The present invention relates to a fusion MHC-Ia open conformer that comprises an HLA heavy chain selected from C08, A25, B58, A30, B53, and C12 covalently linked to an Fc polypeptide sequence, particularly for use in the prophylaxis or treatment of cancer, and for use as immunomodulators.

Human leukocyte antigens (HLA) belong to the classical major histocompatibility complex (MHC) protein family. The HLA complex helps the immune system distinguish the body's own proteins from proteins made by foreign invaders such as viruses and bacteria. Humans have MHC class I molecules comprising the classical (MHC-Ia) HLA-A, HLA-B, and HLA-C, and the non-classical (MHC-Ib) HLA-E, HLA-F, HLA-G and HLA-H molecules. Both categories are similar in their mechanisms of peptide binding, presentation and induced T-cell responses. The most remarkable feature of the classical MHC-la is their high polymorphism, while the non-classical MHC-Ib are usually non-polymorphic and tend to show a more restricted pattern of expression than their MHC-la counterparts.

The HLA nomenclature is given by the particular name of gene locus (e.g. HLA-A) followed by the allele family serological antigen (e.g. HLA-A*02), and allele subtypes assigned in numbers and in the order in which DNA sequences have been determined (e.g. HLA-A*02:01). Alleles that differ only by synonymous nucleotide substitutions (also called silent or non-coding substitutions) within the coding sequence are distinguished by the use of the third set of digits (e.g. HLA-A*02:01 :01). Alleles that only differ by sequence polymorphisms in the introns, or in the 5' or 3' untranslated regions that flank the exons and introns, are distinguished by the use of the fourth set of digits (e.g. HLA-A*02:01:01:02L) (Fig. 1).

A list of MHC-Ia alleles is provided in Table 1. For a complete list of allele subtypes visit the link: http://hla.alleles.org/alleles/class1.html.

Classical MHC-Ia molecules' principle function is to present peptides as part of the adaptive immune response. MHC-Ia molecules are trimeric structures comprising a membrane-bound heavy chain with three extracellular domains (α1, α2 and α3) that associates non-covalently with β2-microglobulin (β2m) and a small peptide which is derived from self-proteins, viruses or bacteria. The α1 and α2 domains are highly polymorphic and form a platform that gives rise to the peptide-binding groove. Juxtaposed to the conserved α3 domain is a transmembrane domain followed by an intracellular cytoplasmic tail.

To initiate an immune response classical MHC-Ia molecules present specific peptides to be recognized by TCR (T cell receptor) present on CD8⁺ cytotoxic T lymphocytes (CTLs), while NK cell receptors present in natural killer cells (NK) recognize peptide motifs, rather than individual peptides. Under normal physiological conditions, MHC-Ia molecules exist as heterotrimeric complexes in charge of presenting peptides to CD8+ T cells and NK cells, however, MHC-Ia molecules may also be present in cells as free-heavy chains lacking β2-microglobulin and peptide, and are referred to as HLA-open conformers (Arosa et al., Trends in Immunology 2007 Mar; 28(3): 115-23) (Fig. 2). The interaction of HLA-open conformers with T cell receptors and NK cell receptors is independent of the peptide and its function is unknown.

Open conformers can be expressed at the cell surface of cells and can be detected with antibodies recognizing linear epitopes of HLA molecules without β2m and peptide (e.g. LA45, L31, HCA2 and HC-10). These antibodies have been used to detect the presence of open conformers in diverse autoimmune patients and healthy individuals (Raine et al., Rheumatology 2006;45:1338-1344). Despite their presence in patients and cell lines little is know of their mode of action. Open conformers have been mostly assessed in Ankylosing spondylitis (AS) +HLA-B27 patients, where HLA-B27 open conformers have been hypothesized to induce autoimmunity, their function in other autoimmune patients has not been yet addressed.

Ciprandi et al. (Allergy 2008, 63, 1335-1338) discuss the association of soluble HLA molecules with allergic rhinitis. Topalian et al. (Cancer Cell 27 (2015), 450-461) review checkpoint blockade in cancer treatment. WO9958557 discloses HLA-B27 dimers in diagnosis of spondyloarthropathy.

Here the inventors disclose for the first time that the classical MHC-Ia (HLA-A, HLA-B and HLA-C) family of molecules when present as open conformers (heavy chains without β2m) are useful therapeutics for their immunomodulatory properties and use in the treatment of cancer.

Overall, modulating the immune contexture of tumors favoring the infiltration of M1 type macrophages, cytotoxic CD8 T-cells, and Th1 cells, and/or reducing the infiltration of MDSCs and M2 type macrophages is a promising therapeutic avenue to treat cancer that is explored here with the use of HLA open conformers proteins in diverse cancer indications.

Amino acid sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rd ed. p. 21).

The term open conformer as used in the present specification refers to an isolated HLA heavy chain molecule not associated to β2-microglobulin either as a monomer or as a dimer (homodimer or heterodimer). Certain embodiments of the open conformers disclosed herein are fusion protein monomers or dimers, wherein the HLA heavy chain is covalently linked to a stabilizing polypeptide region, particularly a crystallizable fragment immunoglobulin domain.

In the context of the present specification the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/). One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1.-2; Gap costs: Linear. Unless otherwise stated, sequence identity values provided herein refer to the value obtained with the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

In the context of the present specification, the term major histocompatibility complex (MHC) is used in its meaning known in the art of cell biology and immunology; it refers to a cell surface molecule that displays a specific fraction (peptide), also referred to as an epitope, of a protein. There a two major classes of MHC molecules: class I and class II. Within the MHC class I two groups can be distinguished based on their polymorphism: a) the classical (MHC-Ia) with corresponding polymorphic HLA-A, HLA-B, and HLA-C genes, and b) the non-classical (MHC-Ib) with corresponding less polymorphic HLA-E, HLA-F, HLA-G and HLA-H genes.

MHC class I heavy chain molecules usually (i.e. when not in open conformer form) occur as an alpha chain linked to a unit of the non-MHC molecule β2-microglobulin. The alpha chain comprises, in direction from the N-terminus to the C-terminus, a signal peptide, three extracellular domains (α1-3, with α1 being at the N terminus), a transmembrane region and a C-terminal cytoplasmic tail. The peptide being displayed or presented is held by the peptide-binding groove, in the central region of the α1/α2 domains.

In the context of the present specification, the term β2-microglobulin domain is used in its meaning known in the art of cell biology and biochemistry; it refers to a non-MHC molecule that is part of the MHC class I heterodimer molecule. In other words, it constitutes the β chain of the MHC class I heterodimer.

In the context of the present specification, the term human leukocyte antigen (HLA) is used in its meaning known in the art of cell biology and biochemistry; it refers to gene loci encoding the human MHC class I proteins. The three major classical MHC-Ia genes are HLA-A, HLA-B and HLA-C, and all of these genes have a varying number of alleles (Table 1). Closely related alleles are combined in subgroups of a certain allele. For example the allele HLA-B57 has more than 100 closely related alleles that vary in one or more amino acids, according to the WHO Nomenclature Committee for Factors of the HLA System, labelled HLA-B*57:01:01 to HLA-B*57:82. The full or partial sequence of all known HLA genes and their respective alleles are available to the person skilled in the art in specialist databases such as IMGT/HLA (http://www.ebi.ac.uk/ipd/imgt/hla/).

