COMBINATION OF HER2/NEU ANTIBODY WITH HEME FOR TREATING CANCER

Abstract

The present invention relates to a method of treating HER2/NEU overexpressing cancers. The inventors discovered that the heme-mediated formation of dimers and in general oligomers of Trastuzumab is associated with an improved complement-mediated cytotoxicity on breast cancer cells. The present data highlight that the sensitivity to heme of Trastuzumab, may have major repercussion on its therapeutic activity. Thus the invention relates to the combination of an HER2/neu antibody with a heme and/or of its oligomers and its therapeutic composition in the HER2/NEU characteristic cancer treatment.

Claims

1. A combination of an HER2/neu antibody with a heme.

2. The combination according to claim 1 wherein the heme is selected from the group consisting of heme a, heme c, heme d, heme d.sub.1, heme o, heme P460, siroheme, and a Fe porphyrine.

3. The combination according to claim 1 wherein the HER2/neu antibody is Transtuzumab.

4. The combination according to claim 1 wherein the HER2/neu antibody and heme form oligomers.

5. A chimeric antigen receptor which comprises at least one VH and/or VL sequence of the HER2/neu antibody combined with a heme.

6. The chimeric antigen receptor of claim 5 which further comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

7. The chimeric antigen receptor of claim 5 comprising an antigen-binding domain comprising a single chain variable fragment (scFv) of the HER2/neu antibody.

8. (canceled)

9. (canceled)

10. The method of claim 14, wherein the combination or the chimeric antigen receptor is administered with at least one additional anti-cancer agent.

11. The method of claim 14, wherein the cancer is selected from the group consisting of breast cancers, cervical cancers, cholangiocarcinomas, extrahepatic colorectal cancers, intrahepatic colorectal cancers, esophageal and esophagogastric junction cancers, gallbladder cancer, gastric adenocarcinomas, head and neck carcinomas, hepatocellular carcinomas, intestinal (small) malignancies, lung cancer (non-small cells), melanomas, ovarian (epithelial) cancers, ovarian (non-epithelial) cancers, pancreatic adenocarcinomas, prostate cancers, unknown primary cancers, uterine cancers, testicular cancers, salivary duct carcinomas, colon cancer and bladder cancer.

12. A therapeutic composition comprising an HER2/neu antibody, a heme and/or a chimeric antigen receptor and at least one excipient.

13. (canceled)

14. A method for treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a combination of a HER2/neu antibody and a heme and/or a chimeric antigen receptor which comprises at least one VH and/or VL sequence of an HER2/neu antibody combined with a heme.

15. The combination according to claim 2 wherein the Fe porphyrine is Fe (III) mesoporphyrin IX, Fe (III) protoporphyrin IX, Fe (III) deuteroporphyrin IX, Fe (III) hematoporphyrin IX, or Fe(III) coproporphyrin I.

Description

FIGURES

[0092] FIG. 1: Exposure of Trastuzumab to heme results in broadening of antigen-binding reactivity. As evident on the graphs the antibody demonstrated heme concentration-dependent binding to structurally unrelated protein antigens (Thyroglobulin, Factor IX, hemoglobin, myoglobin, cytrochrome c). The reactivity of Trastuzumab to insulin is not affected by heme.

[0093] FIG. 2: Self-binding potential of native and heme-exposed Trastuzumab. Evaluation of the binding of native and heme exposed Trastuzumab to immobilized Trastuzumab by ELISA.

[0094] FIG. 3: Functional activity of heme-exposed Trastuzumab. Direct cytotoxicity and complement-dependent cytotoxicity of native and heme-exposed Trastuzumab on human breast cancer cells MDA MB-231 cells (left) and SKBR3 (right). The survival of cancer cells pre-treated or not with Trastuzumab was evaluated by using WST-1 metabolic dye in absence and presence of complement. Each point represents percentage of dead cells from triplicate wells. Here is depicted a representative example of three independent experiments.

[0095] FIG. 4: Functional activity of heme-exposed Trastuzumab. Complement-dependent cytotoxicity of separated fractions of heme-exposed Trastuzumab on human breast cancer cell line SKBR3. The different fractions of heme-exposed Trastuzumab were separated by size-exclusion chromatography. Each point represents percentage of dead cells from triplicate wells. Here is depicted a representative example of three independent experiments. Statistical analyses were performed by using Mann-Whitney test, star indicate: **:p<0.01; ***p<0.001; ****p<0.0001. HM—high molecular weight.

[0096] FIG. 5: Deposition of C3 on the surface of breast cancer cells after incubation with native and heme-exposed Trastuzumab. The antibody at 1 mg/ml was first pre-treated with 14 μM heme; after the tumour cells at density of 1×10.sup.6 were incubated with 10 μ/ml of native and heme-treated antibody. As a source of complement 50% human serum was applied. Following washing of the cells the deposition of activation fragments of C3 component was detected by specific antibody and flow cytometer.

