WO2023166535A1

WO2023166535A1 - Pharmacological conjugates in cancer therapy

Info

Publication number: WO2023166535A1
Application number: PCT/IT2023/050056
Authority: WO
Inventors: Alessandro POGGI
Original assignee: Irccs "Ospedale Policlinico San Martino"
Priority date: 2022-03-01
Filing date: 2023-02-22
Publication date: 2023-09-07
Also published as: IT202200003758A1

Abstract

The present invention relates to pharmacological conjugates of amino bisphosphonates, such as zoledronic acid, with antitumor monoclonal antibodies, such as, for example, cetuximab, the process of preparation thereof and the use thereof in the medical field.

Description

Pharmacological conjugates in cancer therapy

The present invention relates to pharmacological conjugates in cancer therapy.

In greater detail, the invention relates to pharmacological conjugates of amino bisphosphonates, such as zoledronic acid, with antitumor monoclonal antibodies, such as, for example, cetuximab, the process for the preparation thereof and the use thereof in the medical field.

Monoclonal antibodies conjugated to molecules (ADC: antibody-drug conjugates), which a) have cytotoxic effects on tumour cells or b) stimulate immune system activation, represent one of the possible therapeutic options for the treatment of solid and haematologic neoplasms. In fact, the antigenic specificity of a monoclonal antibody directs the activity of the cytotoxic drug prevalently towards cells which express this specificity. In case (a), a classic example is the drug called brentuximab vedotin consisting of an anti-CD30 monoclonal antibody conjugated covalently with monomethyl-auristatin E

(https://www.ema.europa.eu/en/documents/overview/adcetris-epar-medicine- overview_it.pdf https://www.aifa.gov.it/documents/20142/648668/rcp- epar_adcetris_2012-11 -22.pdf). The latter is a microtubular toxin that induces the death of the target cell. The antibody-drug conjugate brentuximab vedotin is used in the treatment of refractory/relapsed Hodgkin’s lymphoma, in systemic anaplastic large cell lymphoma and in cutaneous T-cell lymphoma where the target cell markedly expresses the CD30 antigen. In case (b), the antibody is conjugated to a cytokine that stimulates the immune system, such as, for example, interleukin 2, to respond towards the cell which expresses the antigen to which the antibody reacts (Scott LJ 201 , Thomas A et al 2016). Therefore, the antibody recognises the neoplasm and, besides exerting a direct inhibiting effect on cell growth, brings about an increase in the concentration of a cytokine that activates T cells and natural killer (NK) cells to kill the tumour. Finally, the antibody used as the vector of a cytotoxic drug or immunostimulatory cytokine can activate so-called antibody-dependent cellular cytotoxicity (ADCC), thanks to its fraction called fragment crystallizable (FC) region. This activity can be performed by T cells which express the receptor for the FC fragment of immunoglobulin (FCyRIIIA or CD16) (Monteverde M et al. 2015, Musso A et al. 2014). These are prevalently NK cells, y5 T cells and a minority of CD8+ T cells, which represent the major lymphocytic antitumor effectors (Monteverde M et al. 2015, Musso A et al. 2014, Fuertes MB et al 2021 ). In the group of y5 T cells, the subpopulation called V52 T cells is capable of exerting ADCC and of becoming activated if the target cell produces isopentenyl pyrophosphate (IPP); IPP production is enhanced by the drug zoledronic acid (ZA), which, at a physiological pH, is present in the medium as ZA. ZA is capable of penetrating into the cell and inhibiting the metabolic pathway of cholesterol just after the synthesis of mevalonate (https: //en. Wikipedia, org/wiki/ Mevalonate_pathway, Musso A et al. 2014, Ribot JC et al. 2021 , Li Y et al. 2021 ), thereby bringing about an increase in IPP. The cancer cell presents the accumulated IPP to the V52 T cell, inducing its proliferation in the presence of interleukin 2 (IL2) and the activation of cytotoxic antitumor activity (Musso A et al. 2014, Hermann T et al. 2020).

Bisphosphonates, also called diphosphonates, are drugs having two phosphonate groups. Bisphosphonates are divided respectively into non-nitrogen- containing bisphosphonates and nitrogen-containing bisphosphonates depending on the absence or presence of a nitrogen atom in the structure formula. The latter include amino bisphosphonates, which have the nitrogen atom in an amino group, such as, for example, zoledronate, risedronate, ibandronate, and alendronate.

ZA is an amino bisphosphonate commonly used for the treatment of menopausal osteoporosis (https://it.wikipedia.org/wiki/Acido_zoledronico, Russell RG 2011 , Dhillon S 2016) or osteoporosis associated with bone metastases of solid tumours; the drug has an anti-osteoporotic effect by virtue of its strong tendency to be concentrated in bone and favour the deposition of bone matrix, like other bisphosphonates. Its potential in stimulating V52 T cells is also exploited in the case of multiple myeloma (MM), since it is a localized disease affecting bone: in this disease, the administration of ZA increases the number of V52 T cells in situ and in circulation (Terpos et al. 2021 ).

A limit in the use of ZA to stimulate V52 T cells is precisely its preferential location in bone, if administered as such; consequently, with the exception of tumours located in bone, ZA cannot be proposed in antineoplastic immunotherapy, as it would not be able to activate V52 T cells located in a different tissue. In order to improve the distribution of amino bisphosphonates in tissues other than bone, it has been proposed to use liposomal vesicles loaded with an amino bisphosphonate (ZA or alendronate) (Shmeeda et al. 2013). Thanks to these new formulations, it has been possible to demonstrate that V52 T cells can have a potent antitumor effect against neoplasms that are not necessarily confined to the bone, but also located in other tissues (Shmeeda H et al. 2013). Given these circumstances, it is clear that, in order to stimulate the antitumor activity of V52 T cells, it is necessary to direct ZA into a given tissue, avoiding or limiting the primary location in bone.

In order to direct ZA into tissues other than bone, liposome particles can be used, as mentioned above (Shmeeda H et al. 2013, Salzano G et al. 2016, Porru M et al. 2014, Kolmas J et al. 2017, Hodgins NO et al. 2016, Hodgins NO et al. 2017). Liposomes have small dimensions and a tendency to fuse with cell membranes; furthermore, they are concentrated in the reticuloendothelial system of organs such as the liver and spleen (Clancy et al. 2013). This effect can be reduced by treating the liposome with polyethylene glycol (PEG) and functionalizing it with a specific antibody that directs it towards the desired site (Bendas G 2001 , Puri et al. 2009). However, after repeated infusions of liposomes an anti-PEG immune response or a strong anti-liposome antibody response can be induced (Ishida et al. 2006a, Ishida et al. 2006b, Ishida et al. 2005, Ishida et al. 2001 ). Though extraosseus location of amino bisphosphonate alendronate increases a great deal with liposomal formulations (Shmeeda H et al. 2013), in the case of ZA the results have not been optimal; in fact, the administration of several ZA-loaded liposomal preparations caused the death of the inoculated animal in a short time and the cause of this was not identified (Shmeeda H et al. 2013). Liposomal preparations have high production costs and pose disadvantages as drug carriers, since they are subject to oxidative degradation and must thus be preserved and manipulated in a nitrogen atmosphere. Therefore, there are limitations to the development of a product that may be used as a pharmaceutical preparation in relation to production costs and the quality of the product itself (Tinkle S et al. 2014). In fact, the quality guarantee involves both production processes and the stability of the formulation and systems based on nanoparticles such as liposomes are influenced by i) scalability of the production process, ii) reproducibility and reliability of the final product, iii) lack of equipment and specific experience, iv) chemical instability or denaturation of the encapsulated drug during the production process and v) problems of long-term stability. (Narang AS et al. 2013 and Sercombe L et al. 2015). All these disadvantages have resulted in the fact that, although liposomes are known as a potential drug delivery means, the preparations actually on the market and used are rather limited (Sercombe L et al. 2015).

