EA044391B1 - ANTIBODIES TO SIRPa AND OPTIONS FOR THEIR THERAPEUTIC USE - Google Patents

Description

The invention relates to the field of immunotherapy. The invention relates to new antibodies to

SIRPa, which can specifically reduce the interaction of SIRPa with CD47 without affecting the interaction of SIRPg with CD47.

Targeting immune checkpoints in adaptive immunity has shown significant therapeutic efficacy against numerous cancers, but in a limited proportion of patients. The immune checkpoint on myeloid cells (macrophages, dendritic cells, myeloid-derived suppressor cells (MDSCs), polymorphonuclear cells (PMN)) remains poorly understood, while these cells represent the most common type of immune cells in many solid tumors and are often associated with unfavorable outcome.

Signaling regulatory protein-alpha or SIRPa (also called SIRPa, CD172a, or SHPS-1) is expressed on monocytes, most subsets of tissue macrophages, granulocytes, subsets of dendritic cells in lymphoid tissues, some bone marrow progenitor cells, and to varying levels on neurons , with particularly high levels of expression observed in synapse-rich brain regions such as the cerebellar granular layer and hippocampus. SIRPa is the prototypical member of the SIRP family of paired receptors of closely related SIRP proteins. The gene encoding human SIRPa is a polymorphic gene, several variants of which have been described in humans. The most common protein variants are SIRPa v1 and v2 (accession numbers NP_542970 (P78324) and CAA71403). Polymorphisms in human SIRP result in changes in surface-exposed amino acids, but this does not affect binding to CD47.

The interaction of SIRPa, expressed by myeloid cells, with the ubiquitous CD47 receptor is an important immune checkpoint in the innate immune response, involved in the regulation of myeloid functions. The interaction of SIRPa with CD47 has been widely described and provides a suppressive signal that inhibits phagocytosis of host cells. CD47 is widely expressed at lower levels by most healthy cells, but it is also overexpressed in some cancer cells. Thus, CD47 functions as a don't eat me signal. Both CD47 and SIRPa are also involved in many other interactions. One of the best characterized physiological functions of the CD47-SIRPa interaction is its involvement in maintaining the homeostasis of hematopoietic cells, particularly erythrocytes and platelets. Because CD47 functions as a don't eat me signal and is therefore an important determinant of phagocytosis of host cells by macrophages, the potential contribution of the CD47-SIRPa interaction in the elimination of cancer cells has been intensively studied in recent years. CD47 receptor abundance in tumors has been shown to be inversely associated with overall patient survival and represents a poor prognostic factor for several cancer types.

Currently, the SIRPa/CD47 pathway is the target of various pharmaceutical developments aimed at enhancing phagocytosis by macrophages. In fact, like infected cells, cancer cells carry aberrant loads such as foreign proteins or abnormal levels of normal proteins, and these cells often disrupt innate immune control mechanisms by simultaneously overexpressing immune regulatory molecules. It is becoming apparent that one such mechanism involves CD47, an autologous protein expressed by normal cells. CD47 interacts with SIRPa and leads to the transmission of the don't eat me signal to phagocytic macrophages, which then leave the target cells alone. Overexpression of CD47 by cancer cells renders them resistant to macrophages, even when the cancer cells are coated with therapeutic antibodies, and correlates with poor clinical outcomes in numerous solid cancers and hematological malignancies. In experimental models, particularly xenograft models of human tumors in immunodeficient mice, blocking the CD47/SIRPa pathway with CD47-targeting agents was very effective in promoting tumor clearance by macrophages, as well as reducing cancer cell proliferation and metastasis formation. Blocking the CD47/SIRPa pathway with CD47-targeting agents, by enhancing antibody-dependent phagocytosis by macrophages, has been described to have synergistic effects with depleting therapeutic anticancer antibodies such as Trastuzumab (anti-Her2 antibody), Cetuximab (anti-EGFR antibody) ), Rituximab (anti-CD20 antibody), and Alemtuzumab (anti-CD52 antibody).

However, agents targeting CD47 (anti-CD47 or SIRPa-Fc) have recently been shown to cause hematological toxicity (anemia or thrombocytopenia) related to the physiological role of CD47.

In addition, CD47 also binds to another SIRP family member, SIRP-gamma (also called SIRPg, SIRPy, CD172g, or SIRP-beta 2), which is present on the surface of human T cells but not on human myeloid cells. SIRPg arose from the duplication of the SIRPb gene in eastern hemisphere primates almost 35 million years ago and is limitedly expressed on T lymphocytes, unlike SIRPa, which is expressed on myeloid cells. SIRPg is absent in mice. The SIRPg-CD47 interaction has been shown to mediate cell-cell adhesion, enhance superantigen-dependent T cell-mediated proliferation, and co-stimulate T cell activation (Piccio et al., Blood, 105:6, 2005).

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Due to the high sequence similarity between SIRPa and SIRPg, particularly in the region that interacts with CD47, anti-SIRPa antibodies disclosed in the prior art also bind to SIRPg and have undesirable effects in humans, such as inhibition of T cell proliferation and decreased immune response. Such side effects of anti-CD47 antibodies or non-selective anti-SIRPg antibodies are unpredictable since the known antibodies were tested in mouse models that do not have the SIRPg gene and therefore these side effects were not observed.

There continues to be a significant need in the art for new and improved agents, particularly antibodies, for safe immunotherapy, particularly against cancer, that target innate immune cells and do not adversely affect T cell immune responses. In particular, there is a need to inhibit the SIRPa-CD47 interaction without negatively affecting the SIRPg-CD47 interaction. The inventors of the present invention have made significant progress with the invention disclosed herein.

The object of the present invention is to satisfy this need by providing new agents, in particular antibodies, which solve, in whole or in part, the above problems.

In the present application, the inventors propose new antibodies to SIRPa, in particular, humanized antibodies that antagonize the SIRPa-CD47 interaction, but do not bind specifically to SIRPg and, accordingly, do not adversely affect the SIRPg-CD47 interaction.

Antibodies according to the present invention have, in particular, the following advantages:

they do not cause hematological toxicity due to limited expression of SIRPa (do not bind to human erythrocytes (red blood cells) and platelets);

they reduce tumor growth and modify the tumor microenvironment when used as monotherapy;

they exhibit synergistic effects with checkpoint inhibitors and costimulatory agents;

they induce long-term and durable anti-tumor memory T cell responses;

they provide conditions for immune responses of human T cells, being selective antagonists of the SIRPa-CD47 interaction, without disrupting the CD47/SIRPg interaction. Contrary to expectations, the present inventors obtained such selective antibodies despite the high sequence identity between SIRPa and SIRPg.

These novel antibodies, selected based on these characteristics, are particularly promising for numerous therapeutic applications, particularly in the treatment of cancers, including inflammatory cancers and cancers with infiltrated myeloid cells (particularly infiltrated MDSCs and/or tumor-associated macrophages (THERE)).

Antibodies to SIRPa (With epitopes)

In one aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SLIPVGP ), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

Unless otherwise specified, all technical and scientific terms are used herein to have the meaning commonly understood by one skilled in the art.

For convenience, the meanings of certain terms and expressions used in the description, examples and claims are given.

As used herein, the term antibody includes polyclonal antibodies, monoclonal antibodies, or recombinant antibodies.

As used herein, the term monoclonal antibody is intended to refer to a preparation of antibody molecules, i.e. antibodies that have a common heavy chain amino acid sequence and a common light chain amino acid sequence, as opposed to polyclonal antibody preparations, which contain a mixture of antibodies with different amino acid sequences. Monoclonal antibodies can be produced using several known technologies such as phage, bacterial, yeast or ribosomal display, as well as classical methods such as hybridoma-derived antibodies. Accordingly, the term monoclonal is used to refer to all antibodies derived from a single nucleic acid clone.

Antibodies of the present invention include recombinant antibodies. As used herein, the term recombinant antibody refers to antibodies that are produced, expressed, generated or isolated by recombinant means, such as antibodies that are expressed using a recombinant expression vector transfected into a cell

- 2 044391 owners; antibodies isolated from a recombinant, combinatorial antibody library; antibodies isolated from an animal (for example, a mouse) that is transgenic for human immunoglobulin genes; or antibodies that are produced, expressed, generated, or isolated by any other method in which specific immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are linked to other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies.

As used herein, the term chimeric antibody refers to an antibody in which a variable domain sequence derived from the germ line of a mammalian species, such as a mouse, has been grafted onto a constant domain sequence derived from the germ line of another mammalian species, such as a human.

As used herein, the term humanized antibody refers to an antibody in which hypervariable region (CDR) sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

As used herein, the term antigen-binding antibody fragment refers to a portion of an antibody, i.e. a molecule corresponding to a portion of the antibody structure of the present invention that exhibits antigen-binding activity against SIRPa, possibly in native form; such fragment exhibits an antigen-binding specificity for said antigen that is identical or substantially identical to the antigen-binding specificity of the corresponding four-chain antibody. Preferably, the antigen binding fragments have a binding affinity similar to that of the corresponding 4 chain antibodies. However, an antigen binding fragment that has a reduced antigen binding affinity compared to the corresponding 4-chain antibodies is also included within the scope of the present invention. Antigen-binding ability can be determined by measuring the affinity of the antibody and the target fragment. Such antigen binding fragments may also be referred to as functional antibody fragments.

Antigen-binding fragments of antibodies are fragments that contain their hypervariable domains, designated CDRs (complementarity determining regions), or parts thereof, including the recognition site for the antigen, i.e. extracellular domain of SIRPa, thereby determining the specificity of antigen recognition.

Each variable domain of the light and heavy chains (VL and VH, respectively) of a four-chain immunoglobulin contains three CDRs, designated VL-CDR1 (or LCDR1), VL-CDR2 (or LCDR2), VL-CDR3 (or LCDR3) and VH-CDR1 (or HCDR1), VH-CDR2 (or HCDR2), VH-CDR3 (or HCDR3), respectively.

One skilled in the art can determine the position of the various antibody regions/domains in accordance with standard definitions established for this purpose, including a reference numbering system, see, for example, the Kabat numbering system (KABAT), or by applying the IMGT pearl necklace algorithm. In this regard, it should be noted that for determining sequences according to the present invention, the definition of region/domain boundaries may vary between different reference systems. Accordingly, the regions/domains defined in the present invention include sequences exhibiting approximately ±10% variability in the length or position of the subject sequences within the full-length antibody variable domain sequence.

Based on the structure of four-chain immunoglobulins, antigen-binding fragments can accordingly be determined by comparison with antibody sequences in available databases and known in the prior art, and in particular by comparison of the positions of functional domains in such sequences, noting that the positions framework and constant domains are well defined for various classes of antibodies, especially for IgG, in particular for mammalian IgG. Such comparison also includes data related to the three-dimensional structures of antibodies.

To illustrate specific embodiments of the present invention, antigen binding fragments of an antibody that contain variable domains containing the CDR of the antibody include Fv, dsFv, scFv, Fab, Fab', F(ab') ₂ . Fv fragments consist of the VL and VH domains of the antibody held together by hydrophobic interactions; in dsFv fragments, the VH:VL heterodimer is stabilized by a disulfide bond; in scFv fragments, the VL and VH domains are connected to each other via a flexible peptide linker, forming a single-chain protein. Fab fragments are monomeric fragments that can be produced by papain digestion of an antibody; they contain a complete L-chain and a VH-CH1 fragment of the H-chain, linked through a disulfide bond. The F(ab')2 fragment can be obtained by cleavage of the antibody with pepsin downstream of the disulfide bond in the hinge region; it contains two Fab' fragments and additionally part of the hinge region of the immunoglobulin molecule. Fab' fragments can be generated from F(ab')2 fragments by cutting the disulfide bond at the hinge region. F(ab')2 fragments are divalent, i.e. they contain two antigen-binding sites, similar to the native immunoglobulin molecule

- 3 044391 bowlina; on the other hand, the Fv (VHVL dimer constituting the variable part of Fab), dsFv, scFv, Fab and Fab' fragments are monovalent, i.e. they contain one antigen-binding site. These basic antigen binding fragments of the present invention can be combined to produce multivalent antigen binding fragments such as diabodies, tribodies or tetrabodies. These multivalent antigen binding fragments are also part of the present invention.

As used herein, the term bispecific antibodies refers to antibodies that recognize two different antigens due to the presence of at least one region (eg, derived from the variable region of a first antibody) that is specific for the first antigen and at least a second region (eg, derived from the variable region of a first antibody) that is specific for the first antigen and at least a second region (eg, , derived from the variable region of the second antibody), which is specific for the second antigen. A bispecific antibody specifically binds to two target antigens and is therefore one type of multispecific antibody. Polyspecific antibodies that recognize two or more different antigens can be produced by recombinant DNA techniques or include, but are not limited to, antibodies produced chemically by any convenient method. Bispecific antibodies include all antibodies or antibody conjugates or polymeric forms of antibodies that are capable of recognizing two different antigens. Bispecific antibodies include antibodies that have been reconstituted and restructured while maintaining their bivalent characteristics, and antibodies that have been chemically coupled so that they may have multiple antigen recognition sites for each antigen, such as BiME (bispecific macrophage enhancement antibodies) , BiTE (bispecific antibodies that activate T cells), DART (dual affinity retargetable antibody); DNL (dock-and-lock), DVD-Ig (double variable domain immunoglobulins), HAS (human serum albumin), kih (peaks-to-holes).

Accordingly, the bispecific antibodies of the present invention are directed against SIRPa and the second antigen.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is bispecific.

Several studies aimed at developing therapeutic antibodies have led to the engineering of Fc regions to optimize the properties of antibodies, allowing the creation of molecules that are better suited for the pharmacological activity required of them. The Fc region of an antibody mediates its serum half-life and effector functions such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cell phagocytosis (ADCP). Several mutations located at the border between the CH2 and CH3 domains, such as T250Q/M428L and M252Y/S254T/T256E + H433K/N434F, have been shown to increase FcRn binding affinity and half-life of IgG1 in vivo. However, there is not always a direct relationship between increased FcRn binding and improved half-life. One approach to improve the effectiveness of a therapeutic antibody is to increase its persistence in serum, allowing for higher levels of circulation, less frequent administration, and reduced dosing. Modified Fc regions may be desirable to reduce or increase the effector function of an antibody. Antibodies that target molecules on the cell surface, especially molecules on immune cells, require elimination of effector functions. On the contrary, for antibodies intended for use in oncology, increasing effector functions can improve therapeutic activity. The four human IgG isotypes bind the activating Fcy receptors (FcyRI, FcyRIIa, FcyRIIIa), the inhibitory receptor FcyRIIb, and the first component of the complement system (C1q) with different affinities, resulting in very different effector functions. The binding of IgG to FcyR or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for binding to FcyR and C1q and have unique sequences in IgG2 and IgG4.

As used herein, the term modified antibody refers to a molecule containing an antibody or an antigen-binding fragment thereof, wherein said monoclonal antibody or functional fragment thereof is linked to a functionally different molecule. The modified antibody of the present invention may be a fusion protein or conjugate resulting from any suitable form of attachment, including covalent attachment, grafting, chemical coupling to a chemical or biological group or molecule, such as a PEG polymer or other protecting group or molecule, suitable for protection against protease degradation in vivo, to improve the stability and/or half-life of an antibody or functional fragment. Using similar techniques, especially chemical bonding or grafting, a modified antibody coupled to a biologically active molecule can be produced, said active molecule being, for example, selected from toxins, in particular Pseudomonas exotoxin A, the plant toxin A-chain ricin, or saporin toxicity

- 4 044391 on, especially a therapeutically active ingredient, a vector (including, in particular, a protein vector) suitable for targeting an antibody or functional fragment to specific cells or tissues in the human body, or it may be associated with a tag or linker, especially when used fragments of an antibody, PEGylation of an antibody or functional fragments thereof is a particularly interesting embodiment as it improves the conditions for delivery of the active substance to the host, especially for therapeutic use, PEGylation can be site specific to prevent unwanted interaction with the recognition sites of antibodies or functional fragments, and can be performed using high molecular weight PEG. PEGylation can be achieved by free cysteine residues present in the sequence of the antibody or functional fragment, or by the addition of free cysteine residues in the amino acid sequence of the antibody or functional fragment.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is modified.

Macromolecules of the present invention include antibodies and fragments thereof, and also include artificial proteins, peptides, and any chemical compounds having antigen-binding ability that mimics that of antibodies, which are also referred to herein as antigen-binding antibody mimetics. Suitable proteins include affitins and anticalins. Affitins are artificial proteins that have the ability to selectively bind to antigens. They are structurally derived from the DNA binding protein Sac7d found in Sulfolobus acidocaldarius, a microorganism belonging to the domain Archaea. An affitin library can be generated by randomizing the amino acids on the Sac7d binding surface, for example by creating variants corresponding to random substitutions of the 11 residues of the Sac7d binding interface, and the resulting protein library can be subjected to rounds of ribosomal display, whereby the affinity can be directed to different targets. such as peptides, proteins, viruses and bacteria. Affitins are antibody mimetics and are being developed as tools in biotechnology. They have also been used as specific inhibitors of various enzymes (Krehenbrink et al, J. mol. Biol., 383:5, 2008). One skilled in the art can easily develop anticalins with the desired binding properties using methods known in the art, in particular as described in patent application WO2008068637 and the above-mentioned publication, in particular by creating phage display and/or ribosome display libraries and screening them using the antigen disclosed herein. Anticalins are artificial proteins that are capable of binding to antigens, both proteins and small molecules. They are antibody mimetics derived from human lipocalins, which are a family of natural binding proteins. Anticalins are approximately eight times smaller in size, contain approximately 180 amino acids, and have a mass of approximately 20 kDa (Skerra, Febs J., 275:11, 2008). Phage display libraries of anticalins have been created that allow screening and selection, in particular, of anticalins with specific binding properties. One skilled in the art can easily develop affins with the desired binding properties using methods known in the art, in particular as described in patent EP1270725 B1, patent US8536307 B2 (Schlehuber and Skerra, Biophys. Chem., 96:2- 3, 2002) and the above publication, in particular by creating phage display and/or ribosome display libraries and screening them using the antigen disclosed herein. Both anticalins and affitins can be produced in a number of expression systems, including bacterial expression systems. Accordingly, the present invention relates to affitins, anticalins and other similar antibody mimetics having the characteristics of the antibodies described herein, in particular with respect to binding to SIRPa, inhibition of the interaction of SIRPa with CD47, lack of ability to bind to SIRPg, lack of ability to bind T -cells, lack of ability to inhibit T cell proliferation, lack of ability to inhibit the interaction of SIRPg with CD47, all of which are included within the scope of the present invention as macromolecules according to the present invention.