EXAMPLE

Material & Methods

Immunoblot

[0097] Lysates of Bacillus anthracis or human umbilical vein endothelial cells (HUVEC) were loaded on a 4-12% gradient NuPAGE Novex electrophoresis gel (Invitrogen, Carlsbad, Calif.). After migration, proteins were transferred on nitrocellulose membranes (iBlot gel transfer stacks, Invitrogen) by using iBlot electrotransfer system (Invitrogen). Membranes were blocked overnight at 4° C. in TBS containing Tween 0.1% (TBS-T). Next, the membranes were mounted on Miniblot system (Immunetics, Cambridge, Mass.) and incubated with Trastuzumab (0.2 μM) pre-treated with increasing concentrations of heme (0.15-20 μM) and then incubated for 2 h, at 22° C. Membranes were washed with TBS-T for 1 h before being incubated for 1 h with an alkaline phosphatase conjugated goat anti-human IgG (Southern Biotech, Birmingham, Ala.). Membranes were then washed again for 1 h before revealed with ready-to-use NBT/BCIP substrate solution (KPL Systems, USA).

ELISA

1) Evaluation of Reactivity of Therapeutic Abs to a Panel of Antigens After Heme Exposure

[0098] Ninety-six well microtiter polystyrene plates (NUNC Maxisorp, Roskilde, Denmark) were coated with various antigens—human insulin; human hemoglobin, porcine thyroglobulin; horse cytochrome C; horse myoglobin (all from Sigma-Aldrich, St. Louis, Mo.), and human factor IX (LFB, France), at 10 μg/mL for 2 hours at room temperature. The plates were blocked with PBS containing 0.25% Tween 20 for 1 hour. Trastuzumab or Rituximab was treated at 2 μM in PBS with increasing concentrations of heme (0, 0.078-20 μM) for 5 minutes on ice. IgG was then diluted ten fold (0.2 μM final concentration) in PBS containing 0.05% Tween 20 (PBS-T) and incubated with immobilized antigens for 2 h at room temperature. After washing with PBS-T the plates were incubated for 1 hour with peroxidase-conjugated mouse anti-human IgG (clone JDC-10, Southern Biotech). Immunoreactivities were revealed using the o-phenylenediamine substrate (Sigma-Aldrich).

2) Evaluation of Induction of Antibody Homophilicity by Heme

[0099] Ninety-six well microtiter polystyrene plates (NUNC) were coated with Trastuzumab at 10 μg/mL for 2 hours at room temperature. The plates were blocked with PBS 0.25% containing Tween 20 for 1 hour. Trastuzumab that was biotinylated using EZ-Link™ NHS-LC-Biotin (ThermoFisher Scientific) was treated at 6.7 μM in PBS with 13.7 μM of heme (hemin, Sigma-Aldrich) for 5 minutes on ice. IgG was then diluted two times in PBS-T before incubation on plates for 2 h at room temperature. Following extensive washing, the plates were incubated for 30 min with streptavidin-HRP (Southern Biotech). After extensive washing with PBS-T, the immunoreactivities were revealed using the o-phenylenediamine substrate (Sigma-Aldrich).

[0100] For the evaluation of induction of antibody homophilicity by heme in low ionic strength conditions, same ELISA was performed using low salts concentration buffer (NaCl 15 mM) for the treatment of Trastuzumab with heme. To evaluate the specificity of heme, this method was performed using an analog of heme, zinc protoporphyrin (ZnPP). Trastuzumab was incubated at 6.7 μM in PBS with increasing concentrations of heme or ZnPP (0-64 μM). IgG was then diluted ten times in PBS-T before incubation on the plates for 2 h at room temperature.

Protein Microarray Analyses

[0101] The bindings of Trastuzumab pre-incubated or not with heme were tested against more than 9000 human proteins (ProtoArray Human Protein Microarray v5.0, ThermoFisher Scientific, USA) using antibody specificity biomarker profiling protocol. First, the arrays were equilibrated at 4° C. for 15 min and then incubated with blocking buffer recommended by the manufacturer for 1 h at 4° C. on a circular shaker. After incubation, microarrays were washed once with PBS, 0.1% Tween 20, containing synthetic block (ThermoFisher Scientific) for 5 min. The monoclonal IgG1 (10 μM) was pre-treated or not with heme (20 μM). Following treatment Trastuzumab was further diluted to 33 nM (5 μg/ml) and added in the chamber containing the arrays. Following 90 min of incubation at 4° C. on a circular shaker, each array was washed 5 times for 5 min each at 4° C. To detect Trastuzumab, an Alexa Fluor 647 goat anti-human IgG antibody (ThermoFisher Scientific) was added to the incubation tray at 1 μg/ml for 90 min at 4° C. on a circular shaker. As described previously, the arrays were washed 5 times before being centrifuged in a 50 ml tube at 200×g for 1 minute at room temperature. Scanning of the arrays was performed on the next day with a GenePix 4000B Microarray Scanner. Fluorescence data were acquired by aligning the Genepix Array List onto the microarray using Genepix Pro analysis software. The resulting Genepix Results (GPR) files were imported into Invitrogen's Prospector 5.2 for further analysis.

