Humanized anti-BAG3 antibodies

DESCRIPTION

The present invention provides for a humanized anti-BAG3 antibody or a fragment thereof, pharmaceutical compositions comprising said antibody and its use as a medicament, in particular for use in the treatment of pancreatic tumours or other pathologies of an immune, inflammatory, neoplastic, cardiovascular and/or degenerative nature.

Background of the Invention

BAG3 protein is a 74 kDa cytoplasmic protein which belongs to the family of co-chaperons that interact with the ATPase domain of the protein HSP70 (Heat Shock Protein) through the structural domain known as the BAG domain (amino acids 110-124). Furthermore, BAG3 protein contains a WW domain (Trp-Trp), a proline-rich region (PXXP), and two conserved motifs IPV (He-Pro- Val), which can mediate binding to other proteins. Thanks to the nature of BAG3 protein as an adapter, attributable to the presence of many functional domains, such protein can therefore interact with different proteins.

In humans, bag3 gene expression is constitutive for a few kinds of normal cells, including myocytes, while mutations thereof are associated with diseases of the skeletal and cardiac muscles. Furthermore, BAG3 protein is expressed in many types of primary tumours or tumour cell lines (lymphoid or myeloid leukemias, neuroblastoma, pancreatic cancer, thyroid cancer, breast cancer and prostate cancer, melanoma, osteosarcoma, glioblastoma and tumours of the kidney, colon, ovary, etc.) (Rosati A. et al., 2011).

In normal cell types, such as leukocytes, epithelial cells and glial cells and cells of the retina, bag3 gene expression can be induced by stressors, such as oxidants, high temperatures, lack of serum, heavy metals, HIV-1 infections, etc. These findings indicate that bag3 gene expression regulation is an important component in the cellular response to stress and is correlated with the presence of elements that respond to the transcription factor HSF1 (Heat Shock Transcription Factor), which is activated in various forms of cellular stress in bag3 gene promoter (Franceschelli S. et al., 2008). Moreover, due to the presence of many protein-protein interaction domains in the structure thereof, BAG3 protein influences cell survival in different types of cells, interacting with different molecular partners. (Rosati A. et al., 2011). The first mechanism reported in relation to BAG3 anti-apoptotic activity was identified in osteosarcoma and melanoma cells, where it was observed that BAG3 protein modulates the activation of transcription factor NF-kB and cell survival (Ammirante M. et al., 2010). A different molecular mechanism has been described in glioblastoma cells, where BAG3 protein cooperates in a positive way with HSP70 protein to maintain BAX protein in the cytosol and prevent the translocation thereof into the mitochondria (Festa M. et al., 2011). Finally, in some tumours, BAG3 has been shown to regulate proteins that modulate cell adhesion.

The presence of cytoplasmic BAG3 protein has also been described in many different cellular systems and has been associated, not only with various tumours, but also in pathologies in general related to cell survival.

Furthermore, patent application n. WO2011/067377 describes extracellular BAG3 protein, secreted by some cell types, as a biochemical marker in serum, which is highly specific for the diagnosis of certain pathological conditions, such as cardiac pathologies and pancreatic tumour.

It has recently been reported that BAG3 protein is expressed in 346/346 patients with pancreatic ductal adenocarcinoma (PDAC) and is released by the cells of the pancreatic tumour, but such protein is not expressed in either the surrounding non-neoplastic tissues or in a normal pancreas; likewise, it has been reported that the levels of BAG3 expression are related to patient survival. The results of the study demonstrate that the use of specific siRNA molecules for BAG3 mRNA can silence bag3 gene expression and induce cell death, confirming that BAG3 protein is an important survival factor for pancreatic tumour cells and that the down-regulation thereof, when combined with gemcitabine, may contribute to the eradication of the tumor cells (Rosati A. et al., 2012).