In the light of the above, there is thus an evident need to be able to have new products comprising bisphosphonate compounds, or, even better, amino bisphosphonate compounds, for the treatment of tumours, which are capable of overcoming the disadvantages of the known products comprising bisphosphonates or amino bisphosphonates in this type of therapy.

The solution according to the present invention fits into this context; it aims to provide new conjugates of bisphosphonate compounds, such as amino bisphosphonate compounds, with antitumor monoclonal antibodies for the purpose of directing the bisphosphonate into the tissue of therapeutic interest.

In particular, according to the present invention, among the amino bisphosphonates, use is made of ZA, which has been covalently bonded to a monoclonal antibody, selected from the ones used in cancer therapy, with the aim of determining the specificity of location and action of ZA towards the therapeutic target.

Among antitumor monoclonal antibodies, the epidermal growth factor receptor (EGFR) inhibitor called cetuximab (Cet) (Wong SF 2005, Fasano M et al. 2021 ) is known for the treatment of several tumours such as head and neck cancer (HNC) or colorectal cancer (CRC). However, at present, the CRC treatable with Cet represents a minority fraction (about 10-20%), as Cet can be used only if the neoplastic cells do not show mutations of the signal pathway triggered by EGFR, i.e. so-called CRC with normal EGFR (Wong SF 2005, Fasano M et al. 2021 , Xie YH et al. 2020).

According to the present invention, it has now been shown that it is possible to covalently conjugate amino bisphosphonate ZA to the anti-EGFR monoclonal antibody Cet, here referred to as ZA-Cet ADC. The conjugate according to the invention is capable of: a) reacting with the CRC cells that express EGFR, b) inducing the production of IPP by CRC organoids, c) stimulating the proliferation, in the presence of exogenous IL2, of V52 T cells when incubated with the CRC EGFR+ LS180 line, d) stimulating the expansion of V52 T cells in response to CRC organoids, e) activating cytotoxicity towards CRC organoids.

These demonstrations indicate that ZA, delivered through the Cet antibody, can trigger in the target tumour cell the series of events that are typically induced by soluble ZA and which hesitate in the activation of antitumor T V52 cells (Morita CT et al. 1995, Wesch D et al. 1997, Musso A et al. 2014, Zocchi MR et al. 2017, Jandke A et al. 2020). Furthermore, the effectiveness of the conjugate in inducing the proliferation of V52 T cells is greater than that of soluble ZA.

On the basis of what has been illustrated above and described in the examples, it is thus plausible that the conjugation of Cet with ZA, as it increases the antitumor immune response, is capable of extending the use of Cet also to EGFR- mutated neoplasms. Furthermore, the conjugate according to the present invention makes it possible to provide the effects of both its components, that is, the induction of ADCC by the Cet antibody and the stimulation of the antitumor V52 T cells due to the products derived from the metabolization of ZA by the neoplastic cell; and the activation of ADCC by V52 T cells by means of the CD16 expressed on the latter (Monteverde M et al. 2015, Musso A et al. 2014).

The advantages of using a monoclonal antibody-ZA conjugate derive from the different pharmacokinetics of the conjugated ZA compared to free soluble ZA. The pharmacokinetics of the conjugate should be similar to that of the antibody, thus favouring the continued presence of the antibody-ZA conjugate in circulation rather than at the bone site, as occurs in the non-conjugated formulation (Russel RG 2011 , Droggell SA 2002). In addition, renal clearance should preferably be that of the antibody and not of free ZA (Hedrich WD et al. 2018, Birrer RJ et al. 2019, Abdollahpour-Alitappeh M et al. 2019). In fact, in the case of antibody-drugs conjugates, the half-life of the antibody is what defines the half-life of the antibodydrug conjugate complex. The amount of antibody that becomes located in the target tissue in the case of a solid tumour is around 20% of the amount administered (Chames P et al. 2009, Chamier T et al. 2019), but the renal elimination of a monoclonal antibody is practically absent; thus, the location of the conjugated monoclonal antibody should follow that of the antibody, with an increase in its location in the tumour as a result of the effect of increased permeability, the high vascular density and the rapid angiogenesis which is characteristic in the context of neoplasia (Seki T et al, 2009, Maeda T et al. 2009).

In short, the advantages of the conjugate according to the present invention are the following:

-compared to Cet, the conjugate shows the specific effect of ZA in activating the immune system;

-compared to ZA, the conjugate provides a focused action of ZA on the cancer cell;

-compared to Cet and ZA not linked together, the conjugate provides a simultaneous action of Cet and ZA on the same tumour cell;

- the possibility of treating both the original tumour and metastases, as well as mutated CRC EGFRs;

- the possibility of an industrialization similar to that for monoclonal antibodies.

The mechanisms underlying the effectiveness of the conjugate according to the invention can be assumed based on the following observations. It is known that after having penetrated inside the target cell, ZA influences the cholesterol biosynthesis pathway, bringing about an accumulation of small phosphoantigens such as IPP and dimethylallyl pyrophosphate (https://en.wikipedia.org/wiki/Mevalonate_pathway, Musso A et al. 2014, Ribot JC et al. 2021 , Li Y et al. 2021 ). These small phosphoantigens are capable of inducing the activation of V52 T cells, since they bind to several members of the butyrophilin family (Vavassori et al. 2013) and permit their interaction with the V52 T cell antigen receptor. This triggers the activation of the V52 T cell, resulting in the proliferation thereof, production of cytokines and activation of the cytolytic machinery towards the cell presenting the small phosphoantigens (Vavassori et al. 2013, Poggi A et al, Zocchi et al. 2017, Bonneville M et al. 2010). The experimental results described further below demonstrate that ZA-Cet ADC is capable of stimulating the production of IPP by CRC organoids. This is clear proof of the possibility of using the neoplastic cell as a target of the ZA conjugate according to the present invention. It should be noted that the entity of the production of IPP induced by ZA-Cet ADC is lower compared to that induced by soluble ZA. This can be explained by the fact that, according to the experimental example described further below, the tumour organoids were subjected to stimulation with the control, ZA, Cet or Za-Cet ADC for only 48 hours. However, it is known that it can take antibodies longer to penetrate inside a droplet of geltrex, and hence inside an organoid or a three-dimensional culture, as compared to a drug not linked to an antibody (Ayuso JM et al. 2018). Therefore, the production of IPP induced by ZA-Cet ADC has shown to be lower than that induced by soluble ZA, because the period of stimulation used in the experiment was brief. Furthermore, it can be hypothesized that the processing of ZA-Cet ADC takes place with different mechanisms compared to that of the soluble form. In fact, ZA-Cet ADC can be endocytosed after interaction with the receptor, whereas the soluble form would have to penetrate through non- receptor specific mechanisms (Chen T et al. 2002, Le Louedec et al. 2019, Tan AR et al. 2006, Nilofer A et al. 2012). It remains to be determined whether and where the ZA is separated from the Cet antibody to which it is conjugated in order to perform its activity inside the target cell. In any case, the Cet antibody conjugated with ZA shows a reactivity similar to that of the native Cet antibody with different lines of CRC and is capable of stimulating the activation of V52 T cells, which consequently proliferate in the presence of exogenous IL2. The entity of the proliferation induced in the CRC LS180 line is comparable to that stimulated by soluble ZA and apparently the concentration of ZA linked to Cet which is necessary in order to obtain this effect can be up to 1000 times lower than that of soluble ZA. This data suggests that delivering the ZA specifically towards EGFR+ neoplastic cells after having conjugated it with the Cet antibody considerably increases the ability of the neoplastic cell to present the small phosphoantigens generated by the action of ZA alone in an optimal manner.