Real-Time Kinetic Analyses

1) Evaluation of the Binding of Heme to Trastuzumab

[0102] The binding kinetics and thermodynamics of interaction of Trastuzumab with heme was determined by surface plasmon resonance-based technique (BIAcore 2000, Biacore GE Healthcare, Sweden). Trastuzumab was immobilized on a CMS sensor chip (Biacore) by using amine-coupling kit provided by the manufacturer. The Ab was diluted in 5 mM maleic acid (pH 4) to a final concentration of 10 μg/ml. The achieved immobilization level was 6300 resonance unit (RU). All measurements were performed using HBS-EP (0.01M HEPES, pH 7.4 containing 0.15 M NaCl, 3 mM EDTA and 0.05% Tween 20). Initially, a stock solution of heme (hemin, Frontiers Scientific, Logan, Utah) at 1 mM was prepared in 0.05 N NaOH. Heme was further diluted to 10 μM in the running buffer and 8 two-fold dilutions (10-0.078 μM) were injected at flow rate of 30 μl/min. The association and dissociation phases of the interaction were monitored for 5 min and 10 min, respectively. The binding to the surface of the reference flow cell was subtracted from the binding to the Ab-coated flow cells. The regeneration of the bound-heme was achieved by exposure of the sensor surface to 300 mM imidazole. All binding analyses were performed at temperatures of 5, 10, 15, 20, 25, 30 and 35° C. The BIAevaluation version 4.1 software (Biacore) was used for the estimation of the kinetic rate constants. Calculations were performed by global analyses of the experimental data using the Langmuir binding with drifting base-line model included in the software.

[0103] To evaluate which portion of the Ab was binding to heme, Fab fragments, Fc fragments and the intact Trastuzuman were immobilized on a CMS sensor chip as described above. The achieved immobilization levels were 1500 RU for Fab fragments, 1450 RU for Fc fragments and 4300 RU for the intact Trastuzumab. Heme was diluted at 10 μM in HBS-EP and injected at flow rate of 30 ul/min. The association and dissociation phases of the interaction were monitored for 5 min and 10 min, respectively. Analysis was performed as described above.

2) Evaluation of the Interaction of Heme-Exposed Trastuzumab to Native Trastuzumab

[0104] Trastuzumab was immobilized on a CMS sensor chip (Biacore) by using amine-coupling kit (Biacore) after dilution in 5 mM maleic acid (pH 4) to a final concentration of 10 μg/ml. The achieved immobilization level was 5600 RU. All experiments were performed using HBS-EP. Trastuzumab was diluted to 10 μM in PBS and treated with 20 μM heme. After five minutes incubation on ice, two-fold dilutions of heme-exposed Trastuzumab (1000-1.95 nM) were injected at flow rate of 30 μl/min. The association and dissociation phases of the interaction were monitored for 4 min and 5 min, respectively. The binding to the surface of the reference flow cell was subtracted from the binding to the proteins-coated flow cells. The regeneration of the bound-IgG was achieved by exposure of the sensor surface to 150 mM imidazole. The real-time interaction analyses were performed at 10, 15, 25 and 35° C. The BIAevaluation version 4.1 software (Biacore) was used for the estimation of the kinetic rate constants. Calculations were performed by global analysis of the experimental data using the Langmuir binding with drifting base-line model included in the software.

3) Evaluation of the Binding of Heme-Exposed Trastuzumab to HER-2

[0105] Biotinylated HER-2 mimotope peptide [24] was immobilized on streptavidine (SA) sensor chip (Biacore) to a final concentration of 10 μg/ml. The achieved immobilization level was 500 RU. Trastuzumab (6.7 μM) was pre-treated with 13.404 heme. Native and heme-exposed Trastuzumab were diluted to 10 nM in the running buffer and 8 two-fold dilutions (10-0.078 nM) were injected at flow rate of 30 μl/min. The association and dissociation phases of the interaction were monitored for 5 min and 4 min, respectively. The binding to the surface of the reference flow cell was subtracted from the binding to the proteins-coated flow cells. The regeneration of the bound-Trastuzumab was achieved by exposure of the sensor surface to 1.5 M MgCl2. The BlAevaluation version 4.1 software (Biacore) was used for the estimation of the kinetic rate constants. Calculations were performed by global analysis of the experimental data using the Langmuir binding with drifting base-line model included in the software.

4) Evaluation of the Binding of Heme-Exposed Trastuzumab to FcRn

[0106] Recombinant human FcRn (kindly provided by Dr Sune Justesen, University of Copenhagen, Denmark) conjugated with biotin was immobilized on a straptavidin sensor chip (SA chip, Biacore) at a density of 1500 RU. Fc-γ fragments from Trastuzumab were generated by papain digestion. Native Fc-γ or Fc-γ treated in PBS at 12 μM with equimolar concentration of heme were diluted serially (two-fold each step) from 50 to 0.39 nM in 100 mM Tris-Citrate buffer pH 5.4, containing 0.1% Tween 20. The association and dissociation phases of the interaction were monitored for 4 min and 5 min, respectively. The sensor chip surfaces were regenerated by exposure to 100 mM Tris-Citrate buffer pH 7.4, containing 0.1% Tween 20 for 60 sec. All kinetic measurements were performed at temperature of 25° C. The evaluation of the kinetic data was performed by BIAevaluation version 4.1.1 Software (Biacore).

