EP3555133A1

EP3555133A1 - Combination of anti-cd303 and anti-her2 antibodies

Info

Publication number: EP3555133A1
Application number: EP17826467.7A
Authority: EP
Inventors: Abdessatar Sami Chtourou; Nathalie Fournier; Christophe De Romeuf
Original assignee: LFB SA
Current assignee: LFB SA
Priority date: 2016-12-16
Filing date: 2017-12-15
Publication date: 2019-10-23
Also published as: WO2018109210A1; FR3060395B1; US20190330369A1; FR3060395A1

Abstract

The present invention relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier: a) at least one anti-CD303 antibody; and b) at least one anti-HER2 antibody. The invention also relates to the combination of the two aforementioned antibodies a) and b) to form combination products to be used simultaneously, separately or spread out over a period of time, for the prevention or treatment of HER2-positive cancer.

Description

Combination of anti-CD303 and anti-HER2 antibodies

The subject of the present invention is a novel combination of antibodies, in particular for the prevention or treatment of a HER2-positive cancer.

Breast cancer is the leading cause of death for women between the ages of 35 and 65. The most common breast cancers (95%) are adenocarcinomas, which develop from the epithelial cells of the mammary gland. We distinguish in situ cancers and invasive cancers.

The stage of breast cancer is an important prognostic factor. A lower stage generates less risk of recurrence of cancer (recurrence) and a more favorable prognosis. A higher stage gives rise to a higher risk of recurrence and a less favorable prognosis. The most commonly used system for determining the stage of breast cancer is the TNM classification (Tumor, Nodes, and Metastases). The TNM classification takes into account:

- the size of the primary tumor;

- the number of regional lymph nodes that contain cancerous cells and their location; and

- the spread of cancer, or metastases, to another part of the body.

The most important stage-related factors are lymph node involvement and tumor size.

The size of the breast tumor increases the risk of recurrence:

a large breast tumor (5 cm or more) causes the greatest risk of recurrence;

a breast tumor that is less than 1 cm in size and has not spread to the lymph nodes provides the best prognosis.

Lymph node involvement is also an important prognostic factor for breast cancer, and independent of the size of the tumor. It is evaluated by the presence or absence of cancer in said ganglia, as well as by the number of nodes reached. If the lymph nodes are positive, it gives rise to a higher risk of recurrence and a less favorable prognosis than a breast cancer that has not spread to the lymph nodes (negative lymph nodes). In addition, breast cancer can also spread to the internal mammary lymph nodes without having reached the axillary lymph nodes. In this case, the risk of recurrence is high even if the axillary lymph nodes are negative. Finally, the higher the number of positive lymph nodes, the higher the risk of recurrence.

The International Union Against Cancer (UICC) uses the TNM system to describe the extent of breast cancer, and defines the following stages: Stage 0, Stage I, Stage II A and Stage B, Stage 111A to Stage C and stage IV .

Once detected, different types of treatments can be used to treat breast cancer: surgery, radiotherapy, hormone therapy, chemotherapy and targeted therapies. An individualisation of therapies according to the patients is necessary for an optimal efficiency. Sometimes only one type of treatment is used. In other cases, a combination of treatments is performed to better control the disease. For example, one can perform surgery and then complete the treatment only with chemotherapy, or only with radiotherapy. Several targeted therapies are now used to fight against breast cancer. These therapies, based on monoclonal antibodies or chemical molecules, block specific mechanisms of cancer cells.

However, there is still a need for effective therapy of HER2-positive cancer, particularly breast cancer, especially when the cancer has an invasion of effector cells that can induce tolerance by the immune system, while identifying a therapy that is better tolerated, and has fewer side effects.

The inventors have now discovered that, surprisingly, the combination of an anti-CD303 antibody and an anti-HER2 antibody is particularly effective against HER2-positive tumors, in particular against mammary tumors. In particular, the combination of these two antibodies may have a synergistic effect on the site of the tumor. The present invention thus relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle:

at. at least one anti-CD303 antibody; and

b. at least one anti-HER2 antibody.

The present invention also relates to products containing:

at. at least one anti-CD303 antibody; and b. at least one anti-HER2 antibody,

as combination products for simultaneous, separate or time-course use for its use in the prevention or treatment of HER2-positive cancer.

The invention further relates to

at least one anti-HER2 antibody for use in the prevention or treatment of HER2-positive cancer in combination with at least one anti-CD303 antibody; and

at least one anti-CD303 antibody for its use for the prevention or treatment of a HER2-positive cancer in combination with at least one anti-HER2 antibody.

The invention also relates to a method for treating a HER2-positive cancer, which comprises administering to a patient at least one anti-HER2 antibody and at least one anti-CD303 antibody.

According to the present invention, the term "antibody" refers both to an immunoglobulin, fragments and derivatives of this immunoglobulin. Immunoglobulins are well known to those skilled in the art and consist of an assembly of two dimers each consisting of a heavy chain and a light chain. The multimeric complex is assembled by the binding of a light chain and a heavy chain by a disulfide bridge between two cysteines, the two heavy chains being themselves also linked together by two disulfide bridges.

Each of the heavy chains and light chains consists of a constant region and a variable region. More precisely, each light chain consists of a variable region (V _L ) and a constant region (C _L ). Each heavy chain consists of a variable region (V _H > and a constant region consisting of three constant domains Cm, C _H 2 and C _H 3. The domains C _H 2 and C _H 3 make up the domain Fc. Variable regions of the light chain and of the heavy chain consist of three domains determining the recognition of the antigen (CDR regions, ie Complementary Determining Regions) surrounded by four framework domains (FR or Framework regions).

Anti-CD303 antibodies a) and anti-HER2 b) can be monoclonal or polyclonal. Preferably, they are monoclonal antibodies. The antibodies can be of several isotypes, depending on the nature of their constant region: the constant regions γ, α, μ, ε and δ correspond respectively to immunoglobulins IgG, IgA, IgM, IgE and IgD. Advantageously, the anti-CD303 a) and anti-HER2 b) antibodies are of IgG isotype. Indeed, this isotype shows an ability to generate ADCC activity ("Antibody-Dependent Cellular Cytotoxicity", ie

Antibody-dependent cellular cytotoxicity) in the largest number of individuals (humans). The constant regions γ comprise several subtypes: γ1, γ2, γ3, these three types of constant regions having the particularity of fixing the human complement, and γ4, thus creating the lgG1, IgG2, IgG3, and IgG4 sub-isotypes. Advantageously, the anti-CD303 antibodies a) and anti-HER2 b) are IgG1 or IgG2 isotype, preferably IgG1.

In a particular aspect of the invention, the anti-CD303 a) and anti-HER2 b) antibodies are chosen from murine antibodies, chimeric antibodies, humanized antibodies and human antibodies.

Preferably, the anti-CD303 antibody a) and / or the anti-HER2 b antibody is a chimeric antibody, and more preferably a chimeric antibody selected from a murine / human chimeric antibody or a human / macaque chimeric antibody.

Alternatively, preferably, the anti-CD303 antibody a) and / or the anti-HER2 antibody b) is a humanized antibody, in particular a chimeric antibody whose human and heavy chain constant region is of human origin.

The term "chimeric antibody" refers to an isolated antibody, in which the sequence of each light chain and / or heavy chain that constitutes it comprises or consists of a hybrid sequence derived from at least two distinct animals. In particular, a chimeric antibody contains a wild-type light chain variable region and wild-type heavy chain variable region fused to the human light chain and heavy chain constant regions, respectively. A chimeric antibody can be prepared using genetic recombination techniques well known to those skilled in the art.