All embodiments disclosed herein with respect to antibodies or fragments thereof are carried over, mutatis mutandis, to the macromolecules of the present invention, particularly to antigen-binding antibody mimetics.

As used herein, the term epitope means the portion of an antigen to which an antibody binds. Epitopes of protein antigens can be divided into two categories: conformational epitope and linear epitope. A conformational epitope corresponds to discontinuous regions of the amino acid sequence of the antigen. A linear epitope corresponds to a contiguous sequence of amino acids from an antigen.

According to the present invention, the peptides that are present in SIRPa, and those that are bound by antibodies to SIRPa, are part of an epitope that is specifically recognized by such antibodies.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least two, three, four or five peptides comprising or consisting of an amino acid sequence selected from group consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 3 (KFRKGSPD[DV]/[T] E), within SIRPa, and with at least one peptide containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to peptides containing or consisting of the amino acid sequence set forth in SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE)) , within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ( SLIPVGP), SEQ ID NO: 7 (GRELIYNQKEGH), SEQ ID NO: 8 (KFRKGSPDDVE), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to peptides comprising the sequence set forth in SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 7 ( GRELIYNQKEGH), SEQ ID NO: 8 (KFRKGSPDDVE), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ( SLIPVGP), SEQ ID NO: 9 (ARELIYNQKEGH), SEQ ID NO: 10 (KFRKGSPDTE), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE).

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to peptides comprising the sequence set forth in SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 9 ( ARELIYNQKEGH), SEQ ID NO: 10 (KFRKGSPDTE), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ( SLIPVGP), SEQ ID NO: 11 (GRELIYN), DVE, SEQ ID NO: 12 (HTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to peptides comprising the sequence set forth in SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 11 ( GRELIYN), DVE, SEQ ID NO: 12 (HTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ( SLIPVGP), SEQ ID NO: 13 (ARELIYN), SEQ ID NO: 12 (HTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

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In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to peptides comprising the sequence set forth in SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 13 ( ARELIYN), SEQ ID NO: 12 (HTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), within SIRPa.

Peptides containing the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 correspond to linear epitopes.

These linear epitopes were identified by the present inventors using array-based oligopeptide scanning (sometimes called overlapping peptide scanning or Pepscan analysis). This technique uses a library of oligopeptide sequences from overlapping and non-overlapping segments of a target protein and tests their ability to bind the antibody of interest. Discontinuous epitopes can be mapped with very high reliability and accuracy (Gaseitsiwe et al., 2010 - Clin Vaccine Immunol. Jan; 17(1): 168-175) by combining sequences of non-contiguous peptides from different parts of the target protein and ensuring the conformational rigidity of such combined peptide (eg, using CLIPS scaffolds) (Timmerman et al., 2007, J Mol Recognit, Sep-Oct;20(5):283-99). All of the antibodies tested according to the present invention, including HEFLB, specifically bind to the indicated epitopes.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 70 ( ELIYNQKEGHFPR), SEQ ID NO: 71 (RNNMDFSIRIGN) and SEQ ID NO: 72 (SPRDITLKW), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to a conformational epitope containing or consisting of peptides containing the sequence set forth in SEQ ID NO: 70 (ELIYNQKEGHFPR) , SEQ ID NO: 71 (RNNMDFSIRIGN) and SEQ ID NO: 72 (SPRDITLKW), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 70 ( ELIYNQKEGHFPR) and SEQ ID NO: 71 (RNNMDFSIRIGN).

In one embodiment, the present invention provides an anti-SIRPa antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to a conformational epitope containing or consisting of peptides containing the sequence set forth in SEQ ID NO: 70 (ELIYNQKEGHFPR) and SEQ ID NO: 71 (RNNMDFSIRIGN), within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, as defined above, that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 73 ( YNQK) and SIR, within SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody, or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that specifically binds to a conformational epitope containing or consisting of a peptide containing the amino acid sequence set forth in SEQ ID NO: 73 (YNQK ), and a peptide having the amino acid sequence SIR, within SIRPa.

Peptides containing the sequence shown in SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, and SIRP correspond to conformational epitopes. These conformational epitopes were determined by the present inventors using proteolysis protection procedures (enzymatic digestion: chymotrypsin digestion, trypsin digestion of the antibody-antigen complex immobilized on an affinity chromatography support) followed by mass spectrometry (MALDI-TOF/TOF) for detection and determining the sequence of such peptides of interest, as is well known to one skilled in the art (Van de Water et al., Clinical Immunology and Immunopathology, 1997, vol. 85). The antigen used was human SIRPa (accession numbers NP_542970), and one of the antibodies used according to the present invention was a variant of HEFLB.

An anti-SIRPa antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic of the present invention specifically binds to said conformational epitopes containing or consisting of said peptides in their conformational grouping within native SIRPa.

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The peptide containing the amino acid sequence shown in SEQ ID NO: 73 (YNQK) corresponds to the peptide consisting of amino acids at positions 80-83 in the human amino acid sequence SIRPa, characterized by its accession number NP_542970.

A peptide having the amino acid sequence SIR, peptide SIR, corresponds to the peptide consisting of amino acids at positions 105-107 in the human amino acid sequence SIRPa, which is listed under accession number NP_542970.

As used herein, the term SIRPa refers to a SIRPa protein from a mammalian species, preferably a human SIRPa (eg, accession numbers NP_542970 (P78324) and CAA71403).

As used herein, the term anti-SIRPa antibody refers to an antibody that specifically binds to SIRPa, in particular human SIRP.

Specific binding of an antibody or antigen-binding fragment thereof of the present invention and an epitope (or epitope-containing region) implies that the antibodies exhibit detectable affinity for an epitope (epitope-containing region) on a particular protein or antigen (herein SIRPa). Detectable affinity includes binding with an affinity of approximately ^10'9 M (K _D ) or greater. Preferably, binding is considered specific if the binding affinity is from ^10'9 M to ^10'12 M, optionally from ^10'9 M to ^10'10 M, in particular ^10'10 M. The specificity of the interaction or binding of the binding domain with the target could be easily studied, among other things, by comparing the interaction of said binding domain with a target protein or antigen with the interaction of said binding domain with proteins or antigens other than the target protein.

Affinity can be determined in various ways well known to one skilled in the art. Suitable methods include, but are not limited to, Biacore testing, BLItz™ testing, and Scatchard plotting.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that has a K _D value of less than ^10'9 M, preferably less than ^10'10 M for SIRPa, more preferably less than 1x10' 11 M, in particular, according to research using the Biacore system.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which reduces the interaction of SIRPa with CD47.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which partially or completely, particularly completely, inhibits the binding of CD47 to SIRPa, in particular the binding of human CD47 to human SIRPa.

Such an antibody of the present invention specifically binds to SIRPa and antagonizes the interaction of SIRPa with CD47.

In particular, the anti-SIRPa antagonist antibody of the present invention is capable of reducing or inhibiting the binding of CD47 to SIRPa by at least 50%, 60%, 70%, preferably 80%, more preferably 90%, or most preferably 100%, compared with a negative control molecule in a quantitative binding study.

In particular, the SIRPa antagonist antibody of the present invention is capable of reducing or inhibiting the binding of CD47 to SIRPa by 50-100%, preferably 60-90%, more preferably 70-80%, compared to the negative control molecule, in a quantitative assay binding.

Methods for determining the specificity and affinity of antibodies by competitive inhibition are known in the art (see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Colligan et al. , Current Protocols in Immunology, Green Publishing Assoc, NY (1992; 1993); Muller, Meth. Enzym., 92:589-601 (1983)) and are described in the examples below.

Suitable methods include, but are not limited to, Biacore testing, BLItz™ testing, flow cytometry, and enzyme-linked immunosorbent assay (ELISA).

In one embodiment, the present invention provides an anti-SIRPa antibody as defined above that has an IC ₅₀ value below 500 ng/ml, in particular below 400 ng/ml, 300 ng/ml, more particularly below 200 ng/ml, as defined in quantitative study of the competitive binding of CD47 and antibodies to SIRPa with SIRPa by ELISA.

In one embodiment, the present invention provides an anti-SIRPa antibody as defined above that has an IC ₅₀ value below 500 ng/ml, in particular below 400 ng/ml, 300 ng/ml, more particularly below 200 ng/ml, below 150 ng/ml and even more specifically below 100 ng/ml, determined on human monocytes in a quantitative CD47-anti-SIRPa antibody competitive binding cytometry study.

- 8 044391

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that does not specifically bind to a SIRPg, preferably a human SIRPg.

Such an antibody of the present invention does not affect or prevent the interaction of SIRPg with CD47.

As used herein, the term SIRPg refers to signal regulatory protein gamma (also referred to as SIRP gamma, CD172g or SIRP beta 2) from a mammalian species, preferably human SIRPg.

The reference human SIRPg protein sequence used in the examples of this application corresponds to the sequence associated with accession number Q9P1W8.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which has a KD value greater than 10 ^-9 M, preferably greater than 10 ^-8 M, more preferably greater than 10 ^-7 M in relation to SIRPg, in particular determined using the BLItz™ system.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that does not significantly inhibit or antagonize the binding of CD47 to SIRP-g that does not significantly compete with CD47 for binding with SIRPg.

Such antagonistic effect can be determined using the methods defined in the examples of this application.

According to the present invention, it is intended that an antibody (or an antigen binding fragment or an antigen binding antibody mimetic) does not antagonize the binding of CD47 to SIRPg if the antibody (or an antigen binding fragment or an antigen binding mimetic antibody) does not induce an increase, or induces an increase of less than 1 log , CD47 KD values in a quantitative SIRPg competitive binding assay using the BLItz™ system.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, as defined above, that does not specifically bind to T cells, particularly CD3+ T cells.

In particular, the anti-SIRPa antibody or an antigen-binding fragment thereof or an antigen-binding antibody mimetic according to the present invention does not bind to T cells from a mammalian species, in particular to human T cells.

In particular, an anti-SIRPa antibody (or an antigen-binding fragment thereof or an antigen-binding antibody mimetic) is not considered to bind specifically to human T cells when, in a population of human T cells isolated from peripheral blood mononuclear cells (PBMCs) from a healthy donor, less than 10%, preferably less than 5%, more preferably less than 2%, most preferably less than 1% of the human T cell population is recognized by said anti-SIRPa antibody (or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic).

This effect can be measured using the methods described in the examples of this application.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that does not significantly inhibit the proliferation of T cells, in particular CD3+ T cells, preferably cells from mammalian species and more preferably T - human cells.

In particular, an anti-SIRPa antibody is not considered to significantly inhibit T cell proliferation if T cell proliferation is reduced by less than 30%, preferably less than 20%, more preferably less than 10%, most preferably less than by 5% compared to the negative control.

T cell proliferation can be determined in various ways. For example, T cell proliferation can be measured by incorporating H ³ -thymidine, as described in the examples of this application.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which does not significantly inhibit, antagonize, the binding of surfactant proteins to SIRPa, and which does not significantly compete with surfactant proteins for binding to SIRPa.

As used herein, surfactant proteins are collagen-containing Stipa lectins (calcium-dependent) that contribute significantly to the maintenance of surfactant homeostasis and lung immunity (for review, see Kishore et al., Surfactant proteins SP-A and SP-D: structure, function and receptors, Mol Immunol, 43(9), 1293-315, 2006).

- 9 044391

As used herein, the term surfactant protein refers to a surfactant protein from a mammalian species, preferably a human surfactant protein.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, as defined above, that does not inhibit the binding of human surfactant protein D (SP-D) to SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, as defined above, that does not inhibit the binding of human surfactant protein A (SP-A) to SIRPa.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above that does not antagonize the interaction of surfactant proteins with SIRPa.

Competition between SIRPa and surfactant proteins can be determined using a quantitative competitive binding assay using methods well known to those skilled in the art. Suitable methods include, but are not limited to, Biacore testing, BLItz™ testing, and quantitative ELISA testing.

According to the present invention, an antibody (or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic) is not considered to antagonize surfactant protein binding to SIRPa if said antibody (or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic) does not induce an increase, or induces an increase <1 log _KD value of surfactant protein in a quantitative SIRPa competitive binding assay using the BLItz™ system.

In one embodiment, the present invention provides an anti-SIRPb antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, that binds weakly or does not specifically bind to SIRPb.

As used herein, the term SIRPb refers to a protein SIRPb (also called SIRPe, signal regulatory protein beta 1, SIRP beta 1, a member of the CD172 antigen-like family B or CD172b) from a mammalian species, preferably human SIRPb.

The reference sequence of the human SIRPb protein used in the examples of this application corresponds to the sequence associated with accession number Q5TFQ8-1.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which has a KD value greater than 10 ^-9 M, preferably greater than 10 ^-8 M for SIRPb, particularly as determined by BLItz™ systems.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2 containing or consisting of the amino acid sequence presented in SEQ ID NO: 15 (NIDPSDSDTHYNQKFKD) or SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

HCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 17 (GGTGTMAWFAY), SEQ ID NO: 18 (GGTGTLAWFAY), SEQ ID NO: 19 (GGTGTMAYFAY) or SEQ ID NO: 20 (GGTGTLAYFAY),

LCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of the amino acid sequence set forth in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

18D5/option A/option B.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

- 10 044391

HCDR2, containing or consisting of the amino acid sequence presented in

SEQ ID NO: 15 (NIDPSDSDTHYNQKFKD),

HCDR3, containing or consisting of the amino acid sequence presented in

SEQ ID NO: 17 (GGTGTMAWFAY),

LCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of the amino acid sequence set forth in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

Option C.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2, containing or consisting of the amino acid sequence presented in SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

HCDR3, containing or consisting of the amino acid sequence presented in SEQ ID NO: 17 (GGTGTMAWFAY),

LCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of the amino acid sequence set forth in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

Option E.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2, containing or consisting of the amino acid sequence presented in SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

HCDR3, containing or consisting of the amino acid sequence presented in SEQ ID NO: 18 (GGTGTLAWFAY),

LCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of the amino acid sequence set forth in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

Option F

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2, containing or consisting of the amino acid sequence presented in SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

HCDR3, containing or consisting of the amino acid sequence presented in SEQ ID NO: 19 (GGTGTMAYFAY),

LCDR1, containing or consisting of the amino acid sequence presented in

- 11 044391

SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2, containing or consisting of the amino acid sequence presented in

SEQ ID NO: 22 (RVSNRFS), and

LCDR3, containing or consisting of the amino acid sequence presented in

SEQ ID NO: 23 (FQGTHVPYT).

EF option.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2, containing or consisting of the amino acid sequence presented in SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

HCDR3, containing or consisting of the amino acid sequence presented in SEQ ID NO: 20 (GGTGTLAYFAY),

LCDR1 containing or consisting of the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of the amino acid sequence set forth in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

The anti-SIRPa antibodies of the present invention may comprise a heavy chain variable region containing the amino acid sequence of HCDR1 and/or HCDR2 and/or HCDR3 from the human antibodies of the present invention; and/or a light chain variable region containing the amino acid sequence of LCDR1 and/or LCDR2 and/or LCDR3 from human antibodies according to the present invention.

According to one embodiment of the present invention, the antibody comprises an amino acid sequence variant of one or more CDRs of the subject human antibodies, wherein said variant comprises one or more amino acid insertions at or adjacent to a CDR residue and/or deletions at or adjacent to a CDR residue, and/or substitutions CDR residue(s) (with substitution(s) being the preferred type of amino acid change to produce said variants).

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody as defined above, which comprises a heavy chain variable domain comprising or consisting of an amino acid sequence selected from the sequences set forth in SEQ ID NO: 24. SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, and/or a light chain variable domain containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody as defined above, which comprises a heavy chain variable domain comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, and a light chain variable domain containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33.

The sequences of specific variable domains are given in table. 1 below.

- 12 044391

Table 1

Examples of heavy chain variable domains and light chain variable domains of antibodies according to the invention

Heavy chain variable domain wild antibodies type (chimeric antibody and mouse antibody 18D5) Variable domain QVQLQQPGAELVRPGSSVKLSCKASGYTFTS YWVHWVKQRPIQGLEWIGNIDPSDSDTHYN QKFKDK ASLT VDKS S ST AYMQLS SLTFED S A VYYCVRGGTGTMAWFAYWGQGTLVTVSA EVQLVQSGAEVKKPGESLRISCKASGYTFTS SEQ ID NO: 24 SEQ ID NO: heavy chain humanized version (HA) YWVHWVRQMPGKGLEWIGNIDPSDSDTHY NQKFKDHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAWFAYWGQGTLVTVS S 25 Heavy chain variable domain humanized version (HV) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YNQKFKDHVTLSVDKSISTAYLQLSSLKASD TAMYYCVRGGTGTMAWFAYWGQGTLVTV ss SEQ ID NO: 26 Heavy chain variable domain humanized version (HC) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAWFAYWGQGTLVTVS S SEQ ID NO: 27 Heavy chain variable domain humanized version (NOT) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AM YYC VRGGTGTL AWF A YWGQGTL VT VS S SEQ ID NO: 28 Heavy chain variable domain humanized version (NOT) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAYFAYWGQGTLVTVSS SEQ ID NO: 29 Heavy chain variable domain humanized version (HEE) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTLAYFAYWGQGTLVTVSS SEQ ID NO: thirty Wild type antibody light chain (chimeric antibody and mouse antibody 18D5) DVVMTQTPLSLPVSLGDQASISCRSSQSLVH SYGNTYLYWYLQKPGQSPKLLIYRVSNRFS GVPDRF SSGSGSGTDFTLKISRVEAEDLGVYF CFQGTHVPYTFGSGTKLEIK SEQ ID NO: 31 Light chain variable domain DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS YGNT YLYW YQQRPGQ SPRLLIYRVSNRF SG SEQ ID NO: 32

- 13 044391

humanized variant A (LA) VPDRF SGSGSSGTDFTLKISRVEAED VGVYFC FQGTHVPYTFGGGTKVEIK Light chain variable domain humanized version (LB) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS YGNTYLYWFQQRPGQSPRLLIYRVSNRF SG VPDRF SSGSGSGTDFTLKISRVEAED VGVYYC FQGTHVPYTFGGGTKVEIK SEQ ID NO: 33

The sequences of the variable domains of the antibodies exemplified in the present invention can be determined from the combinations of sequences presented in table. 2.

table 2

Heavy chain variable domain and light chain variable domain of specific antibodies according to the invention

Antibody Variable domain heavy chain Light chain variable domain 18D5 (chimeric antibody and mouse antibody) SEQ ID NO: 24 SEQ ID NO: 31 HALA SEQ Ш NO: 25 SEQ Ш NO: 32 NAEV SEQ ID NO: 25 SEQ ID NO: 33 HBLA SEQ Ш NO: 26 SEQ Ш NO: 32 HBLB SEQ ID NO: 26 SEQ ID NO: 33 HCLA SEQ Ш NO: 27 SEQ Ш NO: 32 HCLB SEQ ID NO: 27 SEQ ID NO: 33 HELA SEQ Ш NO: 28 SEQ Ш NO: 32 HELB SEQ ID NO: 28 SEQ ID NO: 33 HFLA SEQ Ш NO: 29 SEQ Ш NO: 32 HFLB SEQ Ш NO: 29 SEQ Ш NO: 33 HEFLA SEQ ID NO: 30 SEQ ID NO: 32 HEFLB SEQ ID NO: 30 SEQ Ш NO: 33

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody as defined above, which comprises a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody as defined above, which comprises a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a heavy chain variable domain chains containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, preferably a heavy chain variable domain containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 30, more preferably a heavy chain variable domain containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 30,

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody as defined above, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable domain chains containing or consisting of the amino acid sequence set forth in SEQ ID NO: 31, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain containing or consisting of the amino acid sequence - 14 044391 of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 26, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a variable domain heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 26, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 28 and a light chain variable domain, containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable domain containing or consisting of the amino acid sequence set forth in in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence presented in SEQ ID NO: 29, and a light chain variable domain containing or consisting of the amino acid sequence presented in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 30, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33.