In the context of the present specification, the term antibody is used in its meaning known in the art of cell biology and immunology; it refers to whole antibodies including but not limited to immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM), any antigen binding fragment or single chains thereof and related or derived constructs. A whole antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (V_H) and a heavy chain constant region (C_H). The heavy chain constant region is comprised of three domains, C_H1, C_H2 and C_H3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region (C_L). The light chain constant region is comprised of one domain, C_L. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.

The term antibody-like molecule in the context of the present specification refers to a molecule capable of specific binding to another molecule or target with high affinity / a Kd ≤ 10E-8 mol/l. An antibody-like molecule binds to its target similarly to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a designed ankyrin repeat protein (Molecular Partners, Zürich), a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins and a polypeptide derived from tetratricopeptide repeat proteins.

In the context of the present specification, the term crystallizable fragment (Fc) region is used in its meaning known in the art of cell biology and immunology; it refers to a fraction of an antibody comprising two identical heavy chain fragments comprised of a C_H2 and a C_H3 domain, covalently linked by disulfide bonds.

In the context of the present specification, the term dimer refers to a unit consisting of two subunits.

In the context of the present specification, the term homodimer refers to a dimer comprised of two subunits that are either identical or are highly similar members of the same class of subunits. One example for a homodimer would be a dimer consisting of two subunits independently selected from the list of HLA alleles. In certain embodiments, homodimers consist of two identical HLA alleles.

In the context of the present specification, the term amino acid linker refers to a polypeptide of variable length that is used to connect two polypeptides in order to generate a single chain polypeptide. Exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. A non-limiting example of an amino acid linker is the polypeptide GGGGSGGGGS (SEQ ID No. 001) that links an HLA-heavy chain polypeptide with an Fc domain.

In the context of the present specification, the term checkpoint inhibitory agent or checkpoint inhibitory antibody is meant to encompass an agent, particularly a (non-agonist) antibody (or antibody-like molecule) capable of disrupting the signal cascade leading to T cell inhibition after T cell activation as part of what is known in the art the immune checkpoint mechanism. Non-limiting examples of a checkpoint inhibitory agent or checkpoint inhibitory antibody include antibodies to CTLA-4 (Uniprot P16410), PD-1 (Uniprot Q15116), PD-L1 (Uniprot Q9NZQ7), B7H3 (CD276; Uniprot Q5ZPR3), Tim-3, Gal9, VISTA, or Lag3.

In the context of the present specification, the term checkpoint agonist agent or checkpoint agonist antibody is meant to encompass an agent, particularly but not limited to an antibody (or antibody-like molecule) capable of engaging the signal cascade leading to T cell activation as part of what is known in the art the immune checkpoint mechanism. Non-limiting examples of receptors known to stimulate T cell activation include CD122 and CD137 (4-1BB; Uniprot 007011). The term checkpoint agonist agent or checkpoint agonist antibody encompasses agonist antibodies to CD137 (4-1BB), CD134 (OX40), CD357 (GITR), CD278 (ICOS), CD27, CD28.

In the context of the present specification, the term (immune) checkpoint modulatory agent encompasses checkpoint inhibitory agents, checkpoint inhibitory antibodies, checkpoint agonist agents and checkpoint agonist antibodies.

The present invention relates to a fusion MHC-Ia open conformer, wherein said fusion MHC-Ia open conformer comprises or essentially consists of a first monomer or a first and a second monomer, wherein

1. a. said first monomer, or each of said first and second monomer independently of the other monomer, comprises an HLA heavy chain selected from C08, A25, B58, A30, B53, and C12, and
2. b. wherein said first monomer, or each of said first and second monomer are covalently linked to an Fc polypeptide sequence,

In certain embodiments, the fusion MHC-Ia open conformer comprises a first monomer or a first and a second monomer, and each monomer independently of the other monomer comprises a HLA heavy chain.

According to a second aspect of the invention a fusion MHC-Ia open conformer is provided:

• for use as a medicament,
• particularly for use in the treatment or prevention of cancer, or
• particularly for use as an immunomodulatory agent,
  • particularly in a treatment of an infectious disease,
    • more particularly for use in prevention, treatment or therapy of human immunodeficiency virus (HIV), hepatitis A, B, C, virus (HAV HBV, HCV respectively), influenza virus, Respiratory Syncytial Virus (RSV), measles virus, herpes viruses and/or yellow fever virus.

In certain embodiments of the second aspect of the invention or of any above-mentioned alternative to the second aspect of the invention, the cancer is colon cancer or pancreatic cancer.

n aspect of the invention relates to a fusion MHC-Ia open conformer. The fusion MHC-Ia open conformer comprises, or essentially consists of, a first HLA heavy chain monomer or a first and a second HLA heavy chain monomer. Each of these HLA heavy chain monomer independently of the other comprises or essentially consists of a HLA heavy chain selected from C08, A25, B58, A30, B53, and C12. The fusion MHC open conformer additionally comprises an Fc polypeptide sequence. In certain embodiments, the HLA monomer sequence is situated at the N terminus of the fusion MHC open conformer, and the Fc construct is located towards the C terminus. In certain embodiments, an amino acid linker joins the HLA-heavy chain and the Fc fragment.

The fusion MHC-Ia open conformer additionally comprises a polypeptide domain known to metabolically stabilize a polypeptide in vivo. One example of such a stabilizing domain is an Fc (crystallisable fragment) domain of an immunoglobulin, particularly the Fc polypeptide domain of a gamma immunoglobulin. The HLA-heavy chain and the stabilizing domain may optionally be joined by an amino acid linker. An open conformer fusion protein comprising the HLA chain and an immunoglobulin Fc fragment is henceforth termed HLA-Fc open conformer or HLA₂-Fc herein.

The presence of the Fc domain in the fusion protein facilitates increasing the solubility, stability, avidity, half-life, and from a technological point of view, cost-effective production and purification in mammalian systems (protein A or G purification).

In certain embodiments of any one of the aspects of the invention, the isolated MHC-Ia open conformer or fusion MHC-Ia open conformer consists of two subunits independently selected from the above HLA-alleles. In certain embodiments, homodimers consist of two identical HLA-alleles.

In certain embodiments of any one of the aspects of the invention, the isolated MHC-Ia open conformer or fusion MHC-Ia open conformer comprise two identical HLA polypeptide chains. In certain embodiments, the isolated MHC-Ia open conformer or fusion MHC-la open conformer comprises two different HLA polypeptide chains.

In certain embodiments of any one of the aspects of the invention, the isolated MHC-la open conformer or fusion MHC-Ia open conformer additionally comprises a peptide epitope fragment.

In certain embodiments of any one of the aspects of the invention, a peptide epitope fragment is non-covalently attached to the polypeptide within the antigen presenting domain of the HLA peptide chain.