The term "humanized antibody" refers to an antibody derived from an animal other than humans in which heavy chain and light chain sequences other than CDRs have been replaced by corresponding sequences of one or more antibodies. human origin. The antibody is therefore mainly composed of human sequences, but its specificity for the antigen conferred by the CDRs comes from another species. In addition, some of the residues of the skeleton segments (named FR) can be modified to maintain binding affinity (Jones et al., 1986, Verhoeyen et al., 1988, Riechmann et al., 1988). The humanized antibodies according to the invention may be prepared by techniques known to those skilled in the art such as "CDR grafting", "resurfacing", Superhumanization, "Human string content", "FR libraries" technologies. "Guided selection", "FR shuffling" and "Humaneering", as summarized in the review of Almagro et al-2008.

As indicated above, the anti-CD303 antibody and / or the anti-HER2 antibody according to the invention may also be present in the form of an anti-CD303 antibody fragment and / or a fragment of anti-HER2 antibodies, respectively.

The term "fragment" refers in particular to Fab, F (ab) '2 or

Fd.

The term "Fab" refers to an antibody fragment of molecular weight of about 50,000 Da and having antigen binding activity. The Fab fragment consists of the entire light chain (VL + CL) and part of the heavy chain (VH + CH1). It can be obtained in particular by treating IgG with a protease, papain.

The term "F (ab ') 2" refers to a fragment of about 100,000 Da and having antigen binding activity. It corresponds to the association by a disulfide bridge (hinge region) of two Fab fragments described above. It can be obtained by treating IgG with a protease, pepsin.

The term "Fd" corresponds to the portion of the heavy chain that is included in the Fab fragment. The fragment Fd is thus formed of the VH and CH1 domains. As indicated above, the anti-CD303 antibody and / or the anti-HER2 antibody according to the invention may also be present in the form of an anti-CD303 antibody derivative a) and / or a anti-HER2 antibody derivative b), respectively.

The term "derivative" refers in particular to scFv derivatives, and to scFv multimers, such as diabody, triabody or tetrabody.

The term "scFv" (single chain Fv) is a VH: VL polypeptide synthesized using the genes encoding the VL and VH domains and a linker sequence. ScFv includes CDRs maintained in an appropriate conformation, for example using genetic recombination techniques. ScFv can also serve as basic modules for the development of multimeric structures (dimeric: "diabody", trimeric: "triabody", tetrameric: "tetrabody").

The term "diabody" refers to a scFv dimer. This fragment dimer has the property of maintaining the double valence that the parent antibody possesses. Diabody is bivalent, mono or bispecific depending on whether it fixes two identical or different antigens.

The term "triabody" refers to the trivalent association of scFv. A triabody can thus fix three identical or different antigens.

The term "tetrabody" refers to the tetravalent association of scFv. A tetrabody can fix four identical or different antigens.

The term "HER2-positive cancer" refers to a cancer comprising cells that present the HER2 protein on their surface. In particular, said cancer cells overexpress the HER2 protein. HER2-positive cancer is, for example, cancer of the uterus, ovarian cancer, breast cancer or stomach cancer. Preferably, HER2-positive cancer is breast cancer. The term "breast cancer" refers to cancer of the mammary glands, whether it is noninvasive (intracanal ductal carcinoma intracanal (CCIS)) or invasive. The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-CD303 antibody, and at least one anti-HER2 antibody fragment or derivative.

The present invention also relates to products containing at least one anti-CD303 antibody, and at least one anti-HER2 antibody fragment or derivative, as combination products for simultaneous, separate or time-course use, for its use in the prevention or treatment of HER2-positive cancer.

The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-CD303 antibody fragment, and at least one anti-HER2 antibody or one of its fragments or one of its derivatives.

The present invention also relates to products containing at least one anti-CD303 antibody fragment, and at least one anti-HER2 antibody or a fragment thereof or a derivative thereof, as combination products for a simultaneous use, separate or spread over time, for its use in the prevention or treatment of HER2-positive cancer. The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-CD303 antibody derivative, and at least one anti-HER2 antibody or one of its fragments or one of its derivatives.

The present invention also relates to products containing at least one anti-CD303 antibody derivative, and at least one anti-HER2 antibody or a fragment thereof or a derivative thereof, as combination products for one or more simultaneous use, separate or spread over time, for its use in the prevention or treatment of HER2-positive cancer.

The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-HER2 antibody, and at least one anti-CD303 antibody fragment or derivative.

The present invention also relates to products containing at least one anti-HER2 antibody, and at least one anti-CD303 antibody fragment or derivative, as combination products for simultaneous, separate or time-course use, for its use in the prevention or treatment of HER2-positive cancer.

The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-HER2 antibody fragment, and at least one anti-CD303 antibody or one of its fragments or one of its derivatives.

The present invention also relates to products containing at least one fragment of anti-HER2 antibody, and at least one anti-CD303 antibody or a fragment thereof or a derivative thereof, as combination products for a simultaneous use, separate or spread over time, for its use in the prevention or treatment of HER2-positive cancer.

The present invention also relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle, at least one anti-HER2 antibody derivative, and at least one anti-CD303 antibody or one of its fragments or one of its derivatives.

The present invention also relates to products containing at least one anti-HER2 antibody derivative, and at least one anti-CD303 antibody or a fragment thereof or a derivative thereof, as combination products for a simultaneous use, separate or spread over time, for its use in the prevention or treatment of HER2-positive cancer. The present invention thus relates to a pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle:

at. at least one anti-CD303 antibody; and

b. at least one anti-HER2 antibody.

By "pharmaceutically acceptable carrier" is meant a non-toxic medium compatible with a biological system such as a cell, a cell culture, a tissue or an organism. The invention also relates to products each containing one of the two aforementioned active agents a) and b), said active ingredients being combined for simultaneous, separate or time-based use, and in the prevention or treatment of cancer. HER2-positive. Anti-CD303 Antibody

The anti-CD303 antibody is an antibody directed against the CD303 protein. This protein, also originally named BDCA-2, is specifically expressed on the surface of plasmacytoid dendritic cells, and is a type II protein belonging to type C lectins.

The human CD303 antigen (or CD303 protein) is the C member of the 4 ^th type C lectin family (CLEC4 or "C-type lectin domain family 4, member C"). It is a 213 amino acid type II transmembrane glycoprotein (accessible in Uniprot: Q8WTT0), comprising a short cytoplasmic domain without obvious signaling motif (amino acids 1-21), a transmembrane region (amino acids 22- 44), and an extracellular domain (amino acids 45-213).

Plasmacytoid dendritic cells correspond to a subpopulation of dendritic cells, also called DC2. Plasmacytoid dendritic cells are characterized by the Lin- (CD3-, CD19-, CD20-, CD14-, CD56-), HLA-DR +, CD1-1C-, CD123 + and CD45RA + markers. These cells have also been phenotypically characterized: they express the markers CD4, CD303 and BDCA-4. They are present in the lymphoid organs and are also circulating in the blood. They have the ability to secrete type I IFN in the presence of a viral infection. They can promote the growth of tumor cells and their survival, in particular inducing an immunosuppressive environment in the tumor environment, for example by inducing the differentiation of regulatory T cells (Treg). So, these cells exhibit immunosuppressive and / or tumor tolerant properties. The term "immunosuppressive properties" refers to the properties of dendritic cells to develop and maintain immunosuppression in the tumor environment. The term "tolerogenic properties" means that plasmacytoid dendritic cells will not induce an immune response.

The use of anti-CD303 antibodies according to the invention, by virtue of their cytotoxic action, advantageously makes it possible to eliminate tumor-infiltrating plasmacytoid dendritic cells. The immunosuppressive and / or tolerogenic properties with respect to the tumor are thus diminished, advantageously suppressed, which improves the antitumor immunity in situ.

Preferably, the anti-CD303 antibody according to the invention is a monoclonal antibody directed against the ectodomain of the human CD303 antigen (SEQ ID NO: 69).