Moreover, in a recent paper we have reported that PDAC- released BAG3 binds macrophages inducing their activation and the secretion of PDAC supporting factors. We have also identified IFITM-2 as a BAG3 receptor and showed that it signals through PI3K and the p38 MAPK pathways. Finally, we have showed that the use of a mouse monoclonal anti-BAG3 antibody results in reduced tumor growth and prevents metastasis formation in three different mouse models. We have therefore identified a paracrine loop involved in PDAC growth and metastatic spreading, and showed that an anti-BAG3 antibody has therapeutic potential (Rosati A. et al., Nat. Commun. 2015)

Conventional chemotherapy treatments for tumour pathologies, as well as treatments of inflammatory and immune diseases with corticosteroids or NSAIDs (non-steroidal antiinflammatory drugs) pose numerous drawbacks linked to side effects and are not, at present, definitive means of treating such pathologies.

There is therefore an evident need for a new and improved therapeutic treatment which has the advantage of being highly specific and having few or no side effects, as compared with the conventional, commonly known therapies used for the treatment of diseases of an inflammatory, immune, and neoplastic nature described in the present invention.

Definitions

Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those persons skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference; thus, the inclusion of such definitions herein should not be construed to represent a substantial difference over what is generally understood in the art.

The term "antibody" as used herein includes "fragments" or "derivatives", which have at least one antigen binding site of the antibody and/or show the same biological activity.

An antibody preferably comprises at least one heavy immunoglobulin chain and at least one light immunoglobulin chain. An immunoglobulin chain comprises a variable domain and optionally a constant domain. A variable domain may comprise complementarity determining regions (CDRs), e.g. a CDR1, CDR2 and/or CDR3 region, and framework regions.

The term "humanized antibody" refers to an antibody of human origin, whose hypervariable region has been replaced by the homologous region of non-human monoclonal antibodies.

The term "chimeric antibody" refers to an antibody containing portions derived from different antibodies.

The term "recombinant antibody" refers to an antibody obtained using recombinant DNA methods.

The term "scFv fragment" (single chain variable fragment) refers to immunoglobulin fragments only capable of binding with the antigen concerned. ScFv fragments can also be synthesised into dimers (diabodies), trimers (triabodies) and tetramers (tetrabodies) using peptide linkers.

The terms "Fab fragment" (antigen-binding fragment) and "Fab2 fragment" refer to immunoglobulin fragments consisting of a light chain linked to the Fc fragment of the adjacent heavy chain, and such fragments are monovalent antibodies. When the Fab portions are in pairs, the fragment is called Fab2.

The term "hybridoma" refers to a cell producing monoclonal antibodies.

The term "monospecific antibodies" refers to antibodies that all have affinity for the same antigen.

The term "multispecific antibodies" refers to antibodies that have affinity for several antigens. The term "bispecific antibody" refers to an antibody that has affinity for two different antigens.

Description of the figures

Figure 1. ELISA showing the screening for antibody binding activity with a coating using Peptide 2 (BAG3 fragment).

Figure 2. ELISA showing the screening for antibody binding activity with a coating using recombinant full length BAG3 protein.

Figure 3. Flow cytometric evaluation of the ability of the antibody variants to block BAG3 binding to macrophage surface.

Figure 4. Effect of humanized AC-2 antibody variants on tumor growth in vivo.

Figure 5. Effect of humanized AC-2 antibody variant H2L4 on activated fibroblasts within the tumor mass.

Figure 6. Quantification of the AC-2 H2L4 effect on activated fibroblasts number. Disclosure of the invention

It has been surprisingly found by the inventors that specific BAG3 inhibitors are able to induce tumor regression. In particular, the humanized anti-BAG3 antibodies, tested showed a particular affinity for BAG3 protein and can be used in therapy as BAG3 inhibitors, as they block the interaction between BAG3 protein and its receptor on the macrophage surfaces. Furthermore, the experimental data reported in the application demonstrates that said anti- BAG3 antibodies are particularly effective in reducing the activation of fibroblasts and therefore that they can be used in the treatment of all the pathologies wherein fibroblasts are highly activated, such as neoplastic diseases, inflammatory diseases, immune diseases and/or degenerative diseases.