The observation that the Cet conjugated with ZA can also stimulate the expansion of V52 T cells starting from PBMCs suggests that this antibody may also enter cells that do not necessarily express EGFR, since EGFR+ cells are not usually present in peripheral blood. In fact, it is known that the binding with EGFR is mediated by the variable portion of the immunoglobulin, whereas the component of the fragment crystallizable (Fc) region can interact with PBMCs having specific receptors for this portion of the antibody (Bournazos S et al. 2017a, Taylor RP et al. 2015). The monocytes present among PBMCs are the cell type that expresses the highest levels of different Fc receptors and thanks to the latter they can bind the antibodies (Hepburn AL et al. 2004, Bournazos S et al. 2017b). Furthermore, monocytes present among PBMCs represent the circulating cells that are necessary and sufficient in order that the V52 T cells are activated and can consequently proliferate after the presentation of the small phosphoantigens (Morita CT et al. 1995, Wesch D et al. 1997, Musso A et al. 2014, Zocchi MR et al. 2017, Jandke A et al. 2020). It is thus reasonable to think that ZA-Cet ADC can be endocytosed through the engagement of the Fc receptors present on the monocytes and that ZA induces the production of IPP, which in turn triggers the activation of V52 T cells. This V52 T cell activation mechanism, irrespective of the interaction of the Cet antibody-ZA conjugate with EGFR, could reduce its specific antitumor effect. It is necessary to take into consideration that the interaction with the Fc receptors present on accessory cells such as monocytes, macrophages and dendritic cells should also involve the native Cet antibody which is administered in the treatment of CRC or head and neck cancer (Wong SF 2005, Fasano M et al. 2021 ); thus, the pharmacokinetics and the tumour concentration of ZA-Cet ADC should follow that of the native antibody. On the other hand, the activation of monocytes/macrophages due to ZA-Cet ADC could increase the immune response of the V52 T cells.

This phenomenon could thus also be relevant in vivo in the patient and in particular at the tumour site of the CRC or head and neck cancer, i.e. the involvement of antigen-presenting cells could amplify the effect. In this context, it is known that the composition of the tumour microenvironment (TME) is very important in regulating the immune response, in particular in the case of CRC, and that antigen-presenting cells such as macrophages are essential for correctly defining whether and to what extent the TME influences the antitumor response (Fridman W et al. 2012, Church SE et al. 2015). Therefore, it is reasonable to think that ZA-Cet ADC may activate monocytes/macrophages in the patient at the tumour site of the CRC or head and neck cancer.

The demonstration that the organoids of different patients with CRC, incubated with ZA-Cet ADC are capable of activating V52 T cells and that such cells are capable of killing the cells making up the organoids is the “proof of concept” that the ZA-Cet ADC according to the present invention can be used as a therapy in patients with CRC.

As mentioned above, it is known that the Cet antibody is used in the treatment of a subpopulation of CRC characterised by the absence of mutation in the EGFR-mediated activation pathway - a subpopulation that represents around 20% of cases of CRC (Xie YH et al. 2020). Based on the experimental results now obtained for the conjugate according to the present invention, it is plausible that not only this same cohort of patients, but also patients who have a mutated EGFR pathway, may benefit from ZA-Cet ADC (Wong SF 2005, Fasano M et al. 2021 , Xie YH et al. 2020). In fact, the stimulation of V52 T cells should occur irrespective of the biochemical characteristics of the activation pathway, subject only to the condition that EGFR is expressed. In addition, it is reasonable to think that ZA-Cet ADC may also recognise other cells present in the TME expressing EGFR and render them capable of activating V52 T cells. In this regard, it has recently been demonstrated that mesenchymal stem cells (MSCs), such as tumour-associated fibroblasts, which are present at the tumour site, express EGFR and are capable of inhibiting the antitumor activity of T cells owing to the production of inhibiting factors such as [3-transforming growth factor (Costa D et al. 2018). The Cet antibody is capable of activating the antibodydependent cellular cytotoxicity exerted by natural killer cells and this phenomenon counterbalances the inhibitory effect of mesenchymal cells (Costa D et al. 2018). It is reasonable to think that the ZA-Cet ADC according to the present invention may also interact with MSC EGFR+ and that this activates the expansion of V52 T cells. In any case, as occurs for the cells of organoids, ZA-Cet ADC could activate the ADCC of MSCs, i.e. their elimination. Given that these cells inhibit the activation of the antitumor response, ZA-Cet ADC would reduce this inhibitory effect, thus actually increasing the immune response. Thus, whether there is a direct effect on the tumour epithelium or an indirect effect on the inhibitory activity of MSCs, it is plausible that the therapeutic use of the ZA-Cet ADC according to the present invention can be extended to a larger cohort of patients than the one that accesses the native Cet.

At present, to the Applicant’s knowledge and based on what is retrievable in the literature, the conjugate according to the present invention, called ZA-Cet ADC, is unique and different from the various approaches that have been employed to date to direct ZA into the tumour site (Russel RG 2011 , Droggell SA 2002, Tokumaru Y et al. 2019, Kato J et al. 2016, Hedrich WD et al. 2018, Birrer RJ et al. 2019, Abdollahpour-Alitappeh M et al. 2019).

It should also be noted that, in the case of CRC, the combination of the two drugs (soluble ZA and native Cet) has been reported (Tokumaru Y et al. 2019, Kato J et al. 2016) to be more effective than the individual compounds in controlling the growth of CRC also in cases in which the EGFR/RAS-dependent activation pathway was mutated. In the reported cases, ZA was used both because of its ability to treat bone metastases of CRC, where the particular affinity of this drug for bone was exploited (Tokumaru Y et al. 2019), and because of its specific direct antitumor and anti-proliferative cytotoxic activities, which usually manifest themselves at doses larger than 10pM (Kato J et al. 2016); these effects increase synergically in the presence of Cet. However, ZA-Cet ADC, compared to the use of the two drugs used in combination, has the advantage of being directed specifically towards the cells that express EGFR. The effects of the two components of ADC will develop specifically against these cells, namely: blocking of the signal transduction by means of EGFR, ADCC activity mediated by T cells and myelocytes FcyR+, the cytotoxic effect of ZA, and the stimulating effect of ZA for V52 T+ cells.

Overall, therefore, the experimental data shown in the example indicate that the Cet antibody covalently conjugated with ZA effectively combines various antitumor effects mediated by the two drugs individually and associated with the possibility of directing them specifically at the neoplastic cell based on the expression of EGFR specifically recognised by the Cet antibody.

Comparable results were also obtained with the ZA conjugate with another amino bisphosphonate compound, namely Rituximab.

In particular, the experimental data described indicate that ZA-Rit ADC likewise has properties of reactivity and of blocking the proliferation of CD20+ cancer cell lines and, moreover, induces the specific stimulation of V52 T cells, as demonstrated for ZA-Cet ADC in the case of colon cancer. Overall, these data suggest that conjugating ZA to monoclonal antibodies allows the target cell to be precisely identified while simultaneously preserving the functional properties of the antibody. Furthermore, the conjugate is capable of activating V52 antitumor T-cells, suggesting a potential use of the antibody-ZA conjugates in solid and haematologic neoplasms.

These results make it plausible that the advantageous effects obtained with the conjugates according to the invention, between ZA and the cetuximab antibody or Rituximab antibody, can also be obtained with other conjugates according to the present invention comprising bisphosphonate compounds or, better still, amino bisphosphonates, other than ZA and other monoclonal antibodies having a free amino group other than cetuximab or Rituximab.

Therefore, the specific subject matter of the present invention relates to a conjugate of a bisphosphonate compound with a monoclonal antibody having at least one free amino group, wherein said amino group of the monoclonal antibody is linked to at least one phosphonate group of the bisphosphonate compound.

According to one embodiment of the present invention the amino group of the monoclonal antibody is linked to at least one phosphonate group of the bisphosphonate compound without any linker, but rather directly by means of a phosphoramide bond. Several molecules of bisphosphonate can be linked for each monoclonal antibody when the monoclonal antibody has several NH2 groups.

In the conjugate according to the present invention, the bisphosphonate compound can be an amino bisphosphonate compound, preferably an amino bisphosphonate compound without a free NH2 group. For example, the amino bisphosphonate compound can be selected from the group consisting of zoledronate, risedronate, ibadronate, preferably zoledronate.