In one embodiment of the present invention, the antibody or an antigen binding fragment thereof, or an antigen binding antibody mimetic, does not contain a W amino acid substitution at position 33 (W33) in the heavy chain variable domain, which position is identified with respect to the sequences set forth in SEQ ID NO: 24. SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30, and/or does not contain a substitution of amino acid Y at position 39 (Y39) , R at position 55 (R55) and/or F at position 60 (F60) in the light chain variable domain, said positions being identified with respect to the sequences set forth in SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO :33, in particular, does not contain a substitution at position W33 in the heavy chain variable domain and does not contain a substitution at positions Y39, R55 and F60 in the light chain variable domain.

According to the present invention, antibodies can be prepared with any heavy chain and light chain constant domains.

In one embodiment of the present invention, the anti-human SIRPa antibody of the present invention is a humanized monoclonal antibody, particularly wherein the light chain constant domain of the antibody is derived from the human kappa light chain constant domain, more particularly wherein the light chain constant domain consists of sequence set forth in SEQ ID NO: 35, and in which the constant domain of the antibody heavy chain

- 15 044391 is derived from the heavy chain constant domain of an IgG1, IgG2, IgG3 or IgG4 (wild type or mutated variant), in particular from the human IgG4 heavy chain constant domain, more particularly wherein the heavy chain constant domain of an antibody consists of the sequence represented by V

SEQ ID NO: 34.

As will be known to one skilled in the art, the selection of heavy chain constant region IgG isotypes focuses on whether specific functions are required and whether a suitable half-life is required under in vivo conditions. For example, antibodies engineered to selectively eradicate cancer cells typically require an active isotype that allows activation of the complement system and effector cell-mediated cell killing through antibody-dependent cellular cytotoxicity. Both the human IgG1 and IgG3 isotypes (which have a shorter half-life) meet these criteria, particularly the human IgG1 isotype (wild type and variants). In particular, depending on the heavy chain constant domain IgG isotype (particularly wild type and human IgG1 isotype variants), the anti-human SIRPa antibody of the present invention may be cytotoxic to cells expressing SIRPa through the CDC, ADCC and/or mechanisms. or ADF (Salfeld, nature biotechnology, vol. 25, no. 12, 2007; Irani et al. Molecular Immunology, vol. 67, issue 2, part A, 2015). In fact, the crystallizing fragment of immunoglobulin (Fc) interacts with a number of accessory molecules to mediate indirect effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADC), and complement-dependent cytotoxicity (CDC).

Table 3

Examples of Heavy Chain Constant Domain and Light Chain Constant Domain Suitable for Antibodies of the Invention

Constant domain heavy chains (IgG4m- S228P) SEQ ID NO: 34 PREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSPGK Constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK light chain domain (CL kappa) SEQIDNO: 35 VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLS SP VTKSFNRGEC

In one embodiment, the present invention provides an anti-SIRPa antibody as defined above, which comprises a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 56 and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 56 NO: 57, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 36, and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 43, or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 44, or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 37, and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 45, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 38 and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 44, or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 38, and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 45, or a heavy chain a chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 39 and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 44 or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 45, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 40, and a light chain containing or consisting of of the amino acid sequence set forth in SEQ ID NO: 44, or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 40, and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 45, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 41 and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 44, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 41, and a light chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 45, or a heavy chain containing or consisting of the amino acid sequence presented in SEQ ID NO: 42 and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 44, or a heavy chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 42, and a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 45.

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Table 4

Heavy chain and light chain sequences of specific antibodies according to the invention

Heavy chain wild antibodies type (mouse antibody 18D5) T MSSSVTVPSSTWPSQTVTCSVAHPASSTTV DKKLEPSGPISTINPCPPCKECHKCPA PNLEGGPSVFIFPPNIKDVLMISLTPKVTCVV VDVSEDDPDVQISWFVNNVEVHTAQTQTHR EDYNSTIRVVSTLPIQHQDWMSGKEFKCKV NNKDLPSPIERTISKIKGLVRAPQVYILPPPAE QLSRKDVSLTC LVVGFNPGDISVEWTSNGHT EENYKDTAPVLDSDGSYFIYSKLNMKTSKW EKTDSFSCNVRHEGLKNYYLKKTISRSPGK SEQ ID NO: 56 Heavy chain wild antibodies type (chimeric antibody 18D5) QVQLQQPGAELVRPGSSVKLSCKASGYTFTS YWVHWVKQRPIQGLEWIGNIDPSDSDTHYN QKFKDK ASLT VDKS S ST AYMQLS SLTFED SA VYYCVRGGTGTMAWFAYWGQGTLVTVSA ASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSG LYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEV SEQ ID NO: 36

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K Heavy chain humanized version (HA) EVQLVQSGAEVKKPGESLRISCKASGYTFTS YWVHWVRQMPGKGLEWIGNIDPSDSDTHY NQKFKDHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAWFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSS SLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPC PAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 37 Heavy chain humanized version (HV) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YNQKFKDHVTLSVDKSISTAYLQLSSLKASD TAMYYCVRGGTGTMAWFAYWGQGTLVTV SSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSS SLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGV SEQ ID NO: 38

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EVHNAKTKPREEQFNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLS PGK Heavy chain humanized version (HC) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAWFAYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLG TCLV KGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 39 Heavy chain humanized version (NOT) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AM YYC VRGGTGTL AWF A YWGQGTL VT VS S ASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSV VTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEV SEQ ID NO: 40

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K Heavy chain humanized version (HF) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTMAYFAYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLG TCLV KGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 41 Heavy chain humanized option (HEF) EVQLVQSGAEVKKPGESLRISCKASGYSFTS YWVHWVRQMPGKGLEWMGNIDPSDSDTH YSPSFQGHVTLSVDKSISTAYLQLSSLKASDT AMYYCVRGGTGTLAYFAYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLG TKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEV SEQ ID NO: 42

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K Light chain wild antibodies type (mouse antibody 18D5) DVVMTQTPLSLPVSLGDQASISCRSSQSLVH SYGNTYLYWYLQKPGQSPKLLIYRVSNRFS GVPDRF SSGSGSGTDFTLKISRVEAEDLGVYF CFQGTHVPYTFGSGTKLEIKRADAAPTVSIFP R S SEQLT SGGAS VVCFLNNF YPKDINVKWKI DGSERQNGVLNSWTDQDSK DSTYSMSSTLT LTKDEYERHNSYTCEATHKTSSTSPIVKSFNR NEC SEQ ID NO: 57 Light chain wild antibodies type (chimeric antibody 18D5) DVVMTQTPLSLPVSLGDQASISCRSSQSLVH SYGNTYLYWYLQKPGQSPKLLIYRVSNRFS GVPDRF SSGSGSGTDFTLKISRVEAEDLGVYF CFQGTHVPYTFGSGTKLEIKRT V A AP S VFI FPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDS KD STYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC SEQ ID NO: 43 Light chain variable domain humanized variant A (LA) DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS YGNT YLYW YQQRPGQ SPRLLIYRVSNRF SG VPDRF SSGSGSGTDFTLKISRVEAED VGVYFC FQGTHVP YTFGGGTKVEIKRT V A AP S VF IF PPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQ ID NO: 44 Light chain variable domain humanized DVVMTQSPLSLPVTLGQPASISCRSSQSLVHS YGNTYLYWFQQRPGQSPRLLIYRVSNRFSG VPDRF SGSSGSGTDFTLKISRVEAED VGVYYC SEQ ID NO: 45 option (LB) FQGTHVP YTFGGGTKVEIKRT V A AP S VF IF PPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is capable of inducing the differentiation of monocytic myeloid-derived suppressor cells (Mo-MDSC) into non-suppressor cells.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, defined

- 22 044391 above, which is characterized in that said non-suppressor cells secrete pro-inflammatory cytokines such as IL-6, IL-12 and TNF, and do not secrete or secrete at low levels anti-inflammatory cytokines such as IL-10 and FRO-β.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is characterized in that said non-suppressor cells express iNOS.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is characterized in that said non-suppressor cells do not express MHC class II markers and express CD80-CD86 markers.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, which is characterized in that said non-suppressor cells express at least one natural killer cell (NK) cell marker.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, wherein the antibody or antigen-binding fragment thereof is capable of inhibiting the polarization of macrophages into the M2 phenotype and/or promoting the polarization of pro-inflammatory macrophages into the M1 phenotype.

Antibodies of the present invention can modify macrophage polarization to induce a pro-inflammatory environment, i.e. they may inhibit the anti-inflammatory signal provided by M2 macrophages and/or may promote the pro-inflammatory signal provided by M1 macrophages. This approach allows the restoration of an inflammatory environment favorable for the action of effector T cells, in particular for the elimination of cancer cells.

Antibodies to SIRPa

The embodiments mentioned for the antibodies defined above are repeated, with necessary modifications, for the antibodies mentioned in other aspects of the present invention.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 70 (ELIYNQKEGHFPR), SEQ ID NO: 71 (RNNMDFSIRIGn) and SEQ ID NO: 72 (SPRDITLKW), within SIRPa.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope containing or consisting of peptides containing the sequence set forth in SEQ ID NO: 70 (ELIYNQKEGHFPR), SEQ ID NO : 71 (RNNMDFSIRIGN) and SEQ ID NO: 72 (SPRDITLKW), within SIRPa.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of SEQ ID NO: 70 (ELIYNQKEGHFPR) and SEQ ID NO: 71 (RNNMDFSIRIGN).

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope containing or consisting of peptides containing the sequence set forth in SEQ ID NO: 70 (ELIYNQKEGHFPR) and SEQ ID NO : 71 (RNNMDFSIRIGN), within SIRPa.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope comprising at least one peptide selected from the group consisting of the sequences SEQ ID NO: 73 (YNQK) and SIR.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that specifically binds to a conformational epitope containing or consisting of peptides containing the sequence SEQ ID NO: 73 (YNQK) and SIR within SIRPa.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody, which comprises:

a) a heavy chain containing HCDR1, HCDR2 and HCDR3, and/or

b) a light chain comprising LCDR1, LCDR2 and LCDR3, said CDRs being defined as follows:

HCDR1 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 14 (SYWVH),

HCDR2 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 15 (NroPSDSDTHYNQKFKD) or SEQ ID NO: 16 (NIDPSDSDTHYSPSFQG),

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HCDR3 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 17 (GGTGTMAWFAY), SEQ ID NO: 18 (GGTGTLAWFAY), SEQ

ID NO: 19 (GGTGTMAYFAY) or SEQ ID NO: 20 (GGTGTLAYFAY),

LCDR1 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 21 (RSSQSLVHSYGNTYLY),

LCDR2 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 22 (RVSNRFS), and

LCDR3 containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 23 (FQGTHVPYT).

The present invention also provides an anti-SIRPg antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg and/or that does not bind specifically to T cells and/or that does not inhibit proliferation of T cells, and/or does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa, and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit T cell proliferation, and which does not inhibit the binding of CD47 to SIRPg.

Application options

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg , and/or which does not specifically bind to T cells, and/or which does not inhibit the proliferation of T cells, and/or which does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, for use as a drug.

The present invention also provides a method of treating a subject in need thereof, comprising administering to said subject an effective amount of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic, which inhibits CD47 binding to SIRPa and which does not bind specifically to SIRPg, and/or which does not bind specifically to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg.

The present invention also relates to the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or which does not inhibit the proliferation of T cells, and/or which does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, in the manufacture of a medicament.

In another aspect, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg , and/or which does not specifically bind to T cells, and/or which does not inhibit T cell proliferation, and/or which does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg , and which does not specifically bind to T cells, and which does not inhibit T cell proliferation, and which does not inhibit the binding of CD47 to SIRPg, for use in the treatment of any condition that can be improved or prevented by the differentiation of monocytic suppressor cells of myeloid origin (MoMDSC) into non-suppressor cells.

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As used herein, a condition that can be improved or prevented by the differentiation of monocytic myeloid-derived suppressor cells (Mo-MDSCs) into non-suppressor cells corresponds to cancers, including inflammatory cancers and cancers with infiltrated myeloid cells (particularly with infiltrated MDSCs and/or or TAM), infectious disease, trauma, autoimmune disease (such as rheumatoid arthritis, type 1 diabetes, lupus, psoriasis), vaccination, chronic inflammatory diseases (such as inflammatory bowel disease, including Crohn's disease and ulcerative colitis), septic shock, chronic infectious disease (such as disease caused by Pseudomonas or CMV), fibrosis, atherosclerosis or graft dysfunction.

The present invention also provides a method of treating any condition that can be ameliorated or prevented by differentiating myeloid-derived monocyte-derived suppressor cells (Mo-MDSCs) into non-suppressor cells in a subject in need thereof, comprising administering to said subject an effective amount of an anti-SIRPa antibody or an antigen binding fragment, or an antigen binding mimetic antibody as defined above, or an anti-SIRPg antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg, and/or that does not bind specifically to T cells and/or which does not inhibit the proliferation of T cells, and/or which does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit T cell proliferation, and which does not inhibit CD47 binding to SIRPg.

The present invention also relates to the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or which does not inhibit the proliferation of T cells, and/or which does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, in the manufacture of a medicament for the treatment of any condition that can be improved or prevented by the differentiation of monocytic suppressor cells of myeloid origin (Mo-MDSC) into non-suppressor cells.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit T cell proliferation, and which does not inhibit CD47 binding to SIRPg, for use in the treatment of any condition that can be improved or prevented by modifying the polarization of macrophages into pro-inflammatory macrophages .

In fact, SIRPa acts as a checkpoint inhibitor and is involved in macrophage polarization. Specifically, blocking SIRPa induces the proinflammatory macrophage function associated with type 1 macrophages (proinflammatory macrophages type M1=M (IFN-γ)), and inhibits the suppressive activity of macrophages in the tumor, since the proinflammatory profile of macrophages is derived from type 2 macrophages (highly phagocytic). activity of macrophages type M2 = M (IL-4)). Accordingly, a SIRPa antagonist is capable of inhibiting the polarization of macrophages into the M2 phenotype and/or promoting the proinflammatory function of M1 macrophages and can be used in therapy.

As defined herein, a condition that can be improved or prevented by modifying macrophage polarization to preferentially produce pro-inflammatory macrophages corresponds to, for example, solid cancer, hematologic malignancy, infectious disease, trauma, autoimmune disease, vaccination, brain injury, nerve injury, polycythemia, hemochromatosis or chronic inflammatory disease.

The present invention also provides a method of treating any condition that can be improved or prevented by modifying the polarization of macrophages into pro-inflammatory macrophages in a subject in need thereof, comprising administering to said subject an effective amount

- 25 044391 an antibody to SIRPa or an antigen-binding fragment thereof, or an antigen-binding mimetic of an antibody defined above, or an antibody to SIRPa or an antigen-binding fragment thereof, or an antigen-binding mimetic of an antibody that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg, and/or which does not bind specifically to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not binds specifically to T cells, and which does not inhibit T cell proliferation, and which does not inhibit CD47 binding to SIRPg.

In one embodiment, the present invention also provides the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody that inhibits the binding of CD47 to SIRPa and that does not bind specifically to SIRPg, and/or which does not bind specifically to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits CD47 binding to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit T cell proliferation, and which does not inhibit CD47 binding to SIRPg, in the manufacture of a medicament for the treatment of any condition that can be improved or prevented by polarization modification macrophages into pro-inflammatory macrophages.

Modification of macrophage polarization to preferentially produce proinflammatory cells may be beneficial in a number of pathologies or situations. As described above, such a modification is particularly useful in various cancers to restore the antitumor activity of macrophages and/or to prevent the protumor activity of M2 macrophages. Nonphysiological immune responses due to excessive polarization of M2 macrophages also occur in infectious diseases, fibrosis, vaccination, trauma, and chronic inflammatory diseases.

Accordingly, in a specific embodiment, an anti-SIRPa antibody can be used to treat an individual who has a cancer selected from the group consisting of lung cancer, mesotheliomas, ovarian cancer, liver cancer, bladder cancer, brain cancer, breast cancer, types of colon cancer, sarcomas, types of pancreatic cancer, types of head and neck cancer, types of kidney cancer, thymomas, gliomas, melanomas and hematological cancers such as lymphomas (Hodgkin's lymphoma and non-Hodgkin's lymphoma), leukemias, such such as T- and B-cell acute or chronic lymphoblastic leukemia (ALL or CLL) or acute or chronic myeloid leukemia (AML or CML) and myeloma. In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, for use in the treatment of pathologies selected from the group consisting of cancer (in particular, inflammatory cancers and cancers with infiltrated myeloid cells, particularly with infiltrated MDSCs and/or TAMs), infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease (eg, Pseudomonas or CMV disease), fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction.

In one embodiment, the present invention relates to a method for treating a pathology selected from the group consisting of cancer (particularly inflammatory cancers and myeloid cell infiltrated cancers, particularly MDSC and/or TAM infiltrated cancers), infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain damage, nerve damage, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease (eg, Pseudomonas or CMV disease), fibrosis, atherosclerosis, obesity,

- 26 044391 type II diabetes mellitus and graft dysfunction in a subject in need thereof, comprising administering to said subject an effective amount of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic of an antibody defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding an antibody mimetic that inhibits CD47 binding to SIRPa and that does not specifically bind to SIRPg, and/or that does not specifically bind to T cells, and/or that does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg.

