This application claims the benefit of the European Patent Application EP19382675.7 filed on August 2nd, 2019.

Technical Field The present invention is related to therapy. In particular, the present invention refers to bi-specific nanoparticles comprising two different protein populations, such as two different antibodies or immunogenic peptides, more particularly comprising a targeting antibody population and an immune-activating antibody population. The invention also provides a process for their preparation as well as their use in the therapeutic and diagnostic fields.

Background art

Monoclonal antibodies (mAbs) are a family of proteins typically secreted by plasma B cells upon exposure to pathogens. mAbs consist of two heavy chains and two light chains, forming two sub-domains: the Fab domain and the Fc domain. Fab domains are responsible for binding onto specific antigen molecules (via a tertiary structure of polypeptides that comprises the complementarity determining regions or CDRs) while Fc domains engage with receptors on the effector cells (innate immune cells, such as macrophages, natural killer (NK) cells and polymorphonuclear leukocytes) to trigger immune responses. In immunotherapies, these therapeutic proteins function by reducing cell proliferation/inducing tumor cell apoptosis or by eliciting complement activation as well as antibody dependent cellular cytotoxicity, and facilitating the T cell immune response through blockade of immune-suppressive pathways.

In connection with the above, it has been reported that, during the course of a disease, NK cells may become dysfunctional, losing in a great extent the ability to kill infected cells (the permanent stress caused by the infection leads to a chronic inflammation like phenotype or exhausted state that makes them less effective in terms of immune function). Therefore, assisting and enhancing NK response is highly desired.

Immune-targeted therapies based on the use of antibodies to promote NK-mediated responses have been previously developed for cancer cells. Some antibodies with documented ADCC activity have already been approved for the treatment of solid and hematological malignancies. However, although relatively effective in the cancer setting, antibodies need to overcome important limitations, for example the high dosage needed to produce a clinical response or the need to promote an efficient cell-to-cell contact between target and effector cells. Indeed, a contact-dependent response is also needed for NKs to inhibit viral replication, otherwise, infected cells can escape NK scrutiny. Thus, novel strategies designed to engage NK cells will significantly promote and enhance cell killing.

Recent studies have shown that checkpoint blockade efficacy can be further improved through combination with immunotherapies targeting diverse pathways. For example, a clinical trial combining PD-1 and CTLA-4 blockade nearly doubled survival time compared to anti-PD-1 mAb alone. Additional studies in mice have shown similar results, with superior tumor control in mice treated with PD-1 antagonists in combination with CTLA-4 checkpoint blockade and 4-1 BB co stimulation. However, these nonspecific approaches require high concentrations of antibody, as high as 100-200 pg/dose in murine models, and a majority of patients experience significant target side effects, especially when treated with a combination of antibodies.

Improvements in combinatorial immunotherapeutics are thus imperative to enable their use at safe and effective levels.

In this regard, the prior art has already reported the development of nanoparticle platforms for combinatorial immunotherapeutics. These nanoparticles, termed immunoswitch particles (hereinafter also referred as “bifunctional NP”), switch off a particular pathway while simultaneously switching on a co-stimulatory pathway in another cell population. By physically constraining the antibodies on a nanoparticle platform, immunoswitch particles result in synergy between the two immunotherapies and are thus effective at low doses. Concerning the obtaining of nanoparticles conjugated with antibodies, there are three main strategies:

(a) physical adsorption: provides advantages such as simplicity, no need to modify either the Ab or the NP, electrostatic attraction can orientate the antibodies ‘end on’ preserving binding ability; but also some drawbacks such as reversibility, the hydrophobic interaction can cause denaturation of Ab, the electrostatic attraction is weak and pH dependent and there can be a competitive displacement by serum proteins;

(b) Covalent Conjugation (including via linker molecules): provides advantages such as high stability and improved reproducibility, modifications to Ab usually not required, oriented binding possible, use of linker-molecule can avoid hostile reaction conditions, possible to control valency; but also technical drawbacks such as the reaction conditions needed, which may lead to protein unfolding/reduction, a lost of antigen binding capacity, and the fact that binding moieties or linker can significantly affect function; and

(c) Use of adaptor molecules (biotin/streptavidin): provides advantages such as orientated binding, they can resist harsh reaction conditions; but show some drawbacks such as the difficulty to control valency, costs, reversibility of the attachment, toxicity and reduced therapeutic efficacy (Montenegro JM et al., 2013; Ciaurriz P. et al, 2017; and Ahmed M. et al., 2015).

From the several strategies provided by the prior art, bifunctionalized NPs, which are characterized by comprising two different antibody populations, are mainly obtained by using adaptors or linkers (see, for instance, Kosmides et al., 2017 or Li et al., 2017). The use of such “intermediate” moieties can negatively affect to the safety and efficacy of the bifunctionalized NP (as mentioned above) in addition of the increasing complexity of making the nanocostruct and, therefore, can negatively affect to their usability in the therapeutic field.

Therefore, there is still the need of providing safe and effective antibody therapies based on bifunctionalized NPs. On the other hand, immunogenic peptides are substances that may be specifically bound by components of the immune system (antibody, lymphocytes), inducing an immune response, either humoral and/or cell-mediated immune.

The use of antigenic peptides or immunogens for vaccination, compared with traditional vaccines, has advantages such as purity and low risk of adverse effects and disadvantages such as weak immunogenicity and necessitates administration with an adjuvant.

Conjugation of antigenic peptides or immunogens is another widely used strategy for improving vaccine potency. Polysaccharide conjugates in particular have contributed greatly to numerous effective childhood vaccines. Poorly immunogenic peptides and proteins can also become better immunogens when conjugated to protein carriers.

However, the physical characteristics of immunogenic peptides can limit their pharmacokinetic and immunological properties. Summary of the invention

The inventors provide for the first time bifunctionalized nanoparticles coated with proteins P1 and P2 which are preferably selected from immunogenic peptides and antibodies or functional fragments thereof, said protein coating comprising two domains, a domain “A” and a domain “B”, wherein: (a) domain “A” comprises a higher amount of proteins P1, and (b) domain “B” comprises a higher amount of proteins P2, and the proteins forming each one of the domains “A” and “B” being ordered on the nanoparticle surface.

Both the presence of the two well-defined Janus domains, each one substantially formed by proteins of a single type protein population, as well as the highly ordered distribution of the proteins on nanoparticle (NP) surface (which means that they are vertically orientated with respect the NP surface) are critical in the stimulation of the immune system.

As a proof of concept of the above, the present inventors developed a bi- functionalized nanoparticle with two different antibody populations. As it is shown below, the resulting nanoparticle promoted close proximity between targeted cells (e. g., HIV-infected cells) and immune cells (such as NK cells), thus re-directing and potentiating the immune response. This novel molecularly-targeted approach is based on the use of bispecific antibody metallic nanoparticles bearing two different antibody populations: one antibody population which binds to the target cell (such as an infected or cancer cell) and another antibody population which activates the immune system. Thus, the conjugated particle is capable of both (i) multivalent high binding avidity to a target site, and (ii) multivalent presentation of immune-activating ligand, which amplifies immune response. The present inventors also surprisingly found that both protein populations (such as antibody populations) could be successfully conjugated on particle’s surface without negatively affecting their individual stability and functionality. In fact, they found that the polarized antibodies, on nanoparticle surface, retained its individual binding capability and promoted the specific formation of cell-to-cell interactions. As it is shown below, bifunctional nanoparticles (“Bi-NPs”) stimulated the production of IFN-g from NK cells, and triggered a potent ADCC response against HIV-expressing cells mediated by NK cells.

Thus, Bi-NPs may be postulated as a highly attractive tool for achieving a specific and potent immune response against a disease, such as HIV-reactivated cells. In addition to the successful conjugation of the targeting and immune-activating antibody populations, as well as of the retaining of their binding ability (and consequently the efficacy), the present inventors achieved the conjugation avoiding the use of an adaptor or chemical derivatization. As it is shown below, in the case of the conjugate of the invention, the antibodies are in direct contact with the particle, being adsorbed, by coordination bonds (electrostatic interactions), on particle’s surface.

The bifunctionalized NPs of the invention carrying immunogenic peptides can emulate a pathogenic particle, such as a viral particle, due to the small size and the particular order distribution of the immunogens in two well-differentiated domains of the coating, eliciting a complete immune system response and acquiring immune memory.

Therefore the bifunctionalized NPs of the invention can engage T and B cells for immunogenicity (adjuvancy) and presentation (memory). This approach allows to work with extremely low doses and still achieve good immunological memory.

It is the first time that it is reported a stable and efficient bifunctionalized nanoparticle with two different protein populations, in the absence of an adaptor/derivatization mediating the attachment. And this means a great advance in therapeutics and diagnostics fields because, up to now, bifunctionalized nanoparticles had been obtained by chemical modifications with the related potential problems.

Thus, in a first aspect the present invention provides a coated metallic nanoparticle comprising a metallic nanoparticle which is coated with a protein layer comprising two different proteins, P1 and P2, wherein protein P1 is preferably selected from: cell targeting antibodies or functional fragments thereof and immunogenic peptides; and protein P2 is preferably selected from: immune-activating antibodies or functional fragments thereof, and an immunogenic peptide, wherein:

- the metallic nanoparticle has an average particle size of at least 20 nm,

- the proteins are directly attached to the surface of the metallic nanoparticle by electrostatically interactions, - the layer comprises two domains, a domain “A” and a domain “B”, wherein:

-- domain “A” comprises a higher amount of proteins P1 , and -- domain “B” comprises a higher amount of proteins P2, wherein the proteins comprised in domains “A” and “B” are ordered on the nanoparticle surface.

The present invention also provides a coated metallic nanoparticle comprising a metallic nanoparticle which is coated with a layer comprising: a) cell-targeting antibodies or functional fragments thereof; and b) immune-activating antibodies or functional fragments thereof, wherein:

- the metallic nanoparticle has an average particle size of at least 20 nm,

- the antibodies are directly attached to the surface of the metallic nanoparticle by electrostatically interactions, - the layer comprises two domains, a domain “A” and a domain “B”, wherein:

-- domain “A” comprises a higher amount of cell-targeting antibodies or fragments thereof than of immune-activating antibodies or fragments thereof, and

-- domain “B” comprises a higher amount of immune-activating antibodies or fragments thereof than of cell-targeting antibodies or fragments thereof, wherein the antibodies comprised in domains “A” and “B” are ordered on the nanoparticle surface.