In certain embodiments of any one of the aspects of the invention, the first and/or second monomer additionally comprises a peptide epitope fragment.

The fusion MHC-la open conformer comprises only the extracellular HLA-alpha 1, HLA-alpha 2 and HLA-alpha 3 domains. In these embodiments, the transmembrane and intracellular domains of the HLA heavy chains are not included in the therapeutic polypeptide of the invention in order to allow its extracellular expression in recombinant cells. The person skilled in the art can easily identify the respective domains even in previously unknown HLA-sequences by pair-wise sequence alignment with annotated HLA-sequences.

In certain embodiments of any one of the aspects of the invention, the fusion MHC-Ia open conformers comprise an Fc domain. In certain particular embodiments, the Fc domain comprises heavy chain constant regions C_H2 and C_H3 from immunoglobulin type G (IgG), type A (IgA), type D (IgD), type E (IgE) or type M (IgM).

In certain embodiments of any one of the aspects of the invention, the fusion MHC-la open conformers comprise an amino acid linker joining a stabilizing domain, particularly an Fc domain, to the HLA polypeptide. In certain particular embodiments, the amino acid linker comprises 1 to 50 amino acids, particularly 5 to 40 amino acids, more particularly 10 to 30 amino acids, even more particularly 15 to 25 amino acids that link the HLA-heavy chain to the Fc domain as one single polypeptide chain.

In certain embodiments of any one of the aspects of the invention, the isolated MHC-Ia open conformers or fusion MHC-Ia open conformers, are provided as parenteral dosage form, particularly confectioned for injection. In certain embodiments, the immune checkpoint inhibitor agent or agonist agent is provided as parenteral dosage form, particularly confectioned for injection. In certain embodiments, both the MHC-Ia open conformers and the immune checkpoint inhibitor agent or agonist agent are present in the same administration form.

In certain embodiments of the third aspect of the invention, the fusion MHC-Ia open conformer is for use as a medicament.

In certain embodiments of the third aspect of the invention, the fusion MHC-Ia open conformer is for use in the treatment or prevention of cancer, in particular for colon cancer or pancreatic cancer.

In certain embodiments of the third aspect of the invention, the fusion MHC-Ia open conformer is for use as an immunomodulatory agent, particularly for use as negative modulator of regulatory T cells (Treg). In certain embodiments, the fusion MHC-Ia open conformer is for use in the treatment of infectious diseases. In certain embodiments, the fusion MHC-Ia open conformer is for use in the treatment of human immunodeficiency virus (HIV) infection, hepatitis A, hepatitis B, hepatitis C, influenza, respiratory syncytial virus (RSV) infection, measles, herpes and yellow fever.

According to a fourth aspect of the invention, a nucleic acid molecule encoding an Fc open conformer monomer, according to the above aspects of the invention is provided for use in the treatment or the therapy of cancer or for use as an immunomodulatory agent, particularly in a treatment of an infectious disease. Expression of the open conformer in vivo from the nucleic acid molecule will, after dimerization, lead to the fusion protein polypeptide of the invention. The concept of expressing pharmaceutically active polypeptides from nucleic acids encoding them in the patient's body is well known and may confer significant benefits to the patient.

In certain embodiments of the fourth aspect of the invention or any above-mentioned alternative thereof, the cancer is colon cancer or pancreatic cancer.

In certain embodiments, the nucleic acid molecule encodes MHC-Ia open conformers monomers, particularly an Fc open conformer monomer that comprises an amino acid linker and/or an Fc (fragment crystallizable) domain, and is used in the treatment or the therapy of cancer, in particular colon or pancreatic cancer.

According to an alternative aspect of the invention a recombinant expression vector comprising the nucleic acid molecule according to the fourth aspect of the invention (and its alternative aspects) is provided for use in the treatment or the therapy of cancer, in particular colon or pancreatic cancer.

In certain embodiments the recombinant expression vector is a plasmid comprising a promoter that is operable in a mammalian cell, particularly in a human cell. The promoter is operably linked to the nucleic acid molecule of the invention.

According to a fifth aspect of the invention a virus comprising the nucleic acid molecule according to the fourth aspect of the invention (and its alternative aspects) is provided for use in the treatment or the therapy of cancer, in particular colon or pancreatic cancer, or for use as an immunomodulatory agent, particularly in a treatment of an infectious disease. The nucleic acid molecule is under control of a promoter sequence operable in a mammalian cell, particularly in a human cell. In certain embodiments, the virus is an adenovirus, adeno-associated virus, a herpes virus or a lentivirus.

According to a sixth aspect of the invention an in vitro genetically modified host cell comprising the nucleic acid molecule according to the fourth aspect of the invention (and its alternative aspects) is provided.

According to a seventh aspect of the invention, a combination medicament is provided, wherein the combination medicament comprises:

• isolated MHC-Ia open conformers or fusion MHC-Ia open conformers, according to any one of the above aspects or embodiments of the invention, and
• an immune checkpoint modulatory agent selected from
  • ∘ an immune checkpoint inhibitor agent (CPI) selected from:
    • an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA4; also known as CD152) interaction with either B7-1 (CD80) and/or B7-2 (CD86), particularly a polypeptide ligand to CTLA-4 or to cd80 or to cd86 such as for example an antibody,
    • an inhibitor of the interaction of programmed cell death protein 1 (PD-1; also known as CD279) with its ligand PD-L1 (also known as CD274; UniProt ID: Q9NZQ7) and/or PD-L2 (also known as CD273; Uni Prot ID: Q9BQ51), particularly a polypeptide ligand to PD-1 or to PD-L1 or to PD-L2 such as for example an antibody, and
    • an inhibitory polypeptide ligand, particularly an antibody, of T cell immunoglobulin and mucin domain-containing 3 (TIM-3), and
  • ∘ a checkpoint agonist agent, particularly a checkpoint agonist antibody selected to bind to and activate the tumor necrosis factor receptor 4-1BB (also known as CD137 or TNFRSF9).

In certain embodiments, the immune checkpoint inhibitor agent is an inhibitor of interaction of CTLA4 with CD80 or CD86.

In certain embodiments, the immune checkpoint inhibitor agent is ipilimumab (Yervoy; CAS No. 477202-00-9).

In certain embodiments, the immune checkpoint inhibitor agent is an inhibitor of interaction of programmed cell death protein 1 (PD-1) with its receptor PD-L1. In certain embodiments, the immune checkpoint inhibitor agent is selected from the clinically available antibody drugs nivolumab (Bristol-Myers Squibb; CAS No 946414-94-4), pembrolizumab (Merck Inc.; CAS No. 1374853-91-4), pidilizumab (CAS No. 1036730-42-3), atezolizumab (Roche AG; CAS No. 1380723-44-3), and Avelumab (Merck KGaA; CAS No. 1537032-82-8).

In certain embodiments, the immune checkpoint agonist agent is utomilumab (PF-05082566), a fully human IgG2 monoclonal antibody against 4-1BB currently undergoing clinical trials.