Advantageously, the anti-CD303 antibody according to the invention comprises heavy chains comprising three CDR-H (heavy chain CDR according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% of identity with the following sequences, and light chains comprising three CDR-Ls (light chain CDRs according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the following sequences:

i) CDR1 -H-family 1: SEQ ID NO: 1, CDR2-H-family 1: SEQ ID NO: 2,

CDR3-H-family 1: SEQ ID NO: 3, CDR1 -L-family 1: SEQ ID NO: 4, CDR2-L-family 1: SEQ ID NO: 5, CDR3-L-family 1: SEQ ID NO: 6; or

ii) CDR1 -H-family 2: SEQ ID NO: 7, CDR2-H-family 2: SEQ ID NO: 8,

CDR3-H-family 2: SEQ ID NO: 9, CDR1-L-family 2: SEQ ID NO: 10, CDR2-L-family 2: SEQ ID NO: 1 1, CDR3-L-family 2: SEQ ID NO : 12.

The expression "at least 80% identity" means an identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. The percentages of identity to which reference is made in the context of the present invention are determined on the basis of an overall alignment of the sequences to be compared, that is to say on an alignment of the sequences taken in their entirety over any the length using any algorithm well known to those skilled in the art such as the Needleman and Wunsch-1970 algorithm. This comparison of sequences can be performed using any software well known to those skilled in the art, for example needle software using the parameter "Gap open" equal to 10.0, the parameter "Gap extend" equal to 0.5 and a matrix " Blosum 62 ". The needle software is for example available on the ebi.ac.uk world wide website under the name "Align".

When the CDR or variable region of an antibody has an amino acid sequence of at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 96%, at least 97% , at least 98%, or at least 99% identity with a reference sequence, it may have insertions, deletions or substitutions relative to the reference sequence. When it comes to substitutions, the substitution is preferably carried out by an "equivalent" amino acid, that is to say any amino acid whose structure is close to that of the original amino acid and is therefore unlikely to alter the biological activities of the antibody, or an amino acid whose structure differs but whose intrinsic properties are known to be equivalent to those of the original amino acid and not altering by the biological activities of the 'antibody.

Table 1 below summarizes the amino acid sequences of the CDR-IMGTs of the two families of anti-CD303 antibodies that can be used according to the invention:

Table 1. Amino acid sequences of the CDRs of the two families of anti-CD303 antibodies that can be used according to the invention according to the IMGT nomenclature. In each sequence, X can be any amino acid.

Advantageously, the anti-CD303 antibody according to the invention comprises three CDR-H (heavy chain CDR according to the IMGT nomenclature) having the following amino acid sequences, or sequences having at least 80% identity with the sequences. following, and three CDR-Ls (light chain CDR according to the IMGT nomenclature) with following amino acid sequences, or sequences having at least 80% identity with the following sequences:

i) CDR1 -H-122A2: SEQ ID NO: 13, CDR2-H-122A2: SEQ ID NO: 14, CDR3-H-122A2: SEQ ID NO: 15, CDR1 -L-122A2: SEQ ID NO: 16, CDR2-L-122A2: SEQ ID NO: 17, CDR3-L-122A2: SEQ ID NO: 18; ii) CDR1 -H-102E9: SEQ ID NO: 19, CDR2-H-102E9: SEQ ID NO: 20, CDR3-H-102E9: SEQ ID NO: 21, CDR1 -L-102E9: SEQ ID NO: 22, CDR2-L-102E9: SEQ ID NO: 23, CDR3-L-102E9: SEQ ID NO: 24; iii) CDR1 -H-104Cl2: SEQ ID NO: 25, CDR2-H-104Cl2: SEQ ID NO: 26, CDR3-H-104Cl2: SEQ ID NO: 27, CDR1 -L-104Cl2: SEQ ID NO: 28,

CDR2-L-104C12: SEQ ID NO: 29, CDR3-L-104C12: SEQ ID NO: 30; iv) CDR1-H-1 14D1 1: SEQ ID NO: 31, CDR2-H-1 14D1 1: SEQ ID NO: 32, CDR3-H-1 14D1 1: SEQ ID NO: 33, CDR1 -L-1 14D1 1: SEQ ID NO: 34, CDR2-L-1 14D1 1: SEQ ID NO: 35, CDR3-L-1 14D1 1: SEQ ID NO: 36; or v) CDR1 -H-104E10: SEQ ID NO: 37, CDR2-H-104E10: SEQ ID NO: 38,

CDR3-H-104E10: SEQ ID NO: 39, CDR1 -L-104E10: SEQ ID NO: 40, CDR2-L-104E10: SEQ ID NO: 41, CDR3-L-104E10: SEQ ID NO: 42.

Advantageously, the anti-CD303 antibody according to the invention has heavy and light chains whose variable regions have the following amino acid sequences or sequences having at least 80% identity with the following sequences:

i) 122A2 antibody: heavy chain: SEQ ID NO: 43, light chain: SEQ ID NO: 48,

ii) 102E9 antibody: heavy chain: SEQ ID NO: 44, light chain: SEQ ID NO: 49,

iii) Antibody 104C12: heavy chain: SEQ ID NO: 45, light chain: SEQ ID NO: 50,

iv) Antibody 1 14D1 1: heavy chain: SEQ ID NO: 46, light chain: SEQ ID NO: 51, or

v) Antibody 104E10: heavy chain: SEQ ID NO: 47, light chain:

SEQ ID NO: 52.

Table 2 below summarizes the amino acid sequences of the CDRs and variable regions of the heavy and light chains of the anti-CD303 antibodies according to the invention: 122A2 Antibody

Heavy chain

CDR1 -H-IMGT-122A2 GYTFTDYS (SEQ ID NO: 13)

CDR2-H-IMGT-122A2 ISTYYGDS (SEQ ID NO: 14)

CDR3-H-IMGT-122A2 ARNGNFYVMDY (SEQ ID NO: 15)

QVQLQQSGAELVRPGVSVKISCKGSGYTFTDYSMHWVK QSHAKSLEWIGVISTYYGDSNYNQKFKGKATMTVDKSST

VH-122A2

TAYMELARLTSEDSAIYYCARNGNFYVMDYWGQGTSVTV SS (SEQ ID NO: 43)

Light chain

CDR1 -L-IMGT-122A2 QDISNY (SEQ ID NO: 16)

YTS CDR2-L-IMGT-122A2 (SEQ ID NO: 17)

CDR3-L-IMGT-122A2 QQGNTLPWT (SEQ ID NO: 18)

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKP

VL-122A2 DGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQ

EDIATYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 48)

102E9 Antibody

Heavy chain

CDR1 -H-IMGT-102E9 GYTFTDYS (SEQ ID NO: 19)

CDR2-H-IMGT-102E9 INTETGEP (SEQ ID NO: 20)

CDR3-H-IMGT-102E9 TRNGYYVGYYAMDY (SEQ ID NO: 21)

QIHLVQSGPDLKKPGETVKISCKASGYTFTDYSMHWVKQ APGKGLKWMGWINTETGEPTYADDFKGRFAFSLESSAST

VH-102E9

AFLQINNLKNEDTSTYFCTRNGYYVGYYAMDYWGQGTSV TVSS (SEQ ID NO: 44)

Light chain

CDR1 -L-IMGT-102E9 SSVIY (SEQ ID NO: 22)

CDR2-L-IMGT-102E9 STS (SEQ ID NO: 23)

CDR3-L-IMGT-102E9 QQRRSYPFT (SEQ ID NO: 24)

QIVLTQSPAIMSASPGEKVTITCSASSSVIYIHWFQQKPGT

VL-102E9 SPKLWIYSTSYLASGVPARFSGSGSGTSYSLTISRMEAED

AATYYCQQRRSYPFTFGGGTKLEIK (SEQ ID NO: 49)