In addition to the above, the conjugated particles of the invention may have other potential advantages over conventional mAbs in terms of therapeutic application: deeper tissue penetration, stronger immune-activation due to multivalency, and an easily adaptable platform to generate new types of synthetic particle antibodies by varying the target-binding moiety.

The advantages provided by the conjugated particle of the invention are also partly due, in addition to the particular interaction protein-NP surface, to the size of the metallic NP and to the distribution of the antibodies on particle’s surface, as explained above.

Regarding the particle size, the present inventors found that it also contributed to the stability of the bifunctionalized NP: when the value was lower than 20 nm the conjugated nanoparticle was not stable. Regarding the particular distribution of the proteins, the present inventors found, when they used antibodies, that each population of antibodies was homogenously distributed in the form of highly ordered domains (i.e., highly dense domains). In fact, it was found that the conjugated particles showed two domains, each one essentially made of each antibody-type population, and that the antibodies forming the domain, which were adsorbed to particle’s surface, were ordered (as indirect conclusion in the light of the high antibody density detected on NP surface).

The present inventors have also designed an effective strategy for the successful electrostatic functionalization of metallic NPs with proteins, , allowing the formation ofa functional Hard Antibody Protein Corona. This strategy is based on the concept that the system has to relax to its enthalpy configuration in order to make proteins packed densely and segregately at the surface of the NP into different domains. Otherwise, entropy would drive the proteins to attach randomly onto the NP surface (Random Sequential Deposition (RSD)). The present inventors have developed a particular conjugation medium which promotes such relaxation to enthalpy configuration, thus achieving the spontaneous island growth of the proteins and their consequent segregation into two different domains onto NP surface, giving rise to the so-called Janus Hard Protein Corona. In this regard, the mild conditions of the conjugation medium found by the present inventors allow the system to reach its low energy configuration (enthalpy driven), being the interaction between proteins and proteins - NPs weak enough to stabilize the conjugated particle of the first aspect of the invention. This stability related to crystallinity (or packing density) is also referred as the Crowding effect, where to move one protein implies to move many (all of them packed against it and the other packed against the packed against it and so on).

Thus, in a second aspect the present invention provides a process for preparing the coated metallic nanoparticle as defined in the first aspect of the invention, which comprises the steps of:

(a) providing a dispersion comprising: (i) citrate ions, (ii) borate ions, (iii) proteins P1 , (iv) proteins P2, and (v) metallic nanoparticles having an average particle size of at least 20 nm, wherein:

- one of the protein populations (iii) or (iv) has less affinity towards nanoparticle’s surface in the dispersion than the other protein population, and

- the dispersion has: - a total protein population, which corresponds to the sum of the proteins P1 and P2, at a concentration excess with respect to metallic nanoparticles’ concentration;

- the protein population with less affinity towards nanoparticle surface is at a concentration excess with respect the other antibody population;

- citrate ions are at a concentration in excess with respect to metallic nanoparticles concentration;

- borate ions are at a concentration excess with respect to the total antibody population; - provided that the concentration of the citrate and borate ions confer to:

(i) the dispersed nanoparticles a surface charge equal or lower than - 20 mV when measured with Zetasizer Nano series instrument, following manufacturer’s instructions; particularly lower than -25 mV, particularly from -25 to -45 mV; and (ii) the dispersed proteins have a surface charge Y±(Y 0.1), wherein Ύ” represents the mean of the charge of the free protein P1 and the free protein P2, wherein the “charge of the free protein”, either from P1 or P2 protein, is determined by Malvern’s Zetasizer Nano, following manufacturer’s instructions;

(b) stirring at a temperature that allows the formation of the domains “A” and “B” as defined in the first aspect of the invention, and

(c) optionally isolating the resulting metal nanoparticles from the solution resulting from step (a).

The present invention also provides a process for preparing the coated metallic nanoparticle as defined in the first aspect of the invention, which comprises the steps of:

(a) providing a dispersion comprising: (i) citrate ions, (ii) borate ions, (iii) cell targeting antibodies or functional fragments thereof, (iv) immune-activating antibodies or functional fragments thereof, and (v) metallic nanoparticles having an average particle size of at least 20 nm, wherein: - one of the antibody populations (iii) or (iv) has less affinity towards nanoparticle’s surface in the dispersion than the other, and - the dispersion has:

- a total antibody population, which corresponds to the sum of the cell-targeting antibodies or functional fragments thereof and immune-activating antibodies or functional fragments thereof, at a concentration excess with respect to metallic nanoparticles’ concentration;

- the antibody population with less affinity towards nanoparticle surface is at a concentration excess with respect the other antibody population;

- citrate ions are at a concentration excess with respect to metallic nanoparticles concentration; - borate ions are at a concentration excess with respect to the total antibody population;

- provided that the concentration of the citrate and borate ions confer to:

(i) the dispersed nanoparticles a surface charge equal or lower than - 20 mV when measured with Zetasizer Nano series instrument, following manufacturer’s instructions; particularly lower than -25 mV, particularly from -25 to -40 mV; and

(ii) the dispersed antibodies have a surface charge Y±(Y 0.1), wherein Ύ” represents the mean of the charge of the free cell-targeting antibody and the free immune-activating antibody, wherein the “charge of the free antibody”, either from the cell-targeting antibody or immune-activating antibody, is determined by Zetasizer nano

(b) stirring at a temperature that allows the formation of the domains “A” and “B” as defined in claim 1 on nanoparticles surface, and

(c) optionally isolating the resulting metal nanoparticles from the solution resulting from step (a).

NP-protein interactions are controlled by the conjugation medium where they evolve. The conjugation medium is a mix of citrate and borate species that provides the effective charge to NP surface and protein and, consequently, contributes to their water solubility and electrostatic interactions. Citrate buffer provides negative atoms which cover the surface of the NPs, thus allowing the stabilization of the colloidal NPs in solution, and preventing, in the way the aggregation by electrostatic repulsion of the negatively charge surface of the NPs. The direct negative citrate displacement in citrate stabilized metallic NPs, by positively charged molecules, causes multiple electrostatic bridging, leading to irreversible agglomeration of the NPs. Also, taking into account the Hofmeister series, based on the ion-specific effects of several cations and anions on the physical properties of the proteins, citrate increases the denaturalization of the proteins. Borate buffer, on the contrary, increases protein stability, preventing their denaturalization; but they also salt-out the proteins, leading to aggregation. Therefore, the citrate/borate buffer system is able to generate a situation in which both NPs and proteins are stable enough to not aggregate or denaturalize, respectively; but unstable sufficiently to arouse the interaction between them as a means of improving their situation in this special media.

Initially, the conjugation medium was expected to promote the NP-Ab absorption, which is controlled by electrostatic interactions. But surprisingly, as it is shown below with the embodiment wherein the proteins are antibodies, it was also found that this medium induced the formation of Abs domains on top of the NPs in sub-saturated loading concentration conditions, leaving the NP partially coated and having space for the second Ab domain deposition of the reaming free surface. This can be explained by the different protein affinities (which in both cases is higher than Boltzmann constant) for the NP surface, and by the affinity constants: one of them has higher affinity to NP surface, that starting the crowding firstly.

The relative amount of the NPs and the different proteins also contributes to the electrostatic interactions with the environment, solubility, and, consequently, the affinity between NPs and proteins, which has to be low enough to avoid uncontrolled aggregation but high enough to drive the system towards an ordered conjugation, allowing to reach the maximum ordered association of antibodies before the moment of the uncontrolled random aggregation.

Further, as it is shown below, each one of the domains is essentially made of a single type of protein (such as antibody), which is indicative that the proteins are ordered and not randomly distributed. And this is due to the particular conditions of the process of the invention. In this regard, Examples below show that using the conjugation medium of the invention it is achieved the NP with the characterizing ordered protein domains, such as each one of the domains is essentially made of a particular protein type (as it is concluded from FIG. 8). Without being bound to the theory, the present inventors believe that under the conditions of the process provided in the second aspect of the invention, there is cooperative absorption effect: a protein absorbed to a surface binds more strongly to it if it is surrounded by the same or similar protein, thanks to the possibility of build compact arrangements with identical building blocks. Thus, antibodies of the same nature would pack better, favoring the formation of homogenous coating domains from a mix of coating molecules. This would explain the obtaining of polarized nanoparticles comprising two well-defined distinguishable functional surfaces.

Last, but no least, the medium conditions do not negatively affect to the stability/functionality of the proteins (such as antibodies) dispersed in the conjugation medium such as the resulting bifunctionalized NP efficiently recognizes/activates its target, as it is shown below.

In a third aspect the present invention provides a metallic nanoparticle obtainable by the process as defined in the second aspect of the invention.

As it is illustrated below, the inventors developed a bifunctional polarized metallic NP with NK activating antibodies on one side and anti-HIVgp120 antibodies on the other side. This bifunctionalized NP was able to bind and bring together CD4 T and NK cells thus giving rise to a remarkably increase in the cell doublets, about 7-fold higher, as provided in FIG. 5b. The NP of the invention was not only able to bind and bring together both cells, but also the antibodies adsorbed were efficient in producing IFN-g (by NK cells) and of inducing the ADCC against gp120-coated cells. These data are indicative of the therapeutic value of the NP of the invention.

The results provided herein can be obtained with other antibodies or proteins: just considering the conjugation medium and reaction conditions provided herein the skilled person could be able to develop any other stable and functional polarized metallic NP to be used in therapy or diagnostics, just selecting the desired available antibody for activating pathway and another for targeting the NP to a particular point.

Thus, in a fourth aspect the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the coated metallic nanoparticle as defined in the first or third aspect of the invention together with acceptable pharmaceutical excipients and/or carriers.

In a fifth aspect the present invention provides the NP as defined in the first or third aspect of the invention for use in therapy or diagnostics.

In a sixth aspect the present invention provides the NP as defined in the first or third aspect of the invention for use in the treatment of an infection. This aspect can also be formulated as the use of a NP as defined in the first or third aspect of the invention for the manufacture of a medicament for the treatment of an infection. This aspect can also be formulated as a method of treating an infection, the method comprising administering a therapeutically effective amount of a NP as defined in the first or third aspect of the invention or the pharmaceutical composition as defined in the fourth aspect of the invention to a subject in need thereof.