In certain embodiments, the checkpoint modulatory agent is a polypeptide selected from an antibody, an antibody fragment, and an antibody-like molecule, and the polypeptide is selectively reactive to a checkpoint mediator. In certain embodiments, the checkpoint mediator is selected from CTLA4, PD-1, CD80, CD86, PD-L1, and PD-L2, TIM-3, 4-1BB and 4-1BBL.

Fig. 1

shows the nomenclature of MHC class I molecules.

Fig. 2

shows the schematic representation of HLA-heterotrimers and HLA-open conformers (free-heavy chains). Both forms may exist at the cell surface of antigen presenting cells (APC cells). The inventors propose that the interaction of open conformers with immunoregulatory receptors (KIR's, LIL's, PTPRJ, etc.) is different in affinity and thus modified to induce immune responses that favour anti-tumor immunity.

Fig. 3

shows the schematic representation of HLA-Fc and β2m DNA cassettes and expression of HLA-β2m-Fc molecules from CHO cells. A) alpha 1, 2 and 3 domains of MHC-la heavy chains (HLA-heavy chain) are inserted into a human IgG4-Fc vector cassette; and the human- β2microglobulin inserted in a separate vector cassette. B) Transfections in Chinese hamster ovary cells (CHO) cells are performed using both the HLA-Fc-vector + β2m-vector at a ratio of 1:1 for the extracellular production of the HLA-β2m-Fc protein. Supernatants were collected and HLA-β2m-Fc purified using standard antibody purification protocols. β2m is removed from the HLA-β2m-Fc complex and following HLA-Fc monomers are refolded to form HLA₂-Fc homodimers.

Fig. 4

shows the separation of β2m from the HLA-β2m-Fc complex and purification and refolding of HLA₂-Fc by SEC. A) Chromatography histogram plot of HLA-β2m-Fc molecules in Urea-Tris-BME denaturing buffer show the dissociation of HLA-Fc-free heavy chains from β2m using Sephacryl S-100 HR columns by SEC. B) and C) SDS-page gels stained with coomassie blue show the presence of β2m before and after SEC. B) shows HLA-B2m-Fc molecules before being separated in SEC, and C) show HLA₂-Fc molecules recovered and re-folded following SEC.

Fig. 5

shows the interaction HLA₂-Fc (A25₂-Fc, A30₂-Fc, B27₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-Fc and C12₂-Fc) to different immune regulatory receptors of leukocytes populations by enzyme-linked immunosorbent assay (ELISA). A) hu KIR3DL1, B) hu KIR3DL2, and C) hu KIR3DL3 are expressed in NK cells and subpopulations of T cells. D) LILRB1, and E) LILRB2 expressed mostly in myeloid cells, F) PirB (murine homologue to LILRB) and G) PTPRJ (on leukocytes is preferentially expressed in MDSCs cells and activated T cells)

Fig. 6

shows that HLA₂-Fc molecules (A25₂-Fc, A30₂-Fc, B27₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-Fc, and C12₂-Fc) invariably block mouse CD4⁺ T cell conversion into iTreg. Incubation of HLA₂-Fc in a dose dependent manner with naive CD4⁺ T cells blocks the conversion to iTregs. A-B) HLA₂-Fc molecules blocks the expression of FoxP3 (differentiation marker of Tregs) in optimal culture conditions for iTreg differentiation (10µg/mL) Control HLA-β2m-Fc molecules, isotype, media supplemented with TGFβ and IL-2 and media w/o supplementation demonstrate the specific influence of HLA₂-Fc on iTreg conversion.

Fig. 7

shows that HLA₂-Fc (A25₂-Fc, A30₂-Fc, B27₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-Fc and C12₂-Fc) suppresses lymphoma T cells. A-E) suppression assays to determine the proliferation of cells in the presence of HLA₂-Fc molecules or control HLA-β2m-Fc molecules. HLA₂-Fc suppress human (Jurkat) and mouse (EG.7) lymphoma cell lines in a dose dependent manner (µg/200µL), other cell lines such as Daudi, B cell lymphoma; SK-N-AS, neuroblastoma; and L540, human Hodgkin lymphoma were assessed but not suppression was observed from HLA₂-Fc molecules in optimal culture conditions. Other cell lines such as L428, human Hodgkin lymphoma; L1236, human Hodgkin lymphoma; IMR-5, neuroblastoma; and M130428, Melanoma were also tested but no suppression was observed.

Fig. 8

shows that HLA₂-Fc (A30₂-Fc, B58₂-Fc, and C08₂-Fc) as monotherapy or in combination with PD-1 antibodies can reduce the size of tumors in the C38 murine syngeneic colon carcinoma model. A) Experimental design of injection time points of colon carcinoma cells (C38) and injection of compounds. B) Mean average tumor volume mm³ of A30₂-Fc treated groups (n=5). C) Mean average tumor volume mm³ of B58₂-Fc treated groups (n=5). D) Mean average tumor volume mm³ of C08₂-Fc treated groups (n=5). The experimental design of injection time points of cells and injection of substances was as follow: vehicle PBS Q3Dx6, isotype (10mg/Kg) Q3Dx6; HLA₂-Fc (10 mg/Kg) Q3Dx6; PD-1 biwk × 2 (200 µg); and HLA₂-Fc + PD-1 (Q3Dx6 & biwk × 2, respectively). Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis, *p<0.05, **p<0.01, n.s.= not significant. Q= days between injections; Dx= number of injections; biwk= twice a week.

Fig. 9

shows that HLA₂-Fc (B27₂-Fc and B57₂-Fc) in combination with CTLA4 or PD-1 antibodies reduce the size of tumors in the MC38-OVA or C38 murine syngeneic colon carcinoma model. A) Mean average tumor volume mm³ of B27₂-Fc treated groups (n=6). B) Mean average tumor volume mm³ of B57₂-Fc treated groups (n=6). The experimental design of injection time points of cells and injection of substances was as follow: vehicle PBS Q3Dx6, isotype (10mg/Kg) Q3Dx6; HLA₂-Fc (10 mg/Kg) Q3Dx6; CTLA-4 Q3Dx2 (d1= 100 µg; d4 = 50 µg), PD-1 biwk × 2 (200 µg); HLA₂-Fc + CTLA-4 (Q3Dx6 & Q3Dx2, respectively), and HLA₂-Fc + PD-1 (Q3Dx6 & biwk × 2, respectively). Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis, **p<0.01, ***p<0.001, n.s.= not significant. Q= days between injections; Dx= number of injections; biwk= twice a week.

Fig. 10

shows the in vivo study of A25₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of A25₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; A25₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; A25₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and A25₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis *p<0.05. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 11

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with A25₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 10). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001.

Fig. 12

shows the immune contexture of blood leukocyte analysis from Pan02 pancreatic cancer mice with large tumors treated with A25₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 10). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001.