104C12 Antibody

Heavy chain CDR1 -H-IMGT-104C12 GYTFTDYS (SEQ ID NO: 25)

CDR2-H-IMGT-104C12 ISPYYGDT (SEQ ID NO: 26)

CDR3-H-IMGT-104C12 RNADDYYRFAY (SEQ ID NO: 27)

QVQLQQSGAELVGPGVSVKISCKGSGYTFTDYSMHWVK QSHAKSLEWIGVISPYYGDTNYNQKFKGKATMTVDKSSS

VH-104C12

TAYMELASLTSEDSAIYFCARNDDYYRFAYWGQGTLVTV SA (SEQ ID NO: 45)

Light chain

CDR1 -L-IMGT-104C12 QDINNY (SEQ ID NO: 28)

YTS CDR2-L-IMGT-104C12 (SEQ ID NO: 29)

CDR3-L-IMGT-104C12 QQGKTLPWT (SEQ ID NO: 30)

DLQMTQTPSSLSASLGDRVTISCRASQDINNYLSWYQEK PDGTFKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTVRNLE

VL-104C12

QEDIGTYFCQQGKTLPWTFGGGTKLEIR (SEQ ID NO: 50)

114D11 Antibody

Heavy chain

CDR1 -H-IMGT-1 14D1 1 GYTFTDSS (SEQ ID NO: 31)

CDR2-H-IMGT-1 14D1 1 INTETGGP (SEQ ID NO: 32)

CDR3-H-IMGT-114D1RNAGYYVGYYALDY (SEQ ID NO: 33)

QIQLVQSGPELKKPGETVKISCKASGYTFTDSSMHWVQQ APNKGLKWMGWINTETGGPTYADDFKGRFAFSLETSART

VH-1 14D1 1

AYLQ I N N LKN E DTATYFCARNG YYVGYYALDYWGQGTS V TVSS (SEQ ID NO: 46)

Light chain

CDR1 -L-IMGT-1 14D1 1 SSVFY (SEQ ID NO: 34)

CDR2-L-IMGT-1 14D1 1 STS (SEQ ID NO: 35)

CDR3-L-IMGT-1 14D1 1 QQRRSYPYT (SEQ ID NO: 36)

QIVLTQSPAIMSASPGEKVTITCSASSSVFYMHWFQQKPG

VL-1 14D1 1 TSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAE

DAATYYCQQRRSYPYTFGGGTKLEIK (SEQ ID NO: 51)

104E10 Antibody

Heavy chain

CDR1 -H-IMGT-104E10 GYTFTDYS (SEQ ID NO: 37) CDR2-H-IMGT-104E10 INTETGEP (SEQ ID NO: 38)

CDR3-H-IMGT-104E10 ARNGYYVGYYAMDY (SEQ ID NO: 39)

QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQ APGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSATT

VH-104E10

AYLQINNFKNE DTAT YFC AR YYVG NG YYAM D YWGQGTS VTVSS (SEQ ID NO: 47)

Light chain

CDR1 -L-IMGT-104E10 SSVIY (SEQ ID NO: 40)

CDR2-L-IMGT-104E10 STS (SEQ ID NO: 41)

CDR3-L-IMGT-104E10 QQRRSYPYT (SEQ ID NO: 42)

QIVLTQSPAIMSASPGEKVTMTCSASSSVIYMHWFQQKP

VL-104E10 GTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEA

EDAATYYCQQRRSYPYTFGGGTKLEIK (SEQ ID NO: 52)

Table 2. Amino acid sequences of the CDR1, CDR2, and CDR3 heavy and light chains according to the IMGT nomenclature, and VH and VL fragments of the anti-CD303 antibodies according to the invention.

Preferably, the anti-CD303 antibody according to the invention has a human constant region, preferably a human constant region of IgG1 isotype.

The preferred constant sequence sequences of human heavy or light chains, SEQ ID NO: 53 and SEQ ID NO: 54, of lgG1 isotype, are shown in Table 3 below.

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL

TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK

Region

VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP

constant of

EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV

heavy chain

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

human

TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP

favorite (lgG1)

PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG (SEQ ID NO: 53)

Region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA

constant of

LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ

light chain

GLSSPVTKSFNRGEC (SEQ ID NO: 54)

human favorite (lgG1)

Table 3 \. Preferred human heavy and light chain constant region sequences SEQ ID NO: 53 and SEQ ID NO: 54.

Thus, the anti-CD303 antibody according to the invention advantageously comprises the heavy and light chains described in Table 4 below.

Table 4. Amino acid sequences of the heavy and light chains of the anti-CD303 antibodies according to the invention. Anti-HER2 Antibody

The anti-HER2 antibody is an antibody directed against the growth factor receptor for the human epidermis (Human Epidermal Growth Factor Receptor 2).

HER-2, also known as HER2 / neu or ErbB2, is a member of the epidermal growth factor receptor family. HER2 is a plasma membrane-bound tyrosine kinase receptor that can dimerize with itself and with other members of the epidermal growth factor receptor family (HER1, HER2, HER3 and HER4). Dimerization, in turn, leads to the activation of various intracellular biological pathways. HER2 is an oncogene that is overexpressed in a variety of cancers, including breast, ovarian, stomach and uterine cancers. Overexpression of HER2 in cancer (HER2-positive cancer or "HER2 + cancer") is associated with a poor prognosis.

HER2 is the target of trastuzumab (Herceptin®), a monoclonal antibody that binds to the IV domain of the extracellular segment of the HER2 / neu receptor. Trastuzumab was approved by the FDA in 1998 and has been used for the treatment of HER2 + breast cancer and HER2 + gastric cancer.

The anti-HER2 antibody according to the invention can therefore be trastuzumab. However, trastuzumab in its classical version is not therapeutically effective in a large number of patients with HER2 + cancers. Thus, preferably, the anti-HER2 antibody according to the invention is trastuzumab highly galactosylated, optionally highly fucosylated.

Trastuzumab has the following heavy chain sequence SEQ ID NO: 65:

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRW GGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 65)

And it has the following light chain sequence SEQ ID NO: 66:

MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

(SEQ ID NO: 66)

Preferably, the anti-HER2 antibody is present in the form of an anti-HER2 monoclonal antibody composition, preferably in the form of a trastuzumab composition, said composition comprising glycan structures at the Fc glycosylation sites (Asn). 297 according to the US numbering) having a galactose content of more than 60%. The EU numbering is generally used to designate a residue in an immunoglobulin heavy chain constant region (see US numbering reported in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The typical position of the glycosylated residue in an antibody is asparagine at position 297 ("Asn297") according to the EU numbering. It should be noted that one of the anti-HER2 monoclonal antibodies described herein may be partially or fully purified.

Anti-HER2 antibodies can be glycosylated with N-glycan at the Asn297 site of the heavy chain of the Fc region. Since the antibodies generally comprise two heavy chains, they have up to two N-glycans on the Fc region. A variety of oligosaccharide profiles have been observed at this level and generally, oligosaccharides found at this site include galactose, N-acetylglucosamine (GIcNAc), mannose, sialic acid, N-acetylneuraminic acid (NeuAc or NANA). ), N-glycolylneuraminic acid (NGNA) and fucose. The N-glycans found on Asn297 generally have a common base structure consisting of an unbranched chain of a first N-acetylglucosamine (GIcNAc), which is attached to the asparagine of the antibody, a second GIcNAc that is attached to the first GIcNAc and a first mannose that is attached to the second GIcNAc. Two additional mannoses are attached to the first mannose of the GIcNAc-GIcNAc-mannose chain, to complete the core structure (or pentasaccharide core) and provide two "arms" for additional glycosylation. In addition, the fucose residues can be attached to the first Asn297-linked GIcNAc.