Thermodynamic Analyses

[0107] For evaluation of the activation thermodynamic parameters of the interactions between heme and Trastuzumab, as well as the interaction of heme-exposed Trastuzumab to native Trastuzumab, the Eyring's approach was applied. The kinetic rate constants obtained at different temperatures were used to build Arrhenius plots. The values of slopes of the Arrhenius plots were calculated by using a linear regression analysis of the experimental kinetic data and substituted in Equations 1-4,

Ea=−slope×R   (Eq. 1)

[0108] Where the “slope”=∂1n(ka/d/∂(1/T), and where Ea is the activation energy. The enthalpy, entropy, and Gibbs free energy changes characterizing the association phase were calculated using Equations 2-4,

ΔH=Ea−RT   (Eq. 2)

1n(ka/d/T)=ΔH/RT+ΔS/R+1n(k′/h)   (Eq. 3)

ΔG=ΔH−TΔS   (Eq. 4)

where T is the temperature in Kelvin, k′ is the Boltzmann constant, and h is Planck's constant. All activation thermodynamic parameters were determined at the reference temperature of 25° C. (298.15 K).

[0109] To evaluate the changes of the thermodynamic parameters at equilibrium following equation were applied—

Geq=G‡a−G‡d,

Heq=H‡a−H‡d,

TSeq=TS‡a−TS‡d

Size-Exclusion Chromatography

[0110] Molecular composition of the native and heme-exposed Trastuzumab was compared by using FPLC Akta Purifier (GE, Healthcare), equipped with Superose 6 10/300 column. IgG was diluted to 6.7 μM in PBS and exposed to 13.4 μM of heme or heme analogues. In another experiment, Trastuzumab (6.7 μM) was pre-treated with potassium cyanide (KCN, 10 mM final concentration) before treatment with heme (13.4 μM). One ml of each native or heme-exposed Ab was loaded on column equilibrated with the corresponding buffer. The flow rate of 0.5 ml/min was used. Chromatograms were recorded by using UV detection of protein at wavelength of 280 nm and at 400 nm for heme detection. The obtained fractions were collected separately in order to test their individual therapeutic effect subsequently.

Transmission Electron Microscopy

[0111] Trastuzumab was dialyzed to HBS buffer and diluted to 1 mg/ml (6.7 μM). Heme (1 mM stock in 0.05 N NaOH) was added to the Ab solution to final concentration of 13.4 μM. Different concentrations of native and heme-exposed Trastuzumab were first assessed—150 μg/ml; 30 μg/ml and 7.5 μg/ml. Six microliters of each sample are placed on a carbon-copper grid (300 mesh) for 1 min at room temperature after a standard glow discharge procedure (2 mA, 0.3 mBar, 40 sec). After adsorption, the excess is removed by blotting using a Whatman grade 5 paper. Grids are then stained with uranyl acetate 2%, one drop quickly and the second one for 1 minute at room temperature. They are finally blotted with Whatman grade 5 paper and air-dried. Specimen are then observed under a 200 kV F20 (FEI) transmission electron microscope and acquisition of the images is carried out using a 2 k×2 k USC1000 camera (GATAN). All observations are made at magnifications ×50 000 and ×62 000.

Absorbance Spectroscopy

[0112] Absorbance spectra were measured by using UNICAM Helios b, UV-vis spectrophotometer. Trastuzumab was diluted to 2 μM in PBS and titrated with increasing concentrations of heme (0.125-64 μM). Aliquots of heme stock solution (1 mM in 0.05 M NaOH) were added both to cuvette containing Trastuzumab and to a reference cuvette, containing PBS only. After addition of each heme aliquot and incubation for 2 min in dark, the absorbance spectra in the wavelength range 350-700 nm were recorded. The spectra were scanned at rate of 300 nm/min. All measurements were performed at room temperature, in quartz cuvettes with optical path of 1 cm.

Fluorescence Spectroscopy

[0113] Quenching of intrinsic tryptophan fluorescence of Trastuzumab by heme was measured by using Hitachi F-2500 fluorescence spectrophotometer (Hitachi Instruments Inc., UK). The Ab was diluted to 0.1 μM in PBS and titrated with increasing concentrations (0, 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2 μM) of heme, added as aliquots from a stock solution. A wavelength of 295 nm was used to selectively excite tryptophan residues. Excitation and emission slits were both adjusted to 10 nm. The emission spectra of Trastuzumab was measured in the wavelength range 300-450 nm, at scan speed of 300 nm/min. Quartz cuvette with 1 cm optical path was used in the experiment. All measurements were performed at room temperature.