In one embodiment, the present invention provides the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody that inhibits the binding of CD47 to SIRPa and that does not specifically bind with SIRPg, and/or which does not specifically bind to T cells, and/or which does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits CD47 binding to SIRPa and which does not bind specifically with SIRPg, and which does not bind specifically to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, in the manufacture of a medicament for the treatment of a pathology selected from the group consisting of cancer (in particular, types inflammatory cancers and cancers with infiltrated myeloid cells, particularly infiltrated MDSCs and/or TAMs), infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock , chronic infectious disease (eg, disease caused by Pseudomonas or CMV), fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction.

In one embodiment, the present invention provides an anti-human SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, for the uses defined above, wherein said anti-human SIRP antibody or antigen-binding fragment thereof, or an antigen-binding antibody mimetic according to the present invention administered to a patient with symptoms of a SIRPa-positive tumor.

In one embodiment, the present invention provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits CD47 binding to SIRPa and which does not bind specifically to SIRPg , and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, for use in vaccination.

In one embodiment, the present invention provides a method of vaccinating a subject comprising administering to said subject an effective amount of an anti-SIRPa antibody or antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and/or which does not bind specifically to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits binding CD47 with SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit T cell proliferation, and which does not inhibit the binding of CD47 to SIRPg.

In one embodiment, the present invention provides the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody that inhibits the binding of CD47 to SIRPa and that does not specifically bind with SIRPg, and/or which does not specifically bind to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg,

- 27 044391 in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, for the manufacture vaccines.

Suppressor myeloid cells limit the effectiveness of vaccination, especially in young children. Therefore, anti-SIRPa/g agents will limit the benefit that anti-SIRPa agents provide in developing a vaccine response by preventing T cell responses to vaccination.

The antibody or antigen-binding fragment thereof of the present invention can be administered to a subject by various routes, for example, intravenously (IV), subcutaneously (SC), or intramuscularly (IM).

The antibody or antigen binding fragment thereof can be administered alone or in combination with another therapeutic agent, for example, a second human monoclonal antibody or antigen binding fragment thereof. In another example, the antibody is administered together with another agent, for example, an immunosuppressive agent, an erythropoiesis-stimulating agent (ESA), in combination with therapeutic cell compositions, and the like.

In one embodiment, the present invention provides an anti-SIRPa antibody or antigen-binding fragment thereof, or an antigen-binding antibody mimetic, for use as defined above, wherein said anti-SIRPa antibody or antigen-binding fragment is combined with a second therapeutic agent.

In particular, the anti-SIRPa antibodies of the present invention can be combined with several other potential strategies to overcome tumor immune evasion mechanisms using agents in clinical development or already marketed (see Table 1 from Antonia et al. Immuno -oncology combinations: a review of clinical experience and future prospects. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 20, 6258-6268, 2014):

1) reversing the inhibition of adaptive immunity (blocking T cell checkpoint pathways), for example, using an anti-CTLA4, anti-PD1, or anti-PD-L1 molecule;

2) switching adaptive immunity (enhancing T cell co-stimulatory receptor signaling using agonist molecules, particularly antibodies), for example, by targeting CD137 (4-1BB) using agonist molecules, including agonistic antibodies to CD137 (anti -4-1BB) or CD137 ligands (4-1BB);

3) improving the function of innate immune cells;

4) activation of the immune system (increasing the effector function of immune cells), for example, through vaccine-based strategies.

Administration of the second therapeutic agent may be simultaneous with the administration of the anti-SIRPa antibody or may not be simultaneous. Depending on the nature of the second agent, coadministration may be achieved using a combination drug product (product), also known as a combo. A combo is a fixed-dose combination that includes two or more active pharmaceutical ingredients combined into a single unit dosage form that is manufactured and distributed in fixed doses. However, the dosage schedule and/or route of administration may also vary.

According to a preferred embodiment of the present invention, said second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, cell therapy agents (such as CAR T cells), antibiotics and probiotics.

In particular, immunotherapeutic agents that can be used in accordance with the present invention are selected from the group consisting of therapeutic vaccines (DNA, RNA or peptide vaccines), blockers or immune checkpoint activators, in particular adaptive immune cells (T-cells). lymphocytes or B lymphocytes) or immunoconjugates such as antibody-drug conjugates.

As used herein, the term immunotherapeutic agents refers, in particular, to agents that can convert tumor vaccines exhibiting interesting biological phenomena into effective therapeutic agents, including: T cell growth factors to increase the number and repertoire of naïve T cells, growth factors to increase the number of dendritic cells (DCs), agonists to activate DCs and other antigen presenting cells (APCs), adjuvants that provide and complement cancer vaccines, agonists to activate and stimulate T cells, T cell checkpoint blockade inhibitors, T growth factors cells to enhance the proliferation and survival of immune T cells, agents to inhibit, block or neutralize cancer cells and immunosuppressive cytokines derived from immune cells.

There are many immune checkpoint blockers or activators known in the art. In connection with the present invention, examples of immune checkpoint blockers or activators of adaptive immune cells (B or T lymphocytes) that are suitable for use include agonists directed against PDL1, PD1, CTLA4, CD137, CD2, CD28, CD40 ,

HVEM, BTLA, CD160, TIGIT, TIM-1/3, LAG-3, 2B4, OX40 and CD40, CD40-L, TLR agonists, ICOS agonists, ICOS-L and B cell receptor agonists, in particular, anti-PDL1, anti-PD1 and anti-CD137 agonists.

The immunotherapeutic agent may also be an antibody targeting a tumor antigen, particularly selected from the group consisting of an anti-Her2 antibody, an anti-EGFR antibody, an anti-CD20 antibody, an anti-CD19 antibody, and an anti-CD52 antibody.

The antibody may be provided at an effective dose of from about 1 ng/kg body weight to about 30 mg/kg body weight or more. In a specific embodiment of the present invention, the dose may range from 1 μg/kg to about 20 mg/kg, optionally from 10 μg/kg to 10 mg/kg, or from 100 μg/kg to 5 mg/kg.

The term effective dose or effective dosage or effective amount is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term effective dose is intended to include an amount sufficient to cure or at least partially control a disease and its complications in a patient already suffering from the disease. Amounts or dosages effective for a given use will depend on the condition being treated, the antibody construct delivered, the therapeutic situation and objectives, the severity of the disease, prior therapy, the patient's medical history and response to the therapeutic agent, route of administration, size (body weight , body area or organ size) and/or condition (age and general health) of the patient, as well as the general condition of the patient's own immune system. The appropriate dose can be adjusted so that it can be administered to the patient once or over multiple administrations, and in order to obtain optimal therapeutic effect.

Dosing for these purposes may be repeated as needed, such as daily, twice weekly, weekly, twice monthly, monthly, or as needed during relapses.

In one aspect, the present invention also provides an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic that inhibits the binding of CD47 to SIRPa and that does not specifically bind to SIRPg, and/or which does not specifically bind to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit CD47 binding to SIRPg, in particular, which inhibits CD47 binding to SIRPa and which does not bind specifically to SIRPg , and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, for use in a diagnostic test, in particular in personalized medicine, more particularly in an auxiliary diagnostic test.

According to one embodiment, the present invention relates to a diagnostic method, in particular in personalized medicine, more particularly in an auxiliary diagnostic test, using an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic of an antibody defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof. fragment, or antigen-binding antibody mimetic, that inhibits CD47 binding to SIRPa and that does not specifically bind to SIRPg, and/or that does not specifically bind to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit CD47 binding with SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not bind specifically to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg,

In one embodiment, the present invention provides the use of an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody as defined above, or an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding mimetic antibody that inhibits the binding of CD47 to SIRPa and that does not specifically bind with SIRPg, and/or which does not specifically bind to T cells, and/or does not inhibit T cell proliferation, and/or does not inhibit the binding of CD47 to SIRPg, in particular, which inhibits the binding of CD47 to SIRPa and which does not bind specifically to SIRPg, and which does not specifically bind to T cells, and which does not inhibit the proliferation of T cells, and which does not inhibit the binding of CD47 to SIRPg, in the manufacture of a medicament for a diagnostic test, in particular in personalized medicine, more particularly in an auxiliary diagnostic test.

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According to one aspect, the present invention also relates to an in vitro or ex vivo diagnostic method, in particular to a diagnostic method suitable for use in personalized medicine, more particularly in assisted diagnostics, wherein said anti-SIRPa antibody or antigen-binding fragment thereof or its antigen-binding mimetic according to the present invention is used to detect SIRPa ⁺ cells in a sample previously taken from a subject, and optionally to quantify the expression of SIRPa.

According to one aspect, the present invention also relates to the use of an anti-SIRPa antibody or an antigen binding fragment thereof or an antigen binding mimetic according to the present invention in the manufacture of a medicament suitable for use in a diagnostic test, in particular for use in a personalized medicine, or in an adjuvant diagnostic test.

In one aspect, the present invention also provides at least one anti-human SIRPa antibody or antigen binding fragment thereof, or an antigen binding mimetic antibody of the present invention as a means for determining the expression and/or level of expression of SIRPa in a biological sample previously collected from an individual.

In one aspect, the present invention also provides a method, in vitro or ex vivo, for determining in a subject SIRPa-positive cells in a biological sample previously collected from said subject, comprising:

i) determining in vitro the expression and/or expression level of SIRPa in a biological sample previously collected from the subject using an anti-human SIRPa antibody or an antigen binding fragment thereof, or an antigen binding antibody mimetic according to the present invention.

According to one aspect, the present invention also relates to the use, particularly under in vitro or ex vivo conditions, of at least one anti-human SIRPa antibody or antigen binding fragment thereof, or an antigen binding mimetic antibody according to the present invention in a method in which SIRPa is used as a biomarker that is predictive of response to treatment in a subject, in particular in a subject suffering from cancer.

According to one aspect, the present invention also relates to a method, in vitro or ex vivo, for predicting the response of a subject suffering from cancer to treatment, in particular using an anti-human SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic according to the present invention, comprising determining the level of expression of SIRPa in a tumor sample previously obtained from a subject, in particular, when treated with an antibody to human SIRPa or an antigen binding fragment thereof, or an antigen binding mimetic antibody according to the present invention, and comparing the level of expression of SIRPa with a value representing the level of SIRPa expression in a population of non-responding subjects, wherein a higher level of SIRPa expression in the subject's tumor sample is representative of a subject that will respond to treatment.

In one aspect, the present invention also provides a method for determining, in vitro or ex vivo, the presence of SIRPa ⁺ cells in a sample previously collected from a subject, which includes determining the presence of SIRPa as a biomarker that is predictive of the subject's response to treatment, in particularly, a response from a subject diagnosed with cancer, said method comprising:

determining the level of expression of SIRPa in a tumor sample previously collected from a subject, particularly when treated with an anti-human SIRPa antibody or an antigen binding fragment thereof, or an antigen binding mimetic antibody, as defined in any one of claims 1 to 13, and comparing the level of expression of SIRPa with a value representing the level of SIRPa expression in a population of non-responder subjects, with a higher level of SIRPa expression in a subject's tumor sample indicating a patient who will respond to treatment.

Compositions

According to another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen binding fragment thereof as defined above and a pharmaceutically acceptable carrier.

As used herein, the term pharmaceutical composition includes a composition suitable for administration to a subject or patient, such as a mammal, especially a human. In general, the pharmaceutical composition is sterile and generally free of contaminants that are capable of causing an undesirable response in the subject (eg, the compound(s) in the pharmaceutical composition are of pharmaceutical grade). The pharmaceutical compositions may be intended for administration to subjects or patients in need thereof through a number of different routes of administration, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, and the like.

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As used herein, the term pharmaceutically acceptable carrier includes an excipient, diluent, carrier and adjuvant that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic and is neither biologically nor otherwise objectionable and includes an excipient, diluent, carrier and an adjuvant, which are acceptable for veterinary use as well as pharmaceutical use in humans. As used herein, a pharmaceutically acceptable carrier includes one or more of such excipient, diluent, carrier and adjuvant.

In particular, the present invention relates to a pharmaceutical composition which contains as an active ingredient an antibody or an antigen binding fragment thereof as defined above and a pharmaceutically acceptable carrier.

Combination product

According to another aspect, the present invention relates to a therapeutic agent, in particular to a combination product, which contains as active ingredients: an anti-SIRPa antibody or an antigen-binding fragment thereof, or an antigen-binding antibody mimetic as defined above, and a second therapeutic agent, wherein said active ingredients are formulated for monotherapy, sequential or combination therapy, in particular for combined or sequential use.

In particular, the present invention relates to a combination product containing an anti-SIRPa antibody or an antigen-binding fragment or an antigen-binding antibody mimetic thereof, as defined above, and a second therapeutic agent for simultaneous, separate or sequential administration of the drug.

In one embodiment, the present invention provides a combination product as defined above, wherein the second therapeutic agent is selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, cell therapy agents, immunotherapeutic agents, antibiotics, and probiotics.

In one embodiment, the present invention provides a combination product as defined above, wherein said immunotherapeutic agent is selected from the group consisting of therapeutic vaccines, immune checkpoint blockers or activators, in particular adaptive immune cells (T and B lymphocytes) and antibody-drug conjugates.

In one embodiment, the present invention provides a combination product as defined above, wherein said adaptive immune cell (T and B) immune checkpoint blocker or activator is selected from the group consisting of agonists directed against PDL1, PD1, CTLA4, CD137, CD2, CD28, CD40, HVEM, BTLA, CD160, TIGIT, TIM1/3, LAG-3, 2B4, OX40 and CD40, CD40-L, TLR agonists, ICOS-directed agonists, ICOS-L and agonists directed against B cell receptors, in particular selected from the group consisting of agonists directed against PDL1, against PD1 and against CD137.

According to one embodiment of the present invention, said immunotherapeutic agent is an antibody targeting a tumor antigen, particularly selected from the group consisting of an anti-Her2 antibody, an anti-EGFR antibody, an anti-CD20 antibody, an anti-CD19 antibody, an anti-CD52 antibody.

In one aspect, the present invention provides a combination product as defined above for simultaneous, separate or sequential use in the treatment of any condition that can be improved or prevented by differentiating monocytic myeloid-derived suppressor cells (Mo-MDSC) into non-suppressor cells.

In one embodiment, the present invention provides a method of treating any condition that can be improved or prevented by differentiating myeloid-derived monocyte-derived suppressor cells (Mo-MDSCs) into non-suppressor cells in a subject in need thereof, comprising simultaneous, separate or sequential administration to said subject an effective amount of the combination product as defined above.

In one embodiment, the invention relates to the use of a combination product as defined above in the manufacture of a medicament for the treatment of any condition that can be improved or prevented by differentiating monocytic myeloid-derived suppressor cells (Mo-MDSC) into non-suppressor cells.

In one aspect, the present invention provides a combination product as defined above for simultaneous, separate or sequential use in the treatment of any condition that can be improved or prevented by modifying the polarization of macrophages into pro-inflammatory macrophages.

In one embodiment, the present invention provides a method of treating any condition that can be ameliorated or prevented by modifying macrophage polarization to proinflammatory macrophages in a subject in need thereof, comprising simultaneous, separate, or sequential administration to said subject of an effective amount of a combination product as defined above.

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In one embodiment, the present invention relates to the use of a combination product as defined above in the manufacture of a medicament for the treatment of any condition that can be improved or prevented by modifying the polarization of macrophages into pro-inflammatory macrophages.

In one aspect, the present invention provides a combination product as defined above for simultaneous, separate or sequential use in the treatment of a pathology selected from the group consisting of cancer, infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerves, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease (eg, disease caused by Pseudomonas or CMV), fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction, or for use in vaccination.

In one embodiment, the present invention provides a method for treating a condition selected from the group consisting of cancer, infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease disease (eg, Pseudomonas or CMV disease), fibrosis, atherosclerosis, obesity, type II diabetes mellitus, and graft dysfunction in a subject in need thereof, comprising simultaneous, separate, or sequential administration to said subject of an effective amount of the combination product defined above.

In one embodiment, the present invention relates to the use of a combination product as defined above in the manufacture of a medicament for the treatment of a condition selected from the group consisting of cancer, infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerve injury , polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease (eg, disease caused by Pseudomonas or CMV), fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction, or for use in vaccination.

Nucleic acids

According to another aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody or antigen binding fragment thereof as defined above.

As used herein, the nucleic acid molecule may be double-stranded or single-stranded, linear or circular. The nucleic acid molecule is preferably included in a vector, which is preferably contained in a host cell.

In one embodiment, the present invention provides an isolated nucleic acid molecule encoding an antibody or antigen binding fragment thereof as defined above, wherein said nucleic acid molecule contains or consists of at least one sequence selected from SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69.

In one embodiment, the present invention provides an isolated nucleic acid molecule encoding an antibody or an antigen binding fragment thereof as defined above, wherein said nucleic acid molecule comprises a sequence encoding a heavy chain variable domain of said antibody, preferably comprising

SEQ ID NO: 58, SEQ ID NO: 59 or SEQ ID NO: 60,

SEQ ID NO: 61 or SEQ ID NO: 62 and

SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66, and/or a sequence encoding a light chain variable domain of said antibody, preferably comprising

SEQ ID NO: 67

SEQ ID NO: 68, and

SEQ ID NO: 69.

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Table 5

Sequences encoding the heavy chain variable domain CDRs and the light chain variable domain CDRs of antibodies according to the invention

Nucleic acid sequences encoding HCDR1 (corresponding to the amino acid sequence SEQ GO NO: 14) AGCTATTGGGTGCAC TCTTATTGGGTGCAC TCCTATTGGGTGCAC SEQ GO NO: 58 SEQ GO NO: 59 SEQ GO NO:

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60 Nucleic acid sequence encoding HCDR2 (corresponding to amino acid sequence SEQ III NO: 15) AACATCGACCCCAGCGACTC TGATACCCATTACAATCAGA AGTTTAAGGAC SEQ ID NO: 61 Nucleic acid sequence encoding HCDR2 (corresponding to amino acid sequence SEQ III NO: 16) AACATCGACCCCAGCGACTC TGATACACACTACTCCCCTA GCTTTCAGGGC SEQ ID NO: 62 Nucleic acid sequence encoding HCDR3 (corresponding to amino acid sequence SEQ III NO: 17) GGAGGAACCGGAACAATGG CTTGGTTTGCTTAC SEQ ID NO: 63 Nucleic acid sequence encoding HCDR3 (corresponding to amino acid sequence SEQ III NO: 18) GGAGGAACCGGCACACTGG CTTGGTTCGCTTAC SEQ ID NO: 64 Nucleic acid sequence encoding HCDR3 (corresponding to amino acid sequence SEQ III NO: 19) GGAGGAACCGGAACAATGG CTTACTTCGCTTAT SEQ ID NO: 65 Nucleic acid sequence encoding HCDR3 (corresponding to amino acid sequence SEQ III NO: 20) GGAGGAACCGGCACACTGG CTTACTTCGCTTAT SEQ ID NO: 66 Nucleic acid sequence encoding LCDR1 (corresponding to amino acid sequence SEQ III NO: 21) AGGTCCAGCCAGTCCCTGGT GCACAGCTATGGCAACACAT ACCTGTAT SEQ ID NO: 67 Nucleic acid sequence encoding LCDR2 (corresponding to amino acid sequence SEQ III NO: 22) AGGGTGTCTAATCGGTTCTC C SEQ ID NO: 68 Nucleic acid sequence TTTCAGGGCACCCATGTGCC SEQ ID NO:

acids encoding LCDR3 (corresponding to amino acid sequence SEQ ID NO: 23) ATACACA 69

In particular, the present invention relates to a nucleic acid molecule containing or consisting of a sequence selected from SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55.

