC / protamine, Figure 7) or functionalized 3.C11 (3.C11 mAb_SP batch#1 and #2, Figure 8). The conjugated 3.C11 antibody showed specific binding to the 33mer and the glia-a2 peptide compared to the glia-a1a peptide (Figure 7 and Figure 8). Despite the observed increase in background signal, the fully functionalized 3.C11 still bound preferentially to the 33mer (Figure 8B).

[0394] Analysis of siRNA knockdown efficiency of costimulatory molecules

[0395] After incubation of Raji cells with siRNA targeting the mRNA of CD40, CD80 or CD86, gPCR analysis showed a decrease in relative mRNA levels of CD40, CD80 and CD86 compared to a mock siRNA (Figure 9A). Flow cytometry analysis of the cell surface protein expression of CD80 after treating Raji cells with siRNA targeting the mRNA of CD80 showed a decrease in CD80 surface protein expression compared to a mock siRNA (Figure 9B). The data in Figure 9 clearly demonstrates the effective downregulation of all three targets in Raji cells 24 hours after transfection. Depending on the target we achieved up to about 60% reduction of relative mRNA levels. In the case of CD80, we demonstrate that a 30% downregulation in mRNA level translates into a 30% reduction of CD80 on the surface of Raji cells.

[0396] Conjugating the same anti-CD80 siRNA to the 3.C11 antibody (forming an ADC with a CD80 siRNA as a payload, referred to as “ADC-siCD80” here), we demonstrate a concentration dependent reduction in CD80 mRNA levels in Raji cells resulting in a maximum of 50% reduction of CD80 mRNA levels 24 hours after treatment with 110nM CD80-ADC as compared to an ADC with a non-targeting siRNA as payload (“ADC-siNT”) (Figure 10).

[0397] These data show that siRNA can be used to decrease the mRNA levels of costimulatory molecules (CD40, CD80, and CD86) in a cellular model, and this can translate to a decrease in cell surface protein expression, and that siRNA can be conjugated to an ADC and be used as a payload for reduction of mRNA levels in APCs, as exemplified for CD80-ADC.

[0398] Similar effects could also be achieved with alternative payloads such as plasmid DNA encoding shRNA targeting CD80 mRNA, which has been shown to conjugate to the 3.C11 antibody and thus form an ADC (Figure 11A), or a DNA expression cassette harbouring all components necessary for expression of a cshRNA targeting CD80 mRNA, which has been shown to reduce the cell surface expression of CD80 protein when transfected into Raji cells (Figure 11 C) and has also been shown to conjugate to the 3.C11 antibody to form an ADC (Figure 11 B).

[0399] Example 3 - Validation of an in vitro model to study T cell activation by different disease-associated peptides, confirming the inhibition of target T cell and bystander T cell activation by the ADC

[0400] Materials and Methods

[0401] T cell activation

[0402] For generation of T cell lines with stable expression of the 33mer / a2 specific T cell receptor (TCR) (#364) or the 33mer / a1a specific TCR (#380) (to generate the Jurkat TIB152 T cell lines #364 and #380 as used herein) 50,000 Jurkat TIB152 T cells were transduced with Lentivirus particles including the coding region for the corresponding TCR (Genscript) with MO1100 (multiplicity of infection; 100 virus particles / cell). The cells were incubated for 3 days, before surface expression of the TCR was validated using an antibody recognizing the murine constant region present in the recombinant TCR (TCR beta Monoclonal Antibody (H57-597, ThermoFisherScientific, #367-5961-82). Depending on the frequency of H57-597 positive cells within the initial population, FACS was used to reach >80% of H57-597 positive cells within the T cell population.

[0403] T cell activation was measured by CD25 and CD69 surface expression on Jurkat TIB152 T cells and by IL-2 secretion by Jurkat TIB152 T cells. 50,000 viable APCs (Raji cells or K562-CIITA cells) were pulsed with 50pM of either 33mer peptide or gliadin-a1a peptide. All experiments included a negative control (no peptide) and a positive control (Cell Stimulation Cocktail, (12-myristate 13-acetate (PMA)) eBioscience, #00497093). Cells were washed 3x and 40,000 viable Jurkat TIB152 #364 T cells or Jurkat TIB152 #380 T cells were added. The APCs and T cells were co-cultured for 18 hours.