Therefore, a first embodiment of the present invention relates to an antibody or fragment thereof which binds to the BAG3 protein and which comprises:

a) a heavy chain amino acid sequence as encoded by SEQ ID NO: 12 or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 80% thereof, and

b) a light chain amino acid sequence as encoded by SEQ ID NO: 20 or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 80% thereof. As used herein, "sequence identity ¹¹ between two polypeptide sequences, indicates the percentage of amino acids that are identical between the sequences, preferably over the entire length of the amino acid sequences as encoded by SEQ ID NO: 12 and SEQ ID NO: 20. Preferred polypeptide sequences of the invention have a sequence identity of at least 85%, more preferably 90%, even more preferably 93%, 95%, 96%, 97%, 98% or 99%.

In a preferred embodiment of the present invention said amino acid sequence having a sequence identity of at least 80% with respect to SEQ ID N. 12 is selected from SEQ ID N. 14, SEQ ID N: 16 or SEQ ID N. 18.

In a further preferred embodiment said amino acid sequence having a sequence identity of at least 80% with respect to SEQ ID N. 20 is selected from SEQ ID N. 22, SEQ ID N: 24 or SEQ ID N. 26.

In a preferred embodiment the antibody of the present invention is the antibody wherein the heavy chain amino acid sequence is encoded by SEQ ID NO. 18 and the light chain amino acid sequence is encoded by SEQ ID NO 22 or SEQ ID N. 26.

In a preferred embodiment, the heavy chain amino acid sequence or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 80% thereof, comprises the CDRs regions having the following amino acid composition: H-CDR1 comprises the amino acids GFNIKDTYMY (SEQ ID N. 3), H-CDR2 comprises the amino acids GVDPANGNTRYDPKFQG (SEQ ID N. 4), H-CDR3 comprises the amino acids DGAMDY (SEQ ID N. 5) and the light chain amino acid sequence or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 80% thereof, comprises the CDRs regions having the following amino acid composition: L-CDR1 comprises the amino acids KSSQSLLYSSNQKNYLA (SEQ ID N. 6), L-CDR2 comprises the amino acids WASTRES (SEQ ID N. 7) and L-CDR3 comprises the amino acids QQYYTYPLT (SEQ ID N. 8).

A further embodiment of the present invention, is an antibody or a fragment thereof which binds to the BAG3 protein and which comprises:

a) a heavy chain nucleotide sequence as encoded by SEQ ID NO: 11 or at least the variable domain thereof or a nucleotide sequence having a sequence identity of at least 80% thereof, and

b) a light chain nucleotide sequence as encoded by SEQ ID NO: 19 or at least the variable domain thereof or a nucleotide sequence having a sequence identity of at least 80% thereof. As used herein, "sequence identity" between two nucleotide sequences, indicates the percentage of nucleotides that are identical between the sequences, preferably over the entire length of the nucleotide sequences as encoded by SEQ ID NO: 11 and SEQ ID NO: 19. Preferred nucleotide sequences of the invention have a sequence identity of at least 85%, more preferably 90%, even more preferably 93%, 95%, 96%, 97%, 98% or 99%.

In a preferred embodiment of the present invention said nucleotide sequence having a sequence identity of at least 80% with respect to SEQ ID N. 11 is selected from SEQ ID N. 13, SEQ ID N: 15 or SEQ ID N. 17.

In a further preferred embodiment said amino acid sequence having a sequence identity of at least 80% with respect to SEQ ID N. 19 is selected from SEQ ID N. 21, SEQ ID N: 23 or SEQ ID N. 25.

In a preferred embodiment the antibody of the present invention is the antibody wherein the heavy chain amino acid sequence is encoded by SEQ ID NO. 17 and the light chain amino acid sequence is encoded by SEQ ID NO 21 or SEQ ID N. 25.

The antibody or fragments thereof according to the present invention may be any antibody of natural and/or synthetic origin, e.g. an antibody of mammalian origin. Preferably, the constant domain, if present, is a human constant domain. The variable domain is preferably a mammalian variable domain, e.g. a humanized or a human variable domain.