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Table 6

Sequences encoding heavy chain variable domains and light chain variable domains of antibodies according to the invention

Nucleic acid sequence encoding the heavy chain variable domain wild antibodies type (chimeric antibody and mouse antibody 18D5) CAGGTGCAGCTGCAGCAGCCAGGAGCTGA GCTGGTGAGGCCTGGCTCCAGCGTGAAGC TGTCCTGCAAGGCTAGCGGCTACACCTTCA CAAGCTATTGGGTGCACTGGGTGAAGCAG CGGCCAATCCAGGGCCTGGAGTGGATCGG CAACATCGACCCCAGCGACTCTGATACCC ATTACAATCAGAAGTTTAAGGACAAGGCC TCTCTGACCGTGGATAAGTCTTCCAGCACA GCTTATATGCAGCTGTCTTCCCTGACATTC GAGGATTCCGCCGTGTACTATTGCGTGAG GGGAGGAACCGGAACAATGGCTTGGTTTG CTTACTGGGGCCAGGGCACCCTGGTGACA GTGTCTGCT SEQ ID NO: 46 Nucleic acid sequence encoding the heavy chain variable domain humanized version (HA) GAGGTGCAGCTGGTGCAGAGCGGAGCTGA GGTGAAGAAGCCAGGCGAGTCTCTGAGGA TCTCCTGCAAGGCTAGCGGCTACACCTTCA CATCTTATTGGGTGCACTGGGTGCGGCAG ATGCCAGGCAAGGGCCTGGAGTGGATCGG CAACATCGACCTAGCGACTCTGATACCC ACTACAATCAGAAGTTTAAGGACCATGTG ACCCTGTCTGTGGATAAGTCCATCAGCAC AGCCTATCTGCAGCTGTCCAGCCTGAAGG CCTCCGATACAGCTATGTACTATTGCGTGA GGGGAGGAACCGGAACAATGGCTTGGTTC SEQ ID NO: 47

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GCTTACTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC Nucleic acid sequence encoding the heavy chain variable domain humanized version (HV) GAGGTGCAGCTGGTGCAGTCCGGAGCTGA GGTGAAGAAGCCAGGCGAGTCTCTGAGGA TCTCCTGCAAGGCTTCTGGCTACTCCTTCA CCAGCTATTGGGTGCACTGGGTGCGGCAG ATGCCAGGCAAGGGCCTGGAGTGGATGGG CAACATCGACCTAGCGACTCTGATACAC ACTACAATCAGAAGTTTAAGGACCATGTG ACCCTGAGCGTGGATAAGTCCATCAGCAC AGCCTATCTGCAGCTGTCCAGCCTGAAGG CCTCTGATACCGCTATGTACTATTGCGTGA GGGGAGGAACCGGAACAATGGCTTGGTTC GCTTACTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC SEQ ID NO: 48 Nucleic acid sequence encoding the heavy chain variable domain humanized version (HC) GAGGTGCAGCTGGTGCAGTCTGGCGCCGA GGTGAAGAAGCCAGGCGAGAGCCTGAGG ATCTCTTGCAAGGCTAGCGGCTACTCTTTC ACCTCCTATTGGGTGCACTGGGTGCGGCA GATGCCAGGCAAGGGCCTGGAGTGGATGG GCAACATCGACCCCAGCGACTCTGATACA CACTACTCCCCTAGCTTTCAGGGCCATGTG ACCCTGTCCGTGGACAAGTCTATCTCCA CA GCCTATCTGCAGCTGTCCAGCCTGAAGGC CAGCGATACCGCTATGTACTATTGCGTGA GGGGAGGAACCGGAACAATGGCTTGGTTC GCTTACTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC SEQ ID NO: 49 Nucleic acid sequence encoding the heavy chain variable domain GAGGTGCAGCTGGTGCAGTCTGGCGCCGA GGTGAAGAAGCCAGGCGAGAGCCTGAGG ATCTCTTGCAAGGCTAGCGGCTACTCTTTC ACCTCCTATTGGGTGCACTGGGTGCGGGCA GATGCCAGGCAAGGGCCTGGAGTGGATGG GCAACATCGACCCCAGCGACTCTGATACA SEQ ID NO: 50

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humanized version (NOT) CACTACTCCCCTAGCTTTCAGGGCCATGTG ACCCTGTCCGTGGACAAGTCTATCTCCACA GCCTATCTGCAGCTGTCCAGCCTGAAGGC CAGCGATACCGCTATGTACTATTGCGTGA GGGGAGGAACCGGCACACTGGCTTGGTTC GCTTACTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC Nucleic acid sequence encoding the heavy chain variable domain humanized version (NOT) GAGGTGCAGCTGGTGCAGTCTGGCGCCGA GGTGAAGAAGCCAGGCGAGAGCCTGAGG ATCTCTTGCAAGGCTAGCGGCTACTCTTTC ACCTCCTATTGGGTGCACTGGGTGCGGCA GATGCCAGGCAAGGGCCTGGAGTGGATGG GCAACATCGACCCCAGCGACTCTGATACA CACTACTCCCCTAGCTTTCAGGGCCATGTG ACCCTGTCCGTGGACAAGTCTATCTCCA CA GCCTATCTGCAGCTGTCCAGCCTGAAGGC CAGCGATACCGCTATGTACTATTGCGTGA GGGGAGGAACCGGAACAATGGCTTACTTC GCTTATTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC SEQ ID NO: 51 Nucleic acid sequence encoding the heavy chain variable domain humanized version (HEE) GAGGTGCAGCTGGTGCAGTCTGGCGCCGA GGTGAAGAAGCCAGGCGAGAGCCTGAGG ATCTCTTGCAAGGCTAGCGGCTACTCTTTC ACCTCCTATTGGGTGCACTGGGTGCGGCA GATGCCAGGCAAGGGCCTGGAGTGGATGG GCAACATCGACCCCAGCGACTCTGATACA CACTACTCCCCTAGCTTTCAGGGCCATGTG ACCCTGTCCGTGGACAAGTCTATCTCCA CA GCCTATCTGCAGCTGTCCAGCCTGAAGGC CAGCGATACCGCTATGTACTATTGCGTGA GGGGAGGAACCGGCACACTGGCTTACTTC GCTTATTGGGGCCAGGGCACCCTGGTGAC AGTGTCTTCC SEQ ID NO: 52 Subsequence GACGTGGTCATGACCCAGACACCACTGAG SEQ ID NO:

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nucleic acid encoding a light chain variable domain wild antibodies type (chimeric antibody and mouse antibody 18D5) CCTGCCCGTGTCCCTGGGCGATCAGGCCTC TATCTCCTGCAGGTCCAGCCAGTCCCTGGT GCACAGCTATGGCAACACATACCTGTATT GGTACCTGCAGAAGCCAGGCCAGTCCCCC AAGCTGCTGATCTACAGGGTGTCTAATCG GTTCTCCGGCGTGCCTGACAGGTTCTCCGG CTCTGGCTCCGGCACCGATTTCACACTGAA GATCAGCAGGGTGGAGGCTGAGG ACCTGG GCGTGTATTTCTGTTTTCAGGGCACCCATG TGCCATACACATTTGGCTCTGGCACCAAGC TGGAGATCAAG 53 Nucleic acid sequence encoding the light chain variable domain humanized variant A (LA) GACGTGGTCATGACACAGAGCCCACTGTC TCTGCCTGTGACCCTGGGACAGCCAGCCTC TATCTCCTGCAGGTCCAGCCAGTCCCTGGT GCACAGCTATGGCAACACATACCTGTATT GGTACCAGCAGAGGCCCGGACAGAGCCCA AGGCTGCTGATCTACAGGGTGTCTAATCG GTTCTCCGGCGTGCCTGACAGGTTTAGCGG CTCTGGCTCCGGCACCGATT TCACACTGAA GATCTCTAGAGTGGAGGCTGAGGATGTGG GCGTGTATTTCTGTTTTCAGGGCACCCATG TGCCATACACATTTGGCGGCGGCACCAAG GTGGAGATCAAG SEQ ID NO: 54 Nucleic acid sequence encoding the light chain variable domain humanized version (LB) GACGTGGTCATGACACAGAGCCCACTGTC TCTGCCTGTGACCCTGGGACAGCCAGCCTC TATCTCCTGCAGGTCCAGCCAGTCCCTGGT GCACAGCTACGGCAACACATACCTGTATT GGTTCCAGCAGAGGCCCGGACAGAGCCCA AGGCTGCTGATCTATAGGGTGTCTAATCG GTTCTCCGGCGTGCCTGACAGGTTTAGCGG ATCTGGATCCGGAACCGACTTCACCCTGA AGATCTCTAGAGTGGAGGCTGAGGATGTG GGCGTGTACTATTGTTTCCAGGGCACCCAT GTGCCATACACATTTGGCGGCGGCACCAA SEQ ID NO: 55

GGTGGAGATCAAG

Vectors

According to another aspect, the present invention relates to a vector containing a nucleic acid molecule as defined above.

As used herein, the term vector is a nucleic acid molecule used as a carrier for transferring genetic material into a cell. The term vector includes plasmids, viruses, cosmids and artificial chromosomes. Typically, constructed vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is typically a nucleotide sequence, usually a DNA sequence, that contains an insert (transgene) and a larger sequence that serves as the backbone of the vector. Modern vectors may include additional design features,

- 38 044391 in addition to the transgenic insert and backbone: promoter, genetic marker, antibiotic resistance gene, reporter gene, targeting sequence, protein purification tag. Vectors, called expression vectors (expression constructs), are specifically designed to express a transgene in a target cell and usually have control sequences.

Host cells

According to another aspect, the present invention relates to an isolated host cell containing a vector as defined above.

As used herein, the term host cell is intended to include any single cell or cell culture that can be or has been the recipient of vectors, exogenous nucleic acid molecules and polynucleotides encoding the antibody construct of the present invention; and/or the recipient of the antibody construct itself. Introduction of the corresponding material into a cell can be accomplished by transformation, transfection and the like. The term host cell is also intended to include the progeny or potential progeny of a single cell. Suitable host cells include prokaryotic or eukaryotic cells and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, e.g. mouse, rat, rabbit, macaque or person.

Sets

According to another aspect, the present invention provides a kit comprising: an antibody or antigen binding fragment thereof as defined above, a nucleic acid molecule encoding said antibody or antigen binding fragment, a vector containing said nucleic acid molecule, and/or a cell containing said vector.

For convenience, the antibody of the present invention may be provided in a kit, i.e. in a packaged combination of reagents.

As used herein, the term kit means two or more components (one of which corresponds to an antibody or antigen binding fragment thereof, nucleic acid molecule, vector, or cell of the present invention) packaged together in a container, administered to a recipient, or otherwise presented. Accordingly, a kit can be described as a collection of products and/or tools that are sufficient to achieve a specific purpose and that can be marketed as a single unit.

The kit may contain one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size and material containing the antibody construct of the present invention at an appropriate dosage for administration. The kit may further contain instructions for use (for example, in the form of an information leaflet or instructions for use), means for administering the antibody construct of the present invention, such as a syringe, pump, infuser or the like, means for reconstituting the antibody construct of the present invention and/or means for diluting the antibody construct of the present invention.

According to one embodiment, the present invention relates to a kit as defined above for a single unit dosage form. The kit of the present invention may also comprise a first recipient containing a dried/lyophilized antibody construct and a second recipient containing an aqueous formulation. Some embodiments of the present invention provide kits containing single and multi-chamber prefilled syringes (eg, liquid syringes and lyophilized syringes).

Antigens

In one aspect, the present invention relates to a polypeptide, in particular an antigen, comprising or consisting of a human SIRPa epitope sequence consisting of SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E).

In one embodiment, the present invention relates to a polypeptide, in particular an antigen, comprising or consisting of a human SIRPa epitope sequence consisting of SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), wherein said polypeptide contains up to 300 amino acids.

According to one embodiment of the present invention, the polypeptide of the present invention contains from 50 to 300, preferably from 100 to 250 amino acids.

In one embodiment of the present invention, the polypeptide of the present invention contains up to 50, 100, 150, 200, 250, or 300 amino acids.

In one embodiment, the present invention relates to a polypeptide, in particular an antigen, comprising or consisting of a human SIRPa epitope sequence consisting of SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), and at least one sequence human SIRPa epitope consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) or SEQ ID NO: 6 ( YPQRLQLTWLE).

- 39 044391

In one embodiment, the present invention provides a polypeptide, particularly an antigen, comprising or consisting of human SIRPa epitope sequences consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO : 3 (KFRKGSPD[DV]/[T]E), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and

SEQ ID NO: 6 (YPQRLQLTWLE).

In one embodiment, the present invention relates to a polypeptide, particularly an antigen, comprising or consisting of at least one peptide selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72. wherein said polypeptide consists of a sequence containing up to 300 amino acids.

According to one embodiment, the present invention relates to a polypeptide, in particular an antigen, containing or consisting of a peptide containing the amino acid sequence presented in SEQ ID NO: 73 (YNQK), and/or a peptide having the amino acid sequence SIR within SIRPa, wherein said polypeptide consists of a sequence containing up to 300 amino acids.

Antibody production method

According to one aspect, the present invention also relates to a method for producing an antibody, in particular an antibody according to the present invention, comprising immunizing a non-human animal, in particular a non-human mammal, against at least one antigen as defined above, and, in specifically, collecting the resulting serum from said immunized non-human animal to obtain antibodies directed against said antigen.

In particular, the present invention also relates to a method for producing an antibody, comprising immunizing a non-human animal against an antigen containing or consisting of a human SIRPa epitope sequence consisting of SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E) and, in particular, collecting the resulting serum from said immunized non-human animal to obtain antibodies directed against said antigen.

In particular, the present invention also relates to a method for producing an antibody, comprising immunizing a non-human animal against an antigen containing or consisting of a human SIRPa epitope sequence consisting of SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E) , and at least one human SIRPa epitope sequence selected from the group consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G/ARELIYNQKEGH), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) or SEQ ID NO: 6 (YPQRLQLTWLE), and in particular, collecting the resulting serum from said immunized non-human animal to obtain antibodies directed against said antigen.

In particular, the present invention also relates to a method of producing an antibody, comprising immunizing a non-human animal against an antigen containing or consisting of human SIRPa epitope sequences consisting of SEQ ID NO: 1 (SLIPVGP), SEQ ID NO: 2 (G /ARELIYNQKEGH), SEQ ID NO: 3 (KFRKGSPD[DV]/[T]E), SEQ ID NO: 4 (QHTVSFTCESHGFSPRDITLKWF), SEQ ID NO: 5 (ICEVAHVTLQG) and SEQ ID NO: 6 (YPQRLQLTWLE), and, in particular, collecting the resulting serum from said immunized non-human animal to obtain antibodies directed against said antigen.

Antibody selection method

In one aspect, the present invention relates to a method for selecting an antibody of the present invention, an antigen binding fragment or a mimetic of the present invention, comprising or consisting of at least one of the following steps:

a) testing (for example, in accordance with the method described in examples 1-3) the ability of an antibody, antigen-binding fragment or mimetic of said antibody to bind to SIRPa, in particular to the antigen defined above;

b) testing (for example, in accordance with the method described in examples 7 and 8), the ability of an antibody, antigen binding fragment or mimetic of the specified antibody to bind to SIRPb;

c) testing (eg, in accordance with the method described in Examples 9 and 10) the ability of an antibody, antigen binding fragment or mimetic of said antibody to bind to SIRPg;

d) testing (eg, in accordance with the method described in Examples 4 and 5), the ability of an antibody, antigen binding fragment or mimetic of said antibody to inhibit the binding of human CD47 to human SIRPa;

e) testing (for example, in accordance with the method described in example 12) the ability of an antibody, an antigen binding fragment or a mimetic of the specified antibody to bind to T cells;

f) testing (eg, in accordance with the method described in Example 13) the ability of an antibody, antigen binding fragment or mimetic of said antibody to inhibit T cell proliferation;

g) testing (eg, in accordance with the method described in example 11) the ability of an antibody, antigen binding fragment or mimetic of the specified antibody to inhibit the binding of human CD47 to human SIRPg;

- 40 044391 and optionally including the following step:

selecting an antibody, antigen binding fragment or mimetic of said antibody that specifically binds to SIRPa, in particular the antigen defined above, and that significantly inhibits the binding of CD47 to SIRPa and that does not bind specifically to human SIRPg, and/or that does not bind specifically to Human T cells, and/or which does not significantly inhibit the proliferation of human T cells, and/or which does not significantly inhibit the binding of human CD47 to human SIRPg; more specifically, which specifically binds to SIRPa, in particular to the antigen defined above, and which significantly inhibits the binding of CD47 to SIRPa and which does not bind specifically to human SIRPg and which does not bind specifically to human T cells, and which does not inhibit significantly the proliferation of human T cells, and does not significantly inhibit the binding of human CD47 to human SIRPg.

The method for selecting an antibody according to the present invention may preferably be performed as an addition to the method for producing an antibody according to the present invention.

The following drawings and examples are provided to provide those skilled in the art with a complete disclosure and description of the methods for making and using the present invention and are not intended to limit the scope of the invention as claimed by the present inventors, and it is also understood that the following experiments do not represent all experiments or the only experiments performed. Although the present invention has been described with reference to specific embodiments, those skilled in the art will understand that various modifications may be made and equivalents may be substituted without departing from the precise spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation, material, composition, method, method step or steps to achieve the objects, spirit and scope of the present invention. All such modifications are intended to be included within the scope of the appended claims.