[0404] To measure IL-2 secretion, the cell culture supernatant was harvested, and an antihuman IL-2 ELISA was performed according to the manufacturer’s protocol (Human IL-2 ELISA MAX Deluxe Set, BioLegend, #431804) and the absorbance at 450nm was acquired using a Varioskan Lux Microplate reader (ThermoFisherScientific).

[0405] To measure CD25 and CD69 surface expression, the cell pellet was washed 3x in PBS containing 2% FCS. The cells were stained with anti-CD3 (1:50 dilution, MiltenyiBiotec, #130080401), anti-CD69 (1 :50 dilution. BD Biosciences, #555531) and anti-CD25 (1:50 dilution, BioLegend, #302610) antibody. Samples were recorded on the Novocyte Advanteon flow cytometer and data analyzed using FlowJo.

[0406] In the assay to validate co-factor dependency, Raji cells were seeded as above and incubated with the EC50 peptide concentration of the 33mer peptide or gliadin-a1a peptide (for example as determined from Figures 12B and 12D) and Jurkat TIB152 #364 and #380 T cells were added as described above. The co-culture (APCs and T cells) were incubated in the presence of or absence of increasing concentrations of CD80 blocking antibody (Human B7-1 / CD80 Antibody, R&D systems, #MAB140) and IL-2 secretion was measured as described above.

[0407] In vitro T cell activation assay

[0408] Primary APCs (CD14+ or CD19+ cells) were isolated from DQ2.5-positive donor peripheral blood mononuclear cells (PBMCs) using the APC Mouse Anti-Human CD14 (BD biosciences, #555399) and the APC Mouse Anti-Human CD19 (BD Biosciences, #555415) in combination with the EasySep™ APC Positive Selection Kit II (Stem Cell, #17681), according to manufacturer’s instructions.

[0409] 50,000 human APCs (Raji cells) or primary (CD14 / 19+) APCs were pulsed (“first pulse”) with 10nM of 33mer, gliadin-a1 a, or a non-pulsed control for 24 hours. Cells were further incubated with 50nM of the CD80-ADC or a non-targeting control ADC for 24 hours. Cells were washed 3x with PBS containing 2% FCS to remove excess ADC. 42 hours after the ADC was added, cells were washed 3x with PBS containing 2% FCS and pulsed (“second pulse”) with the 33mer (3pM) or the gliadin-a1a peptide (25pM) for 6 hours before the corresponding T cells were added (i.e. Jurkat TIB152 #364 T cells were added to the samples where the second pulse peptide was the 33mer (the “index peptide”), and Jurkat TIB152 #380 T cells were added to the samples where the second pulse peptide was the gliadin-a1a peptide). The cell culture supernatant was harvested 20 hours after the T cells were added and IL-2 secretion was assessed as previously described.

[0410] Results

[0411] Validation of an in vitro system to analyse T cell activation by different disease- associated peptides

[0412] After confirming APC lines such as Raji cells express T cell activation co-factors such as CD80 (Figure 6), and confirming that CD80 expression can be depleted using siRNA when conjugated to the 3.C11 antibody in an ADC (Figure 10), the effect of the ADC on T cell activation by APCs presenting different disease-associated antigens was then investigated.

[0413] Proving the ADC’s ability to successfully downregulate CD80 expression and thereby modulate the APCs ability to activate not only the T cells specific for the index peptide (i.e. target T cells) but also bystander T cells (i.e. T cells recognising a different disease- associated peptide), requires an in vitro model consisting of co-factor dependent (e.g. CD80- dependent) T cell lines specific for the relevant peptides. Thus, we established the stable expression of the 33mer / a2 specific T cell receptor (TCR) (#364) and the 33mer / a1a specific TCR (#380) in Jurkat TIB152 T cells. T cell specificity has been demonstrated by peptide specific activation of the T cell lines using the T cell activation markers CD69 and CD25. APCs pulsed with 33mer resulted in activation of both #364 and #380 T cell lines, while APCs pulsed with gliadin-a1a peptide resulted in activation of the #380 T cell line only (Figures 12A and 12C). The dose-dependent secretion of IL-2 from the Jurkat TIB152 #364 and #380 T cell lines incubated with APCs, pulsed with the 33mer or the gliadin-a1a peptide respectively, has also been shown (Figures 12B and 12D). As expected, treatment of 33mer- pulsed APCs with the naked 3.C11 antibody was shown to inhibit the secretion of IL-2 from, and thus activation of, the #364 T cell line (Figure 12B).