Antibodies or fragments thereof according to the invention may be polyclonal or monoclonal antibodies. Monoclonal antibodies are preferred. In particular the antibodies of the present invention are preferably selected from the group consisting of recombinant antibodies, humanized or fully human antibodies, chimeric antibodies, multispecific antibodies, in particular bispecific antibodies, or fragments thereof.

Monoclonal antibodies may be produced by any suitable method such as that of Kohler and Milstein (1975) or by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using techniques described in Clackson et al. (1991). Humanized forms of the antibodies may be generated according to the methods known in the art, (Kettleborough C.A. et al., 1991), such as chimerization or CDR grafting. Alternative methods for the production of humanized antibodies are well known in the art and are described in, e.g., EP 0239400 and WO 90/07861. Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display, yeast display, and the like.

According the present invention "chimeric antibody" relates to antibodies comprising polypeptides from different species, such as, for example, mouse and human. The production of chimeric antibodies is described, for example, in WO 89/09622.

The term antibody includes "fragments" or "derivatives", which have at least one antigen binding site of the antibody.

According to a preferred embodiment the antibody or fragment thereof may be a Fab fragment, a Fab' fragment, a F(ab') fragment, a Fv fragment, a diabody, a ScFv, a small modular immunopharmaceutical (SMIP), an affibody, an avimer, a nanobody, a domain antibody and/or single chains.

The antibody of the invention may be preferably of the IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD, and IgE antibody-type. It will be appreciated that antibodies that are generated need not initially possess such an isotype but, rather the antibody as generated can possess any isotype and that the antibody can be isotype-switched.

A further embodiment of the present invention is a vector comprising the nucleic acid coding for the antibody of the invention. Said vector is selected from a phage, a plasmid, a viral or a retroviral vector. Preferably, the vector of the invention is an expression vector wherein the nucleic acid molecule is operatively linked to one or more control sequences allowing the transcription and optionally the expression in prokatiotic and/or eukaryotic host cells.

A further embodiment of the present invention is a host comprising the vector of the invention, selected from a prokaryotic or eukaryotic cell, preferably a mammalian or a human cell, or a non-human transgenic animal.

A further embodiment of the present invention is a method for the preparation of the antibody or a fragment thereof disclosed above, comprising culturing the host of the invention under conditions that allow synthesis of said antibody and recovering said antibody from said culture.

A further embodiment is an antibody or a fragment thereof obtained by the method disclosed above.

Preferably, the antibodies of fragments thereof according to the present invention are humanized antibodies.

A further embodiment of the present invention is the use of the aforesaid antibody or a fragment thereof as medicament, preferably in the treatment of a particular pathological state which involves the activation of macrophages. Such pathological state can be chosen from: neoplastic diseases, inflammatory diseases, immune diseases or degenerative diseases.

Preferably, such neoplastic diseases may be either pancreatic tumour or bladder tumor, more preferably pancreatic tumour. Preferably, said inflammatory diseases can be chosen from diseases related to inflammation of the skin, nerves, bones, blood vessels or connective tissues, and more preferably, psoriasis, arthritis, neuritis or connectivitis.

Preferably, said immune diseases can be chosen from autoimmune diseases such as rheumatic diseases, connective tissue diseases, neuromuscular diseases, endocrine diseases, gastrointestinal diseases, haematologic diseases, skin diseases or vasculitis, and more preferably, rheumatoid arthritis, multiple sclerosis, connectivitis, lupus erythematosus, endometriosis or ulcerative colitis.

Preferably, said degenerative diseases can be chosen from neurodegenerative diseases and muscular degenerative diseases, and more preferably Alzheimer's disease, Parkinson's disease, or muscular dystrophy.

A further aim of the present invention is a pharmaceutical composition comprising the aforesaid antibody or fragment thereof in association with at least one pharmaceutically acceptable excipient or carrier.

A further embodiment of the present invention is the use of said composition as a medicament.