The invention also provides a diagnostic kit comprising a NP as defined in the first or third aspect of the invention. Brief description of the drawings

FIG. 1. Characterization of the bispecific gold nanoparticles (BiAb-AuNPs). (a) Representative UV-Vis spectra of the BiAb-AuNPs compared to naked AuNPs, wherein line (1) represents naked AuNPs, and line (2) represents the NPs of the invention (b) Z-potential measurements of BiAb-AuNPs, naked AuNPs, and the single antibodies A32 and 3G8 (n=3, mean with SD). (c) Representative Dynamic light scattering (DLS) characterization of the BiAb-AuNPs (line “2”) compared to naked nanoparticles (line “1”).

FIG. 2. Thermogravimetry experiment to determine the amount of antibodies conjugated. Weight loss reported by TGA, from 30°C to 600°C, in an antibody coated 50 nm AuNP, reporting a decrease in mass of about 10% of the total weight. The loss of mass between 200 and 450 °C was previously attributed to Ab sublimating from an AuNP Surface. Results of the TGA analysis show the weight loss of AuNPs functionalized with mouse IgG.

FIG. 3 shows the targeting capabilities of the A32-monoconjugated and the 3G8- monoconjugated AuNPs. Cells were cultured with the nanoconjugates for 20 minutes and the binding capacity of the conjugated nanoparticles were assessed by flow cytometry a) CEM.NKR CCR5+ coated with the HIV-1 BaL gp120 recombinant protein were cultured in different conditions, including the free antibody A32, naked nanoparticles and the monoconjugated A32-AuNPs. The capacity of the A32-AuNPs to recognize its cognate antigen was assessed using an anti-human secondary antibody that detects the A32 antibody b) Primary isolated NK cells were cultured in different conditions, including the free antibody 3G8, naked nanoparticles and the monoconjugated 3G8-AuNPs. The capacity of the 3G8-AuNPs to recognize its cognate antigen was assessed using an anti-mouse secondary antibody that recognize the 3G8 antibody.

FIG. 4 represents the isothermal absorption curves for the A32 and 3G8 antibodies. Zeta potential (mV) versus the antibody concentration is plotted a) A32-AuNPs saturation curve. The arrow indicates the saturating concentration for the A32 antibody b) 3G8-AuNPs saturation curve. The arrow indicates the saturating concentration for the 3G8 antibody.

FIG. 5. BiAb-AuNPs induce cell-to-cell contact between HIV-expressing cells and NK cells. A) Representative flow cytometry plots showing cell doublets corresponding to primary CD4+ T cells (CD3+) and primary NK cells (CD56+) after 20 minutes co- culture in the presence of the different nanoconjugates. Double positive cells for the markers CD3 and CD56 denote pairs of cells formed by CD4-HIV+ and NK cells. Controls corresponding to medium alone and non-conjugated (naked) nanoparticles are shown. Different ratios for the generation of BiAb-AuNPs and different total antibody concentrations on the nanoparticles are shown b) Summary graph for the induction of cell-to-cell contacts in the presence of different nanoconjugates. Median and ranges (min-max) are shown. 10:100 BiAbAuNPs represent a suboptimal bispecific nanoconjugate prepared with 10% of the saturating concentration for the A32 antibody and 100% for the 3G8 antibody. Statistical analysis consisted on a paired t test. *p<0.05. FIG. 6 Activation and functional assessment of NK cells induced by the BiAb-AuNPs.

Representative flow cytometry plots and summary graphs for different markers of NK cell activation and function after co-culturing the cells for 4.5h with the free antibodies A32 (5 pg/ml) and 3G8 (5 pg/ml), irrelevant-AuNPs and the BiAb-AuNPs (10 pg/ml). Positive controls consisted in adding PMA (10 ng/ml) and ionomycine (1 mM) to the culture a) Percentage of INF-T+ cells b) Percentage of CD107a+ cells c)

Percentage of double positive CD107+ INF-T+ cells d) Percentage of HLA-DR+ cells. All graphs represent median with range (min-max) for each condition. Statistical analysis consisted on a Friedman test (Dunn’s multiple comparisons test). *p<0.05.

FIG. 7. NK-mediated cytotoxic response against HIV-expressing cells promoted by the BiAb-AuNPs. a) Representative flow cytometry plots for NK-mediated cytotoxicity assays. CEM-NKR.CCR5 cells were double-stained with PKH67 and eF670, and coated with the HIVBal gp120 recombinant protein. Cells were co-cultured with primary NK for 4h at 1 : 10 target/effector ratio and in the presence of different nanoconjugates at the following antibody doses: 10 pg/ml for irrelevant AuNPs, 10pg/ml for BiAb-AuNPs and 5 pg/ml for each free antibody. Loss of the eF670 marker was used to determine the percentage of dead cells in an eF670 versus PKH67 plot b) ADCC activity of NK cells in the presence of different nanoconjugates at the following antibody doses: 10 pg/ml for irrelevant AuNPs, 10 pg/ml for BiAb- AuNPs and 5 pg/ml for each free antibody. Statistical comparisons were performed using Wilcoxon matched-pairs signed rank test. Median with range (min-max) are shown. A p value of <0.05 was considered statistically significant (*p<0.05). c) ADCC cytotoxic response against latently-infected ACH-2 cells after viral reactivation. PHA and PMA-reactivated ACH-2 cells were cultured with primary NK cells at ratio 1:10 target/effector for 4h in the presence or absence of the BiAb-AuNPs at 10 pg/ml of antibody burden. Cytotoxicity was calculated as the disappearance of ACH-2 target cells using flow cytometry count beads. Data is normalized to the control condition in absence of the BiAb-AuNPs. Mean with SD of 4 independent experiments is shown.

FIG. 8. Representation of the distribution of antibodies on the surface of bispecific Janus AuNPs by TEM. AuNPs of 60 nm were functionalized with anti-HSA IgG and anti-HSA IgG following the invention method. Then, these Janus AuNPs were exposed to smaller conjugates as labels: AuNPs of 25 nm functionalized with BSA and of 15 nm with HSA. After 24 hours the complexes were visualized by TEM. The upper panel shows a hemispherical distribution of the labels, with three BSA-AuNPs distributed in one side of the Janus AuNP and one HSA-AuNPs in the opposite side. The same can be observed in the lower panel but with an inverted proportion of the labels, further confirming the cooperative absorption achieved by the invention method.

FIG. 9 Distribution of the cell doublets. Primary CD4 ⁺ T cells (CD3 ⁺ ) and primary isolated NK cells (CD56 ⁺ ) were co-cultured in the presence of BiAb-AuNPs (2.5 pg/ml of total antibody) or naked AuNPs for 20 minutes. Cell doublets were identified by flow cytometry using the FSH-H and FSH-A plot. The total cell doublets were distributed in T-T cells (CD3 ⁺ CD3 ⁺ ), NK-NK (CD56 ⁺ CD56 ⁺ ) or T-NK (CD3 ⁺ CD56 ⁺ ) doublets and the percentages are plotted. Data shows median with range of n=3 independent experiments. The increment (D) of T-NK cell doublets after the addition of the BiAb-AuNPs is shown.

FIG. 10. Comparison of polarized BiAb-AuNPs (in orange) and non-polarized BiAb-AuNPs (in grey). Percentages of cell-to-cell attachment efficiency for both nanoconjugates are represented. Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition. The definitions given herein are included for the purpose of understanding and expected to be applied throughout description, claims and drawings. In addition, for the purposes of the present invention, any ranges given include both the lower and the upper end- points of the range. Ranges given, such as temperatures, times, weights, and the like, should be considered approximate, unless specifically stated.

The present invention provides, in a first aspect, metallic nanoparticles completely coated with a monolayer comprising proteins P1 (such as cell-targeting antibodies or functional fragments thereof or immunogenic peptides) as well as proteins P2 (such as immune-activating antibodies or functional fragments thereof or immunogenic peptides), being proteins P1 and P2 different.

The term "nanoparticles" refers to dispersed particles having an average particle size equal or higher than 20 nm at the end of the dispersion preparation. As used herein, “average particle size” refers to particle size measurements based on number average particle size measurements, which can be routinely obtained by laser light scattering methods such as static or dynamic light scattering (SLS or DLS, respectively) or, alternatively, by Transmission Electron Microscopy (TEM). In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the average particle size is determined by TEM or DLS. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the average particle size is comprised from 30-150 nm. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the average particle size is comprised from 40-80 nm. Advantageously, the lower limit is the minimum allowing the formation of two domains and the upper limit determines the solubility when it is dispersed in the conjugation medium.

The metallic nanoparticle comprises one or more metals in elemental or alloy form. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the metal is selected from the group consisting of silver, gold, copper, nickel, cobalt, molybdenum, palladium, platinum, tin, zinc, titanium, chromium, tantalum, tungsten, iron, rhodium, iridium, ruthenium, osmium, aluminium and lead. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below the metallic nanoparticles comprises silver, copper, molybdenum, aluminium, gold, copper, or a combination thereof. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below the metallic nanoparticle comprises gold. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below the metallic nanoparticle consists of gold.

The metallic nanoparticles may be prepared from metal precursor particles by means of an additional step such as a reduction step, for example the reduction of metal oxides to metals. Metal precursor nanoparticles may be selected from the groups of metal oxides, metal salts or metal hydroxides. Illustrative non-limitative examples of metal oxide nanoparticles are based on silver oxide, tin oxide, titanium oxide, zirconium oxide, wolfram oxide, molybdenum oxide, cadmium oxide, copper oxide or zinc oxide. Also doped metal oxide nanoparticles such as ZnO:AI, Sn02:F or Sn02:Sb may be used.

Particular metal hydroxide particles are based on copper hydroxide, titanium hydroxide, zirconium hydroxide, wolfram hydroxide, molybdenum hydroxide, cadmium hydroxide or zinc hydroxide.

Particular metal salts include inorganic acid salts, such as nitrates, carbonates, chlorides, phosphates, borates, sulfonates and sulfates, and organic acid salts, such as stearate, myristate or acetate. Gold nanoparticles may be prepared, for example, by a citrate reduction protocol as previously described by Bastus, N.G. et al. , 2011, based on a kinetically controlled seeded growth strategy via the reduction of HAuCU by sodium citrate.