According to the present invention, the monoclonal antibody used in the conjugate can be an antitumor monoclonal antibody, for example an antitumor monoclonal antibody selected from the group consisting of cetuximab, Rituximab, anti-Her2, anti-CD38 or anti-BCMA.

According to a particular embodiment of the present invention, the conjugate can be between the amino bisphosphonate zoledronate and the antitumor monoclonal antibody cetuximab, said zoledronate and cetuximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of cetuximab.

According to a further embodiment of the present invention, the conjugate can be between the amino bisphosphonate zoledronate and the monoclonal antibody Rituximab, said zoledronate and Rituximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of Rituximab.

The present invention also relates to a pharmaceutical composition comprising or consisting of a conjugate as defined above, as an active ingredient, together with one or more excipients and/or adjuvants.

Furthermore, the present invention relates to the conjugate as defined above or the pharmaceutical composition as defined above for use as a medicament.

The subject matter of the present invention further relates to the conjugate as defined above or the pharmaceutical composition as defined above, for use in the treatment of cancer. The types of cancer that may be treated depend on the monoclonal antibody linked to the bisphosphonate or, even better, to the amino bisphosphonate, for example B and CLL lymphomas in the case of anti- CD20, such as, for example, Rituximab, multiple myeloma in the case of anti-CD38 or anti-BCMA, breast cancer in the case of an anti-her2 conjugate, head-neck cancer or colorectal cancer in the case of Cetuximab. The present invention further relates to a process for the preparation of a conjugate as defined above, comprising the following steps: a) reacting the bisphosphonate compound with a suitable reagent to activate at least one phosphonate group of said bisphosphonate compound for reaction with a free amino group of a monoclonal antibody; b) reacting the bisphosphonate compound obtained in step a) with a monoclonal antibody having a free amino group in order to obtain the conjugate.

According to the present invention, step a) of the process can be carried out by reacting said bisphosphonate compound with 1 -ethyl-3-3- dimethylaminopropyl carbodiimide and imidazole to obtain an O-acylisourea intermediate of the bisphosphonate compound followed by a phosphorimidazolide of the bisphosphonate compound. In this manner, the phosphonate group of the bisphosphonate compound is activated in the form of phosphorimidazolide for the subsequent reaction with the monoclonal antibody.

According to the process of the present invention, both step a) and step b) can be carried out in a medium comprising imidazole, preferably at pH 6±0.2.

As mentioned above, in the process according to the present invention, the bisphosphonate compound can be an amino bisphosphonate compound, preferably an amino bisphosphonate compound without a free NH2 group, such as, for example, zoledronate, risedronate, ibadronate, preferably zoledronate.

Furthermore, in the process of the present invention the monoclonal antibody can be an antitumor monoclonal antibody such as cetuximab, Rituximab, anti-Her2, anti-CD38 or anti-BCMA.

According to one embodiment of the process of the present invention, the amino bisphosphonate is zoledronate and the antitumor monoclonal antibody is cetuximab, said zoledronate and cetuximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of cetuximab.

According to a further embodiment of the process of the present invention, the amino bisphosphonate is zoledronate and the monoclonal antibody is Rituximab, said zoledronate and Rituximab being linked together by a phosphoramide bond between a phosphonate group of zoledronate and at least one free amino group of Rituximab.

The present invention will now be described, by way of non-limiting illustration, according to a preferred embodiment thereof, with particular reference to the figures in the appended drawings, in which:

Figure 1 shows a schematic representation of the synthesis of ZA- Cet ADC: ZA was linked to EDC and the complex obtained was reacted with imidazole, thereby generating an intermediate ester which, upon dissociating, generates isourea and a highly reactive phosphorimidazolide; the latter, in the presence of the free amino groups of the lysine of cetuximab, generates a phosphoram idate between ZA and Cet, thus freeing imidazole. A covalent bond is created in the absence of a linker, so the molecular weight of ZA-Cet ADC differs approximately from the native antibody based on the amount by weight of linked ZA as shown in figure 2.

Figure 2 shows the results of the MALDI analysis of ZA-Cet ADC and a comparison with native Cet: the Cet and ZA-Cet ADC, as indicated in the diagram in figure 1 , were subjected to MALDI analysis. The top graph presents, at the peak of Cet, a molecular weight of 152,247.816, compared to the peak in the bottom graph given by ZA-Cet ADC, with a peak molecular weight of 153,355.381 . The difference in molecular weight between cetuximab and the ADC is given by the molecules of ZA covalently bonded to Cet. The two peaks corresponding to the light chains (50,931.964 for Cet and 51 ,367.187 for ZA-Cet) and heavy chains (76,288.754 for Cet and 76,914.926 for ZA-Cet) of the antibody are present in the left part of each graph.

Figure 3 shows the results of the analysis of the reactivity of ZA-Cet ADC with stabilized CRC cell lines: the stabilized CRC cell lines Caco-2, HT29 and SW620 were used to demonstrate reactivity with ZA-Cet ADC. The cells were incubated with a dose of 2pg/ml as the final concentration of the Cet antibody or ZA- Cet ADC and the reactivity with EGFR was detected by adding a second antibody against human immunoglobulin, Cet being an antibody provided with human FC. Each panel of the figure shows the labelling with the negative control (histogram at the far left of every single sub-panel) and indicated as ctrneg (second reagent only), the reactivity of the Cet antibody (indicated as Cet) and the reactivity with ZA-Cet ADC (indicated as ZA-Cet ADC, histograms with hatching). The reactivity of Cet and ZA-Cet ADC almost overlap. The reactivity of the antibody conjugate is specific and limited to cancer cells that express EGFR.

Figure 4 shows the results of the induction of the expansion of V62 T+ cells from PBMCs or of the CRC LS180 line after incubation with ZA-Cet

ADC: peripheral blood mononuclear cells (PBMCs) were incubated with the different stimuli indicated in the panel on the left (soluble ZA 1.0, 0.5 pM, Cet-ZA conjugate: ZA-Cet ADC 0.5nM or a subsequent 1 :2 dilution); after 24 hours of incubation, 30ILI of recombinant IL2 were added to favour proliferation of the activated cells; the presence of V52 T cells in the culture was evaluated by indirect double immunofluorescence staining with antibodies specific for the V52 receptor and CD3 antigen. The samples were analysed by flow cytometry (10⁴ events/sample). The percentages of CD3+V52+ T cells were determined at various time intervals (7-14- 21 days) in different donors indicated in the upper part of the panel (n=2-6). The results are presented as the mean of the observed values and the extreme values obtained are indicated. CTR indicates the percentage of V52 cells present in the cultures incubated with the medium alone plus added IL2. The CRC LS180 cell line, expressing EGFR, was incubated with the various stimuli indicated (soluble ZA 1 pM or with ZA-Cet ADC 0.5nM and the subsequent 1 :2, 1 :4, 1 :8, 1 :16 dilutions thereof) with T cells isolated from PBMCs highly purified by means of negative selection. After 24 hours of incubation, 30ILI of recombinant IL2 were added to favour the proliferation of the activated cells; the presence of V52 T cells in the culture was evaluated at the specified times as described above. CTR represents the percentage of V52 cells in the population of T cells cultured with IL2 without LS180, whilst LS180 CTR represents the condition under which the T cells were cultured with LS180 and IL2 alone in the absence of other stimuli.