Symbols for drawings

Fig. 1. Results of studies of the binding of antibodies to SIRPa by ELISA (human SIRPa-His was used as a coating and the human kappa chain was detected).

Evaluation by ELISA method on immobilized SIRPa-His chimeric antibodies (♦), HALA (□), HFLA (*), HFLB (+), HEFLA (▲), HEFLB (), SIRP29 (Δ), Kwar23 (o) in Fig. A; HCLA (•), HCLB (x), HELA (◊), HELB (-) in Fig. IN; HALB (-), HBLA (_), HBLB () in Fig. C. Detection was performed using a donkey anti-human protein antibody and the signal was detected colorimetrically at 450 nm using TMB substrate. ED ₅₀ represents the concentration of the specified antibody required to achieve 50% signal in a given study. Binding of clone m18D5 () (n=4), commercial clone SE5A5 (▲) (n=7), clone 6G10 (V) (n=3) and clone 12D7 (□) (n=4) in Fig. D.

Fig. 2. Study of the affinity of antibodies to SIRP on recombinant human SIRP protein using the Biacore system.

The recombinant SIRPa-His protein was immobilized on the CM5 chip at a concentration of 5 μg/ml (500 units), and the indicated antibodies were added at various concentrations. Values were measured after an association period (ka) of 3 min followed by a dissociation period (kd) of 10 min to determine the affinity constant (KD).

Fig. 3. Study of the binding of antibodies to SIRPa on human monocytes (homozygous for the SIRPa variant 1 (v1/v1)).

(A, B) Evaluation on human v1/v1 monocytes (previously stained with human Fc receptor binding inhibitory antibody) of chimeric antibody (♦), HALA (□), HFLA (*), HFLB (+), HEFLA (▲), HEFLB (), SIRP29 (Δ), Kwar23 (o) using cytofluorometry. Detection was performed using phycoerythrin (PE)-labeled mouse anti-human Fc antibody on a CantoII cytometer, values corresponding to the percentage of monocytes stained. ED ₅₀ represents the concentration of the specified antibody required to achieve 50% signal in a given study. In fig. And the percentage values of stained monocytes v1/v1 are shown. In fig. B shows the average fluorescence intensity (MFI) of v1/v1 monocytes.

(C, D) Flow cytometry (FACS) binding study of anti-SIRPa antibodies on human monocytes: various anti-SIRPa antibodies were tested: m18D5 () (n=1), SE7C2 (▲) (n=2), 12D7 (□) ( n=2), 6G10 (♦) (n=4): In Fig. C shows the mean fluorescence intensity (MFI) of various antibodies as a function of dose. In fig. D shows the percentage of stained monocytes as a function of antibody dose. Statistical analyzes were performed whenever possible.

(E, F, G) Binding of SIRPa variants by anti-human SIRPa antibodies in the population. The ability of various anti-human SIRPa antibodies to bind SIRPa variants was measured in 32 volunteers by FACS using a PE-conjugated anti-mouse IgG antibody. All clones were tested at a concentration of 10 μg/ml: m18D5 (), 12D7 (▼), 6G10 (♦) and commercial antibodies SE5A5 (□), SE7C2 (Δ). In fig. E presents the results for volunteers homozygous for option 1 (n=16). In fig. F are represented by re-

- 41 044391 results for volunteers homozygous for option 2 (n=8). In fig. G presents the results for volunteers heterozygous for the V1/V2 variant (n=8).

Fig. 4. Competition of antibodies to SIRPa with CD47 on SIRPa.

(A) ELISA assessment of chimeric antibody (♦), HFLA (*), HFLB (+), HEFLA (▲), HEFLB (), SIRP29 (Δ), Kwar23 (o) at various concentrations incubated with a fixed concentration of biotinylated CD47 -Fc (6 μg/ml), on immobilized SIRPa-His. Detection was performed using peroxidase-streptavidin to detect the CD47 molecule, and the signal was detected colorimetrically at 450 nm using TMB substrate. The results of the second experiment are given with IC ₅₀ values. The IC ₅₀ is the concentration of the specified antibody required to inhibit 50% of the signal in a given assay.

(B) Study of the antagonistic effect of antibodies to SIRPa on the interaction of SIRPa-CD47 by ELISA: the dose dependence of the response of various antibodies to SIRPa was tested: clone m18D5 () (n=1), commercial antibody SE5A5 (▲) (n=2) and m12D7 (□) (n=2). The figure shows the percentage of CD47-positive SIRPa-CD47 interactions measured by ELISA in a dose-dependent antibody response study to human SIRPa.

Fig. 5. Competition of antibodies to SIRPa with CD47 on human monocytes.

(A, B) Evaluation of chimeric antibody (♦), HFLA (*), HFLB (+), HEFLA (▲), HEFLB () at various concentrations incubated with a fixed concentration of biotinylated CD47-Fc (10 μg/ml), on human monocytes (v1/v1) using cytometry. The CD47 molecule was detected using phycoerythrin-streptavidin, and the signal was recorded using a CantoII cytometer. The IC ₅₀ is the concentration of the specified antibody required to inhibit 50% of the signal in a given assay. In fig. And the percentage of positive cells is given. In fig. B shows the average fluorescence intensity values.

(C) FACS study of the antagonistic effect of anti-SIRPa antibodies on the SIRPa-CD47 interaction: the dose-dependent response of various anti-SIRPa antibodies was tested: clone m18D5 () (n=1), commercial antibody SE7C2 (▲) (n=2) and m12D7 (□) (n=2). In fig. C shows the percentage of CD47 positive cells measured by FACS after competition with anti-human SIRPa antibodies.

Fig. 6. (A) BLItz™ assay of the affinity of the SIRP antibody for the recombinant SIRPa protein after preincubation in the presence or absence of SP-D ligand. The recombinant SIRPa-His protein was immobilized on the NINTA biosensor at a concentration of 10 μg/ml, after which the SP-D ligand was added at a concentration of 100 μg/ml (saturating concentration). Anti-SIRPa antibody was then added at a concentration of 20 μg/mL, and affinity values were established after an association period of 120 s (ka) followed by a dissociation period of 120 s (kd) to determine the affinity constant (KD). (B) Affinity assay of SIRP antibody for recombinant human SIRPa protein preincubated with mouse 18D5 antibody using the BLItz™ system. Recombinant SIRPa-His protein was immobilized on the NINTA biosensor at a concentration of 10 μg/ml, and anti-SIRPa antibody was added at a concentration of 20 μg/ml (saturating concentration). SP-D ligand was then added at a concentration of 100 μg/mL, and affinity values were established after an association period of 120 s (ka) followed by a dissociation period of 120 s (kd) to determine the affinity constant (KD).

Fig. 7. (A) Affinity study of anti-SIRP antibodies against recombinant human SIRPb protein using the BLItz™ system.

Recombinant SIRPb-His protein was immobilized on the NINTA biosensor, and the indicated antibodies were added at a concentration of 20 μg/ml. Values were established after an association period of 120 s (ka) followed by a dissociation period of 120 s (kd) to determine the affinity constant (KD). (B) Anti-SIRP antibody binding assay (human SIRPb-His was used as a coating and human kappa chain was detected). The binding of HEFLB (), SIRP29 (Δ), Kwar23 (o), B4B6 (♦) and control IgG4 () to immobilized SIRPb-His was assessed by ELISA. Detection was performed using a donkey anti-human protein antibody, with the exception of B4B6, which was detected using a mouse antibody, and the signal was detected colorimetrically at 450 nm using TMB substrate.

Fig. 8. (A) Affinity study of anti-SIRP antibodies against recombinant human SIRPg protein using the BLItz™ system.

The recombinant SIRPg-His protein was immobilized on the NINTA biosensor and the indicated antibodies were added at a concentration of 10 μg/ml. Values were established after an association period of 120 s (ka) followed by a dissociation period of 120 s (kd) to determine the affinity constant (KD). (B) Binding of anti-SIRP antibodies to SIRPg (human SIRPg-His was used as a coating and human kappa chain was detected) was quantitatively examined by ELISA. Binding of HEFLB (), SIRP29 (Δ), Kwar23 (o), LSB2-20 (•) and control IgG4 () on immobilized SIRPg-His was assessed by ELISA. Detection was performed using a donkey anti-human protein antibody, and the signal was detected colorimetrically at 450 nm using TMB substrate.

- 42 044391

Fig. 9. Study of the affinity of CD47 for recombinant human SIRPg protein, pre-incubated with antibodies to SIRP, using the BLItz™ system. Recombinant SIRPg-His protein was immobilized on the NINTA biosensor at a concentration of 10 μg/ml, and the indicated antibodies were added at a concentration of 20 μg/ml (saturating concentration). CD47Fc was then added at a concentration of 100 μg/ml, and affinity values were established after an association period of 120 s (ka) followed by a dissociation period of 120 s (kd) to determine the affinity constant (KD).

Fig. 10. Geometric mean fluorescence intensities measured by flow cytometry on (A) human peripheral CD3+ T cells, (B) erythrocytes, or (C) platelets after staining with various monoclonal antibodies and detection using a secondary fluorescently labeled anti-IgG antibody.

Fig. 11. Percentage of positive human peripheral CD3+ T cells after staining with various monoclonal antibodies. The table shows the percentage of positive cells in experiments with two repetitions.

Fig. 12. Human T cells isolated from peripheral blood mononuclear cells of healthy volunteers were stimulated using (A) (C) CD3+ and CD28 specific beads in a 1:1 ratio for 3 days or (B) (D) using allogeneic dendritic cells (DCs) at a ratio of 5 T cells: 1 DC for 5 days, or (E) using various concentrations of crude protein derivative of tuberculin (PPD) for 5 days. Antibodies were added on day 0 of cultivation. Proliferation was measured by incorporation of H ³ -thymidine during the last 12 hours of culture.

Fig. 13. Murine CD8+ T cells were isolated from splenocytes of naïve mice. CD8 T cells were stimulated using CD3+ and CD28 specific beads in a 1:1 ratio for 3 days. Antibodies were added on day 0 of cultivation. Proliferation was measured by incorporation of H ³ -thymidine during the last 12 hours of culture.

Fig. 14. Human T cells isolated from peripheral blood mononuclear cells of healthy volunteers were stimulated using allogeneic dendritic cells (DCs) at a ratio of 5 T cells: 1 DC for 5 days.

Antibodies were added on day 0 of cultivation. Proliferation was measured by incorporation of H ³ thymidine during the last 12 hours of culture.

Fig. 15. (A) Antitumor effect of antibodies to SIRPa (clone P84) when administered intravenously three times a week for 4 weeks (300 μg/injection) in combination with two injections (4 and 8 days) of mAb to 41-BB ( clone ZNZ, 100 μg/injection) or without the indicated antibody, or injections (twice weekly) of anti-PDL-1 antibody (clone 10F.9G2, 200 μg/injection, treatment for 4 weeks) or without the indicated antibody in the orthotopic model mouse hepatomas (2.5x10 ⁶ Hepa 1.6 cells, which were administered by injection into the portal vein on day 0). Mice were considered cured if their survival period was three times longer than the time during which all control mice died. (B) Tumor-infiltrating cells were examined at day 13 after tumor inoculation. (C) Tumor-infiltrating cells and spleen cells were examined at day 13 after tumor inoculation. (D) Mice previously cured in a hepatoma model by injection of a combination of anti-SIRPa antibody and anti-4-1BB antibody, or SIRPa mutant mice treated with anti-4-1BB antibody, were reinfected by injection of Hepa 1.6 cells into the spleen (2, 5x10 ⁶ cells/mouse). At the same time, naïve mice also received injections administered in a similar manner to compare tumor development with mice after reinfection. After this, the mice received no treatment. (E) Mice previously treated in a hepatoma model with a combination of anti-SIRPa antibody and anti-4-1BB antibody were reinfected by injecting Hepa 1.6 cells into the spleen (2.5 x 10 ⁶ cells/mouse), and their spleens were harvested after 30 days after re-infection. Splenocytes and T-cell splenocytes were isolated. Naïve mice received intravenous injections of vehicle, whole splenocytes (10 x 10 ⁶ /mouse) or splenocyte-purified T cells (2.5 x 10 ⁶ /mouse), and all mice received injections of Hepa 1.6 cells into the spleen (2.5 x 10 ⁶ cells / mouse ). After this, the mice did not receive treatment and were considered cured if their survival period was three times longer than the time during which all control mice died.

Fig. 16. Mice previously treated in a hepatoma model by injection of a combination of anti-SIRPa antibody and anti-PDL1 antibody were reinfected by injecting Hepa 1.6 cells into the spleen (2.5 x 10 ⁶ cells/mouse). At the same time, naïve mice also received injections administered in a similar manner to compare tumor development with mice after reinfection. After this, the mice received no treatment.

Fig. 17. Antitumor effect of the antibody to SIRPa (clone P84) when administered i.p. three times a week for 4 weeks (200 μg/injection) in an orthotopic model of murine mammary carcinoma (0.25x10 ⁶ 4T1 cells injected into the mammary gland) . Tumor development was assessed by measuring the tumor diameter and calculated using the formula: =(0.52*(d2)) ^A 1.5. Mice were euthanized when the size of the developed tumor was approximately 1000 mm ³ in accordance with ethical guidelines.

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Fig. 18. Study of the phenotype of immune cells in the spleen, tumor and lymph nodes of mice treated with anti-SIRPa antibody (clone P84) or control mAb intraperitoneally three times a week for two weeks (200 μg/injection) in an orthotopic mouse mammary carcinoma model (0 ,25x106 4T1 cells injected into the mammary gland). The study of immune cells was carried out two weeks after tumor inoculation.

Fig. 19. Anti-SIRPa antibody (clone P84), anti-CD47 antibody (clone MIAP410) and non-targeting isotype control antibody were administered at a concentration of 12 mg/kg intraperitoneally on days 0 and 2 to C57B1/6 mice. Blood samples were collected on days 0 and 3 in EDTA-containing tubes, and complete blood count was performed using an XS-800i hematology analyzer (Sysmex). Hemoglobin level (left) and percentage hematocrit (right) were assessed on day 3. Dashed lines represent normal range values in C57B1/6 mice for each parameter.

Fig. 20. (Top panel) Flow cytometry study of anti-SIRPa mAb (HEFLB, gray) and anti-CD47 mAb (B6H12, black) in comparison with control mAb (dashed line) on human platelets freshly isolated from the blood of healthy donors. (Bottom panel) Measurement of human platelet aggregation in the presence of 50 μg/ml control mAb, anti-SIRPa antibody (HEFLB), anti-CD47 antibody (B6H12), or anti-integrin-aIIb antibody as a positive aggregation inhibitor control using an optical aggregometer. Antibodies were assessed on non-activated and ADP-activated platelets.

Fig. 21. Allogeneic CD4+ T cells were cultured in a 1:1 ratio with CD14+ myeloid cells extracted from (A) fresh or (B) frozen ovarian cancer ascites in the presence of 10 μg/ml control antibody (white), anti-SIRPa, anti-HEFLB (black), or anti-CD47 mAb (gray). Proliferation was measured on day 5 by incorporation of H ³ -thymidine. (C) In another embodiment, allogeneic T cells were cultured in a 5:1 ratio with allogeneic dendritic cells and varying ratios of CD14+ myeloid cells extracted from ovarian cancer ascites in the presence of 10 μg/ml of the antibody indicated in (A).

Examples

In the following examples, antibody 18D5 (or m18D5) corresponds to mouse antibody 18D5, chimeric antibody corresponds to mouse/human chimeric antibody 18D5, and antibodies HALA, HALB, HBLA, HBLB, HCLA, HCLB, HELA, HELB, HFLA, HFLB, HEFLA, and HEFLB correspond to specific humanized variants of 18D5. Antibodies 6G10 and 12D7 belong to the Applicant; these antibodies were generated in the same manner as m18D5 and are used as controls. These control antibodies are mouse monoclonal antibodies IgG2a to human SIRPa.

In addition, commercial antibodies were used for comparison. The first antibody is an anti-SIRPa antibody called SE7C2 (Santa Cruz sc-23863); the second antibody is an antibody capable of recognizing both SIRP α/β and named SE5A5 (BioLegend BLE323802); and the third antibody is an anti-human SIRPa antibody called Kwar23 (Creative Biolabs). An anti-human SIRPa antibody called SIRP29 from the University of Toronto described in PCT publication WO2013056352 was used for comparison.

Example 1. Binding studies of antibodies to SIRPa on SIRPa by ELISA.

Method: binding activity of antibodies to SIRPa was assessed by ELISA. To perform a quantitative ELISA study using a chimeric antibody, humanized antibodies, SIRP29 and Kwar23, recombinant human SIRPa (Sino Biologicals, Beijing, China; catalog number 11612-H08H) were immobilized on plastic at a concentration of 0.5 μg/ml in carbonate buffer ( pH 9.2) and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG antibody (Jackson Immunoresearch, USA, catalog number 709-035-149) was added and detected by conventional methods.

To perform a quantitative ELISA study using mouse antibodies, recombinant human SIRPa (Sino Biologicals, Beijing, China; catalog number 10975-Н08Н) was immobilized on plastic at a concentration of 0.5 μg/ml in carbonate buffer (pH 9.2) and added purified antibody to measure binding. After incubation and washing, peroxidase-labeled goat anti-mouse Fc chain antibody (Jackson Immunoresearch, catalog no. 115-036-071) was added and detected by conventional methods.

Results: As shown in FIG. 1A, 1B and 1C, the binding activity of various antibodies to SIRPa on SIRPa, measured by ELISA, was characterized by effective concentrations (EC ₅₀ ) of 2.9 ng/ml for the chimeric antibody, 3.9 ng/ml for HALA, 5.1 ng/ml for HFLA, 4.0 ng/ml for HFLB, 7.1 ng/ml for HEFLA, 4.4 ng/ml HEFLB in the first experiment, 4.06 ng/ml for chimeric antibody, 5.60 ng/ml ml for HCLA, 5.59 ng/ml for HCLB, 4.61 ng/ml for HELA, 4.13 ng/ml for HELB in the second experiment and 2.74 ng/ml for chimeric antibody, 2.53 ng/ml for HALB, 2.68 ng/ml for HBLA, 2.95 ng/ml for HBLB in the third experiment. The results obtained indicate that the antibodies of the present invention effectively bound SIRPa, as measured by ELISA, compared to other known anti-SIRPa antibodies, SIRP29 (3.7 ng/ml) and Kwar23 (3.3 ng/ml). Received

- 44 044391 results indicate that the epitope recognized by all antibodies according to the present invention is accessible when SIRPa is applied to a plastic well.