As mentioned above, the monolayer comprises proteins P1 and P2.

In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, P1 and P2 are different immunogenic peptides. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, P1 and P2 are immunogenic viral peptides, particularly from the viral capside. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, one of P1 and P2 is an immunogenic peptide, particularly an immunogenic viral peptide, more particularly from the viral capside, and the other is an antibody. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, proteins “P1” and “P2” are different antibodies. In another embodiment, optionally in combination with any of the embodiments provided above or below, proteins “P1” are cell-targeting antibodies or functional fragments thereof and proteins “P2” are immune-activating antibodies or functional fragments thereof.

The term “immunogenic peptide” have the same meaning, in the present application, as “immunogenic protein” and can be interchangeably used.

The term "antibody or a functional fragment thereof" is to be understood as any immunoglobulin or fragment thereof able to selectively bind the target protein. The term "fragment thereof” encompasses any part of an antibody having the size and conformation suitable to activate the immune system or cell-target. Suitable fragments include F(ab), F(ab'), and Fv. An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies).

The antibodies referred in the present invention are monoclonal (MAb). There are well known means in the state of the art for preparing and characterizing monoclonal antibodies. Monoclonal antibodies can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody producing cells. Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation. The immunogen is injected into animals as antigen. The antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen. In the present invention, the term “cell-targeting antibody” means an antibody which interacts with a surface cell component of a particular cell, i.e, the target cell, which can be a microorganism causing the disease (either a virus, bacteria etc.) or a “diseased” cell. There are a huge amount of antibodies well-known in the state of the art as behave as targetings towards a particular cell. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets a malignant cell (i.e. , a “diseased cell”) , such as tumoral or infected cell. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to a viral infected cell. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to an HIV-infected cell. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which is an anti-gp120 antibody, such as the A32. Alternatively, in one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to the microorganism causing the disease. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to a virus particle or virion. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to a HIV virus particle or virion epitope. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the cell-targeting antibody is one which targets to a HIV virus particle or virion epitope and is selected from 2G12, VRC01 among others.

In the present invention, the term “immune-activating antibody” means an antibody which raises the activation of the immune system by interacting with any of the components involved in the regulation of the system. There are a huge amount of antibodies well-known in the state of the art as activating immunological cells. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the immune activating antibody is a NK- cell activating antibody. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the

NK-cell activating antibody is selected from: anti-CD16, anti-CD27, and anti-NKG2D, among others.

The cell-targeting and immune-activating antibodies can be selected from those currently available in the market or they can be prepared following routine protocols. Moreover, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody producing cells. Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation. The immunogen is injected into animals as antigen. The antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen.

As mentioned above, the proteins (such as antibodies or immunogenic peptides) are directly and electrostatically interacting with nanoparticle’s surface. As it has been explained in detail above, this is due to the negative charge of the metallic nanoparticles and the positive charge of the proteins (such as antibodies or immunogenic peptides), the appropriate balance between positive and negative charges being “regulated” by the particular conditions of the conjugation medium. It is also highlighted, again, that even those charges the proteins (such as antibodies or immunogenic peptides) are stable and active, being capable of performing the corresponding function, either the targeting to a cell or the activation of the immune response.

The NPs of the invention are coated by a layer comprising two protein-based domains, a domain “A” and a domain “B”. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the NPs of the invention are completely coated with the antibodies or immunogenic peptides. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the metallic nanoparticle is coated with an antibody or immunogenic peptide monolayer.

In the present invention by a “monolayer” is to be understood a layer with a thickness corresponding to the one of a single molecule vertically orientated with respect to NP surface. That is, the proteins, which are electrostatically adsorbed on NP surface, are considered as being highly-ordered distributed when the thickness of the layer corresponds to the length of the protein forming the domain: this will indicate that the protein is vertically orientated with respect to NP surface. The thickness of the monolayer can be easily determined as the difference between the average size of the uncoated NP and coated NP, which can be determined by TEM or DLS: if this difference (the thickness) is equal or higher than at least a 70%, at least a 80% or at least a 90% identical to the length of the free protein, this will be indicative that the proteins are highly ordered distributed. In one embodiment, optionally in combination with any of the embodiments provided above or below, the thickness of the domain A corresponds to the length of the free protein P1 and/or the thickness of domain B corresponds to the length of free protein P2. In one embodiment, optionally in combination with any of the embodiments provided above or below, the thickness of the domain A corresponds to the length of the free protein P1 and the thickness of domain B corresponds to the length of free protein P2.

The expression “length of free protein” refers to the full-length of the protein, either the one expected by the aminoacids forming it or the one acquired in the reaction medium. Due to the particular conditions of the process of the invention, the proteins develop affinity towards proteins of the same type: that is, a cell-targeting antibody will tend to aggregate with another cell-targeting antibody and not with an immune-activating antibody. The same will occur with the immunogenic peptides. In this way, the antibodies will be ordered in two main domains, a domain “A” wherein the antibody population is predominantly comprising cell-targeting antibodies, and a domain “B” wherein the antibody population is predominantly comprising immune-activating antibodies. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the monolayer consists of two domains, domain “A” and “B”. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “A” surface is occupied by ordered immunogenic peptide or cell-targeting antibodies or fragments thereof. In one embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “B” surface is occupied by ordered immunogenic peptide or immune-activating antibodies or fragment thereof. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “A” surface is occupied by ordered immunogenic peptide or cell-targeting antibodies, and at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “B” surface is occupied by ordered immunogenic peptide or immune-activating antibodies or fragment thereof. The percentage of domain’s surface can be determined using routine protocols. For instance, it can be indirectly determined from the surface charge of the AuNP conjugated with the two antibody population. Thus, once the metallic NP has been completely coated, its surface charge will correspond to that of the free antibody. The sum of both individual charges will provide the percentage of surface occupied by each antibody. For example, if the functionalization of the NP is performed with an antibody population with a surface charge of -25 mV and another with a surface charge of -35 mV (all the charges measured in equivalent media/conductivity), and the conjugated Bi-NP of the invention shows a surface charge of about -30 mV, this is indicative that both antibody populations are occupying about a 50% of the metallic NP surface. Analogously, the same indirect determination protocol can be followed when P1 and P2 are different immunogenic peptides. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, each one of the domains occupies from 30 to 70% of the whole nanoparticle surface, provided that the sum does not exceed 100% of nanoparticle surface.

The extension of a domain on the NP surface can be determined routine protocols. For instance, it can be determined by aggregation experiments since unless over a 60% of the coating is grown, NPs will aggregate. This can be determined with the Langmuir like isothermal Ab absorption curves. In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below, the surface charge of the coated metallic NP’s surface is comprised from -10 to -40 mV, -25 to -35 mV, from -28 to -34 mV or from -29 to -33.5mV. The surface charge of the nanoparticle can be determined using any routine technique or apparatus, such as the one used in the Examples provided below, Zetasizer Nano series instrument, and merely following manufacturer’s instructions.

In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below: the nanoparticle is a gold nanoparticle with an average particle size from 40 to 80 nm, the cell-targeting antibody is an antibody towards HIV infected cell and the immune- activating antibody is an anti-NK-cell activating antibody, the coated metallic nanoparticle has a surface charge value within the range from -28 to -34 mV or from -29 to -33.5 mV, particularly of -31.4 mV (free antibody), and at least a 75% of the domain “A” surface is occupied by ordered cell-activated antibodies and at least a 75% of the domain “B” surface is occupied by ordered NK- cell activating antibodies.

In another embodiment of the first aspect of the invention, optionally in combination with any of the embodiments provided above or below: the nanoparticle is a gold nanoparticle with an average particle size from 40 to 80 nm, the cell-targeting antibody is an anti-gp120 antibody and the immune-activating antibody is an anti-CD16 antibody, the coated metallic nanoparticle has a surface charge value within the range from -28 to -34 mV or from -29 to -33.5 mV, particularly of -31.4 mV (free antibody), and at least a 75% of the domain “A” surface is occupied by ordered anti-gp120 antibodies and at least a 75% of the domain “B” surface is occupied by ordered anti- CD16 antibodies.

In a second aspect the present invention provides a process for the preparation of the bifunctionalized NP of the first aspect of the invention.

In a first step the process of the invention comprises providing a dispersion comprising citrate ions, borate ions, immunogenic peptides, or alternatively, cell targeting antibodies or functional fragments thereof, immune-activating antibodies or functional fragments thereof, and metallic nanoparticles having an average particle size of at least 20 nm

All the definitions and particular embodiments for the nanoparticles and antibodies provided under the first aspect of the invention, are also embodiments of the nanoparticles and antibodies referred in the process of the second aspect of the invention. The concentration ratio between cell-targeting antibodies and the immune-activating antibodies is dependent on their affinity towards NPs. The higher affinity, the lower amount is required. The affinity towards NP can be determined by routine methods, such as by isothermal adsorption monoconjugation curves by incubating, separately, each one of the antibodies with the metal nanoparticle in a buffered solution as the one also referred in the present invention as “conjugation medium” and measuring the zeta potential as well as conductivity using Zetasizer Nano series instrument (following manufacturer’s instructions) and then analyzing the minimum concentration of antibody needed to promote a stable and surface-saturated monoconjugate, indicated by the less negative Z-potential value. The immunogenic peptide or antibody requiring a lower concentration to achieve the sufficient coating (to avoid the aggregation) conferring stability, is the one with higher affinity. In one embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the targeting antibody has more affinity towards NP than the immune-activating antibody. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the antibody with less affinity towards NP surface is NK-cell activating antibody. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the immune-activating antibody is in excess concentration with respect to the targeting antibody. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the antibody with less affinity is at a concentration which corresponds to the 100% of its saturation value, being understood the “saturation value” as the one providing the most positive surface charge of the nanoparticles, corresponding to the full coating of the nanoparticles. In another embodiment, optionally in combination with any of the embodiments provided above or below, the immune activating antibody is at a concentration ratio with respect to the targeting antibody from 2: 1 to 10: 1 or from 3:1 to 8: 1.