A preferred embodiment of the present invention is the use of the composition in the treatment of neoplastic diseases and diseases of an inflammatory, immune and/or degenerative nature, preferably neoplastic disease, selected from pancreatic tumor or bladder tumor.

The composition of the present invention can be formulated in a form suitable for oral administration or in a form suitable for parenteral or topical administration.

In a preferred embodiment of the present invention, said oral form can be chosen from the following: tablets, capsules, solutions, suspensions, granules and oily capsules.

In a further preferred embodiment of the present invention, said topical form can be chosen from the following: cream, ointment, ointment, solution, suspension, eye drops, pessary, nebuliser solution, spray, powder, or gel.

In a further preferred embodiment of this invention, said parenteral form can be either an aqueous buffer solution or an oily suspension. Said parenteral administration include administration by intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intranodal, or intrasplenic means.

Preferably the pharmaceutical composition according the present invention comprises a further active principle, selected from antimetabolites, camptothecins or taxanes.

More preferably said active principles are selected from: Gemcitabine, 5-fluorouracil,

Irinotecan, Oxaliplatin, Albumin-bound paclitaxel, Capecitabine, Cisplatin, Paclitaxel,

Docetaxel or Irinotecan liposome.

EXAMPLES

Example 1 - Chimerization and humanization of AC2 antibody.

AC-2 murine antibody is produced by a hybridoma isolated from the hybridoma mother clone n° PD02009 deposited on the 17/12/2002 at the Centra Biotecnologie Avanzate di Genova and disclosed in WO03/0S5908.Total RNA was extracted and RT-PCR performed to clone and sequence the variable regions of the antibody using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).

Based on sequence information of the variable region, heavy chain and light chain of AC-2 murine antibody (SEQ ID No. 1 and SEQ ID N. 2 for the amino acid sequences and SEQ ID 9 and SEQ ID N. 10 for the nucleotide sequences), different humanized variants of said region have been obtained by gene synthesis using standard procedures.

Sequences coding for the antibody variants were cloned in Evi-5 expression vector (Evitria AG, Switzerland) and expressed in CHO-K1 cells.

For antibody chimerization, the murine constant regions were replaced with the human constant regions. One chimeric versions of the heavy chain (HC) was made in an IgGl context.

For antibody humanization, Complementarity Determining Regions (CDRs) from the murine were grafted into a human antibody framework.

Twenty four humanized versions of the heavy chain (HC) were made in an IgGl and LC- kappa context. Each version is characterized by specific point mutations in the FR.

Example 2 - Screening for antibody binding activity Chimeric Ab and humanized variants were tested in parallel for the ability to bind to BAG3, using an indirect ELISA test.

Material and Methods

96 well microplate (NUNC Maxisorp) were coated using a specific sequence (Pep2) within the BAG3 full-length protein (from aa 385 to aa 399) recognized by the murine antibody (AC-2). The plate was incubated overnight with 1μg/ml of Pep2 (SO μΐ/well) in a phosphate buffer (PBS, pH 7.4). Then, the plate was washed two times with a detergent solution (0,05% Tween-20 in PBS), and the blocking of non-specific sites was performed for one hour at room temperature using, for each well, 150 μΐ of a solution of 0.5% fish gelatin (Sigma) in phosphate buffer (PBS, pH 7.4). Plates were washed two times with the washing buffer and then the antibodies were loaded on. In some of the wells, scalar dilutions of chimeric and humanized variants of the antibody, 500 ng/ml (50 μΐ/well) were loaded in duplicates. Antibodies were diluted in a solution of 0.5% fish gelatin, 0.05% Tween in PBS (BSA/Tween). The plate was incubated for 2h at room temperature and then washed for 5 times with washing buffer. Samples were then incubated 30 minutes at room temperature with 50 μΐ/wetl of HRP-conjugated anti-mouse IgG or HRP-conjugated anti-human IgG as the secondary antibody. After 6 washes with the washing buffer, the peroxidase substrate, tetramethylbenzidine (TMB), was added to the wells (50 μΐ/well). The colorimetric reaction was blocked after 10 minutes by addition of sulfuric acid 0.5 M (25 μΐ/well) and the optic density values (OD) were detected using a spectrophotometer at the wavelength of 450nm (Figure 1). Chimeric and humanized variants of the antibody were tested also using plates coated with recombinant BAG3 full-length protein (rBAG3) by indirect ELISA test using the same protocol described above (Figure 2).