Fig. 13

shows the in vivo study of A30₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of A30₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; A30₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; A30₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and A30₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 14

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with A30₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 13). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 15

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with A30₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 13). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 16

shows the in vivo study of B27₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of B27₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; B27₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; B27₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and B27₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 17

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with B27₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 16). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 18

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with B27₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 16). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 19

shows the in vivo study of B53₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of B53₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; B53₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; B53₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and B53₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 20

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with B53₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 19). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 21

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with B53₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 19). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 22

shows the in vivo study of B57₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of B57₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; B57₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; B57₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and B57₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 23

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with B57₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 22). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 24

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with B57₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 22). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 25

shows the in vivo study of B58₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of B58₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; B58₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; B58₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and B58₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 26

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with B58₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 25). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 27

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with B58₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 25). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 28

shows the in vivo study of C08₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of C08₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; C08₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; C08₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and C08₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 29

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with C08₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 28). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 30

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with C08₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 28). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 31

shows the in vivo study of C12₂-Fc in combination with PD-1 and 4-1BB antibodies in large tumors of the pancreatic Pan02 syngeneic mouse model. A) Mean average tumor volume in mm³ of C12₂-Fc treated animals (n=6). B) % Δtumor inhibition of treated mice groups compared to control. The experimental design of injection time points of substances was as follow: isotype (5 mg/Kg) biwk × 2; C12₂-Fc (5 mg/Kg) biwk × 2; 4-1BB antibody (1 mg/Kg) biwk × 2 injections; C12₂-Fc + 4-1BB (5 mg/Kg and 1 mg/Kg, respectively) biwk × 2; PD-1 antibody (5 mg/Kg) biwk × 2; and C12₂-Fc + PD-1 (5 mg/Kg each) biwk × 2. Tumor volumes are expressed as mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post-hoc analysis. Δtumor inhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm³), to the end volume of the treatment (e.g. 1000 mm³) compared to isotype. biwk= twice a week.

Fig. 32

shows the immune contexture of Tumor Infiltrating Lymphocytes (TILs) analysis from Pan02 pancreatic cancer mice with large tumors treated with C12₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 31). Relevant leukocytes analysed infiltrating the tumor: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and the CD8+/Treg ratio. B) Granulocytes, Macrophages, Macrophage M1-type, Macrophage M2-type, and Myeloid Derived Suppressor Cells (MDSCs). C) M1/M2 macrophage ratio, Monocytes, Natural killer cells (NK), and Natural KillerT cells (NKT). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Fig. 33

shows the immune contexture of blood leukocyte analysis from treated Pan02 pancreatic cancer mice with large tumors treated with C12₂-Fc, 4-1BB and PD-1 by flow cytometry (continuation of experiment in Fig. 31). Relevant leukocytes analysed present in the blood: A) CD3+ T cells, CD4+ T cells, Regulatory T cells (Treg), CD8+ T cells, and CD8+/Treg ratio. B) Th1 cells (CD4+ T cells IFNy+), Natural Killer cells (NK), and Natural Killer T cells (NKT). C) Monocytes, Granulocyte-Myeloid Derived suppressor cells (G-MDSCs), and Monocytic-Myeloid Derived Suppressor cells (M-MDSCs). Leukocytes % are expressed as box plots showing sample maxima and minima, and each group analysed by one-way ANOVA followed by Dunnet post-hoc analysis *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

The inventors surprisingly found that MHC-Ia open conformers interact with diverse immunoregulatory cell surface receptors present in NK cells, NKT cells, T cells, macrophages and MDSC cells with unique binding or stronger affinity than their control MHC-Ia heterotrimers. HLA class I-a open conformers can be used as a therapeutics to target diseases were white blood cells impair the development of protective immunity, as is the case of cancer and infectious diseases.

Additionally, they discovered a novel in vivo mode of action with injections of HLA₂-Fc as monotherapy or combinatorial approaches using checkpoint modulatory agents. HLA₂-Fc therapy alone or in combinatorial therapies can modulate the infiltration of diverse sets of leukocytes into tumors as determined by the increased infiltration of macrophages M1/M2 ratio, increased NK cells, NKT cells, CD3+ T cells, and CD8+T cells, and reduction of MDSCs.

Furthermore, they observed that systemically by blood analysis HLA₂-Fc therapy increase the expansion of NKT cells and in some cases Th1 cells, indicating the presence of a biomarker that can be used for therapy efficacy in pre-clinical and clinical settings. Interestingly, they also observed that monotherapy with 4-1BB increases systemically the expansion of CD3+, CD4+, CD8+ T cells and Tregs in the blood of animals, indicating a potential side effect of hyper activation of the immune system by 4-1BB. Diverse combinations of HLA₂-Fc + 4-1BB reduced significantly the presence of blood CD3+, CD4+, Treg, and CD8+ T cells, indicating a positive combinatorial approach in case of unwanted lymphocyte expansion on the blood of treated patients with agonistic antibodies.

Overall, the mode of action of MHC-Ia open conformers, particularly when present as fusion proteins comprising an Fc immunoglobulin fragment, alone or in a combinatorial approach with antagonistic/agonistic antibodies is of undoubted relevance as immunomodulatory agents, and can be useful for its translation in the treatment of cancer.

HLA open conformers can be used as a therapeutic to target diseases where immunomodulation is a therapeutic approach, as is the case of cancer and infectious diseases.

MHC-Ia open conformers bind to immunoregulatory receptors expressed in diverse types of white blood cells with unique binding or different affinity than their HLA-β2m-Fc control counterparts

The inventors determined if MHC-Ia open conformers interact with specific immunomodulatory receptors by enzyme-linked immunosorbent assay (ELISA). Results demonstrated that MHC-la open conformers interact uniquely to KIR3DL2, and PTPRJ (for exception of HLA-C-β2m-Fc) and display different affinities to KIR3DL1, KIR3DL3, LILRB1, LILRB2, and Pirb immmunoregulatory receptors than their HLA-β2m-Fc control counterparts (Fig. 5 A-G). This data shows for the first time that MHC classical alleles (HLA-A, HLA-B & HLA-C) (MHC-Ia) have a similar binding pattern to immunoregulatory receptors when they are present as open conformers.

The influence of MHC-Ia molecules on naive CD4⁺ T cells for iTreg conversion was analysed with 10 µg/mL of HLA₂-Fc (A25₂-Fc, A30₂-Fc, B27₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-Fc and C12₂-Fc), HLA-β2m controls (A25-β2m-Fc, A30-β2m-Fc, B27-β2m-Fc, B53-β2m-Fc, B57-β2m-Fc, B58-β2m-Fc, C08-β2m-Fc and C12-β2m-Fc), isotype, and PBS, incubated with naive CD4⁺ T cells in optimal culture conditions for iTreg conversion. MHC-la open conformers demonstrated invariably to down modulate the induction of FoxP3 (Fig. 6) and thus conversion of naive CD4+ T cells into iTregs.

MHC-la open conformers impair the proliferation of leukaemia T cells.

The inventors determined the effect of MHC-la open conformers (A25₂-Fc, A30₂-Fc, B27₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-Fc and C12₂-Fc) with the blocking of proliferation in different tumor cell lines. Results demonstrated that MHC-la open conformers modulate invariably the proliferation of lymphoma T cell lines, when compared to their control counterparts HLA-β2m-Fc (Fig. 7) or isotype IgG4 (data not provided), indicating its potential application to the treatment of lymphoma as a targeted therapy.