In one embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion is prepared at a temperature from 1 °C to room temperature. Advantageously, working at this range of temperature it is achieved a modulation in the speed of formation of antibody domains: it is avoided protein degradation and it helps to the correct domain formation during the conjugation. Depending on the nature of the proteins (either antibodies or immunogenic peptides) and metallic NPs, the temperature will have to be adjusted within this range. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion is prepared at a temperature from 1 °C to 15°C. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion is prepared at a temperature from 2 °C to 10°C, such as 4 °C. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the NPs concentration in the dispersion is comprised from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion has a concentration of citrate ions from 5 to 20 times the citrate concentration needed to completely coat the NPs included in the dispersion. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion has a concentration of citrate ions from 5 to 10 times the citrate concentration needed to completely coat the NPs included in the dispersion. In another embodiment of the process of the second aspect of the invention, the concentration of citrate ions is comprised from 0.1 to 15 mM, from 0.2 to 10 mM. The use of different borate concentrations correspond to the different isoelectric points of different proteins (either antibodies or immunogenic peptides), taking into account that NPs and proteins (such as antibodies or immunogenic peptides) have to be mildly unstable in order to slowly dissipate energy and reach an enthalpic conformation rather than rapidly dissipating energy leading to entropic states (the higher the z potential of the antibody, the higher the borate concentration) while maintaining it close to 30 mV. In another embodiment, optionally in combination with any of the embodiments provided above or below, the concentration of citrate ions is 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3 mM. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion comprises: metallic NP at a concentration from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml, citrate ions at a concentration from 0.1 to 15 mM, and borate ions at a concentration of 2.2 mM.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion has a concentration of borate ions from 5 to 10 times the borate concentration needed to completely coat the total population of proteins (such as antibodies or immunogenic peptides). In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion has a concentration of borate ions from 2 to 15 mM or from 3-10 mM. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion has a concentration of borate ions of 3, 4, 5, 6, 7, 8, 9, or 10 mM.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion comprises: metallic NP at a concentration from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml, citrate ions at a concentration from 0.1 to 15 mM and borate ions at a concentration from 3 to 10 mM. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion comprises: metallic NP at a concentration from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml, citrate ions at a concentration from 0.1 to 15 mM borate ions at a concentration from 3 to 10 mM. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the dispersion comprises: metallic NP at a concentration from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml, citrate ions at a concentration from 0.1 to 15 mM borate ions at a concentration from 3 to 10 mM.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is performed by first mixing citrate and borate buffers and then adding the proteins (such as immunogenic peptides or antibodies) to the resulting mixture. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a) is performed by: (a.1) preparing a mixture of citrate buffer, borate buffer and immunogenic peptides or antibodies; and (a.2) adding to the resulting mixture from (a.1), the metallic NPs. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the citrate buffer is sodium citrate.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, the borate buffer is prepared using sodium tetraborate decahydrate.

In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a.2) is performed by mixing the solution resulting from step (a.1) with a solution comprising metallic NPs, at a volume ratio from 1:1 to an excess of the immunogenic peptides or antibodies solution resulting from step (1) vs the metallic NP solution. Advantageously, the conjugation speed is regulated to appropriately aggregate antibodies on NP surface to provide the monolayer comprising both protein domains. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a.2) is performed by mixing the solution resulting from step (a.1) with a solution comprising metallic NPs, at a volume ratio from 1:1 to 20:1, particularly 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20: 1. In another embodiment of the process of the second aspect of the invention, optionally in combination with any of the embodiments provided above or below, step (a.2) is performed by mixing the solution resulting from step (a.1) with a solution comprising metallic NPs, at a volume ratio of 9:1.

The invention also provides a diagnostic kit comprising a NP as defined in the first or third aspect of the invention. All the embodiments provided under the first and third aspect of the invention are also embodiments of the NPs referred in the kit of the fourth aspect of the invention.

In one embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit comprising means for detecting whether the immunogenic peptides or antibodies bound to the target. In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, diagnostic kit further comprises a reactor, a reaction solution, a buffer, and general tools.

The “detection means” can be selected from the group consisting of an enzyme- linked immunosorbent assay, an enzyme immunoassay, a florescence immunoassay, a luminescence immunoassay, and a radioimmunoassay, but it is not restricted to these. The diagnostic kit can be prepared as an ELISA kit or a strip kit.

In another embodiment of the fourth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the NP as defined in the first or third aspect of the invention is used as a label. The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of metallic nanoparticle as defined in the first or third aspect of the invention.

The expression "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed. The particular dose of the peptide administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations.

The expression "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and non-human animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. Examples of suitable pharmaceutically acceptable excipients are solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (the bifunctionalized NP of the invention) into association with a excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprisinga predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one- half or one-third of such a dosage.

The relative amounts of the active ingredient (i.e. , the bifunctionalized NP), the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. In one embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pharmaceutical composition is a vaccine. In another embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pharmaceutical composition is a vaccine and the proteins P1 and P2 coating the NP surface are immunogenic peptides, particularly viral immunogenic peptides. In another embodiment of the fifth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the pharmaceutical composition in a vaccine comprising immunogenic peptides, particularly viral immunogenic peptides, and one or more adjuvants. Excipients and adjuvants that can be incorporated to a vaccine are well- known by the skilled person in the art and will be selected, in such a way that they do not negatively affect the immunological activity of the bifunctionalized nanoparticles of the invention.

In another aspect, the present invention provides the NP as defined in the first or third aspect of the invention for use in the treatment of an infection. All the embodiments provided under the first aspect of the invention are also embodiments of the use referring to said NP. In one embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the infection is treated by inducing antibody-dependent cellular cytotoxicity. In an alternative embodiment of sixth aspect of the invention, the infection is treated by eliciting an humoral response. In one embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the infection is a viral infection. In one embodiment of the sixth aspect of the invention, optionally in combination with any of the embodiments provided above or below, the infection is HIV.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of’. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Reference signs related to drawings and placed in parentheses in a claim, are solely for attempting to increase the intelligibility of the claim, and shall not be construed as limiting the scope of the claim. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

A) Materials and methods Ethics statement

The study protocol was approved by the Comite d’Etica d’lnvestigacio Clinica (Institutional Review Board number PR(AG)350/2017) of the Hospital Universitari Vail d’Hebron, Barcelona, Spain. Samples were obtained from healthy adults, who all provided written informed consent, and were prospectively collected and cryopreserved in the Biobanc (register number C.0003590). All samples received were totally anonymous and untraceable.

Cells, antibodies and reagents

Cell lines CEM.NKR CCR5+ and ACH-2 (obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, from Dr. Alexandra Trkola and Dr. Thomas Folks, respectively), were used. PBMCs from healthy donors were obtained anonymously from the Banc de Sang I Teixits, Barcelona, Spain.

CD4+ T cells and NK cells were isolated from cryopreserved PBMCs of healthy donors using commercial kits and the corresponding manufacturer’s instructions (MagniSort Human CD4+ T Cell Enrichment; Affymetrix, and MagniSort™ Human NK cell Enrichment; eBioscience).

PBMCs from healthy donors were isolated by Ficoll-Paque density gradient centrifugation and cryopreserved in liquid nitrogen. Cells were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS; Gibco, Life Technologies, Inc.), 100 U/ml penicillin, and 100 pg/ml streptomycin (Life Technologies, Inc.) (R10; control medium), and maintained at 37°C in a 5% CO2 incubator.

The A32 antibody (targeting antibody to HIV gp120 epitope) was obtained through the AIDS Research and Reference Program, NIAID, NIH (Cat#11438) from Dr. James E. Robinson.

BaL gp120 recombinant protein was also obtained through the NIH AIDS Reagent Program.

The anti-CD16 clone 3G8 was purchased from Stemcell. Synthesis and conjugation of antibodies to gold nanoparticles

- Chemicals

Sodium citrate tribasic dihydrate (³99%), gold (III) chloride trihydrate HAuCU · 3H2O (99.9% purity) and sodium tetraborate decahydrate were purchased from Sigma- Aldrich. Fetal Bovine serum, FBS, (research grade, sterile filtered) and Dulbecco’s Modified Eagle Medium, DMEM, (with 1000 mg/ml_ glucose and sodium bicarbonate, without L-glutamine, sodium pyruvate, and phenol red, liquid, sterile-filtered) were purchased from Sigma. All reagents were used as received without further purification and all glass material was sterilized and dehydrogenated in an oven prior to use. Milli-Q water was used in the preparation of all solutions. - Gold nanoparticle synthesis

Aqueous solutions of sterile endotoxin free 40 nm citrate-stabilized AuNPs were synthetized according to previously developed seeded-growth method following a citrate reduction protocol as previously described [Bastus, N.G. et al., 2011] AuNPs were obtained after different sequential steps of growing, yielding about 5-10 ¹⁰ NPs/mL (33 micrograms/mL). After purification by centrifugation, to discard by products and contaminants, NPs were resuspended in a solution of 2.2 mM sodium citrate and stored at 4°C in the dark. A main UV-Vis Spectroscopy absorption peak at 532 nm was identified.

All the particles were used within 20 days after their synthesis. - Design of a conjugating media

In order to appropriately conjugate the AuNPs with the mixture of Abs under cooperative regime, the inventors had to design an appropriate media.

To that end, sodium tetraborate decahydrate and sodium citrate solution. The concentration of sodium citrate was fixed at 2.2 mM for the nanoparticles stability whereas a range of borate buffer between 3 and 10 mM could be used to make a compatible medium for antibodies. - Preparation of the antibody mixture

Antibodies A32 (targeting antibody) and anti-CD16 (NK-activating) were dispersed in a conjugating media consisting of a mixture of 4 mM Borate and 2.2 mM SC solution. The Ab concentrations in the resulting mixing solution were of 3 pg/ml of the anti- gp120 (A32) Ab, and 20 pg/ml of anti-CD16 (3G8). The difference in Ab concentration was directly related to the different Ab affinity for the AuNP surface previously evaluated in isothermal absorption curves obtained by preparing different antibodies concentration and measuring the zeta potential and conductivity using the Zetasizer Nano series instrument (following manufacturer’s instructions). The results are provided in FIG. 4, wherein it is shown that A32 has more affinity towards NPs than 3G8.

The NP preparation obtained as disclosed above at a concentration of 2.2 mM, was added to the antibodies solution, at a volume ratio antibodies solution:NP of 1:1 or 9:1 by gently drop-wise addition with very mild agitation (100 rpm, standard hot plate), and left for 48 hours at 4 °C.