Results

Binding of the different antibody variants was detected in ELISA using peptide 2 as coating (Fig.l) or recombinant full length BAG3 (Fig. 2). The following antibody variants showed higher binding ability and were chosen for further analysis: H1L2, H1L3, H1L4, H2L2, H2L3, H2L4, H3L2, H3L3, H3L4, H4L2, H4L3, H4L4.

Example 3 - Humanized Antibody variants KD determination Materials and Methods

Binding experiments were performed on Octet Red96 at 2S°C. Antibodies were captured on dip and read AHC (anti-human IgG Fc capture) sensors, followed by binding of Ag (E. Coli rBAG3) at variable concentrations. Binding of antigen to the antibodies was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (KD) was calculated from the observed ka and kd.

Full kinetic analysis was performed using analyte concentrations from ΙΟΟηΜ to 0. The analysis was performed using analyte concentrations of 100 nM and run with serial dilutions in assay buffer, 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0 nM. Chi square (χ ² ) analysis was carried out between the actual Sensorgram (colored line) and the sensorgram generated from the Fortebio Octet analysis software (red line) to determine the accuracy of the analysis. 2 value within 1-2 is considered significant (accurate) and below 1 is highly significant (highly accurate).

Note: 2x 8 sensors were used for 1 full kinetic analysis, sensors were regenerated and used multiple times and captured with BAG-H-L, BAG-H1-L2, BAG-H1-L3, BAG-H1-L4, BAG- H2-L2, BAG-H2-L3, BAG-H2-L4, BAG-H3-L2, BAG-H3-L3, BAG-H3-L4, BAG-H4-L2, BAG-H4-L3, BAG H4-L4 to perform 13 full kinetics.

Results

Affinity for the different variants calculated as above is reported below. The AC -2 murine antibody showed a KD (M) of 10 ^'12 while all human variants showed a KD (M) of 10 ^'9 (see Table 1). However, this difference did not impact on variants biological activity as will be described.

Table 1. Affinity of AC-2 antibody variants

Example 4 - Evaluation of the ability of the antibody variants to block BAG3 binding to macrophage surface

BAG3 binds to the surface of macrophages and we have shown that binding is specifically inhibited by the murine anti-BAG AC-2 antibody that sequesters BAG3 protein. Here we tested the ability of chimeric and humanized variants of the antibody to block binding to the macrophage surface.

Materials and Methods

J774 A.1 cells (1 x 106/ml) were incubated with blocking solution (PBS containing 5% FBS/0.1% NaN3) and with FcR blocking mouse (Miltenyi Biotec-cod. 130-092-575) (1μΙ/1χ106 cells) for 30 minutes at 4°C. Then, 1x105 cells were incubated with FITC-rBAG3 protein (40 μg/ml) alone or in presence of anti-BAG3 mouse antibody (AC2) (3200 μg/ml) or murine IgGl (3200 μg/ml), or with the chimeric and humanized variants of the antibody (3200 gg/ml), or human IgG (3200 μg/ml) in blocking solution, for 30 minutes on ice. After incubation, cells were washed with PBS and analyzed by flow cytometry.

Results

Flow cytometric evaluation of fluorescent BAG3 binding to the surface of J774 A.1 macrophage cell line. Data are reported as mean fluorescence intensity (Figure 3). On the basis of these results, the H4L2, H4L3, H4L4 and H2L4 humanized antibody variants were chosen. Example 5 - Evaluation of the ability of humanized AC-2 antibody variants to block primary monocyte activation

The H4L2, H4L3, H4L4 and H2L4 humanized antibody variants were selected, purified by means of Protein A capture (HiTrap Protein A HP, GE Healthcare), and further tested for their ability block activation of primary human monocytes.