The basic structure of both arms is also referred to as the "antenna". The most common type of N-glucan units of the antibodies present in the plasma is of the complex type, ie composed of more than one type of monosaccharide. In the biosynthetic pathway for this N-glycan motif, several GIcNAc transferases bind the GIcNAc residues to the mannoses of the glycan nucleus, which can be extended by galactose, sialic acid and fucose residues. This glycosylation pattern is called the "complex" structure.

A second glycosylation pattern found on the "arms" of the N-glycan base structure is a "high mannose" motif, which is characterized by additional mannoses (either branched or unbranched).

A third glycosylation pattern is a hybrid structure in which one of the arms is replaced by the mannose while the other arm is complex. A "galactosylated" anti-HER2 antibody, as used herein, refers to any anti-HER2 antibody that has at least one galactose monosaccharide in one of its N-glycans. The galactosylated antibodies comprise antibodies wherein both N-glycans each have complex-like units on each of the N-glycan moieties, antibodies where both N-glycans have a single-arm complex motif pattern of N-glycans. glycans, antibodies that have a single N-glycan with complex-type units on each of the N-glycan arms, and antibodies that have a single N-glycan having a complex-type motif on only one of the N-glycan arms. N-glycan patterns. Galactosylated antibodies, which include at least one galactose monosaccharide, include the forms G1 (galactose), G1 F (galactose, fucose), G2 (two galactoses), and G2F (two galactoses, one fucose). In addition, N-glycan which comprises at least one galactose monosaccharide may be sialylated or unsilylated. It should further be noted that N-glycans may also contain additional galactose residues, such as alpha-Gal, in one or more arms of the complex glycan unit, potentially resulting in N-glycan with four galactose moieties.

A "highly galactosylated" anti-HER2 antibody as used herein refers to an anti-HER2 antibody that comprises at least two galactose monosaccharides in the N-glycan moieties. Highly galactosylated antibodies include antibodies wherein both N-glycans each have complex-like units on each of the N-glycan moieties, antibodies where both N-glycans have a complex-type motif on a single arm of the motifs. N-glycans, and antibodies that have N-glycan with a complex type motif on each of the N-glycan arms. Thus, the highly galactosylated anti-HER2 antibodies according to the invention correspond to the forms in which the N-glycans each comprise a galactose in the glycan unit (for example G1 or G1 F), in which at least one N-glycan comprises two galactoses. in the glycan unit (eg G2 or G2F) and in which 3 or 4 galactoses are in the glycan unit (for example: (i) an N-glycan with a G1 glycan unit and an N-glycan with a G2 glycan unit or G2F, or (ii) two N-glycans with G2 or G2F). More preferably, the highly galactosylated anti-HER2 antibody comprises at least three galactose monosaccharides in the glycan units. In some embodiments, the highly galactosylated anti-HER2 antibody comprises at least four galactose monosaccharides in the glycan units. The highly galactosylated anti-HER2 antibody composition according to the invention preferably comprises a population of anti-HER2 antibodies (preferably trastuzumab) in which the degree of galactosylation of the antibodies in the population is at least 60%, preferably at least 70%. Preferably, the highly galactosylated anti-HER2 antibody composition according to the invention comprises a highly galactosylated anti-HER2 (preferably trastuzumab) antibody population in which the level of fucosylation is in addition at least 50%, preferably at least 60%. Preferably, the composition of high-galactosylated anti-HER2 antibodies, optionally highly fucosylated, according to the invention is a trastuzumab composition produced in mammary epithelial cells of a non-human mammal, preferably in a transgenic non-human mammal. Typically, the non-human mammal is selected from a goat, a sheep, a buffalo, a camel, a cow, a pig, a rabbit, a buffalo, a horse, a rat, a mouse and a llama. More particularly, trastuzumab can be produced by transgenic goats, more specifically produced in the milk of transgenic goats.

An example of such transgenic goats is obtained as follows: Trastuzumab-producing goats are generated using traditional microinjection techniques (see for example US 7,928,064). The cDNAs encoding the heavy and light chains (SEQ ID NO: 67 and SEQ ID NO: 68) were ligated with the casein beta expression vector to yield BC2601 HC and LC BC2602 constructs. In these plasmids, the nucleic acid sequence encoding trastuzumab is under the control of a promoter facilitating the expression of trastuzumab in the mammary gland of goats. The prokaryotic sequences are removed and the DNA is microinjected into pre-implantation embryos of the goat. These embryos were then transferred to pregnant pseudo-females. The resulting offspring was screened for the presence of the transgenes. Descendants carrying both chains were identified as transgenic founders. Then, at the appropriate age, the founders were raised. As a result of pregnancy and parturition, they were milked. The highly galactosylated, highly fucosylated trastuzumab composition was thus purified from the milk. Advantageously, reference is made to the production of transgenic anti-HER2 antibodies as described in the patent application published under the number WO2014 / 125377 ("Highly Galactosylated Anti-HER2 Antibodies and Their Uses"). The anti-CD303 and anti-HER2 antibodies according to the invention may, each independently, be produced in a host cell, a transgenic non-human animal or a transgenic plant comprising at least one nucleic acid encoding such an antibody, its fragments or derivatives, or a vector containing such a nucleic acid.

Preferably, the anti-CD303 antibody and / or the anti-HER2 antibody according to the invention are produced by transgenesis, in particular in a transgenic non-human animal or a transgenic plant.

The host cell may be of prokaryotic or eukaryotic origin, and may especially be chosen from bacterial cells, insect cells, plants, yeast or mammals. The antibody according to the invention can then be produced by culturing the host cell under appropriate conditions. A host cell according to the invention can in particular be obtained by transformation of a cell line by the expression vector (s) of the heavy and light chains of an antibody according to the invention, and separation of the different cellular clones. obtained. The transformed cell line is preferably of eukaryotic origin, and may in particular be chosen from insect, plant, yeast or mammalian cells. Cell lines suitable for the production of antibodies include the lines selected from: SP2 / 0; YB2 / 0; IR983F; human myeloma Namalwa; PERC6; the CHO lines, in particular CHO-K-1, CHO-Led O, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr-, or CHO line deleted for the two alleles coding for the FUT8 gene and / or the GMD gene; Wil-2; Jurkat; Vero; Molt -4; COS-7; 293-HEK; BHK; K6H6; NSO; SP2 / 0-Ag 14, P3X63Ag8.653, embryonic duck cell line EB66® (Valneva); rat hepatoma lines H4-II-E (DSM ACC3129), and H4-ll-Es (DSM ACC3130) (see WO2012 / 041768), NM-H9D8 (DSM ACC2806), NM-H9D8-E6 (DSM ACC 2807), and NM H9D8-E6Q12 (DSM ACC 2856) (see WO2008 / 028686).

A non-human transgenic animal according to the invention can be obtained by direct injection of the gene (s) of interest into a fertilized egg (Gordon et al. 1980). A transgenic non-human animal can also be obtained by introducing the gene (s) of interest into an embryonic stem cell and preparing the animal by a chimera aggregation method or a chimeric injection method ( see Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994), Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)). A non-human transgenic animal can also be obtained by a cloning technique in which a nucleus, in which the gene (s) of interest has been introduced, is transplanted into an enucleated egg (Ryan et al 1997, Cibelli et al., 1998, WO00 / 26357) . A transgenic non-human animal producing an antibody of interest can be prepared by the above methods. The antibody can then be accumulated in the transgenic animal and harvested, in particular from the milk or eggs of the animal. For the production of antibodies in the milk of transgenic non-human animals, methods of preparation are described in particular in WO90 / 04036, WO95 / 17085, WO01 / 26455, WO2004 / 050847, WO2005 / 033281, WO2007 / 048077. Methods for purifying proteins of interest from milk are also known (see WO01 / 26455, WO2007 / 106078). Non-human transgenic animals of interest include the mouse, rabbit, rat, goat, cattle (especially cow), and poultry (including chicken).