As shown in FIG. 1D, the binding activity of various anti-SIRPa antibodies on SIRPa, measured by ELISA, had an effective dose ( _ED50 ) of 0.16 nM (24 ng/ml) for SE5A5 and 0.06 nM (9 ng/ml) for clone m18D5 . Clones 6G10 and 12D7 did not appear to bind to SIRPa based on ELISA results. The results indicate that clone m18D5 efficiently bound SIRPa by ELISA compared to a commercial antibody and indicate that the epitope recognized by this clone is accessible when SIRPa is applied to a plastic well compared to clones 6G10 and 12D7.

Example 2: Measuring the affinity of anti-SIRPa antibodies against SIRPa using a biosensor.

Method: recombinant human SIRPa (Sino Biologicals, Beijing, China, catalog number 11612-Н08Н) was immobilized on a CM5 sensor chip (GeHealthcare, France) at a concentration of 5 μg/ml (500 units of derivation), and various concentrations of antibodies were added at speed flow 40 µl/min. The study was performed using the BIAcore 3000 system (Biacore, GeHealthcare). Values were measured after a 3 min association period (ka) followed by a 10 min dissociation period (kd) to determine the affinity constant (KD).

Results: As shown in FIG. 2, the antibodies of the present invention have a higher affinity (KD) for SIRPa (from 1.93x10'1 M to ^3.67x10'10 M), which is equivalent to the affinity of known antibodies to SIRPa, SIRP29 and Kwar23, and exceeds the affinity of commercial antibodies to SIRPa, SE7C2 and SE5A5.

Example 3. Study of SIRPa binding on human monocytes using cytofluorometry.

Method: To measure the binding of antibodies to SIRPa on human monocytes, human Fc receptor binding inhibitor (BD Pharmingen, USA; catalog number 564220) was first added and incubated for 30 min at room temperature to block human Fc receptors on human monocytes to reduce nonspecific binding. Antibodies were then incubated for 30 min at 4°C and washed before staining for 30 min at 4°C using PE-labeled anti-human Fc IgG antibody (BioLegend; USA; catalog no. 409303). For mouse antibodies, PE-labeled anti-mouse IgG antibody was used (Jackson Immunoresearch; catalog no. 715116-151). Samples were examined on a BD LSRII or Canto II cytofluorimeter.

Results: As shown in FIG. 3, the results indicate strong binding of the anti-SIRPa antibodies of the present invention to human monocytes and more efficient binding (measured based on MFI (median fluorescence intensity)) than the binding of the known anti-SIRPa antibodies, Kwar23, SE7C2 and SE5A5.

Example 4. Quantitative study of competitive binding of CD47 and antibodies to SIRPa by ELISA to assess antagonistic activity.

Method: To conduct a quantitative competitive binding assay by ELISA, recombinant human SIRPa (Sino Biologicals, Beijing, China, catalog number 11612-Н08Н) was immobilized on plastic at a concentration of 0.5 μg/ml in carbonate buffer (pH 9.2). For the chimeric antibody, humanized antibodies, SIRP29 and Kwar23, purified antibody (at various concentrations) were mixed with biotinylated human CD47Fc (AcroBiosystems interchim, France, catalog number CD7-H82F6) at a final concentration of 6 μg/ml (fixed concentration) to measure competitive binding for 2 hours at 37°C. After incubation and washing, peroxidase-labeled streptavidin (Vector laboratoring, USA; catalog number SA-5004) was added to detect binding to biotin-CD47Fc and detected by conventional methods.

When using mouse antibodies, purified antibody (at various concentrations) was mixed with 0.04 μg/ml CD47Fc (Sino Biologicals, Beijing, China, catalog number 12283-H02H) to measure competitive binding for 2 h at 37°C. After incubation and washing, peroxidase-labeled donkey anti-human Fc chain antibody (Jackson Immunoresearch, catalog no. 709-035-149) was added to detect CD47Fc binding and detected by conventional methods.

Results: As shown in FIG. 4, the antibodies of the present invention had antagonistic activity against the SIRPa-CD47 interaction. In particular, it was observed that the chimeric antibody, HFLA, HFLB, HEFLA and HEFLB had better antagonistic activity compared to the antagonistic activity of SIRP29 and the commercial anti-SIRPa antibody, SE5A5.

Example 5. Quantitative study of competitive binding of CD47 and humanized antibodies to SIRPa on human monocytes using cytofluorometry to assess antagonistic activity.

Method: To measure the competition between CD47 and humanized anti-SIRPa antibodies on human monocytes, purified antibody was added to monocytes and incubated for 15 min at 4°C, then mixed with biotinylated human CD47Fc (AcroBiosystems Interchim, France; catalog number CD7-H82F6) at a final concentration of 5 μg/ml and incubated for 30 min at 4°C to measure competing binding antibody. After incubation and washing, PE-45 044391 labeled streptavidin (BD Biosciences, USA, catalog number 554061) was added and incubated for 15 min at 4°C to detect 6uotuh-CD47Fc binding and examined on a BD LSRII or Canto II cytofluorimeter.

To measure the competition between CD47 and mouse anti-human SIRPa antibodies on human monocytes, purified antibody was added to monocytes for 15 min at 4°C, then mixed with CD47Fc (Sino Biologicals, Beijing, China, catalog number 12283-H02H) at a final concentration 5 μg/ml and incubated for 15 min at 4°C to measure competing binding antibody. After incubation and washing, FITC-labeled anti-human Fc antibody (Beckman Coulter, catalog no. IM1627) was added and incubated for 15 min at 4°C to detect CD47Fc binding and assayed on a BD LSRII or Canto P cytofluorimeter.

Results: As shown in FIG. 5, the antibodies of the present invention have antagonistic activity against the SIRPa-CD47 interaction on human monocytes.

Example 6: Competitive Binding Study in the Presence of SP-D Using the BLItz™ System.

Method: This method was performed using the BLItz™ system (Forte Bio, USA, catalog number C22-2 No. 61010-1).

Condition A: SIRPa + anti-SIRPa antibody + surfactant protein D (SP-D). At the first stage, the recombinant protein SIRPa (His) (Sino Biologicals, Beijing, China, catalog number 11612-Н08Н) was immobilized at a concentration of 10 μg/ml through a histidine tail on a Ni-NTA biosensor (Forte Bio, USA, catalog number 18-0029) within 30 s. In the second step, anti-SIRPa antibodies were added at a concentration of 20 μg/ml (saturating concentration) for 120 s. Then human SP-D (R&D Systems, USA, catalog number 1920-SP-050) was associated at a concentration of 100 μg/ml, in competition with antibodies to SIRPa, for 120 s.

SP-D dissociation was carried out in kinetic buffer for 120 s. Data analysis was performed using BLItz™ pro 1.2 software, which calculated the association constant (ka) and dissociation constant (kd), and determined the affinity constant KD (ka/kd).

Condition B: SIRPa + surfactant protein D (SP-D) + anti-SIRPa antibody. At the first stage, the recombinant protein SIRPa (His) (Sino Biologicals, Beijing, China, catalog number 11612-Н08Н) was immobilized via a histidine tail at a concentration of 10 μg/ml on a Ni-NTA biosensor (Forte Bio, USA, catalog number 18-0029) within 30 s. In the second step, human SP-D (R&D Systems, USA, catalog number 1920-SP-050) was added at a concentration of 100 μg/ml for 120 s. Anti-SIRPa antibodies were then bound at a concentration of 20 μg/ml (saturating concentration) for 120 s. The anti-SIRPa antibody was dissociated in kinetic buffer for 120 s. Data analysis was performed using BLItz™ pro 1.2 software, which calculated the association constant (ka) and dissociation constant (kd), and determined the affinity constant KD (ka/kd).

Results: As shown in FIG. 6, binding of the anti-SIRPa antibody, 18D5, did not block the binding of SP-D to SIRPa, and binding of SP-D did not block the binding of 18D5 to SIRPa. Therefore, the antibody of the present invention does not inhibit the interaction of SIRPa and SP-D.

Example 7: Affinity study of anti-SIRPa antibodies against SIRPb using the BLItz™ system.

Method: This method was performed using the BLItz™ system (Forte Bio, USA catalog number C22-2 No. 61010-1). Recombinant hSIRPb-His (Antibodies-online, USA, catalog number ABIN3077231) was immobilized via a histidine tail at a concentration of 10 μg/ml on a Ni-NTA biosensor (Forte Bio, USA, catalog number 18-0029) for 30 s. Anti-SIRPa antibody was then bound at a concentration of 20 μg/ml for 120 s. The anti-SIRPa antibody was dissociated in kinetic buffer for 120 s. Data analysis was performed using BLItz™ pro 1.2 software, which calculated the association constant (ka) and dissociation constant (kd), and determined the affinity constant KD (ka/kd).

Results: As shown in FIG. 7A, the antibodies of the present invention had lower affinity for SIRPb compared to SIRPa. In particular, it was noted that the chimeric antibody, HFLA, HFLB, HEFLA, HEFLB had reduced affinity for SIRPb compared to SIRP29 and Kwar23.

Example 8. Binding studies of antibodies to SIRP on SIRPb by ELISA.

Method: For quantitative ELISA activity testing, recombinant hSIRPb-His (Antibodies-online; USA; catalog number ABIN1466557) was immobilized on plastic at a concentration of 1 μg/ml in carbonate buffer (pH 9.2) and purified antibody was added to measure binding . After incubation and washing, peroxidase-labeled donkey anti-human IgG antibody (Jackson Immunoresearch, USA, catalog number 709-035-149) was added and detected by conventional methods.

Results: As shown in FIG. 7B, antibodies to SIRPa had low affinity for SIRPb. It should be noted that detection was performed using donkey anti-human antibody for all antibodies except B4B6 (detected using mouse antibody), this

- 46 044391 may explain the fact that the signal obtained for the anti-SIRPb antibody, B4B6, is lower than the signal obtained for the anti-SIRPa antibodies.

Example 9: Testing the affinity of anti-SIRPa antibodies against SIRPg using the system

BLItz™.

Method: This method was performed using the BLItz™ system (Forte Bio, USA catalog number C22-2 No. 61010-1). Recombinant hSIRPg-His (Sino Biologicals, Beijing, China, catalog number 11828-Н08Н) was immobilized via a histidine tail at a concentration of 10 μg/ml on a Ni-NTA biosensor (Forte Bio, USA, catalog number 18-0029) for 30 s. Anti-SIRPa antibody was then bound at a concentration of 20 μg/ml for 120 s. The anti-SIRPa antibody was dissociated in kinetic buffer for 120 s. Data analysis was performed using BLItz™ pro 1.2 software, which calculated the association constant (ka) and dissociation constant (kd), and determined the affinity constant KD (ka/kd).

Results: As shown in FIG. 8A, the anti-SIRPa antibodies of the present invention had low affinity for SIRPg. This affinity is slightly weaker than the affinity of known antibodies to SIRPa, SIRP29 and Kwar23. However, the kinetic properties differed between antibodies to SIRPa, SIRP29 and Kwar23, with antibodies to SIRPa having a high dissociation rate constant (Kd) compared to SIRP29 and Kwar23. In particular, the HFLB antibody had the lowest affinity for SIRPg with a KD value of 1.036 x 10' ⁷ M, which is a 2 log difference compared to the KD values for SIRP29 and Kwar23.

Example 10. Study of the binding of antibodies to SIRPa to SIRPg by ELISA.

Method: For quantitative ELISA activity testing, hSIRPg-His (Sino Biologicals, Beijing, China, catalog number 11828-H08H) was immobilized on plastic at a concentration of 1 μg/ml in carbonate buffer (pH 9.2) and purified antibody was added to measure binding. After incubation and washing, peroxidase-labeled donkey anti-human IgG antibody (Jackson Immunoresearch, USA, catalog number 709-035-149) was added and detected by conventional methods.

Results: As shown in FIG. 8B, the anti-SIRPa antibody, HEFLB, did not bind to SIRPg, whereas the known anti-SIRPa antibodies, SIRP29 and Kwar23, showed significant binding to SIRPg.

Example 11. Study of competition with CD47 for binding to SIRPg using the BLItz™ system: SIRPg + anti-SIRPa antibody + CD47.

Method: This method was performed using the BLItz™ system (Forte Bio, USA, catalog number C22-2 No. 61010-1). In the first step, hSIRPg-His (Sino Biologicals, Beijing, China, catalog number 11828-Н08Н) was immobilized at a concentration of 10 μg/ml through the histidine tail on a Ni-NTA biosensor (Forte Bio, USA, catalog number 18-0029) for 30 With. In the second step, anti-SIRPa antibody was added at a concentration of 20 μg/ml (saturating concentration) for 120 s. Human CD47Fc (Sino Biologicals, Beijing, China, catalog number 12283-H02H) was then associated at a concentration of 100 μg/ml, in competition with anti-SIRPa antibodies, for 120 s. Dissociation of CD47Fc was carried out in kinetic buffer for 120 s. Data analysis was performed using BLItz™ pro 1.2 software, which calculated the association constant (ka) and dissociation constant (kd), and determined the affinity constant KD (ka/kd).

Results: As shown in FIG. 9, the anti-SIRPa antibody, HEFLB, of the present invention did not compete with CD47 for binding to SIRPg. In contrast, other known SIRP29 antibodies, and in particular kwar23, competed with CD47 for binding to SIRPg.

Example 12. Study of binding to erythrocytes by flow cytometry.

Method: The experiment was carried out to study the binding of antibodies to SIRPa on human blood cells. CD3-positive T lymphocytes, red blood cells and platelets were extracted from purified blood from healthy volunteers. Cells were then stained for 30 min at 4°C with 10 μg/ml of each antibody tested, washed, and then stained with a secondary fluorescently labeled anti-IgG antibody for an additional 30 min at 4°C. After washes, cells were examined on a CANTO II flow cytometer (BD Bioscience).

Results: As shown in FIG. 10, T cells, red blood cells, and platelets were positive for CD47, which is ubiquitously expressed, and the cells were stained with the B6H12 antibody. SIRP29 and Kwar23 antibodies, such as LSB2.20 (a SIRPy-specific antibody), bind to T cells known to express SIRPy. However, the humanized anti-SIRPa antibody, 18D5, did not bind to T cells (similar results were obtained using four different humanized 18D5 variants tested). Red blood cells and platelets do not express SIRPa and, therefore, they were not detected by the humanized antibody 18D5 and other anti-SIRPa antibodies. This result demonstrates the specificity of the humanized antibody 18D5 against SIRPa on living cells compared to known SIRPa antibodies.

As shown in FIG. 11, T cells did not stain with humanized 18D5 antibody (similar results were obtained using the five different humanized 18D5 variants tested) and chimeric 18D5 (data not shown), whereas more than 70% of T cells stained

SIRP29 and Kwar23.

Example 13 Proliferation of human CD3+ T cells.

Method: Human PBMCs were isolated from the buffy coat of healthy volunteers. CD4 or CD8 T cells were selected by positive selection using AutoMACS (Miltenyi) and seeded in a 96-well plate (50,000 cells/well). Proliferative signals were provided using microbeads coated with anti-CD3/CD28 agents (LifeTechnologies) at a ratio of 1 bead: 1 T cell for three days or allogeneic mature dendritic cells obtained in vitro at a ratio of 5 T cells: 1 mature DC for 5 days or with different concentrations of crude tuberculin protein derivative (PPD) for 5 days. Antibodies targeting the SIRPa/CD47 pathway were added at the start of the proliferation assay at a saturating concentration (10 μg/ml). Proliferation was measured by incorporation of H ³ -thymidine during the last 12 hours of culture.

Results: As shown in FIG. 12, anti-SIRPa, HALA, and HEFLB variants did not inhibit T cell proliferation when T cells were stimulated with CD3+/CD28-specific beads (A) (C) or allogeneic dendritic cells (B) (D) or PPD ( E), while the anti-SIRPa antibody, Kwar23, inhibited T cell proliferation when T cells were stimulated with allogeneic dendritic cells. As expected, anti-CD47 and anti-SIRPg were inhibitors of T cell proliferation.

Example 14 Proliferation of mouse CD8+ T cells.

Method: splenocytes were isolated from naïve mice. CD8 T cells were selected by positive selection using AutoMACS (Miltenyi) and seeded in a 96-well plate (50,000 cells/well). Proliferative signals were provided using CD3/CD28-specific microbeads (LifeTechnologies) at a ratio of 1 bead: 1 T cell for three days. A mouse anti-SIRPa antibody (P84) and an anti-CD47 antibody (MIAP310) targeting the SIRPa/CD47 pathway were added at the start of the proliferation assay at a saturating concentration (10 μg/ml). Proliferation was measured by incorporation of H ³ -thymidine during the last 12 hours of culture.

Results: As shown in FIG. 13, anti-SIRPa antibody or anti-CD47 antibody did not inhibit mouse T cell proliferation. This result is explained by the fact that mice do not express the SIRPg gene. Therefore, mice can be used as a model to predict the effects of a specific anti-SIRPa antibody that does not bind to SIRPg in in vivo conditions. In contrast, the preclinical in vivo efficacy of anti-CD47 or non-selective anti-SIRPa antibodies, particularly on adaptive immunity and memory T cell production, does not have prognostic value in humans.

Example 15 Proliferation of Human T Cells

Method: Human PBMCs were isolated from the buffy coat of healthy volunteers. CD4 or CD8 T cells were selected by positive selection using AutoMACS (Miltenyi) and seeded in a 96-well plate (50,000 cells/well). Proliferative signals were provided using microbeads specific for CD3/CD28 (LifeTechnologies) at a ratio of 1 bead: 1 T cell for three days, or allogeneic mature dendritic cells obtained in vitro at a ratio of 5 T cells: 1 mature DK within 5 days. Antibodies were added at the start of the proliferation assay at a saturating concentration (5 μg/ml for anti-CD47 and anti-SIRPa antibodies and 2.5 μg/ml for anti-PD-1/PD-L1 and recombinant 4-1BBL). Proliferation was measured by incorporation of H ³ thymidine during the last 12 hours of culture.

Results: As shown in FIG. 14, anti-PD-1/PD-L1 and recombinant 4-1BBL had a stimulatory effect on human T cell proliferation, while anti-CD47 had a negative effect on human T cell proliferation. Specifically, anti-CD47 antibody prevented the immunostimulatory efficacy of agonist agents directed against PD1/PD-L1 or 4-1BB on human T cells. The anti-SIRPa antibody, HEFLB, had no significant effect on T cell proliferation.

Example 16 Antitumor effects in mice.