Figure 5 shows that ZA-Cet ADC induces the expansion of V62 T cells in the co-cultures between T cells and organoids from patients with CRC: Panel A. Double immunofluorescence analysis of the cells recovered from the cultures of highly purified autologous T cells from peripheral blood: the first three contour diagrams refer to the T cells, the second three contour diagrams represent the T cells in co-culture with the organoid OMCR16-005TK. The cultures were analysed after 21 days, after labelling the cells with anti-CD3 antibodies (x axis) and anti-V52 antibodies (y axis). The data are presented as fluorescence intensity in arbitrary units (on a decimal logarithmic scale of red fluorescence versus fluorescence read in the far-red region). Every sub-panel can be divided into 4 quadrants where the CD3-V52- cells (bottom left quadrant), CD3+V52- cells (bottom right quadrant), CD3-V52+ cells (top left quadrant) or CD3+V52+ cells (top right quadrant) are represented. The percentage of double positive cells, which represent the V52 T cells, is indicated in the top right quadrant. The various stimuli used are represented by ZA (ZA, 1 pM) or Cet (4pg/ml) or ZA-Cet ADC (ZA-Cet ADC, 4pg/ml). The first panel represents the control condition, i.e. the T cells cultured on their own. The control, with only Cet, of the cultures of T cells co-cultured with the organoid OMCR16-005TK is represented in the fourth sub-panel. Panel B. Titration curve for the Cet antibody-ZA conjugate in the in vitro expansion of the population of V52 T cells in response to the organoid OMCR16-005TK. Allogenic T cells derived from a healthy donor were used. The various concentrations are indicated on the x-axis. Panel C. Percentage of the population of V52 T cells evaluated as in panel A, after co-culture of T cells derived from healthy donors and organoids derived from patients with CRC stimulated with ZA-Cet ADC (4pg/ml). The percentages were evaluated at the various times indicated (0-7-14-21 days). Panel D. Evaluation of the percentage of V52 cells obtained from the co-culture between cells of patients with CRC and the organoids derived from the tumour of the same patients. The percentages were evaluated at the days indicated as described in panel A. Every symbol present in the panels C and D indicates a specific donor of allogenic (C, n=9) or autologous (D, n=6) T cells.

Figure 6 shows that V62 T cells are capable of killing the cells making up the organoids derived from patients with CRC in the presence of ZA-Cet ADC: Panels A and B. Viability of organoids of CRC cells incubated with populations of V52 T cells activated under an allogenic (A) or autologous (B) condition at the different effectortarget tumour ratios indicated (2.5:1 , 5:1 , 10:1 , 20:1 ). Panels C and D. Viability of organoids of CRC cells incubated with populations of allogenic (C) or autologous (D) V52 T cells at the effector: target ratio of 1 :1 in the absence of stimuli (CTR) or with soluble ZA (1 pM) or with ZA-Cet ADC (4pg/ml) or with the native Cet antibody (4pg/ml). Every point corresponds to a specific healthy donor of T cells (under an allogenic condition A n=7 and C n=6) or patient with CRC (autologous condition B and D, n=4).

Figure 7 shows the results of the MALDI analysis and the functional effects of ZA-Rit ADC and the comparison with native Rit: the native Rit and ZA-Rit ADC produced in a similar manner to ZA-Cet ADC, as indicated in figure 1 , replacing the Cet antibody with the Rit antibody, were subjected to MALDI analysis (panel A). The peaks related to the complete Rit antibody (peak on the right 147,309.546) are shown in the top part and those of the light and heavy chains on the left. The two sub-panels represent two preparations of ZA-Rit ADC which differ because of a different DAR (drug antibody ratio), i.e. the different amount of Za linked to Rit obtained by varying the amount of ZA added in the conjugation reaction. The reactivity of the ZA-Rit ADC conjugate at 2pg/ml (black line of the central subpanel) is represented in panel B on the two lines CD20+ Raji and Karpas compared with the native Rit antibody (on the right) or the human anti-lg alone (control). The effect on the proliferation of the two lines indicated is represented in panel C. It shows a decrease in the proliferation of the two lines in the presence of ZA-Rit ADC which is similar to the native Rit for Raji and lower for Karpas. Panel D. Evaluation of the expansion of V52 T cells from peripheral blood in the presence of ZA-Rit ADC at various doses (20-2-0.2-0.02pg/ml) compared with that obtained with ZA 1 pM evaluated at day 10 of the culture. Za-Rit ADC induces an expansion effect comparable to that of soluble ZA at the doses of 20 and 2.0 pg/ml). The percentage of V52 T cells present at time zero (V52 dO) is shown in the sub-panels on the left. The amounts in pM of ZA linked to the antibody conjugate are also indicated. The data refer to two distinct donors of PBMCs.

EXAMPLE 1: Preparation of the ZA and Cet conjugate according to the present invention and study of its effectiveness in tumours

MATERIALS AND METHODS

Conjugation of ZA to Cet to formulate the compound ZA-Cet ADC

The ZA (p.m. 272.09) was purchased from Selleckchem (Houston Texas, USA), whereas the Cet was obtained as a leftover part of the dose used for the administration of the drug Erbitux^R to patients with CRC, kindly supplied by the Pharmacology Unit of the San Martino Polyclinic Hospital in Genoa. Erbitux^R is dialyzed to eliminate the excipients. The reaction medium is a 0.1 M imidazole solution at pH6±0.2 (con HCI 1 N). The solutions containing zoledronic acid (ZA) or EDC are prepared in the solution with imidazole. The zoledronic acid (ZA) is incorporated into the structure of the protein by exploiting the phosphoric groups present therein, in accordance with the reactions reported by Ghosh SS et al. 1990, Shabarova ZA et al. 1983 Shabarova ZA 1970 and Itumoh EJ et al. 2020 for the conjugation of peptides to the phosphoric groups of deoxyribonucleic acid. The following reactions can be defined: zoledronic acid and 1 -ethyl-3-3- dimethylaminopropyl carbodiimide (EDC, Sigma/Aldrich), with a molar excess of EDC relative to ZA of 100 times (e.g. 1 mg/ml ZA with 100mg/ml EDC), at room temperature, which generates an O-acylisourea as an intermediate that reacts with imidazole (99% Sigma) in solution and generates a phosphorimidazolide. The phosphorim idazolide reacts with the free amino groups of the Cet antibody added to the solution with a molar excess of ZA of 585 times (e.g. approximately 1 mg ZA and 1 mg of Cet), which generates a highly reactive intermediate that dissociates, giving rise to the phosphoram idate compound consisting of ZA linked to Cet; imidazole is released in the next 4h at room temperature. The ZA-Cet ADC is purified from the rest of the free components (imidazole, EDC and ZA) by dialysis with a Slide-A-Lyzer Cassette (Thermo Fisher) at 10000 MWCO for 24h.

The chemical conjugation of ZA to Cet (figure 1 ) was performed as per an order from the company Nanovex Biotechnologies SL (Parque Tecnologico de Asturias, Edificio CEEI, 33428 Llanera, Asturia, Spain). The product of conjugation was subjected to analysis by MALDI (matrix-assisted laser desorption/ionization) mass spectrometry using an Ultraflex Extreme MALDI spectrometer (Broker) after a quality control of correct operation. The mass spectra were recorded in the m/z interval 20000 and 200000. The peaks of the light chains, heavy chains and the whole antibody as well fall in this interval. The MALDI analysis was performed as per order in the laboratories of the Fondazione Toscana Life Sciences (53100 Siena, Italy). Quantification with inductively coupled plasma mass spectrometry (ICP-MS) was performed using the 8900 ICP-QQQ device of Agilent Technologies to determine the amount of phosphorus coupled to the antibody. The samples were digested in 2% HNO3. This technique provides information about the content of ZA coupled to the antibody by calculating P, whereas the analysis of S relates to the concentration of the protein (i.e. of the antibody). It is important to observe that, for the analysis by ICP-MS and the conjugation of ZA to Cet, use was made of the same preparation that was administered to the patient after dialysis of the excipients (sodium chloride, glycine, polysorbate 80, citric acid monohydrate and sodium hydroxide). The dialysis was performed with the Slide-A-Lyzer Dialysis Cassette 10,000 MWCO for 24 hours from a 5mg/ml starting solution. These analyses were performed as per an order from Nanovex Biotechnologies SL (Asturia, Spain). ZA- Cet ADC, as an antibody conjugate, was later used in the experiments, indicating the concentration of the antibody used in pg/ml as is usually done with the native Cet. In the case of stimulation experiments with soluble zoledronate, this was indicated in pM as for every chemical compound. In the case of the expansion of V52 T cells from PBMCs or with the CRC LS180 line, for that preparation of ZA-Cet ADC, the molarity of ZA linked to Cet is indicated in order to compare it with the amount of soluble zoledronate used in the same experiments.