A transgenic plant according to the invention may be chosen from any plant that allows the production of antibodies. Many antibodies have already been produced in transgenic plants and the technologies necessary to obtain a transgenic plant expressing an antibody of interest and to the recovery of the antibody are well known to those skilled in the art (see Stoger and al-2002, Fisher et al-2003, Ma et al. 2003, Schillberg et al. 2005). It is also possible to influence the glycosylation obtained in plants to obtain glycosylation close to that of natural human antibodies (without xylose), but with, in addition, low fucosylation, for example with the aid of small interfering RNAs (Forthal et al. al, 2010).

The anti-CD303 antibody and the anti-HER2 antibody are present, according to the invention, either in a pharmaceutical composition or as combination products.

They can therefore be combined with pharmaceutically acceptable excipients, and optionally extended release matrices, such as biodegradable polymers.

The pharmaceutical composition or the combination products may be administered orally, sublingually, subcutaneously, intramuscularly, intravenously, intraarterially, intrathecally, intraocularly, intracerebrally, transdermally, pulmonally, locally or rectally. The antibodies can then be administered in unit dosage form, in admixture with conventional pharmaceutical carriers. Unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, the forms sublingual and oral administration, aerosols, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, intrathecal implants, intranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical composition or combination products contain a pharmaceutically acceptable carrier for a formulation that can be injected. It may be in particular isotonic, sterile, saline solutions (with monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium or magnesium chloride and the like, or mixtures of such salts), or freeze-dried compositions which, when adding sterilized water or physiological saline as appropriate, allow the constitution of injectable solutions.

Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions, oily formulations, including sesame oil, peanut oil, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it must be injected by syringe. It must be stable under the conditions of manufacture and storage and must be preserved from contaminations of microorganisms, such as bacteria and fungi.

The dispersions according to the invention can be prepared in glycerol, liquid polyethylene glycols or mixtures thereof, or in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerine, propylene glycol, polyethylene glycol, and the like), suitable mixtures of these, and / or vegetable oils. The proper fluidity can be maintained, for example, by the use of a surfactant, such as lecithin. Prevention of the action of microorganisms may be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid or thimerosal. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be caused by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin. Sterile injectable solutions are prepared by incorporating the active ingredients in the required amount in the appropriate solvent with several of the other ingredients listed above, if appropriate, followed by sterilization by filtration. In general, the dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the other required ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization. During formulation, the solutions will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, but drug release capsules and the like can also be used. For parenteral administration in an aqueous solution for example, the solution should be suitably buffered and the liquid diluent rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, a dose may be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of appropriate liquid, or injected at the proposed site of the infusion. Certain dosage variations will necessarily occur depending on the condition of the subject being treated.

The level of therapeutically effective dose specific for a particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disease, the activity of the specific compound employed, the specific composition used, the age, the body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the rate of excretion of the specific compound used, the duration of treatment, or the drugs used in parallel. The invention is now illustrated by the following examples.

EXAMPLES

Advantageously in the context of the present invention, the anti-CD303 antibody used is a chimeric or humanized antibody comprising the CDRs 122A2 sequences. The anti-HER2 antibody preferably used below is a trastuzumab antibody produced in transgenic goat's milk, for example as described in WO2014 / 125377). Example 1 Impact of the Elimination of pDC on ADCC Activities and Phagocytosis of Trastuzumab (Herceptin®)

1 .1 - Principle of the study

The pDCs are responsible for the differentiation of T regulators (Moseman EA, 2004, Martin-Gayo E, 2010) inter alia by the ICOS / ICOSL interaction, (Ito, T., 2007, Faget, J. 2012, Faget, J 2013). The T thus differentiated regulators exert mechanisms of immunosuppression on the functions of the other cells of the immune system, in particular the NK cells, via the cell / cell contact but also via the secretion of immunomodulatory cytokines such as IL-10, IL-35 and TGF-β (Liu, C 2016).

By cascade effect, it is thus possible to study the impact of the removal of pDCs on the activity of a tumor-specific anti-cancer agent involving pDC activation in situ. In particular, the protective effect of anti-CD303 antibodies targeting pDCs can be demonstrated by the correlative study of the decrease or elimination of cytokines IL-10 and TGF-β in the tumor environment and its impact on the effector functions of an anti-cancer agent administered, specific for the tumor (anti-HER2 antibody such as Herceptin®). Indeed, according to this model, the anti-CD303 antibodies depleting the pDCs lead to a limitation of the immunosuppressive effects of regulatory T and thus a limitation of their secretion of IL-10 and TGF-β. This limitation of secretion of IL-10 and TGF-β is correlated with a better ADCC of the NK cells, validating the indirect stimulatory effect of the anti-CD303 antibodies on the anti-cancer action of the anti-HER-2 antibodies.

1 .2- Effect of IL-10 and TGF-β on the ADCC activity of trastuzumab

In order to test the ADCC of trastuzumab in a tumor context involving activation of pDC on the one hand or by eliminating pDC on the other hand, the following protocol is put in place:

CD16-transfected NK effector cells ("NK-CD16") were cultured at 3x10 ⁵ cells / ml in IL-2-containing culture medium.

On D-1, NK-CD16 cells were cultured in culture medium without IL-2, in the absence or in the presence of IL-10 (5, 50 or 120 ng / ml) and TGF-β (5 , 50 or 120 ng / ml). At OJ, BT-474 cells (breast tumor cells) (35000 cells / well) expressing HER-2 are incubated in a flat-bottomed 96-well plate with said NK-CD16 cells, with an E / C ratio (Effector - NK / Target - BT-474) equal to 5/1 and 5000 ng / ml of anti-Her2 antibody (trastuzumab).

After 16 hours of incubation at 37 ° C, the supernatant is collected.

The negative control corresponds to the same protocol in which trastuzumab is replaced by an anti-FVIII chimeric antibody produced in YB2 / 0.

The target cell lysis induced by the anti-HER-2 antibodies is measured chromogenically by quantifying the intracellular enzyme lactate dehydrogenase (LDH) released in the supernatant by the lysed target cells (Roche Diagnostics - Cytotoxicity Detection Kit LDH).

The percentage of lysis is calculated according to the following formula:

% lysis = [(ER - SR) / (100 - SR)] - [(NC - SR) / (100 - SR)]

Where ER and SR respectively represent the experimental (ER) and spontaneous (SR) release of LDH, and NC represents the natural cytotoxicity of NK cells.

The results (% lysis) are expressed as a percentage, 100% being the value taken as a reference, obtained with NK-CD16 cells in the presence of trastuzumab and in the absence of IL-10 and TGF-β (i.e. Trastuzumab alone).

The results of ADCC are detailed in Table 1 below:

Table 1 These results show that the ADCC activity of trastuzumab is inhibited in the presence of IL-10 and TGF-β compared to control without these cytokines.

Based on an arbitrary value of 100% for trastuzumab alone, the average percentage of ADCC is 63% in the presence of IL-10 and TGF-β at a concentration of 5000 ng / ml of trastuzumab.

Conclusion: By cascade effect, the comparison of the percentage of lysis observable in the absence of cytokines IL-10 and / or TGF-β, with the percentage of lysis observable in the presence of these same cytokines, makes it possible to evaluate the impact of depletion of pDCs present at the tumor site. It has thus been demonstrated that anti-CD303 antibodies indirectly potentiate the effect of anti-cancer antibodies against HER2.

2- Effect of IL-10 and TGF-β on Trastuzumab-induced phagocytosis

The monocytes are differentiated into CD16 + macrophages (M2 like) for 2 days in RPMI 1640 + 10% FCS + M-CSF 50 ng / ml for 48 hours.