Circular Dichroism

[0114] The CD spectra measurements were performed with JASCO-J710 Spectrometer. Data pitch and slit were both set to 1 nm. The data were recorded at scan speed on 10 nm/min in the range of 260-185 nm. Before the measurements Trastuzumab was dialyzed against 10 mM Na phosphate buffer pH 7.4 and diluted in the same buffer to a final concentration of 1 μM. The Ab was exposed or not to 10 μM concentration of heme. All spectra were acquired at 20° C. in quartz cuvettes with 1 mm optical path.

Molecular Docking

[0115] Molecular docking studies were performed using the Autodock (version 4.2.6) software tool [25]. The X-ray crystallographic structure of Trastuzumab was acquired from the protein data bank (PDB) at a resolution of 2.08 Å (4 HKZ). Protein L and protein A fragments were removed from the file. The model was prepared by adding Gasteiger charges and optimizing torsion angles, and saved in PDBQT format. All water molecules were removed from the macromolecule and polar hydrogen atoms were added. A blind molecular docking method was used to dock heme on Fab fragment of Trastuzumab. A second docking was made on variable region of Trastuzumab for more precise analysis. The first grid was generated around the whole structure, the second one was calculated based on following coordinates (X=−16.139, Y=−17.886 and Z=9.161) in order to encompass the entire variable site. Lamarckian genetic algorithm (LGA) was selected for freezing, docking with default parameters in Autodock. The ten best conformations were selected and their energies calculated.

Cell Lines

[0116] MDA-MB-231 cells were cultured in DMEM F-12 medium and supplemented with 10% heat-inactivated FBS, 1% Penicilin-Streptomycin. SKBR3 cells were culture in Mc Coy's 5A medium and supplemented with 10% heat-inactivated FBS, 1% Penicilin-Streptomycin. All the cells were maintained in an incubator at 37° C. with 5% CO2 and their viability was controlled by Trypan Blue.

Flow Cytometry—Evaluation of Trastuzumab Binding to Breast Cancer Cells

[0117] MDA-MB-231 or SKBR3 cells were blocked in medium 10% FCS for 15 minutes and washed in PBS for 10 minutes. Cells were re-suspended in PBS and treatments were added. Trastuzumab diluted at 10 mg/ml in PBS was treated or not with 50 μM heme. Cells were incubated with a final concentration of Trastuzumab of 10 μg/ml. As controls, same resulting quantities of heme were added in separate tubes. Moreover, another IgG1 (Rituximab, Roche) was treated with heme at the same concentrations as a control. The cells were treated for 1 hour at 37° C. before washed two times in PBS. To detect the bound IgG, FITC-conjugated rabbit anti-human IgG (Southern Biotech) was incubated for 30 minutes at room temperature. Cells were washed once with PBS before analyses by using LSRII BD Flow Cytometer (BD Immunocytometry Systems, San Jose, Calif.). A total of 10 000 gated events were analyzed per sample.

Direct and Complement-Mediated Cytotoxicity

[0118] Cells were treated on 96-wells plates with flat bottom and low evaporation lid (Coster, USA). MDA-MB-231 or SKBR3 breast cancer cells were plated and left for 2 hours to adhere. Trastuzumab diluted to 1 mg/ml was treated or not with 20 μM heme. The Ab was diluted to a final concentration of 3.7 μg/ml. As a source of complement, baby rabbit complement (AbD Serotec) was added to obtain a dilution of ×5 (MDA-MB-231 cells) or ×8 (SKBR3 cells). As control, heat inactivated fetal calf serum was added at the same ratios. To evaluate the amount of alive cells, WST-1 dye (Roche) was added after 5 days of incubation at 37° C. The absorbance at 450 nm and 690 nm was read after 2 hours incubation at 37° C.

Antibody-Mediated Cellular Cytotoxicity

[0119] PBMCs were isolated from healthy donor blood (Établissement Français du Sang [EFS] Cabanel, Paris, France) by density gradient using Ficoll-Paque Plus (GE-Healthcare, UK), and used as a source of NK cells. PBMCs were seeded in a 24-wells plate and stimulated with IL-2 (100 UI/ml) overnight at 37° C. and 5% CO2 in RPMI 1640 medium with L-glutamine and 10% of heat-denatured FBS (Gibco, Life Technologies, USA). The next day, 5000 breast cancer cells/well prepared in FBS-free medium were transferred to a 96-well plate with round bottom. Trastuzumab (1 mg/ml) was pre-incubated or not with heme (20 μM) and was then diluted to 10 μg/ml before being added to the cancer cells. The plate was incubated for 30 min at 37° C. PBMCs were then mixed with cancers cells at different E:T ratios (from 60:1 to 3.75:1) and co-cultured for 4 h at 37° C. After the incubation, the cytotoxic effects of PBMCs on cancer cells were determined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega).