Similarly, following the same reaction scheme, the conjugate of the commercial anti-CD20 antibody Rituximab (Mabthera^R) with ZA, called ZA-Rit ADC, was produced and subjected to MALDI-TOF as described for ZA-Cet ADC.

Culture of CRC cell lines and immunofluorescence assays

CRC cell lines Caco-2, HT29 and SW620, supplied by the Cell Bank of the San Martino Polyclinic Hospital in Genoa, were purchased as follows: Caco-2 (cat no.86010202, Sigma Aldrich), HT29 (HTB-38™) from the American Tissue Culture Collection (ATCC), and SW620: (CCL-227™) ATCC. Furthermore, use was made of the cell lines: Raji (CCL-86™) ATCC Burkitt’s lymphoma and Cellosaurus Karpas 299 anaplastic large cell lymphoma (CVCL_1324), as CD20+ is reactive with the anti-CD20 Rituximab (MabThera^R). The cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin (Varesano et al. 2018). For the immunofluorescence assay, 10⁵ cells were incubated with 2pg/ml of Cet or ZA-Cet ADC at 4°C for 30m in. After incubation, the samples were washed by centrifugation with the addition of 3ml of medium/sample. The cells were then incubated with 2pg/ml of anti-human IgG antiserum conjugated with the fluorochrome Alexa Fluor 488 for 30m in at 4°C. Control samples were instead incubated with anti-human IgG antiserum. After incubation, the samples were washed and resuspended in 0.5ml of culture medium and analysed by means of a CyAn ADP or Cytoflex flow cytometer equipped with red laser to evaluate labelling with Alexa Fluor 488. 10⁴ events were analysed and the data are shown in histograms showing the relationship between the intensity of antibody reactivity (X- axis) expressed in arbitrary units of fluorescence detected in the green region (FL1 ) with the number of events analysed (Y-axis).

Generation of tumour organoids from CRC

The patients from whom the organoids were derived were recruited by the Oncological Surgery and Implantable Systems unit after their informed consent was obtained (Ethics Committee of the San Martino Polyclinic Hospital no 4/2011 and Ethics Committee of the Region of Liguria PR163REG2014). The tumours were staged according to the IIICC TNM classification. The pathological tissues were collected at the invasive front of the neoplasm by an expert pathologist. The tissue was chopped up with scissors under conditions of sterility and digested enzymatically for 45min at 37°C in Leibovitz medium (GIBCO), with 0.5mM EGTA, penicillin 100IU/ml and streptomycin 100pg/ml (GIBCO), gentamycin 5pg/ml (SIGMA), and collagenase I and II from Clostridium with approximate activity of 200IU/ml. The collagenases were used at different concentrations for the digestion of biopsies drawn from different portions of the colon. At the end of digestion, the tissue suspension was washed in RPMI1640 (GIBCO) with 10% bovine serum (Euroclone, tested ESC cat. ECS0196L) and passed through a 100pm sieve (Sarstedt, cat. 83.3945.100). The tumour fragments were then cultured after incorporation in Geltrex (LDEV-free, cat. A143302 GIBCO); 5-7 5pl domes were plated in a 24-well plate (Euroclone cat. ET3024). After their polymerisation, which took place at 37°C for 20min, 500pl of culture medium were added to each well. After 48 hours the culture medium was replaced with the medium for organoid expansion; no reference is available for the composition of the culture medium, as it is completely new and exploits the efficiency of the Fujii-Sato medium described previously (Fujii M et al. 2016). The medium was changed every two days and the cultures were divided after gentle trypsinization, which never induced separation into individual cells in new geltrex domes. The organoids for the experiments of stimulating the expansion of V52 T cells were obtained from the geltrex domes after incubation with PBS without Ca2+ and Mg2+, resuspension and seeding in plates with 96 Il-bottom wells (Sarstedt). The number of cancer cells contained in the organoids plated in each well was between approximately 10,000 and 20,000. This number was determined by means of a manual cell count or an automated one with a Cytoflex flow cytometer (Beckman Coulter) after complete trypsinization of the plated organoids. Similarly, the organoids were used as a target for experiments on the cytotoxicity exerted by V52 T cells in Il-bottom wells; the neoplastic cells were identified by labelling with propidium iodide (PI, SIGMA), or 7 actinomycin D (7AAD, SIGMA).

Evaluation of the production of small phosphoantigens by HPLC/TOF-MS

The production of IPP by the organoids after treatment with soluble ZA or ZA-Cet ADC for 48 hours was evaluated in cell extracts dissolved by stirring in MilliQ double-distilled water, with 250pl of Na3VO4, and clarified by centrifugation in a microcentrifuge (Eppendorf, 13,000 rpm, 3 minutes) by HPLC/TOF-MS as previously described (Jauhiainen M et al. 2009, Zocchi MR et al. 2017) with some modification (Zocchi MR et al. 2017). The calibration curve for IPP was generated by diluting the standard with a known concentration (0.1 -15pM) in MilliQ water with 250pl Na3VO4. The content of IPP was obtained with HPLC/MS-TOF using an Agilent 1200 chromatography system (with degasser G1379B, capillary pump G1376A and autosampler G1377A). The complete negative mass spectra were recorded with Agilent Mass Hunter software in a mass range of m/z 60-500. The graphs of the spectra obtained were processed with Agilent Mass Hunter analysis software, version B.02.00. The content of IPP was obtained by extracting the ion current peak (EIC m/z 244.99 [M-H]). The results are presented as pmol of IPP extracted with acetonitrile/ total protein content in each cell lysate. The method used precludes the IPP isomers from being distinguished as dimethylallyl pyrophosphate (DMAPP). The identity of the parent ion present in our cell extracts was verified through the formation of fragmented ions (m/z 79, m/z 159, m/z 177 and m/z 227); the negative MS/MS spectra were generated with the Agilent 1100 LC/MSD Trap mass spectrometer, equipped with an orthogonal electrospray source and an ion trap analyser as described (Zocchi MR et al. 2017).

Experiments on V62 T cell proliferation stimulation with CRC cells

ZA, used as a positive control in V52 T cell proliferation experiments, was solubilised in DMSO following the directions provided by the manufacturer. The amount of ZA used to stimulate the activation and expansion of V52 T cells varied between 0.5-5pM in accordance with previously reported data (Puri et al., 2009, Jauhiainen, M et al., 2009, Di Mascolo, D. et al., 2019). At these concentrations, the dilution of the DMSO present in the culture was lower than 1 : 10³ (1 :2x10³-1 :2x10⁴); at this dilution DMSO does not induce cytotoxic effects, as demonstrated by means of an assay with crystal violet/propidium iodide (data not shown). The fraction of peripheral blood mononuclear cells (PBMCs) was obtained from healthy adult blood donors, after informed consent signed at the time of the donation, and samples of venous blood derived from patients with CRC, by density gradient centrifugation (Pancoll human, density 1.077 g/ml, PAN Biotech, Munich, Germany) as described (Zocchi MR et al. 2017). In order to obtain the populations of V52 T cells, used in assays of cytolysis directed at CRC intestinal organoids, the T cells (including TCRa[3>95% and TCRyb <5%, usually) were isolated from PBMCs using the specific negative separation kit by StemCell Technologies (Vancouver, Canada supplied by Voden, Milan). The separation provided a pure population with T cells > 95% and the contaminated component consisted of B cells, whereas monocytes were virtually absent (<0.5%). The T cells thus isolated were maintained in culture in RPMI 1640 complete medium overnight in an incubator at 37°C with 5% CO2 to favour the adhesion of any residual monocytes. The following day, the T cells were recovered and cultured with the organoids seeded in plates with 96 Il-bottom wells, in 200pl of RPMI 1640 medium supplemented with 10% FBS, Penicillin/Streptomycin and L-Glutamine and in the presence of serial dilutions of ZA, or ZA-Cet ADC or the Cet antibody alone; the cultures were maintained in an incubator at 37°C in a humidified atmosphere with 5% CO2. After 24h of co-culture and when necessary, in relation to cell growth (approximately every 3 days), 10OpI of medium were eliminated and replaced with fresh medium supplemented with human recombinant interleukin 2 (IL2) (final concentration 30IU/10ng/ml, Miltenyi Biotec Italy, Bologna). At day 10-14 the cultured cells were divided and the culture wells duplicated; this was carried out over time whenever necessary in relation to cell growth (usually every 3-4 days). The percentage of V52 T cells was determined at various incubation times by double indirect immunofluorescence using the anti- TCR V52 monoclonal antibody (mAb) called y5123R3 ( IgG 1 ) in association with the anti-CD3 antibody JT3A 289/11/F10 (lgG2a) (Zocchi MR et al. 2017, Varesano S et al. 2018, Di Mascolo D et al. 2019) and analysis by flow cytometry (Beckman-Coulter CyAN ADP or Beckman-Coulter Cytoflex).