SKBR3 cells and macrophages are labeled with PKH-67 (green fluorescence) and PKH-26 (red fluorescence), respectively.

The SKBR3 cells are opsonized with 10 μg / ml of the Trastuzumab antibody or with a control antibody and then incubated with the macrophages (1 .105 of each cell / well) in the absence or in the presence of different concentrations of IL-10 (5 , 50 or 120 ng / ml) alone, TGF-β (5, 50 or 120 ng / ml) alone and IL-10 + TGF-β.

After 3h incubation at 37 ° C, the cells are placed on a counting cell (Mallassez) and observed with a fluorescence microscope.

The percentage of phagocytosis is evaluated by counting the number of macrophages (at least 100 macrophages) containing SKBR3 cells.

Conclusion: By cascade effect, the comparison of the percentage of phagocytosis observed in the absence of cytokines IL-10 and / or TGF-β, with the percentage of lysis observable in the presence of these same cytokines, makes it possible to evaluate the impact of depletion of pDCs present at the tumor site. It can thus be demonstrated that anti-CD303 antibodies indirectly potentiate the effect of anticancer anti-HER2 antibodies.

Example 2 Effect of an Anti-CD303 Antibody on the Activation of Regulatory T Cells (Treq) 2.1 - Role of anti-CD303 on the phenotype and expansion of Treq in PBMC

Mononucleated cells (PBMC) are isolated from a blood tube taken from anti-coagulant. PBMCs do not contain pDCs. Treg cells are identified and phenotypically characterized by flow cytometry based on 3 markers: CD4, CD25 and Fox-P3.

Different amounts of the anti-CD303 antibody (from 1 ng to 10 μg / ml) are added to the PBMCs in the presence of IL-2 (500 U / ml). The number of Tregs and their phenotype is monitored over time (1 to 4 days).

Under the same conditions, beads coated with anti-CD3 / anti-CD28 to stimulate proliferation T are added in a ratio Treg / beads of 4/1 to verify the activation of Treg.

Conclusion: It can thus be demonstrated that anti-CD303 antibodies, in the absence of pDC, do not have a direct impact on the expansion and immunosuppressive phenotype of regulatory T.

2.2- Role of anti-CD303 on phenotype and expansion of purified Treq in the presence of pDC

Treg cells (CD4 ⁺ , CD25 ⁺ ) are purified from PBMC by a two-step process: depletion of CD4 negative cells (CD8, CD14, CD15, CD16, CD19, CD36, CD56, CD123 positive cells) , TCRy / δ, and CD235a) then positive selection of CD25 ⁺ cells.

Purified pDCs or pDC lines, e.g. obtained according to the method described in Maeda T et al., Int J Hematol. 2005 Feb; 81 (2): 148-54 (such as the CAL-1 line or a new line generated by retransfection of CD303 into Cal-1 and selection of a line stably expressing more than 40,000 CD303 antigenic sites per cell, preferably between 40,000 and 50,000) are added in a Treg / pDC ratio of 100, 10 and 1. Different amounts of the anti-CD303 antibody (from 1 ng to 10 μg / ml) are added to the Treg / pDC mixture in the presence of IL-2 (500 U / ml). The number of Tregs and their phenotype is monitored over time (1 to 4 days).

Under the same conditions, beads coated with anti-CD3 / anti-CD28 to stimulate proliferation T are added in a ratio Treg / beads of 4/1 to verify the activation of Treg. A negative control in the absence of pDC is established, in order to check the impact (expected neutral) of the anti-CD303 directly on the Tregs.

Conclusion: The observation over several days of the expansion and differentiation of purified Tregs in the presence of pDC, after administration of anti-CD303 antibodies may make it possible to show that the administration of anti-CD303 is effective in reducing or suppress the immunosuppressive properties of pDCs.

Example 3: Depletion of human pDCs via an anti-CD303 antibody in vivo in the treatment of breast cancer

3.1 -Generation of a model of reproductive study of a pathological situation in humans

In order to study the effect of an anti-CD303 antibody in the treatment of breast cancer, the mouse model 'humanized tumor mouse model' (HTM) can be used. It is characterized by the development of a mature human immune system and the growth of human breast cancer cells that have been previously co-transplanted with human hematopoietic stem cells. Very recently, the efficacy of Trastuzumab / Herceptin treatment has been proven in this model (Wege AK et al, Oncotarget 2017, 8 (2): 2731-2744).

This model advantageously brings together several elements that are relevant for the reproducibility of the in vivo conditions: presence of human pDCs which alone express on their surface the target CD303, presence of infiltration of human Treg cells, presence of human tumor cells expressing on their surface HER2 , trastuzumab / Herceptin target molecule and an immunocompetent murine host (NK effector cells for ADCC activity). The model previously described in the article Wege et al (Int J Cancer 201 1, 129: 2194-2206) combines all these characteristics.

This model has been adapted to make it compatible with BRGSF ™ or BRGSF ™ -A2 mice (BALB / c, Rag2tm1 Fwa, IL-2Ryctm1 Cgn, SIRPocNOD, Flk2tm1 lrl, Tg (HLA-A / H2-D / B2M) 1 Bpe) characterized by the absence of T, B, and NK murine cells, and expressing only HLA class HLA-A2.1 human HLA. Said BRGSF ™ mice can be generated according to the procedure described by Legrand N, Huntington ND, Nagasawa M et al. (Functional CD47 / signal regulatory protein alpha (SIRP (alpha)) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo. Proc.Natl.Acad.Sci.USA 201 1; 108: 13224-13229). They acquire the genotype (HLA-A / H2-D / B2M) ^{1 Bpe} by transgenesis. (Pascolo, S., N. Bervas, JM Ure, AG Smith, FA Lemonnier, and B. Perarnau, 1997. HLA-A2.1 -restricted education and cytolytic activity of CD8 (+) T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 single-stranded transgenic H-2Db beta2m double knockout mice, J Exp Med 185: 2043-2051).

3.2 - Method that can be used to test the activity of the anti-CD303 antibody

Briefly, newborn mice from the immunodeficient mouse line BRGSF ™ -A2 (BALB / c Rag2 ^{tm1 Fwa} IL-2Ry _c ^tm1 ^tm1 ^C9N SIRPoc ^NOD Flk2 ^lrl Tg (HLA-A / D H2- / B2M) ^{1 Bpe} are irradiated (3 Gy) during their first 192 hours of life and 24 hours after transplantation by intrahepatic injection with 1 x 10 ⁵ human CD34 ⁺ cells isolated from umbilical cord blood (RBC) in the presence or not of tumor cells 3x10 ⁶ BT474-Luc (expressing luciferase for monitoring bioluminescence). the BT474 cells are cells of human origin derived from a breast carcinoma expressing at their surface the receptor HER2 / Neu, the target of the Trastuzumab antibody Within the framework of this particular model, the BT474 cells were, before their administration, modified by lentiviral transduction in order to make them luminescent thanks to the constitutive expression of luciferase. modification allows a control the cross-over time of the taking of tumors through analysis of bioluminescence while not sacrificing animals and thus better adjust the start of processing window. Eleven to twelve weeks after the co-administration of the human cells, the mice are tested for their degree of humanization by analysis of the composition of cells present in their blood (human and murine) by flow cytometry and then divided into five groups. different (see Table 2 below): a control group without injection of BT474 cells treated with an isotype antibody (ie an anti-Factor VIII antibody) (this group serves as a negative control for tumor 1), an injection control group of BT474 cells treated with an isotype antibody (ie an anti-Factor VIII antibody) (group 2), a group treated with transgenic trastuzumab (TTG) (group 3), a group treated with the anti-CD303 antibody (group 4) and a group treated with the combination of anti-CD303 and TTG antibody treatments (group 5). The doses used and the treatment frequencies are shown in Table 2 below:

Treatment begins 14 weeks after humanization (ie injection of human CD34 + cells isolated from umbilical cord blood (CB) in the presence or absence of 3x10 ⁶ BT474-Luc tumor cells) and lasts 19 weeks.