Results

Heme Induces Antigen-Binding Polyreactivity of Trastuzumab

[0120] Previously, it was reported that Trastuzumab acquires reactivity towards protein and lipid autoantigens after heme exposure [19]. We first aimed to characterize the extent of the new binding specificity of heme-exposed Trastuzumab. To this end, we compared the reactivity of native and heme-exposed Trastuzumab to bacterial and endothelial antigens using immunoblot analyses (Data not shown). The results showed that exposure to heme results in a concentration-dependent increase of binding to the bacterial and autoantigens. These results were further supported by ELISA assay where reactivity of the Ab to a panel of unrelated polypeptide antigens was characterized. Heme-exposed Trastuzumab demonstrated binding to most of them, in a heme concentration dependent manner (FIG. 1). To investigate a broader spectrum of target recognition, we compared the reactivity of native and heme-exposed Trastuzumab to more than 9,000 human proteins using ProtoArray® technology. As expected, native Trastuzumab demonstrated a binding to very few proteins. Strikingly, heme-exposed Trastuzumab showed a considerable gain of reactivity by binding to a large number of human proteins (Data not shown). Quantitative analyses of the binding indicated that among the most strongly recognized targets of the heme-exposed Trastuzumab there were both intracellular and extracellular proteins (Data not shown). It is also important to note that heme alone was able to bind to numerous human proteins, but to a lower extent as compared to the antibody exposed to heme.

[0121] Next, we investigate the binding of the antibody to its cognate target, HER2/neu, upon heme exposure. The binding of heme-exposed Trastuzumab to HER2/neu was assessed by flow cytometry analyses using two different human breast cancer cell lines—SK-BR-3 and MDA-MB-231 (Data not shown). SK-BR-3 line has a high level of expression of HER2/neu, whereas MDA-MB-231 has a very low level of expression of HER2/neu. Accordingly, the binding of native Trastuzumab to MDA-MB-231 cells was characterized with a low intensity. No significant difference was observed in the binding of the heme-exposed Ab (Data not shown). The binding of Trastuzumab to high-HER2/neu expressing cell line showed also no difference between native and heme-exposed Ab (Data not shown). Finally, the binding of the native and heme-exposed Trastuzumab to its cognate target was determined by surface plasmon resonance (SPR)-based technique (Data not shown). HER2/neu mimotope was immobilized on a sensor chip and the binding of increasing concentrations of native or heme-exposed Trastuzumab was measured. Although heme-exposed Trastuzumab showed a lower binding response, both forms of the Ab demonstrated a considerable binding to HER2/neu mimotope, with binding affinities in the same order (KD values of 3.8 nM and 6.6 nM for the native and heme-treated Ab, respectively). To investigate whether heme exposure affects the Fc portion of Trastuzumab, the binding of heme-exposed Trastuzumab to recombinant human neonatal Fc receptor (FcRn) was determined by SPR-based technique. Estimation of the kinetic parameters showed that native and heme-exposed Trastuzumab bind to the receptor with identical binding affinities (Data not shown).

[0122] Taking together, these data demonstrated that Trastuzumab acquires polyreactivity upon heme exposure without affecting its ability to bind to HER2/neu and to interact with FcRn receptor.

[0123] In addition, the potential of other compounds, derivatives of heme, to induce polyreactivity of Trastuzumab was assessed. Thus, the exposure of Trastuzumab to Fe (III) mesoporphyrin IX, Fe (III) deuteroporphyrin IX and Fe (III) coproporphyrin I, resulted in an appearance of novel antigen-binding specificities (data not shown). In contrast free iron ions or protoporphyrin IX structure devoid of metal ion were not able to modify the specificity of Trastuzumab. These result clearly demonstrate that the most potent inducer of polyreactivity of Trastuzumab is Fe (III) deuteroporphyrin IX, whereas Fe (III) protoporphyrin IX has the lowest potential to uncover reactivity towards bacterial antigens (data not shown). These results indicate that Fe-containing porphyrin molecular system is indispensable for uncovering the polyreactivity of Trastuzumab.

Heme Binds with a High Affinity to Variable Region of Trastuzumab

[0124] Our results indicated that exposure of Trastuzumab to heme induces a new pattern of antigen recognition. To understand the mechanism of these changes in the antigen binding function of the Ab, we investigated the interaction between heme and Trastuzumab. Absorbance spectroscopy revealed that the exposure of the Ab to heme resulted in an increased absorbance intensity of heme in high and low energy regions of the spectrum (Data not shown). These changes in the spectral characteristics of heme are consistent with a specific binding of the tetrapyrrole compound to the protein molecule. Besides the absorbance spectroscopy, the ability of heme to interact with Trastuzumab was investigated by using fluorescence spectroscopy. To this end, the quenching of the intrinsic tryptophan fluorescence of the Trastuzumab was measured as a function of the concentration of heme. Exposure to heme resulted in a concentration-dependent decrease in the fluorescence signal of the therapeutic Ab (Data not shown). Furthermore, the binding of heme to Trastuzumab was investigated by circular dichroism spectroscopy (Data not shown). Considerable changes of the circular dichroism ellipticity curves were observed after exposure of the Ab to heme. This result indicates that exposure to heme resulted in alteration in the secondary structure of the IgG. This data is in accordance with data from absorbance and fluorescence spectroscopy demonstrating that heme interacts and binds directly to the Ab.