Assays on cytotoxicity towards CRC organoids

The experiments on the cytotoxicity exerted by V52 cells towards the organoids derived from patients with CRC were carried out by applying an assay with crystal violet staining of live cells made to adhere after the cytotoxic event.

Briefly, the organoids in geltrex droplets (3pl domes) in plates with 96 flatbottom wells (1 dome/well) and tested in a co-culture with autologous or allogenic V52 cells at different effector/target ratios (10:1 -5:1 -2:1 ) for 3 days to enable both the penetration of the cells into the domes (24-36 hours as evaluated in preliminary experiments) and the development of cytotoxic activity. The number of tumour cells contained in the dome was evaluated by means of a manual count and with a flow cytometer (Cytoflex, Beckman Coulter or Miltenyi MACS Quant) in order to be able to correctly calculate the number of cells to be added. As is described in detail elsewhere (Varesano et al. 2018, Di Mascolo et al. 2019), after incubation of the plated organoids the entire content of the culture well was transferred from the original plate into a plate which permitted the adherence of the neoplastic cells at 37°C with 5% CO2. After 48 hours, the wells were stained with crystal violet as described and the staining intensity was evaluated with a reading, after elution of the stain, at a wavelength of 594nm (VICTORX5, Perkin Elmer). The ODs obtained under the conditions of the organoid and V52 T cell co-culture were compared with the ODs obtained in the cultures of organoids alone, considered as 100% viability.

Statistical analysis

A statistical analysis was carried out using a two-tailed Student’s t-test. The p values are shown in the figure legends or reported in the text and the results are expressed as the mean±SD. The number of cases analysed is indicated in the figure legends or in the text. The experiments usually refer to 3-6 experiments using cell samples derived from different donors.

RESULTS

Conjugation of ZA to humanised anti-EGFR Cet monoclonal antibody and demonstration of successful conjugation

The conjugation of ZA to the Cet antibody is based on the possibility of linking ZA according to the reactions described in the literature for the conjugation of nucleic acids to proteins. The method exploits the possibility of forming a phosphoram idate. Figure 2 shows a comparison between the mass spectrum of the native Cet and that of the ADC derived after conjugation with ZA. The analysis by MALDI determined that the mass spectrum of both samples is characterised by the presence of the singly charged species at around 152 kDa and around 153 kDa, respectively, for Cet in native form and the Cet-ZA complex analysed. The other peaks are attributable to multiple charged chemical species. The increase in molecular weight observed for ZA-Cet ADC compared to the native antibody is around 1300 units: a variation is shown in the molecular weight compared to the native form and the link between ZA and the antibody is a covalent bond. As the link between ZA and the Cet antibody is direct, in the preparation analysed the drug antibody ratio (DAR) is 4.7 molecules of ZA per molecule of antibody.

The evaluation by ICP-MS determined, in the same preparation as analysed by MALDI, the amount of P (phosphorus) and S (sulphur), indicated in table I. Table I

ICP-MS evaluation of the content of P and S in ZA-Cet ADC

The samples indicated in table I were subjected to ICP-MS in order to determine the content of sulphur (associated with the Cet protein) and phosphorus (associated with ZA) in the different preparations. The Cet purified of excipients is the one used for conjugation with zoledronate.

The amount of P in the initial preparation of Cet, purified of excipients, is particularly low; thus, the variations in the content of P measured by ICP-MS can be assumed as an indicator that conjugation with ZA (rich in P) has taken place. In fact, it may be observed that the amount of P (attributable to ZA) in the preparation of ZA-Cet ADC is 886 times the amount present in the preparation of Cet alone after dialysis to remove the excipients. The amount of S (ascribable to the Cet protein) is nearly identical to the initial one. These data indicate overall that the ZA was covalently conjugated with the Cet antibody.

Demonstration of the reactivity of ZA-Cet ADC with CRC cell lines

The CRC cell lines Caco-2, HT29, and SW620 were used to demonstrate, by indirect immunofluorescence assays (Varesano et al. 2018), whether the reactivity of ZA-Cet ADC with positive EGFR cells (Caco-2 and HT29) or negative EGFR cells (SW620) was similar to that of the native non-conjugated antibody. The cell lines were labelled with Cet or ZA-Cet ADC, followed by the second human anti-lg reagent conjugated with the fluorochrome Alex Fluor 488. Control samples were incubated only with the second reagent. As indicated in Figure 3, the reactivity of the two compounds was comparable (ZA-Cet ADC histogram with hatching of the various sub-panels). Though the data are not shown, the titrations of Cet and ZA-Cet ADC, assayed on the HT29 line, were comparable, indicating that the zone of equivalence between the EGFR expressed by HT29 and the two reagents was comprised between 20pg/ml and 0.2pg/ml. It is important to note that ZA-Cet ADC does not react with the SW620 cell line, which does not express EGFR, in accordance with the data obtained for the native Cet. These data demonstrate that the conjugation with ZA does not alter the reactivity of Cet with EGFR, which retains its antigenic specificity.

ZA-Cet ADC induces the expansion of V62 T cells present in peripheral blood

In order to demonstrate the ability of ZA-Cet ADC to induce the proliferation of V52 T cells, PBMCs were incubated with this compound. A control stimulation was carried out using soluble ZA with the same donors. Briefly, and as described in detail (Zocchi MR et al. 2017), the PBMCs were incubated with the different compounds at different concentrations (figure 4); after 24 hours IL2 was added to favour the growth of activated cells, and the percentage of V52 T cells was analysed by indirect immunofluorescence using an antibody specific for the V52 receptor in association with an anti-CD3 antibody (Zocchi MR et al. 2017). This analysis was carried out at different times (7-14-21 days) and unstimulated samples represent the control conditions. As shown in figure 4 (panel on the left), a strong increase is observed in the percentage of V52 T cells present in the cultures of PBMCs stimulated with ZA-Cet ADC. In fact, the percentage increases from 0.5-4%, found in 6 different healthy donors, to a mean value of 25% after 7 days of culture, a value that increases to 55% after a total of 14 days. This expansion is comparable to that induced by soluble ZA. It is important to note that the calculated dose of ZA present in ZA-Cet ADC which is capable of inducing a stimulation is 0.5nM, compared 0.5pM in the case of soluble ZA (difference of 1 :1000). This would suggest that ZA-Cet ADC is capable of stimulating the expansion of V52 T cells from PBMCs with a much greater efficiency (1000x) than free ZA.

ZA-Cet ADC is capable of inducing proliferation of V62 T cells in response to the LS180 cell line.