The in vivo proof of concept of MHC-la open conformers as immunomodulatory therapeutic molecules for cancer therapy was demonstrated using a validated pre-clinical syngeneic murine C38 and MC38-OVA colon carcinoma models (Figure 8 and 9), and in the pancreatic (Pan02) cancer mouse model (Figure 10, 13, 16, 19, 22, 25, 28 and 31).

A valid strategy, from a therapeutic point of view, is to produce MHC-la open conformers molecules in stable format (Fc fusion), to increase solubility, stability, avidity, half-life, and from a technological point of view, cost-effective production and purification in mammalian systems. HLA-β2m-Fc complex was successfully produced by inserting the alpha 1, 2 and 3 domains of HLA-A25, HLA-A30, HLA-B27, HLA-B53, HLA-B57, HLA-B58, HLA-C08 and HLA-C12 into a human IgG4-Fc vector cassette (Fig. 3A), together with a human-β2m vector, necessary for extracellular production of the HLA-β2m-Fc protein (Fig. 3A,B). Transfections in Chinese hamster ovary cells (CHO) cells were performed using both the HLA-Fc-vector + β2m-vector at a ratio of 1:1. Supernatants were collected and HLA-β2m-Fc purified using standard antibody purification protocols (Recombinant Protein Purification Handbook, principles and methods. 2009. GE Healthcare, 18-1142-75). Separation of β2m from HLA-Fc free-heavy chains was performed using denaturing conditions by SEC (Fig. 4A), or dialysis (data not shown). Refolding of HLA₂-Fc was assessed using the dilution method in refolding buffer and analysed SDS page (Fig. 4B,C) or by western blot (data not shown).

The in vivo proof of concept study using HLA₂-Fc (A30₂-Fc, B27₂-Fc, B57₂-Fc, B58₂-Fc, and C08₂-Fc) as immunomodulatory therapeutic molecules was demonstrated in the C38 and MC38-OVA murine colon carcinoma cancer models as monotherapy or in combination with a murine CTLA4 or murine PD-1 antibody.

Following established protocols C38 or MC38-OVA fragment tumours were subcutaneously injected in the flank of syngeneic mice. Once the tumour reached 60 mm³ (between 1-2 weeks after transplantation of tumors), mice were distributed according to their tumor volume. A30₂-Fc, B27₂-Fc, B57₂-Fc, B58₂-Fc, and C08₂-Fc was injected i.p. six times every 3^rd day (Q3Dx6), CTLA4 was injected two times (Q3Dx2), and PD-1 injected 4 times twice a week (biwk × 2) (Fig. 8A).

Selected HLA₂-Fc can synergize and enhance anti-tumor responses in syngeneic C38 and MC38-OVA colon cancer mouse models (Fig. 8 & 9) either as monotherapy (C08₂-Fc) (Fig. 8D) or in combination with checkpoint antibodies, such as PD-1 + A30₂-Fc (Fig. 8B), B58₂-Fc (Fig. 8C), B57₂-Fc (Fig. 9B) and CTLA4 + B27₂-Fc (Fig. 9A).

For the pancreas (Pan02) cancer mouse model, following established protocols Pan02 cells were injected at 1×10⁵ in the right flank of syngeneic mice respectively. Once the tumors had reached 300 mm3 (approximately 3 weeks after injection of cells) mice were statistically distributed according to their tumor volume. To note that large tumors are harder to treat than smaller tumors, but are useful for further analysis of tumor infiltrating lymphocytes (TILs). Furthermore large tumors are closer to a clinical setting where interventions with immunomodulators are performed in large size tumors of patients.

In pancreas (Pan02) data demonstrated that HLA₂-Fc combination with PD-1 antibody can significantly reduce large Pan02 tumors in combination with A25₂-Fc (Fig. 10A-B), B27₂-Fc (Fig. 16A-B), C08₂-Fc (Fig. 28A-B), and C12₂-Fc (Fig. 31A-B), whereas PD-1 monotherapy showed no therapeutic effect. Other HLA₂-Fc combinations with PD-1 did not demonstrate statistical significance, however % Δtumor inhibition was observed in combination B57₂-Fc (Fig. 22). Additionally, combo therapy of HLA₂-Fc with 4-1BB antibody demonstrated to significantly reduce the tumor size or several HLA₂-Fc combo therapies (for exception of A30₂-Fc and C08₂-Fc) when compared to isotype. The most striking tumor reductions (p<0.01) were observed with B53₂-Fc (Fig. 19A-B), B57₂-Fc (Fig. 22A-B), and B58₂-Fc (Fig. 25A-B). 4-1BB monotherapy was not significantly different when compared to isotype control. Monotherapy with C08₂-Fc (Fig. 28A-B) showed significant tumor reduction (p<0.01) compared to isotype.

The tumor immune contexture of pancreas (Pan02) mice demonstrated the influence of HLA₂-Fc therapy towards diverse sets of tumor infiltrating leukocytes as observed with the infiltration of macrophages M1/M2 ratio, increased NK cells, NKT cells, CD3+ T cells, and CD8+T cells, and reduction of MDSCs, with variations for each HLA₂-Fc as observed in A25₂-Fc (Fig. 11A-C), A30₂-Fc (14A-C), B27₂-Fc (17A-C), B53₂-Fc (20A-C), B57₂-Fc (23A-C), B58₂-Fc (26A-C), C08₂-Fc (29A-C), and C12₂-Fc (32A-C). Systemically analysis of leukocytes from the blood demonstrated only few changes when compared to their control monotherapy counterparts in NKT cells and Th1 cells for some cases, A25₂-Fc (Fig. 12A-C), A30₂-Fc (15A-C), B27₂-Fc (18A-C), B53₂-Fc (21A-C), B57₂-Fc (24A-C), B58₂-Fc (27A-C), C08₂-Fc (30A-C), and C12₂-Fc (33A-C).

The present invention demonstrates for the first time that the family of classical MHC-la molecules when produced as heavy chains without β2m (HLA-A, HLA-B and HLA-C open conformers and their corresponding HLA₂-Fc fusion proteins) have immunomodulatory properties that differ from their control HLA-β2m counterparts. Using as non-limiting examples diverse sets of HLA alleles the inventors provide data demonstrating that invariably MHC-la molecules, when present as open conformers are immunomodulatory agents with unique properties as demonstrated by the modulation of leukocytes present in the tumor microenvironment and in the blood. Furthermore its use is not only limited to modulatory agents, but also for its use as therapeutics for the treatment of cancer as demonstrated in pre-clinical cancer mouse models of colon cancer and pancreatic cancer either as monotherapy or in combination therapy with checkpoint inhibitor antibodies (e.g. CTLA4 and PD-1) and checkpoint agonistic antibodies (e.g. 4-1BB).

Interaction of HLA₂-Fc with diverse immunoregulatory receptors (KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, LILRB2, PTPRJ and Pirb) distributed in diverse white blood cells (e.g. NK, NKT, CD4+ T-cells, macrophages and MDSCs) demonstrates that the multitasking nature of the molecules paves a new way of modulating the immune system with HLA open conformers.