[0414] The co-factor dependency of the Jurkat TIB152 #364 and #380 T cell lines was verified by performing a T cell activation assay in the presence of increasing concentrations of an anti-CD80 blocking antibody, showing reduced T cell activation in a dose-dependent manner (Figure 13). This shows activation of the Jurkat TIB152 #364 and #380 T cell lines is dependent on CD80.

[0415] In vitro validation of ADC for inhibition of target T cell and bystander T cell activation

[0416] The in vitro assays were performed as outlined in Figure 14. In short, human APC lines or primary APC cells were pulsed (“first pulse”) with either the 33mer peptide (the index peptide) or the gliadin-a1a peptide or a non-pulsed control and incubated with the CD80- ADC or a non-targeting control ADC. Cells were washed to remove excess ADC before the cells were pulsed (“second pulse”) with the 33mer peptide or the a1 a peptide for 6 hours before the corresponding T cells were added (i.e. when the “second pulse” used the 33mer peptide the #364 T cell line was added, and when the second pulse used the gliadin-a1a peptide the #380 T cell line was added).

[0417] Inhibition of target T cell activation was assessed using the Jurkat TIB152 #364 T cells, which are activated by interaction with the 33mer peptide but are not activated by the gliadinala peptide (Figure 15A-E). In these samples the “second pulse” was always with the 33mer peptide (for specific activation of the #364 T cells).

[0418] APCs pulsed (“first pulse”) with 33mer peptide, treated with the CD80-ADC, pulsed (“second pulse”) with 33mer and incubated with Jurkat TIB152 #364 T cells (the “CD80- 33mer” sample in each of Figures 15A-E) led to a significantly reduced IL-2 secretion (reduced T cell activation) as compared to APCs that were treated with the CD80-ADC and had a “first pulse” of gliadin-a1a peptide (Figure 15A, “CD80-a1a” sample) or of a nonpulsed control (Figure 15B, “CD80-NP” sample), and also a significantly reduced IL-2 secretion (reduced T cell activation) as compared to APCs that were treated with the nontargeting control ADC (i.e. conjugated to a non-targeting control siRNA) and had a “first pulse” of 33mer peptide (Fig 15C, “NT-33mer” sample), or of gliadin-a1a peptide (Figure 15D, “NT-a1a” sample) or of a non-pulsed control (Figure 15E, “NT-NP” sample).

[0419] Inhibition of bystander T cell activation was assessed using the Jurkat TIB152 #380 T cells, which are activated by the gliadin-a1a peptide (Figure 15F-J). In these samples the “second pulse” was always with the gliadin-a1a peptide (for specific activation of the #380 T cells). The same results as for the target T cell (Jurkat TIB152 #364 T cells, Figures 15A-E as described above) was also observed for the Jurkat TIB152 #380 T cells activated (“second pulse”) with the gliadin-a1a peptide (Figure 15F-J), i.e. the specific inhibition of Jurkat TIB152 #380 T cell activation by the gliadin-a1a peptide using the CD80-ADC.

[0420] The same results for the inhibition of target T cell activation (Jurkat TIB152 #364 T cells) and bystander T cell activation (Jurkat TIB152 #380 T cells) was shown using primary APCs (CD14 / 19+ cells) from a DQ2.5 positive donor (Figures 16A-E for Jurkat TIB152 #364 T cells and Figures 16F-J for Jurkat TIB152 #380 T cells).