After that, the resulting mixture was centrifugated at 5000xg for 40 minutes to precipitate the NPs and redispersed them in clean fresh Borate-sodium citrate medium at the same concentrations as specified above under this same section, thus achieving a dispersion of the Bi-NP functionalized with A32 and anti-CD16. Analogously, and for comparative purposes, bispecific bifunctionalized AuNPs were obtained with IgG mouse (Sigma-Aldrich) and IgG rat (Sigma-Aldrich), instead of A32 and anti-CD16. Just the concentration of the antibodies during the preparation of the mixture was changed to 18 pg/ml of each one. These IgGs showed a saturation concentration of about 5 pg/ml. Hereinafter these NPs are referred as “Irre-AuNPs”. In addition, a previously reported antibody-adsorption conjugation protocol to generate the A32-3G8 bispecific gold nanoparticles (Ciaurriz, P., et al. 2017) was reproduced. This protocol, which lead to a random disposition of the antibodies on the surface of gold nanoparticles (non-polarized), was performed to compare the activity of these generated random bispecific nanoparticles with that of the invention (with ordered disposition of antibodies), in terms of specific cell doublets formation.

Briefly, 133 pi of 15 mM borate buffer pH 8.7 was added to 1 ml_ of AuNPs obtained as disclosed above. 3 pg/ml of A32 and 20 pg/ml of 3G8 were added and allowed to react under agitation for 30 mins. Afterwards, sucrose was incorporated to a final concentration of 5% and incubated for 30 min. Then, 160 pL of 3% bovine serum albumin (BSA) were added and shaken for 10 min. Thereafter, the sample was centrifuged (7,500g for 30 min) to remove unbound antibodies and AuNPs were re suspended in 1 ml_ of 2 mM borate buffer pH 8.7 containing 5% sucrose, 2% glycerol, 0.5% BSA, and 0.01% Tween. The washing step was repeated and the conjugate was re-suspended in 100 pl_ of the mentioned borate buffer.

- Physicochemical characterization of Bi-AuNPs

Physicochemical characteristics of synthesized nanoparticles before and after conjugation were assessed by different methodologies; the size and the monodispersity of the synthesized NPs were assessed by transmission electron microscopy (TEM).

To characterize the different conjugates, the samples were evaluated by UV-Vis spectrophotometry (Cary 60 UV-Vis Spectrophotometer, Agilent), Dynamic Light Scattering (DLS) and zeta potential experiments. DLS (for measuring particle size) and zeta potential experiments were conducted in a Zetasizer Nano series instrument (Malvern). The particles size and zeta potential were measured simultaneously three times. Previously to the characterization experiments, samples were centrifuged at 5.000xg for 40 mins to remove unbound antibodies.

- Experiment to assess the distribution of antibodies onto Janus AuNPs

With the aim to directly observe if the method of the invention leads to the segregation of a mix of antibodies in two different domains, a straightforward experiment was performed.

On the one hand, AuNPs of ~ 60 nm were doubly conjugated with rabbit anti-HSA IgG (18 pg/ml) and rabbit anti-BSA IgG (18 pg/ml). On the other hand, smaller AuNPs of 25 nm were functionalized with BSA (5 mM) and of 15 nm functionalized with HSA (5 mM). The bispecific conjugates were incubated with the AuNPs functionalized with BSA and the AuNPs functionalized with HSA for 24 hours. Then, the sample containing the three types of AuNPs was purified from the excess of smaller AuNPs by decantation and after that, the TEM grids were prepared by dipping. Finally, the sample was analyzed by Transmission Electron Microscopy.

Conjugates were visualized using an 80-keV TEM JEOL 1010 equipped with an Orius (Gatan) CCD Camera. TEM grids consist on an ultrathin-formvar coated 200 mesh copper grid covered with a layer of carbon (Ted Pella, Monocomp, Madrid, Spain). . The TEM grids were prepared by the dipping procedure, which consists in the direct immersion of the grids in the AuNPs solution and letting them dry in open atmosphere overnight.

- Calculation of the number of antibodies adsorbed to NP surface

The theoretical calculation of the number of Ab molecules conjugated to the spherical AuNP was done assuming that the functionalized AuNP could be approximated by a perfect sphere, and that the factor of occupation of Abs on top of the AuNPs could be considered 0.8. Regarding this, AuNPs of approximately 40 nm were used at concentration 5*10 ¹⁰ NPs/ml. Therefore, by applying equation number 1, the total available surface for functionalization was obtained as 5026.55 nm ² /NP, which multiplied by the total number of nanoparticles gave 2.51*10 ¹⁴ nm ² .

A ( sphere ) = 4 x p x R _{u NP} (1 )

On the other hand, considering one antibody having a diameter of 14 nm, the fingerprint was calculating using equation 2, obtaining 153.94 nm ² /Ab.

Therefore, by dividing the total available NPs surface between the space occupied by one antibody, it was found a space for 1.63*10 ¹² antibodies. As the number of nanoparticles was known, each nanoparticle should be coated by 32 antibodies. However, different factors such as steric hindrance, efficiency at packing and certain degree of functionality loss can have an impact in the conjugation, being loaded a lower number of antibodies. According to these criteria and supporting results from TGA experiment (FIG. 2) the different doses were selected to be employed in the assays.

- Thermogravimetry experiment

The concentration of antibodies in the AuNPs was also estimated by thermogravimetry, where the mass loose between 200 °C and 400 °C is attributed to Ab sublimation. Results of the TGA analysis showed the weight loss of AuNPs functionalized with mouse IgG. A decrease of weight of about 10% corresponds with the Abs (FIG. 2). With the reported literature values of antibody density per surface area (150 nm ² per antibody) on dense antibody coatings (Saha B. et al. , 2014), it was obtained a number of 32 Abs per NP approximately. Considering the density of Ab to be 1.04 and that of Au 19.3 g/ml, a 10 nm corona of antibodies in a 40 nm diameter Au solid NP accounts for about the 10% of the total weight, as TGA data shows (FIG.2), corroborating the above calculations. The anisotropy of the signal with respect to Abs is likely due to the different thermal degradation of the Ab fraction on direct contact with the NP surface.

Thermogravimetric analyses (TGA) were carried out on a Pyris TGA 8000 under N2 flow with a temperature range from 30 °C to 600 °C at a heating rate of 5 °C min-1. Considering that the limit of detection is a loss in mass of 5 pg of Ab, a total volume of 417 pi of AuNPs was conjugated with mouse IgG producing 0.269 mg of bioconjugates. The purified bioconjugates were deposited in a ceramic crucible, allowing the excess of water to evaporate during 24 hours in a laminar flow cabinet. The measurements of TGA were carried out by the ICN2 (Institut Catala de Nanociencia i Nanotecnologia) technical services.

- Cell-to-cell contact assays

CD4+ T cells were coated with 1 pg of recombinant gp120 protein during 1 hour at room temperature. After extensive washes, CD4+ T cells were mixed with NK cells in a 1:1 ratio and stained with anti-CD56 (PE; Beckton Dickinson) and anti-CD3 (PE- Cy7; Beckton Dickinson) antibodies for detection of NK cells and CD4 T cells, respectively. After washing, unconjugated AuNPs, Bi-AuNPs of the invention or comparative bispecific AuNPs conjugated with IgG mouse and IgG rat, both obtained as explained previously, were added. After 20 minutes incubation at room temperature, cells were washed and fixed with PFA (2%). Samples then were acquired on a LSR Fortessa flow cytometer (Becton Dickinson) and data were analyzed using FlowJo V10 software.

- Confocal Microscopy

Attachment of Bi-AuNPs to their targets and subsequently promotion of dual cell conjugates were assessed by confocal microscopy.

First, CEM.NKR CCR5+ cells were stained with 2 mM of the membrane lipid marker PKH67 (Sigma-Aldrich) according to the manufacturer instructions. Cells were coated with 1 pg of HIV-1 BaL gp120 recombinant protein for 1h at room temperature. Then, NK cells isolated from PBMCs of healthy donors were obtained as described above. NKs and PKH67-labelled CEM.NKR CCR5+ cells were mixed at 1:1 ratio and incubated for 20 mins with Bi-AuNPs (2.5 pg/ml of total antibody burden ) of the invention (prepared as disclosed above) and attached to coverslips previously coated with 0.1 mg/ml of poly-L-lysine (Sigma-Aldrich) and fixed with PFA (2%) for 15 minutes at room temperature. Cells were finally stained with DAPI (1:5,000 dilution) (Thermo Fisher) and mounted with Fluoromount G (eBioscience).

Preparations were imaged with an Olympus BX61 microscope. ImageJ software was used for image compositions. Note that Au NPs of 40 nm do not reflect or diffract light because they are below the visible light resolution limit but they are still good at dispersing light, especially if aggregated, and this light scattering/dispersion can be observed in the confocal microscope.

- NK cell activation assay NK cell activation and cytotoxicity profile after stimulation with Bi-AuNPs was evaluated using PBMCs of healthy donors.

Cytotoxicity was assessed by measurement of CD107a and IFN-g. Briefly, PBMCs were cultured in R10 medium with 10 pg/ml Bi-AuNPs, 10 pg/ml Irre-AuNPs, 5 pg/ml of A32, 5 pg/ml of 3G8 or 10 ng/ml PMA plus 1 mM ionomycin (positive control) for 4.5 hours in a 96-well plate at 37°C and 5% CO2. CD107a-PE-Cy5 (H4A3;

Beckton Dickinson), BD GolgiPlug Protein Transport Inhibitor (Beckton Dickinson) and BD GolgiStop Protein Transport Inhibitor containing monensin (Beckton Dickinson) were also added to each well at the recommended concentrations.

Cells were then washed and stained with a viability dye (LIVE/DEAD Fixable Violet dead cell stain; Thermo Fisher) for 20 minutes at room temperature.