[0125] The interaction of heme with Trastuzumab was further investigated by SPR (Data not shown). Heme bound to immobilized Trastuzumab with a high affinity (KD value of 100 nM). As can be deduced by the slow dissociation observed on real time binding profiled (Data not shown), Trastuzumab formed stable complexes with heme. The interaction between heme and Trastuzumab was further investigated at different temperatures. The association rate constant increased with an increase of the temperature. The dissociation rate constant was also sensitive to temperature. However, the augmentation of the temperature resulted in decrease in the dissociation rate i.e. in an increase in the stability of the intramolecular interaction. The temperature dependencies of the rate constants were further used to evaluate the thermodynamic parameters for the association, the dissociation, and the equilibrium of the interaction of heme with the monoclonal Ab. The change in the entropy during the association was with a negative value (TΔS=−12.4±7.3 kJ mol-l). The apparent value of the changes of enthalpy during association was positive (ΔH=40.2±7.2 kJ mol-l). During dissociation, the apparent values of ΔH and TΔS were both with negative values. At equilibrium heme binding to Trastuzumab was characterized with unfavorable change in the enthalpy (ΔH=87.7±18.9 kJ mol-l) and highly favorable changes in the binding entropy (TΔS=127±18.3 kJ mol-l). These data demonstrated that heme binding to the Ab is entropy-driven and enthalpy controlled process. Overall, the results from the thermodynamic analyses indicate that the binding of heme to Trastuzumab does not require major structural adaptations of the protein. The favorable entropy changes most probably arise from disruption of the solvatation shell of heme.

[0126] Further, we investigated the position of heme binding site in the IgG molecule. To this end, the SPR experiment was conducted using Fab fragments and Fc fragments of Trastuzumab. Heme demonstrated a preferential binding to the immobilized Fab portion of the Ab (Data not shown).

[0127] Finally, the heme-binding site on the Fab fragment of Trastuzumab was predicted by molecular docking using Autodock software (Data not shown). The first four most probable sites of heme binding, based on the binding energy score, on the variable region of Trastuzumab were found to be on the heavy chain variable region. The putative heme-binding site partly overlaps with the CDR H2 loop. Molecular docking analyses predicted that heme is bound to the polypeptide chain in such a way that it remains at large extend exposed to the solvent.

Heme Induces Self-Association of Trastuzumab

[0128] To investigate whether heme binding affects the molecular composition of Trastuzumab we applied size exclusion chromatography. While native Trastuzumab eluted only as monomers, heme-exposed Trastuzumab eluted both as monomeric and oligomeric species. Moreover, it was observed that heme co-localized with the oligomeric forms of Trastuzumab (Data not shown). The effect of heme on the molecular composition of the Ab was concentration-dependent (Data not shown). Further, Fc and Fab fragments of Trastuzumab were exposed to heme and analyzed in the same conditions (Data not shown). Upon heme exposure, Fc fragments remained in a monomeric form. In contrast, heme-treated Fab fragments of Trastuzumab were eluted in two distinct molecular species—monomers and dimers. Heme co-localized with the dimeric species of Fab.

[0129] Next, to further characterize the mechanism of formation of the soluble oligomers of Trastuzumab, heme was pre-treated with cyanide before addition of the Ab (Data not shown). Cyanide anion is a high affinity ligand of heme's iron and as a consequence blocks metal's coordination potential and redox chemistry. The pre-treatment of heme with cyanide inhibited its potential to induce formation of soluble oligomers of the monoclonal Ab (Data not shown). To further substantiate this result, an additional experiment was performed using Zn (II) protoporphyrin IX (ZnPP) instead of heme (Data not shown). Treatment of Trastuzumab with ZnPP failed to induced antibody homophilicity or oligomerization. Taken together these results indicate that the iron in the tetrapyrrole structure of heme plays a crucial role in the formation of the soluble oligomers of Trastuzumab. A close structural analogue of heme—Fe(III)mesoporphyrin IX was also able to induce oligomerization of Trastuzumab. Proteoporphyrin IX (heme analogue devoid of Fe ion) or free iron ions were not able to induce self-association of the antibody (Data not shown). This result indicate that porphyrin molecular system that contains Fe(III) ion is indispensable for triggering of the self-association of Trastuzumab.

[0130] To investigate whether the formation of soluble oligomers induced by heme exposure is typical only for Trastuzumab, we analyzed molecular profiles of five additional therapeutic monoclonal Abs. No formation of oligomers was observed following exposure of these therapeutic Abs to heme (Data not shown).