[0421] These results show that the CD80-ADC significantly inhibits both target T cell activation and bystander T cell activation from both APC lines and primary APCs, which is dependent on the “first pulse” peptide being the 33mer (leading to specific binding of the 3.C11 antibody and internalisation of the conjugate) and the ADC being conjugated to CD80 siRNA (leading to the depletion of the co-factor CD80 expression and inhibition of both target and bystander T cell activation).

[0422] The fact that activation of Jurkat TIB152 #380 T cells by the gliadin-a1a peptide (which is not targeted by the exemplified CD80-ADC, as the 3.C11 antibody used in the conjugate is specific for the 33mer) is also inhibited using the CD80-ADC of the present invention evidences bystander T cell inhibition (by the reduced expression of the costimulatory molecule CD80 on APCs).

[0423] This is also summarised in Figure 17, where the reduction in bystander T cell (Jurkat TIB152 #380 T cells) activation between APC samples treated with the CD80-ADC versus the naked 3.C11 antibody is highlighted with a “1” in the Figure. The T cell activation shown in the samples treated with 3.C11 reflects the binding specificity of the 3.C11 in combination with the pMHC specificity of the T cells. The observed reduction in T cell activation is entirely due to blocking of the TCR / pMHC (signal 1) interaction and therefore a maximum inhibition of >90% can be observed. In a different in vitro assay, preventing the interaction between CD80 with its cognate ligand on T cells (part of signal 2) using a CD80 blocking antibody achieved a maximum inhibition of 40-60% of T cell activation (Figure 13). In comparison, the effect on inhibition of T cell activation seen with ADC mediated downregulation of CD80 is comparable to or even higher than the inhibition of T cell activation observed using the CD80 blocking antibody.

[0424] This shows the ability of the CD80-ADC of the present invention to promote immune tolerization to a gluten-derived peptide and also to address the well-recognised issue of epitope spreading.

Claims

CLAIMS:

1. A conjugate comprising i) an antigen binding protein, for example an antibody, comprising two antigen binding domains that each has the ability to bind to a peptide associated with an MHC class II molecule (pMHCll), wherein said peptide associated with an MHC class II molecule is present on the surface of a target cell, and ii) a silencing molecule that has the ability to target the expression of one or more of CD80, CD86 and CD40.

2. The conjugate of claim 1 , wherein the conjugate is an antibody-drug conjugate (ADC).

3. The conjugate of claim 1 or claim 2, wherein the internalisation of the conjugate delivers the silencing molecule into the target cell, preferably wherein the target cell is an antigen presenting cell.

4. The conjugate of any one of claims 1 to 3, wherein the silencing molecule reduces the cell surface expression of one or more of CD80, CD86 and CD40, preferably CD80 or at least CD80.

5. The conjugate of any one of claims 1 to 4, wherein the silencing molecule comprises: a) one or more inhibitory RNA construct, b) a CRISPR-Cas gene silencing construct, or c) one or more targeted protein degradation (TPD) molecules.

6. The conjugate of claim 5, wherein the inhibitory RNA construct comprises at least one inhibitory RNA molecule or a DNA molecule that encodes at least one inhibitory RNA molecule, preferably wherein the inhibitory RNA molecule is one or more of: siRNA and shRNA.

7. The conjugate of claim 5, wherein the CRISPR-Cas gene silencing construct comprises a nucleic acid molecule that encodes at least one Cas nuclease, and a nucleic acid molecule that comprises, or encodes, at least one gRNA molecule.

8. The conjugate of claim 5, wherein the CRISPR-Cas gene silencing construct comprises a Cas nuclease complexed to a gRNA.

9. The conjugate of claim 7 or claim 8, wherein the Cas nuclease is a Cas9 nuclease.

10. The conjugate of claim 5, wherein the targeted protein degradation (TPD) molecule is one or more proteolysis targeting chimera (PROTAC) molecules.

11. The conjugate of any one of claims 1 to 10, wherein parts i) and ii) of the conjugate are coupled by a linker molecule, preferably wherein the linker molecule comprises a carrier peptide and / or a chemical crosslinker, preferably wherein the carrier peptide is protamine and / or the chemical crosslinker is sulfo-SMCC.