Method: Mice were anesthetized using a xylazine/ketamine cocktail. After laparotomy, Hera 1.6 tumor cells were administered by portal vein injection (2.5x106 cells/100 μl) in PBS. Treatment began 4 days after tumor injection. An agonistic monoclonal antibody to 4-1BB (3H3) was administered by intraperitoneal injection twice on days 4 and 8 after injection of Hepa 1.6 cells (liver carcinoma cells, HCC) in PBS (100 μg/injection). Anti-PDL1 monoclonal antibody was administered by intraperitoneal injection twice a week for 4 weeks in PBS (200 μg/injection). SIRPa antagonist antibody (P84) was administered by intraperitoneal injection three times a week for four weeks in PBS (300 μg/injection).

The antitumor response was assessed in an orthotopic HCC model thirteen days after tumor inoculation. At this time, the tumor and spleen were collected to phenotype immune cells that infiltrated into the tumor or entered the systemic circulation. Splenocytes and non-parenchymal cells (NPCs) of the liver, which are infiltrating immune cells,

- 48 044391 were stained using four different mixtures for flow cytometry recording.

Results: As shown in FIG. 15A, anti-SIRPa alone significantly increased survival in a proportion of mice (28%). When combined with anti-4-1BB or anti-PDL1, anti-SIRPa produced very high response rates in mice that survived even after treatment was stopped.

As shown in FIG. 15B, the combination of an anti-SIRPa antibody with a costimulatory agent (eg, anti-4-1BB) or a T-cell checkpoint inhibitor (eg, anti-PDL1 antibody) modified the tumor microenvironment by reducing the number of regulatory and immunosuppressive immune cells (regulatory T cells, Mo -MDSC), while increasing the accumulation of effector memory CD8+ T cells, in the case of combination with the antibody to 4-1BB. Mo-MDSCs are characterized by high levels of Ly6C expression and the absence of Ly6G in a CD11b-positive and MHC class II-negative population.

As shown in FIG. 15C, the combination of an anti-SIRPa antibody with a co-stimulatory agent (eg, anti-4-1BB) or a T-cell checkpoint inhibitor (eg, anti-PDL1 antibody) modified the cellular composition of the tumor microenvironment and at the periphery in the spleen, reducing the frequency of immature and naïve B-cells. cells, while increasing the accumulation of memory cells and plasmablasts. Similarly, the combination of anti-SIRPa antibody with anti-41BB or anti-PDL1 antibody induced the accumulation of cytolytic (CD27-negative) NK cells in the tumor and in the periphery.

Taken together, our results indicate that anti-SIRPa modified tumor and peripheral immunity, particularly adaptive (T cells, regulatory T cells, B cells) and innate (MDSCs, macrophages, NK cells) immune cells to promote elimination tumors and long-term protection.

Example 17 Antitumor effects in previously treated mice.

Method: Mice previously cured in a hepatoma model by injection of a combination of anti-SIRPa antibody and anti-4-1BB antibody, or SIRPa mutant mice treated with anti-4-1BB antibody, were reinfected by injecting Hepa 1.6 cells into the spleen (2.5 x 106 cells/mouse). Mice were anesthetized using 3% isoflurane in air. An incision was then made on the side of the mice, the spleen was removed, and Hepa 1.6 tumor cells in PBS were injected into the spleen (2.5 x 106 cells/50 μl). At the same time, naïve mice also received injections administered in a similar manner to compare tumor development with mice after reinfection.

Results: As shown in FIG. 15D, all cured mice survived reinfection, and, in contrast, all naïve mice died. This result demonstrates that anti-SIRPa antibody therapy or the absence of SIRPa signals (SIRPa mutant mice) resulted in the induction of memory T cells that were still maintained long-term in cured mice.

Example 18 Antitumor effects of T cell splenocytes or whole splenocytes collected from previously cured mice.

Method: After reinfection, mice treated with a combination of anti-SIRPa antibody and anti-4-1BB antibody were euthanized and spleens were collected. After erythrocyte lysis, splenocytes were extracted and CD3-positive T cells were isolated from a subset of splenocytes using AutoMACS. After anesthesia, mice were injected intravenously with T cell splenocytes (2.5x106 cells/100 μl) or whole splenocytes (10x106 cells/100 μl) or excipient alone (PBS). All mice received Hepa 1.6 cells via the portal vein as described previously (2.5 x 106 cells/100 µl).

Results: As shown in FIG. 15E, splenocytes and isolated T lymphocytes collected from cured mice had a strong positive effect on mouse survival. These results indicate that memory T cells were present among the splenocytes of cured mice after hepatoma treatment and were responsible for long-term adaptive immune memory.

Example 19 Antitumor effects in previously treated mice.

Method: Mice previously treated in a hepatoma model by injection of a combination of anti-SIRPa antibody and anti-PDL-1 antibody were reinfected by injection of Hepa 1.6 cells into the spleen (2.5 x 106 cells/mouse). Mice were anesthetized using 3% isoflurane in air. An incision was then made on the side of the mice, the spleen was removed, and Hepa 1.6 tumor cells in PBS were injected into the spleen (2.5x106 cells/50 μl). At the same time, naïve mice also received injections administered in a similar manner to compare tumor development with mice after reinfection.

Results: As shown in FIG. 16, all cured mice survived re-infection and, on the contrary, all naive mice died. This result suggested that memory T cells were still present in the cured mice. This result demonstrated that anti-SIRPa antibody therapy induced memory T cells that still persisted long-term in the cured mice.

- 49 044391

Example 20. Effect on tumor growth in a breast carcinoma model.

Method: Mice were anesthetized using 3% isoflurane in air. Mice were shaved in the abdominal region, and 4T1 cells in 50 μl of PBS were injected into the mammary gland using an insulin syringe (30 Gauges). SIRPa antagonist antibody (P84) or control antibody in PBS was administered by intraperitoneal injection three times a week for four weeks (200 μg/injection).

Results: As shown in FIG. 17, anti-SIRPa antibody significantly (p<0.01) reduced tumor growth in a breast carcinoma model compared with control antibody.

In fig. Figure 18 shows the results of a study of immune cells two weeks after inoculation. The anti-SIRPa antibody had a positive effect on myeloid and non-myeloid cells (T and NK cells) both in the tumor and in the periphery (spleen) with a strong decrease in the number of regulatory T cells and accumulation of memory T cells.

Example 21. Effect of antibodies to SIRPa on hemoglobin concentration and hematocrit.

Method: anti-SIRPa antibody (clone P84), anti-CD47 antibody (clone MIAP410) and a non-targeting isotype control antibody were administered intraperitoneally on days 0 and 2 at a concentration of 12 mg/kg to C57B1/6 mice. Blood samples were collected on days 0 and 3 in EDTA-containing tubes, and complete blood count was performed using an XS-800i hematology analyzer (Sysmex). Hemoglobin level (left) and percentage hematocrit (right) were assessed on day 3.

Results: As shown in FIG. 19, anti-SIRPa antibody had no toxic effect on hemoglobin concentration and hematocrit. In contrast, anti-CD47 induced a decrease in hemoglobin concentration and a decrease in hematocrit, which was consistent with the anemia observed during phase 1 in humans.

Example 22. Platelet aggregation.

Method: Blood was collected from healthy volunteer donors into Vacuette collection tubes (Greiner Bio-One) containing sodium citrate buffer. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained by centrifugation for 10 min at 200g and 15 min at 3500g, respectively. The working PRP was adjusted to 3x10 ⁸ platelets/l ^-1 . Quantitative inhibition studies: mAbs were preincubated with PRP for a final test antibody concentration of 40 or 50 μg/ml. After 3 min without stirring, platelet aggregation was initiated by adding 5 μM ADP. Aggregation was determined by measuring the light transmission through the sample at 37°C with continuous stirring using a standard optical aggregometer (TA-8V Thrombo-Aggregometer, SD Innovation SAS, Froire, France). The RRR transmittance was set to 100%. Aggregation was recorded with stirring for a total of 5 min. Quantitative induction studies: Platelet aggregation was directly initiated by the addition of MAT (50 μg/ml). Aggregation was recorded with stirring for a total of 10 min.

Results: As shown in FIG. 20, unlike anti-CD47 antibodies, anti-SIRPa antibodies did not bind to human erythrocytes or platelets. Consequently, the anti-CD47 antibody induced platelet aggregation in vitro, whereas the anti-SIRPa antibody did not. Similarly, antibodies to SIRPa did not impair reversible ADP-induced human platelet aggregation, whereas antibodies to integrin a2b completely neutralized it.

Example 23. Proliferation of allogeneic T cells under the influence of SIRPa-blocking CD14+ cells from ovarian cancer ascites.

Method: Allogeneic CD4 T cells were isolated by positive selection using AutoMACS (Miltenyi) from human PBMCs from the buffy coat of a healthy volunteer. CD4 was seeded in a 96-well plate (50,000 cells/well). CD14+ cells were isolated in a similar manner from ascites from a patient with ovarian cancer. CD14+ cells were seeded with allogeneic CD4 T cells in a 1:1 ratio for 5 days. Under some conditions, allogeneic human monocyte-derived dendritic cells (moDCs) whose maturation was induced by LPS were added at a ratio of 1:5 to stimulate T cells, and the immunosuppressive effects of different ratios of CD14+ MDSCs purified from ascites were examined. Antibodies targeting the SIRPa/CD47 pathway were added at the start of the proliferation assay at a saturating concentration (10 μg/ml). Proliferation was measured by incorporation of H ³ -thymidine during the last 12 hours of culture.

Results: As shown in Figures 21A and 21B, fresh and frozen human myeloid cells (TAM) purified from ovarian cancer ascites were hypostimulatory allogeneic human T lymphocytes. Unlike anti-CD47 antibodies, anti-SIRPa antibodies modified the properties of myeloid cells to enable activation and proliferation of human T cells.

As shown in FIG. 21C, human myeloid cells (MDSCs) purified from ovarian cancer ascites could suppress human T cell proliferation induced by allogeneic moDCs at 1:1 and 2:1 myeloid cell to T cell ratios. In contrast to anti-CD47 antibodies, anti-SIRPa antibodies did not enhance immunosuppression induced by human MDSCs.

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Claims (32)

CLAIM 1. An antibody to human signal regulatory protein alpha (SIRPa) or an antigen-binding fragment thereof, which contains: a) a heavy chain variable domain containing HCDR1, HCDR2 and HCDR3, and b) a light chain variable domain comprising LCDR1, LCDR2 and LCDR3, wherein: (i) HCDR1 contains or consists of the amino acid sequence set forth in SEQ ID NO: 14, HCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 15, HCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 17, LCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 21, LCDR2 contains or consists of the amino acid sequence set forth in SEQ ID NO: 22, and LCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 23, or (ii) HCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 14, HCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 16, HCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 19, LCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 21, LCDR2 contains or consists of the amino acid sequence set forth in SEQ ID NO: 22, and LCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 23, or (iii) HCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 14, HCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 16, HCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 20, LCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 21, LCDR2 contains or consists of the amino acid sequence set forth in SEQ ID NO: 22, and LCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 23, or (iv) HCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 14, HCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 16, HCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 17, LCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 21, LCDR2 contains or consists of the amino acid sequence set forth in SEQ ID NO: 22, and LCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 23, or (v) HCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 14, HCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 16, HCDR3 contains or consists of the amino acid sequence presented in SEQ ID NO: 18, LCDR1 contains or consists of the amino acid sequence presented in SEQ ID NO: 21, - 51 044391 LCDR2 contains or consists of the amino acid sequence presented in SEQ ID NO: 22, and LCDR3 contains or consists of the amino acid sequence set forth in SEQ ID NO: 23. 2. An antibody to human SIRPa or an antigen-binding fragment thereof according to claim 1, which does not bind specifically to human SIRPg. 3. The anti-human SIRPa antibody or antigen-binding fragment thereof according to claim 1, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 24 and a light chain variable domain containing or consisting of the amino acid sequence, set forth in SEQ ID NO: 31, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33 or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a variable domain a light chain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of an amino acid sequence set forth in SEQ ID NO: 30 and a light chain variable domain containing or consisting of an amino acid the sequence set forth in SEQ ID NO: 33, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence presented in SEQ ID NO: 27, and a light chain variable domain containing or consisting of the amino acid sequence presented in SEQ ID NO: 33, or a heavy chain variable domain, containing or consisting of the amino acid sequence set forth in SEQ ID NO: 28, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 32, or a heavy chain variable domain containing or consisting of the amino acid sequence set forth in in SEQ ID NO: 28, and a light chain variable domain containing or consisting of the amino acid sequence set forth in SEQ ID NO: 33. 4. The anti-human SIRPa antibody or antigen binding fragment thereof according to any one of claims 1 or 2, which comprises a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a heavy chain variable domain comprising or consisting of from the amino acid sequence shown in SEQ ID NO: 30. 5. An anti-human SIRPa antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2, which comprises a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32 and a heavy chain variable domain containing or consisting of the amino acid sequence sequence shown in SEQ ID NO: 30. 6. An anti-human SIRPa antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2, which comprises a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a heavy chain variable domain containing or consisting of the amino acid sequence shown in SEQ ID NO: 29. 7. An anti-human SIRPa antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2, which comprises a light chain variable domain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32 and a heavy chain variable domain containing or consisting of the amino acid sequence sequence shown in SEQ ID NO: 29. 8. The anti-human SIRPa antibody or antigen binding fragment thereof according to any one of claims 1 to 7, which is a humanized monoclonal antibody or antigen binding fragment thereof, in particular wherein the light chain constant domain of the antibody is derived from the human kappa light chain constant domain. 9. The anti-human SIRPa antibody or antigen binding fragment thereof according to any one of claims 1 to 8, wherein the light chain constant domain consists of the sequence set forth in SEQ ID NO: 35. - 52 044391 10. The anti-human SIRPa antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the heavy chain constant domain of the antibody is derived from a heavy chain constant domain IgG1, IgG2, IgG3 or IgG4. 11. The anti-human SIRPa antibody or antigen binding fragment thereof according to any one of claims 1 to 10, wherein the heavy chain constant domain of the antibody consists of the sequence SEQ ID NO: 34. 12. An anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, which contains a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 42, and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 45. 13. An anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 42, and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 44. 14. An anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 41, and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 44. 15. The anti-human SIRPa antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 41, and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 41 45. 16. The anti-human SIRPa antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, which is a SIRPa antagonist and inhibits the binding of human CD47 to human SIRPa. 17. The anti-human SIRPa antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, which does not inhibit the binding of human CD47 to human SIRPg. 18. An antibody to human SIRPa or an antigen-binding fragment thereof according to any one of claims 1 to 17 for the manufacture of a medicinal product. 19. An anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17 for the prevention or treatment of a disease selected from the group consisting of cancer, in particular inflammatory cancer and cancer with infiltrated myeloid cells, in particular with infiltrated MDSC (suppressor myeloid-derived cells) and/or TAM (tumor associated macrophages), infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease, among others diseases caused by Pseudomonas and CMV, fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction, or for vaccination. 20. A pharmaceutical composition for the prevention or treatment of a disease selected from the group consisting of cancer, infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain damage, nerve damage, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease, fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction, or for use in vaccination, comprising at least one anti-human SIRPa antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 17, and a pharmaceutically acceptable carrier. 21. The pharmaceutical composition according to claim 20, characterized in that the disease is an inflammatory cancer or a cancer with infiltrated myeloid cells, in particular MDSCs and/or TAMs, or a chronic infectious disease caused by Pseudomonas or CMV. 22. A combination product for the prevention or treatment of a disease selected from the group consisting of cancer, infectious disease, chronic inflammatory disease, autoimmune disease, neurological disease, brain damage, nerve damage, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease, fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction, or for use in vaccination, comprising at least one anti-human SIRPa antibody or antigen-binding fragment thereof as defined in any one of claims 1 to 17, and at least one second a therapeutic agent selected from the group consisting of chemotherapeutic agents, radiotherapeutic agents, immunotherapeutic agents, cell therapy agents, antibiotics and probiotics; in particular immunotherapeutic agents selected from the group consisting of a checkpoint blocker or an activator of adaptive immune cells, especially selected from the group consisting of agonists directed against PDL1, PD1, CTLA4, CD137, CD2, CD28, CD40, HVEM, BTLA, CD160, TIGIT, TIM-1/3, LAG-3, 2B4, OX40 and CD40, CD40-L, TLR agonists, ICOS-targeted agonists, ICOS-L and agonists , directed against B cell receptors. 23. The combination product according to claim 22, characterized in that the disease is an inflammatory cancer or a cancer with infiltrated myeloid cells, in particular MDSCs and/or TAMs, or a chronic infectious disease caused by Pseudomonas or CMV. 24. An isolated nucleic acid molecule encoding an antibody to human SIRPa or an antigen-binding fragment thereof according to any one of claims 1 to 17. 25. An expression vector containing a nucleic acid molecule according to claim 24. 26. An isolated host cell containing a nucleic acid molecule according to claim 24 and/or a vector according to claim 25. 27. A method for determining in vitro or ex vivo SIRPa-positive cells in a subject from a biological sample previously taken from said subject, comprising: i) in vitro determination of the expression and/or level of expression of SIRPa in a biological sample previously taken from the specified subject, using an antibody to human SIRPa or an antigen-binding fragment thereof defined in any of claims 1 to 17 to the specified biological sample, and determination of the amount an anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17, which binds to cells present in the biological sample. 28. Use of an anti-human SIRPa antibody or an antigen-binding fragment thereof according to any one of claims 1 to 17 in the manufacture of a compound suitable for use in a diagnostic test, wherein the condition being diagnosed is selected from cancer, an infectious disease, a chronic inflammatory disease, an autoimmune disease, a neurological disease, brain damage, nerve damage, polycythemia, hemochromatosis, trauma, septic shock, chronic infectious disease, fibrosis, atherosclerosis, obesity, type II diabetes mellitus and graft dysfunction. 29. Use according to claim 28, characterized in that said compound is intended for use in personalized medicine or in an auxiliary diagnostic test. 30. Use according to claim 28 or 29, characterized in that the disease is an inflammatory cancer or a cancer with infiltrated myeloid cells, in particular MDSCs and/or TAMs, or a chronic infectious disease caused by Pseudomonas or CMV. 31. An in vitro or ex vivo method for predicting response to treatment in a subject suffering from cancer, comprising determining the level of expression of SIRPa in a tumor sample previously taken from the subject using said anti-human SIRPa antibody or antigen binding fragment thereof as defined in any one of paragraphs. 1-17, and comparing said level of SIRPa expression with a value representing the level of SIRPa expression in a population of non-responders, with a higher level of SIRPa expression in a tumor sample of said subject indicating a patient who will respond to treatment. 32. The in vitro or ex vivo method of claim 31, wherein the subject suffering from cancer is likely to be treated with an anti-human SIRPa antibody or antigen binding fragment thereof according to any one of claims 1 to 17. -