For measuring the activation marker H LA-DR, cells were firstly stained with anti- CD56-FITC (B159; Beckton Dickinson), anti-CD3-PE-Cy7 (SK7; Beckton Dickinson) and anti-HLA-DR-SB600 (LN3; eBioscience) antibodies for 20 minutes at room temperature. Cells were then fixed and permeabilized with Fixation/Permeabilization Solution (Beckton Dickinson) for 20 minutes at 4 °C, washed with BD Perm/Wash buffer and stained with anti-IFN-g AF700 (Life technologies) for 30 minutes at 4°C. After washing, cells were fixed with PFA (2%) and acquired on an LSR Fortessa flow cytometer (Becton Dickinson). Data were analyzed using FlowJo V10 software. - Antibody-dependent cell-mediated cytotoxicity (ADCC) assay

The ADCC assay was performed as described by Gomez-Roman V.R. and colleagues [Gomez-Roman V.R., et al., 2006]

Briefly, CEM-NK ^R .CCR5 cells were used as target cells after being double-stained with PKH67 (Sigma-Aldrich) and eF670 (Labclinics) dyes following manufacturer’s instructions.

Then, cells were coated with 1 pg of the HIV-1 BaL gp120 recombinant protein for 1 hour at room temperature and extensively washed in ice-cold R10 medium.

Target cells were dispensed in U-bottom 96-well plates (5,000 cells/well) and incubated for 15 minutes with a 1:1000 dilution of a plasma obtained from an HIV- infected person with detectable viral load (positive control), 5 pg/ml of free A32, 5 pg/ml of free 3G8, unconjugated AuNPs, monoconjugated A32-AuNP, monoconjugated 3G8-AuNP or Bi-AuNPs.

After incubation, NK effector cells isolated from PBMCs of healthy donors were added at 1:10 target/effector ratio. Plates were centrifuged and incubated for 4 hours at 37°C and 5% CO2. Finally, cells were washed, fixed with PFA (2%), acquired on a LSR Fortessa flow cytometer (Becton Dickinson) and analyzed using FlowJo software.

Target cells were identified in a PKH67-versus side scatter (SSC) plot. Loss of the eF670 marker was used to determine the percentage of killed target cells in an eF670 versus PKH67 plot.

ADCC on virally-reactivated cells was performed as follows; the latently-infected cell line ACH-2 was stimulated to produce HIV by the addition of 10 pg/ml PHA (Phytohemagglutinin; Fisher Scientific) and 10 nM PMA (phorbolmyristate acetate; Abeam) during 17 h at 37°C. Then, cells were subjected to the ADCC assay as described above. ADCC of viral-reactivated cells was calculated as the fraction of cells that disappeared within the target population after the addition of BiAb-AuNPs in comparison to the control condition with targets and NK cells, but lacking the nanoparticles. For assessing the absolute number of cells killed by ADCC, we added to each well flow cytometry particles for absolute cell counting (5*10 ⁴ /ml) (AccuCount

Blank 5.0-5.9 pm, Cytognos). - Statistical analysis

Analysis were performed with GraphPad Prism v6. p values <0.05 were considered statistically significant.

B) Results - Production of Bi-AuNPs

Au NPs of 40 ± 4 nm, with a surface charge of -44 mV and colloidally stable in sodium citrate at 5*10 ¹⁰ NPs/ml were functionalized with mouse IgG anti-human CD16 and human IgG anti-HIV gp120 antibodies, using the reaction conditions and media developed by the inventors and provided in previous sections. After conjugation, AuNPs presented a redshift in the UV-VIS spectra of 4 nm (FIG.

1a), consistent with a fully coating of the NP surface with antibodies.

The surface charge of the bifunctionalized Au NPs was also characterized, reporting a more positive surface charge (-31.4 mV), which is near the charge of free antibodies in the same buffer further indicating total coverage (FIG. 1b). The DLS (Dynamic Light Scattering) hydrodynamic ratio increases upon conjugation correlating well with the UV-Vis spectroscopy measurements. This DLS increase corresponding to a single monolayer of antibodies, already indicates that there is no Ab denaturation. The DLS hydrodynamic ratio increased upon conjugation, showing a size increase attributed to a single monolayer of Abs (Figure 1c). No broadening of the UV-VIS, DLS or Surface charge peaks after the conjugation process was observed, indicating the high stability and the homogeneity of the formed bioconjugates. Otherwise, if there was a dispersion of the conjugation process, the final conjugated peaks would integer the dispersion of the NP core and the dispersion of the NP coating. As the characteristic width of the measured peaks remained constant through the conjugation process, the conjugation could be determined as homogeneous.

- Quantification of Abs per NP:

The inventors then determined how many antibodies were fitted on NPs, and how many of them were still functional (more by shading effects due to dense packing rather than denaturalization, since the later induces protein aggregation leading to greater DLS measurements). The theoretical calculation of the number of Ab molecules bond to the spherical AuNP was done assuming that the bifunctionalized Au NP can be approximated by a sphere, and that the factor of occupation of Abs on top the Au NPs could be considered 0.8 Regarding this, the total number of Abs conjugated (Nmax) estimated was 25 Abs/NP (as it has been explained above):

Thermogravimetric analysis (TGA) of the conjugates was performed after purification in order to correlate the thermal loss in mass, with the loss of the antibodies from the surface of the AuNPs. This reported a number of antibodies of approximately 30 Abs/NP (FIG. 2). - Antibodies conjugated onto monofunctionalized AuNP fully retain their specificity for antigen recognition

In order to assess the impact of the conjugation process on the capacity of both A32 and 3G8 antibodies to recognize their cognate antigen, the specific cell-surface binding affinities of both monoconjugates to their targets was evaluated by flow cytometry:

To do so, CEM.NKR CCR5+ cells, coated with the gp120 recombinant protein, were incubated with free A32 or monoconjugated A32-AuNPs. As expected, free A32 antibody recognized the entire population of gp120-coated cells (median 99.60%), whereas the addition of naked AuNPs and control medium did not result in any positive signal (median 2.48 and 0.66%, respectively). Importantly, monoconjugated A32-AuNPs retained the same binding capability than free antibodies (median 98%), indicating that the conjugation process does not specifically alter antibody targeting capacity (FIG. 3a).

Similarly, it was found that free 3G8 and monoconjugates of 3G8-AuNPs were able to target CD16 in a similar manner (median 58.60 and 51.30%, respectively). Again, negative controls were unsuccessful to recognize the CD16 molecule. (FIG. 3b).

Therefore, it was concluded that the conjugation of A32 and 3G8 antibodies onto gold nanoparticles did not impact their specific capabilities for antigen recognition.

- Generation of Bispecific AuNPs (Bi-AuNPs). After verifying that conjugated antibodies retain their antigen-binding specificity, the inventors prepared the conjugated nanoparticles consisting in the double conjugation of both A32 and 3G8 antibodies as disclosed above. To that end, both antibodies were mixed in a conjugating media to which previously- manufactured AuNPs were added dropwise. The relative amounts of each Ab was varied to compensate for their different affinity for the gold surface extracted from the isothermal adsorption curves monoconjugation curves (FIG. 4). In order to promote the 50/50 coating to maximize cell coupling, the antibody 3G8 was always added at 100% of its saturation concentration value due to its low affinity detected in the calibration curves and, varying the amount of A32 it was observed that concentrations 60:100 A32:3G8 produced the highest number of T cell - NK doublets with 3 fold increase compared to the control condition, consisting in the culture of the gp120-coated CD4 T cells with the NK cells in the absence of any nanoconjugate.

Overall, it was demonstrated that Bi-AuNPs could be successfully generated, maintain their capabilities to recognize the cognate antigen and specifically support the formation of cell doublets consisting in gp120-coated cells and NK cells. - Bi-AuNPs promote cell contact between gp120-coated cells and NK cells

The potential of Bi-AuNPs of the invention to bring closer NK cells expressing the CD16 molecule and gp120-coated CD4 T cells was determined.

Bi-AuNPs of the invention were added to equally mixed CD4 T and NK cell populations, and cell bioconjugates were quantified by flow cytometry using the markers CD3 and CD56 for the identification of CD4 T cells and NK cells, respectively.

The experiments were performed with 2 different doses of conjugates, one containing 2.5 pg/ml of total antibody burden and another 10 pg/ml. FIG. 5a shows the gating strategy used to quantify cell doublets generated by Bi-AuNPs. Naturally occurring doublets were low (median 2.54%) but after the addition of Bi-AuNPs at 2.5 pg/ml,

CD4-NK contacts significantly increased reaching a median of 5.91% (p=0.04). A higher percentage of cell doublets was obtained when the more concentrated Bi- AuNPs (10 pg/ml) were tested, with a 7.4-fold increase compared to control medium (median 16%) (p=0.01) (FIG. 5b). Of note, controls consistent in naked AuNPs did not promote the generation of cell-to- cell contacts.

With typical binding affinities of nM, it is difficult to imagine that a single BiAuNP will be able to bring two cells together, therefore, it is expected more a number of conjugates zipping both cells together, what can be easily observed in the confocal microscope. Thus, it was observed that Bi-AuNPs promoted the specific formation of CEM NKR-NK cell doublets by confocal micrographs showing the presence of the BiAb-AuNPs in between the linkage of HIV-expressing cells (CEM.NK ^R CCR5 ⁺ coated with HIV gp-120 recombinant protein and stained with PKH67 in green, and DAPI nuclei staining in blue) and primary NK cells (DAPI nuclei staining in blue) (data not shown).

- Bi-AuNPs stimulate the production of IFN-g by NK cells

Next, the capability of BiAb-AuNPs of the invention to activate NK cells in culture was determined. PBMCs from healthy donors were incubated with free antibodies, Bi- AuNPs composed of irrelevant IgGs (polyclonal antibody IgG mouse and IgG rat), or PMA plus lonomycin (as a positive control).

BiAb-AuNPs significantly increased the production of IFN-Y (FIG. 6a). Moreover, focusing on NK cells co-expressing INF-g and CD107a, it was found that they did not induce significantly expression of CD107 alone (FIG. 6b), but that they increased the percentage of cells producing both functional markers (median of 22.50, 3.68, 9.03 and 16.20 for BiAb-AuNPs, A32, 3G8 and Irre-AuNPS, respectively) (FIG. 6c). The marker H LA-DR was not affected significantly, indicating that BiAb-AuNPs do not induce global NK cell activation (FIG. 6d). Overall, the data provided herein demonstrates that Bi-AuNPs of the invention induces IFN-g production from NK cells and increases the proportion of cells co expressing the functional markers IFN-g and CD107a.