We have previously shown that BAG3 can activate primary human macrophages. Here we use IL6 secretion as a read out of macrophage activation to show the ability of the H4L2, H4L3, and H4L4 humanized antibody variants to block BAG3 dependent monocyte activation.

Materials and methods

Buffy Coats (CompoFlex® Triple "Top and Top" System -Fresenius Kabi) were obtained from healthy donors and stored overnight at 4°C. PBMC were isolated on standard Ficoll- Hypaque density gradients. Mononuclear cells were isolated and subjected to sequential washes with SO ml of PBS IX in order to eliminate platelets and erythrocytes contaminations; (I: at 1 lOOg for 20 mins; II: at 800g for 10 mins; III: at 400g for 10 mins; IV: at 300g for 10 mins; V and VI: at lOOg for 10 mins). All centrifugations were made with the brake off. Cells were then checked under the microscope and if contaminations were still present, an additional centrifugation at lOOg for 10 mins was performed.

PBMC were then plated in 96 well microplates (2X10 ⁶ cells/ml) and cultured in RPMI without glutamine (ΙΟΟμΙ/weli) without FBS and with FcR Blocking Reagent human (1 μΐ every 1 million of cells) (Miltenyi 130-059-901) for 16 hours (overnight).

The day after, cells were treated for 6h with with rBAG3 (0.S μg/ml). mAb blocking assays were performed incubating the rBAG3 together with different concentrations of AC-2 or the humanized variants.

Treatment was performed without changing the media to the cells, but adding ΙΟΟμΙ of RPMI without FBS to each well containing molecules described above. All molecules were added to 100 μΐ of RPMI without FBS in a sterile tube and molecules were added at twice of their final concentration to the cells. Molecules were pre-incubated for 30 minutes at room temperature in the tubes and then added to the cells. Pre-incubation is necessary for mAb blocking assays. After treatment cell culture medium was collected after centrifugation at 400g for S minutes. Surnatants were analyzed for IL-6 contents in duplicate at 1 : 10 dilution with a human ELISA IL-6 Kit (eBioscience, San Diego, CA). IL-6 concentration were evaluated by comparing the OD of the sample with that of a standard curve of recombinant IL-6.

Results

Results reported in Table 2 are expressed as % of inhibition of IL-6 production induced by rBAG3 by the different antibodies. All variants show inhibitory ability comparable to the murine AC2 anti BAG3 antibody. H4L2 and H4L4 were chosen for further testing in vivo, for their ability block tumor growth in a xenograft tumor model.

Table 2

Example 6 · Effect of humanized AC-2 antibody variants on tumor growth in vivo

Based on in vitro experiments we choose variant H4L2 and H2L4 for the subsequent in vivo validation.

Materials and methods

Mia-Paca 2 cells (2x1ο ⁶ ) were suspended in PBS (200 μΐ) and injected into the right flank of female CD1 mice (6 weeks old; Charles River, Italy). After 10 days mice were divided in four arms consisting of 7 (control) or 6 (treated) mice each in which tumor volume average ranged 80-100 mm3. Control group received vehicle (PBS) i.p. injection every 48 hrs whilst treated groups received i.p. injection every 48 hrs of murine (AC2) or humanized H2L4, H4L2 anti- BAG3 mAbs at the dose of 20 mg/kg in PBS for S weeks. Animals were weighted and tumor volume measured by caliper (Dxd ² /2) once weekly.

Results

Figure 4 reports tumor fold change at week intervals of control (PBS) or animals treated with the indicated antibodies. The ratio is between the average volume at the indicated time point and the average volume at day 0. While tumors of control treated animals show a 4 fold increase, treated animals only increase by 2.S fold.

Example 7 - Impact of humanized AC-2 H2L4 variant on activated fibroblast number within the tumor mass.

Results obtained on tumor growth prompted us to further investigate changes in tumor microenvironment. One of the major player in tumor development are fibroblasts associated to the tumor mass.