The treatment consists of the injection of the products tested intravenously at a dose of 30 mg / kg of body weight in the case of the anti-CD303 antibody every 3 days and once a week at 10 mg / kg of weight. in the case of TTG. The body weight of the mice is determined 3 days before the start of treatment to adjust the dose individually.

Frequent blood samples are taken to test the depletion efficiency of human pDCs by flow cytometry. In addition, an analysis carried out from the beginning of the treatment then every two weeks by bioluminescence is carried out to compare the effectiveness of the different products tested. Finally, a tumor analysis of three animals per group at week 18 after humanization is performed by flow cytometry to check for the presence or absence of human infiltrating pDCs in the tumors. Results:

The impact of the treatment on the human pDC subpopulation as well as the other populations of lymphoid cells (B lymphocytes, T lymphocytes, etc.) present in the blood and the spleen was determined by flow cytometry at different times: , j3 and j7. The results show that treatment with anti-CD303 antibody at a dose of 30 mg / kg in humanized BRGSF-HIS mice induces depletion of human pDCs for at least 7 days in the blood and spleen. In blood, the depletion activity of human pDCs is rapid (> 80% on day 1) and later in the spleen. In the blood and the spleen, depletion of human pDCs was still effective (> 90%) on day 7, ie 3 days after the last injection of anti-CD303 monoclonal antibody. It should be noted that the depletion activity of the anti-CD303 antibody is very specific since it does not significantly affect the other subpopulations of human hematopoietic cells in the organs tested.

Moreover, it should be noted that this model proves to be a model particularly suited to the study of the invention since the search for pDC in sacrificed mouse tumors shows for the first time the presence of pDC infiltrating these tumors. (see Figure 1 which shows the percentage of human pDC in the liver - tumor site - for the negative control (mice not injected with BT474 cells) (CT), and for the mice injected with BT474 cells (BT474)) .

Conclusion:

The adapted HTM murine model can be advantageously used to evaluate the indirect effect of the administration of an anti-CD303 antibody on the effect of the anti-tumor breast agent, the anti-HER2 antibody (trastuzumab), under conditions reproducing a physiological situation in vivo, by comparing in particular the results obtained with the different groups tested.

This model is thus useful to be able to evaluate the benefit, advantageously the synergistic effect, of administering an anti-CD303 antibody in combination with the administration of an anti-HER2 antibody in a breast tumor.

Claims

1. A pharmaceutical composition comprising, in a pharmaceutically acceptable vehicle:

at. at least one anti-CD303 antibody; and

b. at least one anti-HER2 antibody.

2. Products containing:

at. at least one anti-CD303 antibody; and

b. at least one anti-HER2 antibody,

3. The pharmaceutical composition according to claim 1 or products according to claim 2, wherein the antibodies a) and b) are selected from murine antibodies, chimeric antibodies, humanized antibodies and human antibodies.

The pharmaceutical composition according to claim 1 or 3, or products according to claim 2 or 3, wherein the anti-CD303 antibody is a monoclonal antibody directed against the ectodomain of the human CD303 antigen.

The pharmaceutical composition according to claim 1, 3 or 4, or products according to one of claims 2 to 4, wherein the anti-CD303 antibody comprises the following CDRs:

CDR1 -H-IMGT-122A2 GYTFTDYS (SEQ ID NO: 13)

CDR2-H-IMGT-122A2 ISTYYGDS (SEQ ID NO: 14)

CDR3-H-IMGT-122A2 ARNGNFYVMDY (SEQ ID NO: 15)

CDR1 -L-IMGT-122A2 QDISNY (SEQ ID NO: 16)

YTS CDR2-L-IMGT-122A2 (SEQ ID NO: 17)

CDR3-L-IMGT-122A2 QQGNTLPWT (SEQ ID NO: 18) or else CDR2-H-IMGT-102E9 INTETGEP (SEQ ID NO: 20)

CDR3-H-IMGT-102E9 TRNGYYVGYYAMDY (SEQ ID NO: 21)

CDR1 -L-IMGT-102E9 SSVIY (SEQ ID NO: 22)

CDR2-L-IMGT-102E9 STS (SEQ ID NO: 23)

CDR3-L-IMGT-102E9 QQRRSYPFT (SEQ ID NO: 24) or

CDR1 -H-IMGT-104C12 GYTFTDYS (SEQ ID NO: 25)

CDR2-H-IMGT-104C12 ISPYYGDT (SEQ ID NO: 26)

CDR3-H-IMGT-104C12 RNADDYYRFAY (SEQ ID NO: 27)

CDR1 -L-IMGT-104C12 QDINNY (SEQ ID NO: 28)

YTS CDR2-L-IMGT-104C12 (SEQ ID NO: 29)

CDR3-L-IMGT-104C12 QQGKTLPWT (SEQ ID NO: 30) or else

CDR1 -H-IMGT-1 14D1 1 GYTFTDSS (SEQ ID NO: 31)

CDR2-H-IMGT-1 14D1 1 INTETGGP (SEQ ID NO: 32)

CDR3-H-IMGT-114D1RNAGYYVGYYALDY (SEQ ID NO: 33)

CDR1 -L-IMGT-1 14D1 1 SSVFY (SEQ ID NO: 34)

CDR2-L-IMGT-1 14D1 1 STS (SEQ ID NO: 35)

CDR3-L-IMGT-1 14D1 1 QQRRSYPYT (SEQ ID NO: 36) or else

CDR1 -H-IMGT-104E10 GYTFTDYS (SEQ ID NO: 37)

CDR2-H-IMGT-104E10 INTETGEP (SEQ ID NO: 38)

CDR3-H-IMGT-104E10 ARNGYYVGYYAMDY (SEQ ID NO: 39)

CDR1 -L-IMGT-104E10 SSVIY (SEQ ID NO: 40)

CDR2-L-IMGT-104E10 STS (SEQ ID NO: 41)

CDR3-L-IMGT-104E10 QQRRSYPYT (SEQ ID NO: 42)

Pharmaceutical composition according to one of claims 1 or 3 to 5, or products according to one of claims 2 to 5, wherein the anti-HER2 antibody is a trastuzumab composition, said composition comprising glycan structures at the sites of Fc glycosylation having a galactose content of greater than 60%, preferably at least 70%.

7. Pharmaceutical composition according to one of claims 1 or 3 to 6, or products according to one of claims 2 to 6, wherein the anti-HER2 antibody is a trastuzumab composition, said composition comprising glycan structures on the Fc glycosylation sites having a fucosylation level of at least 50%, preferably at least 60%.

8. Pharmaceutical composition according to one of claims 1 or 3 to 7, or products according to one of claims 2 to 7, wherein the anti-HER2 antibody is trastuzumab produced in the milk of transgenic goats.

9. Pharmaceutical composition according to one of claims 1 or 3 to 8, or products according to one of claims 2 to 8, wherein the anti-CD303 antibody and / or the anti-HER2 antibody is selected from the Fab , F (ab) '2, Fd, scFv and scFv multimers.

10. The pharmaceutical composition according to one of claims 1 or 3 to 9, or products according to one of claims 2 to 9, for its use in the prevention or treatment of HER2-positive cancer selected from cancer of the uterus, ovarian cancer, breast cancer and stomach cancer.

1 1. Pharmaceutical composition according to one of claims 1 or 3 to 9, or products according to one of claims 2 to 9, for its use in the prevention or treatment of HER2-positive breast cancer.