- Bi-AuNPs induce a potent ADCC response against gp120-coated cells

After the successful conjugation of A32 and 3G8 antibodies onto AuNPs, after verifying that binding properties are still preserved, and the successful generation of bioconjugates, it was tested the potential of Bi-AuNPs of the invention to promote an ADCC response against HIV-expressing cells. To do so, an ADCC assay was performed by co-culturing NK and gp120-coated cells with Bi-AuNPs. Cell killing was measured by the loss of the marker eFluor670 (FIG. 7a). It was found that Bi-AuNPs were able to promote a highly potent and specific ADCC response, significantly better than the cytotoxicity reached by the addition of irrelevant bispecific AuNPs or free A32 antibody (median ADCC of 29.16% versus 14.90 and 12.71% for Irre-AuNPs and free A32, respectively) (FIG. 7b). The specific killing of Bi-AuNPs translated in a 3.71-fold enhanced cytolytic effect compared to A32. As expected, free 3G8 did not promote ADCC, and combination of free A32 and 3G8 gave similar ADCC values than the A32 antibody alone (FIG. 7b).

- The ordered distribution of antibodies onto AuNPs favors the specific cell coupling. Further, to functionally confirm the polarized nature of the BiAb-AuNPs of the invention, the percentage of cell doublets was compared with the one of naked AuNPs.

BiAb-AuNPs, and contrarily to the unconjugated nanoparticles, favored the formation by 3-fold of specific cell doublets NK-HIV ⁺ as mentioned. Moreover, the BiAb-AuNPs did not increment the percentage of homodoublets of HIV ⁺ or NK cells (FIG. 9). In the absence of AuNP polarization, the expectation would be an increase on the homodoublets due to random distribution of both antibodies on the surface of the NPs. In fact, it was also compared the BiAb-AuNPs of the invention with an already stablished protocol expected to yield antibodies-coated NPs randomly (detailed in the above materials and methods section). It was found that the ordered distribution characterizing the NPs of the invention allows producing 2.95 times more cell doublets than the previously described “random” protocol (FIG. 10).

Nowadays there is not straight forward methods to directly analyze the composition, conformation and distribution of Abs per NP. For that, the inventors conducted another kind of experiment. For these experiments, doubly functionalized AuNPs with anti-HSA and anti-BSA IgGs were exposed to smaller AuNPs of 25 nm functionalized with BSA, and of 15 nm functionalized with HSA, as labels to directly observe the presence and distribution of the two different antibodies on the nanoparticle surface. Then the 3 functionalized AuNPs were incubated together and observed by TEM.

Interestingly, the cooperative absorption approach was confirmed by the observation of polarized nanoparticles with hemispherical distribution of the labels (FIG. 8). These TEM images confirmed that following our described protocol, Janus nanoparticles with ordered domains of antibodies can be obtained. Altogether, our results indicate we have developed a conjugation method which enable the successful conjugation of two different proteins, such as antibodies, in domains, which ultimately translates into a better biological response. REFERENCES CITED IN THE APPLICATION:

Ahmed M. et al., “Lack of in Vivo Antibody Dependent Cellular Cytotoxicity with Antibody Containing Gold Nanoparticles”, Bioconjug Chem., 2015, 26(5), pages 812- 816; Bastus N. G. et al., “Kinetically Controlled Seeded Growth Synthesis of Citrate- Stabilized Gold Nanoparticles of up to 200 nm: Size Focusing versus Ostwald Ripening”, Langmuir, 2011, 27, pages 11098-11105;

Ciaurriz, P., et al., “Comparison of four functionalization methods of gold nanoparticles for enhancing the enzyme-linked immunosorbent assay (ELISA)”, Beilstein J Nanotechnol,, 2017, 8, pages 244-253;

Gomez-Roman, V.R., et al., A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity. J Immunol Methods, 2006. 308(1-2): p. 53-67;

Kosmides A.K. et al., “Dual targeting nanoparticle stimulates the immune system to inhibit tumor growth”, ACS Nano, 2017, 11, p. 5417-5429;

Li W. et al., “One-domain CD4 fused to human anti-CD16 antibody domain mediates effective killing of HIV-1 infected cells”, Sci. Reports, 2017, pages 1-12;

Montenegro JM et al., “Controlled antibody/(bio-) conjugation of inorganic nanoparticles for targeted delivery”, Adv Drug Deliv Rev., 2013,65(5), pages 677-688; Saha, B. et al., "How antibody surface coverage on nanoparticles determines the activity and kinetics of antigen capturing for biosensing", 2014, Anal Chem, 86(16): page 8158-8166

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

1. A coated metallic nanoparticle comprising a metallic nanoparticle which is coated with a layer comprising: a) cell-targeting antibodies or functional fragments thereof; and b) immune-activating antibodies or functional fragments thereof, wherein: - the metallic nanoparticle has an average particle size of at least 20 nm,

- the antibodies are directly attached to the surface of the metallic nanoparticle by electrostatically interactions,

- the layer comprises two domains, a domain “A” and a domain “B”, wherein:

-- domain “A” comprises a higher amount of cell-targeting antibodies or fragments thereof than of immune-activating antibodies or fragments thereof, and

-- domain “B” comprises a higher amount of immune-activating antibodies or fragments thereof than of cell-targeting antibodies or fragments thereof, wherein the antibodies comprised in domains “A” and “B” are ordered on the nanoparticle surface. 2. The coated metallic nanoparticle of clause 1, wherein the average particle size is comprised from 30-150 nm.

3. The coated metallic nanoparticle of any one of the clauses 1-2, wherein at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “A” surface is occupied by ordered cell-targeting antibodies or fragments thereof and at least a 75%, at least a 80%, at least a 85%, at least a 90% or at least a 95% of the domain “B” surface is occupied by ordered immune-activating antibodies or fragment thereof.

4. The coated metallic nanoparticle of any one of the clauses 1-3, wherein the cell targeting antibody is capable of interacting with a component of an infected cell membrane, such as an antibody which interacts with a component of an HIV-infected cell.

5. The coated metallic nanoparticle of any one of the clauses 1-4, wherein the cell activating antibody or fragment thereof is a NK-cell activating antibody, such as an anti-CD16 antibody. 6. The coated metallic nanoparticle of any one of the clauses 1-5, wherein the metallic nanoparticle comprises one or more metals selected from silver, gold, copper, nickel, cobalt, molybdenum, palladium, platinum, tin, zinc, titanium, chromium, tantalum, tungsten, iron, rhodium, iridium, ruthenium, osmium, aluminum and lead. 7. The coated metallic nanoparticle of any one of the clauses 1-6, wherein:

- the nanoparticle is a gold nanoparticle with an average particle size from 30 to 150 nm,

- the cell-targeting antibody is an anti-HIV antibody and the immune-activating antibody is an NK-cell activating antibody,

- the coated metallic nanoparticle has a surface charge value within the range from -10 to -40 mV, particularly from -29 to -33.5mV, when measured with

Zetasizer Nano series instrument, following manufacturer’s instructions, and

- at least a 75% of the domain “A” surface is occupied by ordered anti-gp120 antibodies and at least a 75% of the domain “B” surface is occupied by ordered anti-CD16 antibodies. 8. A process for preparing the coated metallic nanoparticle as defined in any one of the clauses 1-7, which comprises the steps of:

(a) providing a dispersion comprising: (i) citrate ions, (ii) borate ions, (iii) cell targeting antibodies or functional fragments thereof, (iv) immune-activating antibodies or functional fragments thereof, and (v) metallic nanoparticles having an average particle size of at least 20 nm, wherein:

- one of the antibody populations (iii) or (iv) has less affinity towards nanoparticle’s surface in the dispersion than the other, and

- the dispersion has:

- a total antibody population, which corresponds to the sum of the cell-targeting antibodies or functional fragments thereof and immune-activating antibodies or functional fragments thereof, at a concentration excess with respect to metallic nanoparticles’ concentration;

- the antibody population with less affinity towards nanoparticle surface is at a concentration excess with respect the other antibody population; - citrate ions are at a concentration excess with respect to metallic nanoparticles concentration;

- borate ions are at a concentration excess with respect to the total antibody population;

- provided that the concentration of the citrate and borate ions confer to:

(i) the dispersed nanoparticles a surface charge equal or lower than - 20 mV when measured with Zetasizer Nano series instrument, following manufacturer’s instructions; particularly lower than -25 mV, particularly from -25 to -40 mV; and

(ii) the dispersed antibodies have a surface charge Y±(Y 0.1), wherein Ύ” represents the mean of the charge of the free cell-targeting antibody and the free immune-activating antibody, wherein the “charge of the free antibody”, either from the cell-targeting antibody or immune-activating antibody, is determined by Zetasizer nano

(b) stirring at a temperature that allows the formation of the domains “A” and “B” as defined in clause 1 on nanoparticles surface, and

(c) optionally isolating the resulting metal nanoparticles from the solution resulting from step (a).

9. The process of clause 8, wherein the dispersion has:

- a concentration of citrate ions from 5 to 20 times the citrate concentration needed to completely coat the NPs included in the dispersion:; and

- a concentration of borate ions from 5 to 10 times the borate concentration needed to completely coat the total population of antibodies.

10. The process of any one of the clauses 8-9, wherein the dispersion has:

- a concentration of nanoparticles comprised from 10 ^L 9 NPs/ml to 10 ^L 15 NPs/ml; - a concentration of citrate ions from 0.2 to 10 mM, particularly from 1 to 5 mM, particularly 2.2; and

- a concentration of borate ions from 2 to 15 mM, particularly from 3 to 10.

11. The process of any one of the clauses 8-10, wherein step (a) is performed by: a.1. preparing a mixture of citrate buffer, borate buffer and antibodies; and a.2. adding to the resulting mixture from (a.1), the metal NPs.

12. A diagnostic kit comprising the coated metallic nanoparticles as defined in any one of the clauses 1-7 and, optionally, instructions for its use.

13. A pharmaceutical composition comprising coated metallic nanoparticles as defined in any one of the clauses 1-7 together with one or more pharmaceutically acceptable excipients and/or carriers.

14. A coated metallic nanoparticle as defined in any one of the clauses 1-7 for use in therapy or diagnostics.

15. A coated metallic nanoparticle as defined in any one of the clauses 1-7 for use in the treatment and/or prevention of a disease by inducing antibody-dependent cellular cytotoxicity, such as in the treatment and/or prevention of human immunodeficiency virus.