Materials and methods

At the end of the experiment described in example 6 tumors were paraffin embedded and sections analysed by immunofluorescence using an anti- a sma antibody (A2S47, Sigma- Aldrich, at 1:350). Nuclei were counterstained with ^g ml ^'1 Hoechst 33342 (Molecular Probes, Oregon, USA). Images were acquired in sequential scan mode by using the same acquisitions parameters (laser intensities, gain photomultipliers, pinhole aperture, objective * 40, zoom 1) when comparing experimental and control material. Leica Confocal Software and ImageJ were used for data analysis (% of a-sma positive area).

Results

Figure 5 show representative images from 2 PBS- treated and 2 AC-2 H2L4 treated tumors of a-sma staining. The presence of activated fibroblasts associated to the masses grown in the control animals is very high as demonstrated by a-sma protein positivity. Instead, AC-2 H2L4 treated animals are negative. In Figure 6 we have reported a quantification of a-sma positive area by comparing control (PBS-treated) and AC-2 H2L4 treated samples.

These data strongly support the evidence that humanized anti-BAG3 antibodies have an effect on fibroblasts that have an important role not only in neoplastic diseases but also in other pathological processes such as inflammation (Kalluri, 2016).

References

- Ammirante M, Rosati A, Arra C, Basile A, Falco A, Festa M, Pascale M, d'Avenia M, Marzullo L, Belisario MA, De Marco M, Barbieri A, Giudice A, Chiappetta G, Vuttariello E, Monaco M, Bonelli P, Salvatore G, Di Benedetto M, Deshmane SL, Khali li K, Turco MC, Leone A. "IKK {gamma} protein is a target of BAG3 regulatory activity in human tumor growth". Proc Natl Acad Sci U S A. 2010; 107(16): 7497-502.

- Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature 1991 ; 15;352(6336):624-8.

- Festa M, Del Valle L, Khalili K, Franco R, Scognamiglio G, Graziano V, De Laurenzi V, Turco MC, Rosati A. "BAG3 protein is overexpressed in human glioblastoma and is a potential target for therapy". Am J Pathol. 2011 ; 178(6):2504- 12.

- Franceschelli S, Rosati A, Lerose R, De Nicola S, Turco MC, Pascale M. "Bag3 gene expression is regulated by heat shock factor 1". J Cell Physiol. 2008; 215(3):575-7.

- Kalluri R. The biology and function of fibroblasts in cancer. Nature Reviews Cancer.

2016; 16, 582-598

- Kettleborough CA, Saldanha J, Heath VJ, Morrison CJ, Bendig MM. "Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation". Protein Eng. 1991 ; 4(7):773-83.

- Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-497.

- Rosati A, Graziano V, De Laurenzi V, Pascale M, Turco MC. "BAG3: a multifaceted protein that regulates major cell pathways". Cell Death Dis. 2011 Apr 7;2:el41.

- Rosati A, Bersani S, Tavano F, Dalla Pozza E, De Marco M, Palmieri M, De Laurenzi V, Franco R, Scognamiglio G, Palaia R, Fontana A, di Sebastiano P, Donadelli M, Dando I, Medema JP, Dijk F, Welling L, di Mola FF, Pezzilli R, Turco MC, Scarpa A. "Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival". Am J Pathol. 2012 Nov; 181 (5): 1524-9.

- Rosati A, Basile A, D'Auria R, d'Avenia M, De Marco M, Falco A, Festa M, Guerriero L, lorio V, Parente R, Pascale M, Marzullo L, Franco R, Arra C, Barbieri A, Rea D, Menichini G, Hahne M, Bijlsma M, Barcaroli D, Sala G, di Mola FF, di Sebastiano P, Todoric J, Antonucci L, Corvest V, Jawhari A, Firpo MA, Tuveson DA, Capunzo M, Karin M, De Laurenzi V, Turco MC. "BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages". Nat Commun., 6:8695 doi: 10.1038/ncomms969S.