Additionally, HLA₂-Fc molecules demonstrated to block the conversion of naive CD4+ T-cells to iTregs in vitro, pointing out to a mode of action were HLA₂-Fc acts as an immunomodulatory molecule affecting the differentiation and function of iTregs. Targeting iTregs is a strategy for diverse therapeutic indications, such as infectious diseases and cancer.

Overall, the mode of action of HLA₂-Fc as combinatorial approaches with antagonistic/agonistic antibodies is of undoubted relevance in the treatment of cancer, and correlates to the current clinical need in cancer immunotherapy.

HLA₂-Fc molecules emerge as a novel class of immunomodulatory drugs. In vitro and in vivo data points to a mechanism were HLA₂-Fc molecules act as a switch-on mechanism for the activation of anti-tumor immunity. Without wishing to be bound by theory, the inventors hypothesize that the interaction of HLA₂-Fc open conformers with diverse immunomodulatory receptors present in NK, T cells, macrophages and MDSCs, and functional modulation of Tregs participate synergistically and exacerbates the immune response.

In vivo experiments were performed using C38 and MC38-OVA colon carcinoma mouse cell lines.

In vitro experiment cell lines used were: EL4, mouse T cell lymphoma; EG.7, mouse T cell lymphoma; Jurkat, human T cell lymphoma; L428, human Hodgkin lymphoma; L540, human Hodgkin lymphoma; L1236, human Hodgkin lymphoma; Daudi, B cell lymphoma; IMR-5, neuroblastoma; SK-N-AS, neuroblastoma; and M130428, Melanoma.

Lymphocytes populations for iTreg conversion experiments were stained with: CD4 (FITC-BD Bioscience), FoxP3+ (efluor 450- eBioscience), CD3 (PE-Cy7- eBioscience), CD45 (PerCP-eBioscience).

Analysis of tumor infiltrating lymphocytes was performed with the following antibodies: CD45 (Biolegend, clone 30-F11); CD3 (BD Bioscience, clone 145-2011); CD4 (Biolegend, clone GK1.5), CD8 (BD Bioscience, clone 53-6.7), CD25 (Biolegend, clone PC61), FoxP3 (eBioscience, clone FJK-16s), CD335 (Biolegend, clone 29A1.4), F4/80 (Biolegend, clone BM8), CD11b (Biolegend, clone M1/70), Gr-1 (BD Bioscience, clone RB6-8C5), MHCII I-A/I-E (BD Bioscience, clone 2G9), CD206 (Biolegend, clone C068C2) and LID stain (eBioscience).

Analysis of blood leukocytes was performed with the following antibodies: CD45 (Biolegend, clone 30-F11); CD3 (BD Bioscience, clone 145-2011), CD4 (Biolegend, clone GK1.5), CD8 (BD Bioscience, clone 53-6.7), FoxP3 (eBioscience, clone FJK-16s), T-Bet (BD Bioscience, clone 4B10), CD335 (Biolegend, clone 29A1.4), F4/80 (Biolegend, clone BM8), CD115 (Biolegend, clone AFS98), CD11b (Biolegend, clone M1/70), Ly6G (Biolegend, clone 1A8), Ly6C (Biolegend, clone HK1.4) and LID stain (eBioscience).

Checkpoint inhibitor antibodies CTLA4 clone 9H10, PD-1 clone RMP1-14, and agonist antibody 4-1BB clone 3H3 were obtained from Bio X Cell Co.

HC10 mAb (IgG2a) binding to β2m-free heavy chains of MHC-la alleles was a gift from Dr. Hidde Ploegh (MIT, MA).

Recombinant production of HLA-β2m-Fc (A25-β2m-Fc, A30-β2m-Fc, B2705-β2m-Fc, B53-β2m-Fc, B57-β2m-Fc, B58-β2m-Fc, C08-β2m-Fc & C12-β2m-Fc) was achieved by inserting the alpha 1, 2 and 3 domains of HLAs into a human IgG4-Fc vector (InvivoGen), and the human β2-microglobulin (β2m) in a separate vector. Production of recombinant HLA-β2m-Fc was performed by co-transfection of the HLA-Fc-vector sand β2m-vector into Chinese hamster ovary (CHO) cells. Production of HLA-β2m-Fc was outsourced to Evitria AG.

Purification of HLA-β2m-Fc constructs was performed using conventional protocols for antibody purification. Production of HLA₂-Fc was performed with the addition of a denaturing step to remove β2m from the HLA-β2m-Fc complex.

Briefly, the capture step of HLA-β2m-Fc proteins was performed after running supernatants (5 mL/min) through protein-G columns (Amersham Pharmacia). Intermediate purification steps were performed by eluting the selected HLA-β2m-Fc from protein G-columns using elution buffer (100 mM glycine, pH 2.0), and recovering fractions in 8M Urea, 100 mM Tris-HCI pH 8.0. The 1^st Polishing step was to separate HLA-Fc monomers fractions from β2m by either size exclusion chromatography (SEC) using superdex 200 prep grade or Sephacryl S-100 HR (GE Lifescience) with an AKTA system (GE Lifescience), or by dialysis with membranes of 50 KDa pore size (Millipore). The recovered HLA-Fc monomers from both protocols were refolded by the dilution method after pulsation of the HLA-Fc monomers at 3 times with intervals of 8 hours each in 100 times volume of refolding buffer (50 mM Tris-HCl pH8.5, 500 mM L-Arginine, 1 mM EDTA, 0.15 mM NaCl, 1% Sucrose, 0.01% Tween-20). The 2^nd Polishing step by SEC was performed to remove further impurities and to buffer exchange newly recovered fractions of HLA₂-Fc proteins into dilution buffer (PBS, 1% Sucrose, and 0.01% Tween-20). Purified solutions of HLA₂-Fc proteins (A25₂-Fc, A25₂-Fc, B2705₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-F, C12₂-Fc) were filter sterilized using 0.2 µm membranes (Millipore).

Fractions HLA-β2m-Fc complexes and HLA₂-Fc were analysed by gradient 4-20% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and western blot using HC10 (specific for HLA-free-heavy chains) antibodies. β2m western blots were performed with and without denaturing conditions (10 mM DTT) (data not shown).

Competition ELISA assays were performed using Maxisorp (Nunc, Switzerland) 96 well plates coated with 10 µg/mL of selected leukocyte receptors (human KIR3DL1, human KIR3DL2, human KIR3DL3, human LILRB1, human LILRB2, human PTPRJ and mouse Pirb) purchased from Creative Biomart. Receptors were incubated for ON 4°C, blocked with 5% milk powder-TBS 2 hrs. HLA₂-Fc selected constructs (A25₂-Fc, A30₂-Fc, B2705₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-F, and C12₂-Fc) and their controls (A25-β2m-Fc, A30-β2m-Fc, B2705-β2m-Fc, B53-β2m-Fc, B57-β2m-Fc, B58-β2m-Fc, C08-β2m-Fc and C12-β2m-Fc) and isotype IgG4 were added at 10 µg/mL for 2 hrs RT. HRP-conjugated antibodies against human Fc were used as detectors.