12. The conjugate of any one of claims 1 to 11, wherein the antigen binding protein, for example antibody, is monospecific.

13. The conjugate of any one of claim 1 to 12, wherein the pMHCll molecule bound by the two antigen binding domains is HLA-DQ2.5-glia-a2 or HLA-DQ2.5-glia-a1a.

14. The conjugate of any one of claims 1 to 13, wherein the antigen binding domains i) bind to HLA-DQ2.5-glia-a2 and comprise at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises: a variable heavy (VH) CDR1 that comprises the amino acid sequence of GGTVRSRVHA (SEQ ID NO:5) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of IIPIFGTA (SEQ ID NO:6) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDVQRMGMDV (SEQ ID NO:7) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of QDISNW (SEQ ID NO:8) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence;a VL CDR2 that comprises the amino acid sequence of DSS (SEQ ID NO:9) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QQFNSYPLT (SEQ ID NO: 10) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative said sequence; or ii) bind to HLA-DQ2.5-glia-a1a and comprise at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises: a variable heavy (VH) CDR1 that comprises the amino acid sequence of GDSVSSNSAA (SEQ ID NO:23) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of TYYRSKWYN (SEQ ID NO:24) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDRTTGWHPYGMDV (SEQ ID NO:25) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of HDISSY (SEQ ID NO:26) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:27) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QDLNSYPL (SEQ ID NO:28) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative said sequence; or iii) bind to HLA-DQ2.5-glia-a1a and comprise at least one light chain variable domain and at least one heavy chain variable domain, each domain comprising three complementarity determining regions (CDRs), wherein said antigen binding domain comprises:a variable heavy (VH) CDR1 that comprises the amino acid sequence of GDSVSSSSAA (SEQ ID NO:41) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR2 that comprises the amino acid sequence of TYYRSKWYN(SEQ ID NO:42) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VH CDR3 that comprises the amino acid sequence of ARDRTTGWHPYGMDV (SEQ ID NO:43) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; and / or (preferably “and”) a variable light (VL) CDR1 that comprises the amino acid sequence of HDISSY (SEQ ID NO:44) or a sequence containing 1 , 2 or 3 amino acid substitutions, additions or deletions relative to said sequence; a VL CDR2 that comprises the amino acid sequence of AAS (SEQ ID NO:45) or a sequence containing 1 or 2 amino acid substitutions, additions or deletions relative to said sequence; and a VL CDR3 that comprises the amino acid sequence of QNLNSYPL (SEQ ID NO:46) or a sequence containing 1, 2 or 3 amino acid substitutions, additions or deletions relative said sequence.

15. A composition comprising the conjugate of any one of claims 1 to 14 and a diluent, carrier or excipient, preferably a pharmaceutically acceptable diluent, carrier or excipient.

16. A composition comprising at least two different conjugates, wherein each conjugate is as defined in any one of claims 1 to 14.

17. A conjugate or composition as defined in any one of claims 1 to 16 for use in treating or preventing a disease or disorder, preferably wherein the disease or disorder is an autoimmune disease.

18. The conjugate or composition for use of claim 17, wherein the autoimmune disease is one or more of: celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis, and Sjogren's syndrome.

19. A conjugate or composition as defined in any one of claims 1 to 16 for use in suppressing an immune response and / or inducing immune tolerance in vivo.

20. A method of treating or preventing a disease or disorder, said method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition as defined in any one of claims 1 to 16, wherein the disease or disorder is an autoimmune disease.

21. The method of claim 20, wherein said autoimmune disease is one or more of: celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis, and Sjogren's syndrome.

22. A method of suppressing an immune response and / or inducing immune tolerance in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate or composition as defined in any one of claims 1 to 16.

23. The use of a conjugate or composition as defined in any one of claims 1 to 16 in the manufacture of a medicament for use in therapy, preferably wherein said therapy is the treatment or prevention of an autoimmune disease.

24. The use of claim 23, wherein the autoimmune disease is one or more of: celiac disease, rheumatoid arthritis, type I diabetes, multiple sclerosis, and Sjogren's syndrome.

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