[0131] To acquire more details about the self-binding tendency of heme-exposed Trastuzumab, the induction of antibody homophilicity by heme was first evaluated by ELISA (FIG. 2). Biotinylated Trastuzumab was treated with heme and incubated on plates with immobilized native Trastuzumab. Whereas native Trastuzumab showed only negligible self-binding activity, the heme-treated Ab demonstrated a strong binding in a bell-shaped dependent manner, which is typical for interactions of homophilic Abs (FIG. 2). To further characterize the self-binding of Trastuzumab induced by heme, kinetic measurements were performed. The heme-treated Ab bound to itself in a dose-dependent manner (Data not shown). The kinetic analyses confirmed that Trastuzumab is able to bind to itself with physiological relevant affinity (KD value of 92.3 nM at 25° C.). Thermodynamic analyses of self-association of heme-bound Trastuzumab revealed that the process is entropy-driven and enthalpy-controlled (Data not shown). Noteworthy, similar thermodynamic mechanism was observed in the case of heme binding to the Ab (Data not shown). This result suggests that the main driver for self-association of Trastuzumab is heme.

[0132] To characterize the heme-mediated oligomerization of Trastuzumab, a negative stain transmission electron microscopy technique was used. The morphologies of native and heme-bound Trastuzumab were compared (Data not shown). The visualization of native Ab showed a typical appearance of objects containing three globular domains, as expected for an intact IgG molecule (Data not shown). In the case of heme-exposed Trastuzumab, in addition to monomeric structures there were molecular species containing two or three IgG molecules (Data not shown). Importantly, self-binding of Trastuzumab molecules resulted in well-organized species but not in a random aggregation. Next, we used protein A bound to colloidal gold to specifically label Fc fragments of Trastuzumab (Data not shown). This labelling allowed us to confirm that Fc fragments were not involved in the interactions between heme-bound Trastuzumab. In summary these results indicate that formation of supramolecular species of the Ab is due to interactions between the variable regions.

Heme Exposure Increases Tumor Killing Potential of Trastuzumab

[0133] To provide understanding about functional impact of heme binding to the therapeutic Ab, we tested its ability to kill malignant cells. Several mechanisms of action of Trastuzumab have been described and discussed in the literature. The main ones include direct action on cancer cells by blockage of the receptor and antibody-dependent cellular cytotoxicity (ADCC) [21,22,23].

[0134] First, the antibody-dependent cellular cytotoxicity of native and heme exposed Trastuzumab was investigated (Data not shown). In this experiment, the breast cancer cell lines were incubated with the native or heme-exposed Trastuzumab and then incubated in presence of freshly isolated PBMCs. After 4 hours of incubation, the cell lysis was quantified by the release of lactate dehydrogenase. No difference in the cytotoxic potential was observed between the native and heme-exposed Trastuzumab on the HER2/neu low expressing and HER2/neu high expressing cell lines (Data not shown).

[0135] Next, we determined if the direct cytotoxicity and complement-dependent cytotoxicity (CDC) of Trastuzumab were affected by interaction with heme. SK-BR-3 and MDA-MB-231 cells were treated with native Trastuzumab or heme-exposed Trastuzumab in the presence—or not of complement. The SK-BR-3 cells showed a clear cytotoxicity in the presence of Trastuzumab. Interestingly, in the presence of complement, the cytotoxicity was significantly higher in the case of heme-bound Trastuzumab than the native form (p <0.01, Mann-Whitney test; FIG. 3). When we assessed the cytotoxicity of low HER2/neu expressing cancer cell line—MDA-MB-231, in the presence of complement a negligible decrease in the percentage of live cells was observed, but no significant difference was detected between the native and the heme-exposed Ab.

[0136] To exclude a potential cytotoxic action of heme on the cells, both cell lines were also treated with heme alone at identical concentrations as those introduced by heme-bound Trastuzumab. This treatment has no negative impact on the cell proliferation.

[0137] Next, we assessed whether the cytotoxic action of heme-bound Trastuzumab on the SK-BR-3 cells was due to the oligomers induced by heme binding (FIG. 4). To this end, Trastuzumab was treated with heme, and the 3 oligomeric fractions as well as the monomeric one were collected separately by a size-exclusion chromatography. In the same experiment settings as described above, the SK-BR-3 cells were treated with the different molecular species in the presence of complement. The monomeric fraction demonstrated the same cytotoxic effect as the native Trastuzumab. Interestingly, the oligomeric fractions showed the same cytotoxic effect as the heme-treated Trastuzumab, which were significantly different from the monomeric fraction (FIG. 4). Moreover, the first two oligomeric fractions demonstrated an even higher cytotoxic effect than the heme-treated Ab. This result demonstrates that the increased cytotoxic potential of heme-exposed Trastuzumab on the HER2/neu-positive cells is mediated by the dimers and higher molecular species of the IgG.

[0138] Moreover, deposition of C3 on the surface of breast cancer cells after incubation with native and heme-exposed Trastuzumab was assessed. This experiment clearly demonstrate that heme-exposed Trastuzumab has considerably high capacity to activate the complement on the cellular surface and to induce opsonisation of the cancer cells with C3. Thus, the heme-exposed Trastzumab can facilitate cell elimination through phagocytosis.

REFERENCES

[0139] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0140] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896214/

[0141] https://www.ncbi.nlm.nih.gov/pubmed/16458110

[0142] https://www.ncbi.nlm.nih.gov/pubmed/25716732

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