Flow cytometry analysis was performed using a FACS canto II (BD Bioscience) and data were analysed using FlowJo version 7.6.4.

To induce expression of Foxp3 in murine CD4⁺ T cells, we harvested spleen cells from C57BL/6 splenocytes and purified (Mouse Naive CD4⁺ T Cell Isolation Kit- Easy Sep) to obtain CD4⁺ T naive cells. Cells were then cultured for 96 h at 10⁵ cells/200 µL/well in 96-well plates with coated 5 µg/mL anti-CD3mAb (eBioscience), soluble 2 µg/mL anti-CD28 mAb (Biolegend), 10 µg/mL of TGF-β1 (R&D systems) and 100 IU/mL of IL-2 (R&D systems).

Murine naive CD4⁺ T cells in optimal culture conditions for iTreg conversion were incubated in the presence at dose concentrations (5µg/200 µL) of HLA₂-Fc (A25₂-Fc, A30₂-Fc, B2705₂-Fc, B53₂-Fc, B57₂-Fc, B58₂-Fc, C08₂-F, and C12₂-Fc), controls (A25-β2m-Fc, A30-β2m-Fc, B2705-β2m-Fc, B53-β2m-Fc, B57-β2m-Fc, B58-β2m-Fc, C08-β2m-Fc and C12-β2m-Fc) Isotype IgG4, media without differentiation factors and PBS for 72 h. iTreg conversion was measured by flow cytometry.

Cells were plated in round 96-wells plates at a density of 5 × 10⁵ cells / well following the addition of drugs at different concentrations (25, 10, and 5 µg/well) for 1 day. XTT proliferation assay was performed accordingly to the manual instructions (cell proliferation kit II, Roche). Results were obtained with the absorbance of wells at 450 nm using a microtiter plate reader.

C38 or MC38-OVA tumour fragments were injected subcutaneously into the right flanks of syngeneic female C57BL/6 mice at week 6. Pan02 cell lines were injected at 1×10⁵ in the right flank of syngeneic mice C57BL/6 at week 6. Animals were distributed according to their individual tumour volume size and divided into groups displaying no statistical differences between them. For C38 and MC38-OVA experimental treatment began when the tumors had reach ±60 mm³. For pancreas Pan02 experimental treatment began in large tumors of 300 mm³. Tumour diameters were measured using a caliper, and volume was calculated according to the formula, D/2×d² where D and d are the longest and shortest diameter of the tumour in mm, respectively.

The Experimental design of injection of substances was established as follow for colon (C38 and MC38): vehicle (PBS 200 µL) Q3Dx6; isotype (10mg/Kg) Q3Dx6; HLA₂-Fc (10 mg/Kg) Q3Dx6; anti-CTLA4 Q3Dx2 (1st injection 100 µg and 2^nd injection 50 µg); PD-1 biwk × 2 (200 µg); HLA₂-Fc + CTLA-4 (Q3Dx6 and Q3Dx2, respectively); HLA₂-Fc + PD-1 (Q3Dx6 and biwk × 2, respectively). For pancreas (Pan02) the experimental design of injection of substances was as follow: isotype (5 mg/Kg) biwk × 2; HLA₂-Fc (5 mg/Kg) biwk × 2; PD-1 (5 mg/Kg) biwk × 2; 4-1BB (1 mg/Kg) biwk × 2; HLA₂-Fc + PD-1 biwk × 2; and HLA₂-Fc + 4-1BB biwk × 2.

% ΔInhibition is calculated from the ΔT/ΔC tumor growth ratio, which represents the growth of the tumor in % from the beginning of the treatment (e.g. 300 mm3), to the end volume of the treatment (e.g. 1000 mm3) compared to control using the following formula: Mean % Δlnhibition = (mean(C)-mean(C0)) - (mean(T)-mean(T0)) / (mean(C)-mean(C0)) * 100%. Where T = treated group value, T0 - treated group initial value; C - control group value, C0 - control group initial value.

For the analysis of tumor infiltrating lymphocytes the following gating strategies where used: CD45+ for total leukocytes; CD45+ CD3+ for total T cells; CD45+ CD3+ CD4+ for CD4 T helper cell; CD45+ CD3+ CD8+ for CD8 Cytotoxic T cell; CD45+ CD3+ CD4+ FoxP3+ CD25+ for Treg cell; CD45+ CD3- CD11+ Gr-1+ for MDSCs; CD45+ CD3- CD11+ F4/80+ for Macrophages; CD45+ CD3- CD11+ F4/80+ MHCII+ for M1-type macrophages; CD45+ CD3-CD11+ F4/80+ CD206+ for M2-type macrophages; CD45+ Gr-1- F4/80- CD335+ for NK cells; and CD45+ Gr-1- F4/80- CD335+ CD3+ for NKT cells.

For the analysis of blood leukocytes the following gating strategies where used: CD45+ for total leukocytes; CD45+ CD3+ for total T cells; CD45+ CD3+ CD4+ for CD4 T helper cell; CD45+ CD3+ CD8+ for CD8 Cytotoxic T cell; CD45+ CD3+ CD4+ FoxP3+ for Treg cell; CD45+ CD3+ CD4+ T-Bet+ for Th1 cells; CD45+ CD3- CD11+ Ly6C+ Ly6G+ for G-MDSCs and M-MDSCs; CD45+ Ly6C- Ly6G- CD335+ for NK cells; and CD45+ Ly6C- Ly6G- CD335+ CD3+ for NKT cells.

Preparation of tumor and blood samples for flow cytometry were performed using protocols described by eBioscience (https://www.ebioscience.com/media/pdf/best-protocols/cell-preparation-for-flow-cytometry.pdf, accessed Feb 21, 2017). HLA-A HLA-B HLA-C A*01 B*07 B*53 C*01 A*02 B*08 B*54 C*02 A*03 B*13 B*55 C*03 A*11 B*14 B*56 C*04 A*23 B*15 B*57 C*05 A*24 B*18 B*58 C*06 A*25 B*27 B*59 C*07 A*26 B*35 B*67 C*08 A*29 B*37 B*73 C*12 A*30 B*38 B*78 C*14 A*31 B*39 B*81 C*15 A*32 B*40 B*82 C*16 A*33 B*42 B*83 C*17 A*34 B*44 C*18 A*36 B*46 A*43 B*47 A*66 B*48 A*68 B*49 A*69 B*50 A*74 B*51 A*80 B*52 Sequence identifier (length in aa) Amino acid sequence A*25:01:01 HLA00071 (365aa) SEQ ID NO. 002 A*30:01:01 HLA00089 (365aa) SEQ ID NO. 003 B*27:05:02 HLA00225 (362aa) SEQ ID NO. 004 B*53:01:01 HLA00364 (362aa) SEQ ID NO. 005 B*57:01:01 HLA00381 (362aa) SEQ ID NO. 006 B*58:01:01:01 HLA00386 (362aa) SEQ ID NO. 007 C*08:01:01 HLA00445 (366aa) SEQ ID NO. 008 C*12:02:01 HLA00453 (366aa) SEQ ID NO. 009