It was then analysed whether CRC lines that express EGFR could stimulate the expansion of V52 T cells in the presence of ZA-Cet ADC. Therefore, among the various CRC cells, the EGFR+ LS180 line selected was the one that demonstrated to be capable of stimulating the expansion of V52 T cells if incubated with low doses of soluble ZA (1 pM), as described previously (Zocchi MR et al. 2017). For this purpose, the T cells were isolated from PBMCs and were then incubated with the LS180 cells irradiated in a 10:1 ratio as described (Zocchi MR et al. 2017). The presence of V52 T cells was evaluated at various times during the culture by indirect immunofluorescence with specific anti-V52 antibodies as described in the previous paragraph. It was demonstrated that the ZA-Cet ADC added to the purified T cells co-cultured with LS180 induced the proliferation of V52 T cells (figure 4, right panel). In fact, there was a strong increase after only 7 days compared to the control conditions, i.e. the percentage of V52 T cells went from <5% in the control to >20% in the presence of ZA-Cet ADC. Furthermore, in these experiments the entity of the stimulation with an amount of soluble ZA equal to 1 pM was in the same order of magnitude as that of ZA-Cet ADC in an amount of 0.5nM. This indicates that ZA- Cet ADC functions with better efficiency than the soluble form in stimulating the expansion of V52 T cells.

ZA-Cet ADC is capable of inducing the production of small phosphorylated antigens such as IPP

It is well established that the small fraction of V52 T cells in peripheral blood (0.1 -5% in the majority of cases) can be expanded in the presence of monocytes, zoledronic acid and IL2. The amount of ZA that induces the activation of V52 T cells ranges from 0.5pM to 5pM (Zocchi et al. 2017, Di Mascolo et al. 2019). It is likewise well established that ZA brings about the inhibition of farnesyl pyrophosphate synthase (FPPS) of the mevalonate pathway and increases the accumulation of IPP in antigen-presenting cells, such as monocytes (Mo) or dendritic cells (DCs) (Jandke et al. 2020, Zocchi MR et al. 2017). It is commonly accepted that IPP is the stimulus for the proliferation of V52 T cells (Wesch D et al. 1997, Morita CT et al. 1995). Consequently, it was analysed whether incubation with ZA-Cet ADC was capable of stimulating the production of IPP by tumour organoids isolated from patients with CRC. The organoids were derived as described in the materials and methods section; for every experimental point, 60 geltrex droplets containing the organoids were cultured for 48h with 4pg/ml of ZA-Cet ADC. Control samples were prepared by incubating the droplets containing organoids with the culture medium alone for the same time or with soluble ZA 5pM. The data obtained with the organoids called OMCR16-005TK, OMCR19-006TK and OMCR18-059TK are summarised in Table II.

Table II Evaluation of IPP production in organoids of patients with CRC

The tumour organoids called OMCR16-005TK, OMCR19-006TK and OMCR18-058TK were plated in geltrex droplets (60 droplets for each experimental point) and incubated with simple culture medium (CTR) or soluble ZA (5pM) or the native Cet alone (4pg/ml) or ZA-Cet ADC (4pg/ml) for 48 hours. Then the IPP was extracted as indicated in the materials and methods section. The samples were analysed by HPLC/TOF-MS and the amount of IPP present was calculated in comparison to an IPP standard with a known concentration. The data are expressed in nM of IPP.

After the extraction of IPP, the samples were analysed by HPLC/TOF- MS. It can be observed that the production of IPP increases in the presence of ZA- Cet ADC compared to the control in the organoids of the patients. The increase is 50% in the case of OMCR16-005TK (equal to an IPP concentration of 3.9pM versus 2.6pM, antibody conjugate versus the control), whereas in the case of 0MCR19- 006TK the increase is 47% (equal to an IPP concentration of 1.18pM versus 0.63pM, antibody conjugate versus the control), which indicates a similar effect on both organoids. It should be noted that the production induced by the soluble form is greater, equal to 16.3pM versus 2.6pM, ZA versus the control in the case of OMCR16-005TK, and 3.7pM versus 0.6pM in the case of the organoid 0MCR19- 006TK. Analogous results are obtained with the organoid OMCR18-059TK. Overall, these data indicate that ZA-Cet ADC activates the tumour organoids to produce IPP to stimulate the activation of V52 T cells.

ZA-Cet ADC is capable of inducing the proliferation of autologous or allogenic V62 T cells in response to the tumour organoids of patients with CRC

It was then analysed whether the culture of highly purified T cells isolated from PBMCs with organoids, incubated with ZA-Cet ADC in the presence of IL2, could stimulate the expansion of V52 T cells. These experiments were conducted using both T cells isolated from the same patients from whom the organoids were derived and with T cells belonging to healthy donors. The aim was to evaluate whether the patients’ cells could be stimulated in a manner comparable to that of the T cells isolated from healthy subjects. The co-culture consisted of organoids derived from patients and highly purified T cells and was then incubated with nothing else (control indicated as T), soluble ZA (positive control indicated as ZA), ZA-Cet ADC or Cet alone. After 24 h of incubation recombinant IL2 was added to induce the expansion of the activated cells. The results shown in panel A illustrate the culture analysed after 21 days. As may be noted, the cultures of T cells alone did not induce the expansion of V52 T cells. In contrast, ZA-Cet ADC (4pg/ml) induced a strong expansion of V52 T cells (last frame of panel A), comparable to that obtained with soluble ZA (1 pM) in the culture performed in the presence of the organoid OMCR16-005TK. Incubation with the native Cet did not bring about any expansion of the V52 T cells (figure 5). In another series of experiments, the dose of ZA-Cet ADC with 50% efficiency (EC50) (panel B, with the organoid OMCR16- 005TK) was determined to be around 1 pg/ml. Furthermore, the expansion of V52 T cells in co-cultures of T cells isolated from healthy donors with different tumour organoids (OMCR16-005TK, OMCR19-006TK, OMCR19-010TK, OMCR18-003TK, OMCR19-10TK, OMCR19-011TK) is presented in panel C; the same expansion is evaluated starting from autologous T cells (isolated from the same patients from whom the organoids were obtained) in panel D. The percentage of V52 T cells increases progressively as the stimulation proceeds over time, usually reaching a maximum value after 21 days: it may be noted that the expansion is very marked both when allogenic T cells (panel C) and autologous T cells (panel D) are used. Overall, these data indicate that ZA-Cet ADC stimulates V52 T cells in all cases analysed, suggesting that all patients may respond to this new formulation of ZA. The entity of the stimulation is comparable to that obtained with T cells of healthy donors, suggesting that there is no intrinsic deficiency of T cells in the patients and that an allogenic condition is not necessary to induce an optimal expansion.

ZA-Cet ADC is capable of activating the cytotoxic activity of V62 T cells

It was then evaluated whether V52 T cells generated with ZA-Cet ADC could recognise and eliminate cancer cells in the form of organoids. For this purpose, the organoids were incubated with V52 T cells from healthy donors (figure 6A) or patients with CRC (figure 6B) at an effector/target cell ratio of 10:1 -5:1 -2:1 and the cytotoxic activity was evaluated after 72 hours of incubation, the organoids having been transferred from the original plate into a replicate plate where adherence was permitted. After an additional 48h, the cultures were stained with crystal violet and after elution of the stain, an evaluation was made of viability in reference to the control condition, represented by the culture of organoids on their own. As shown in figure 6, the V52 T cells were capable of reducing the viability of the organoids to a greater extent as the ratio (panels A and B) between the lymphocyte and tumour organoid increased. This effect was evident either when allogenic V52 T cells derived from healthy donors (figure 6A) or autologous ones derived from patients (figure 6B) were used, indicating that the recognition was independent of the expression of a different HLA of class I on the organoid. In addition, the use of the anti-EGFR ZA-Cet ADC further reduced organoid viability (panels C and D, under allogenic or autologous conditions, respectively). This effect was greater than the one exerted by the native Cet antibody and comparable to the one induced by soluble ZA (figure 6 panels C and D). These data indicate that V52 T cells can be activated to kill the cells making up organoids if suitably stimulated with ZA-Cet ADC.

EXAMPLE 2: Preparation of Z/ and Rituximab conjugate according to the present invention and study of its effectiveness in cancer

The conjugate was prepared by means of the process described above for the ZA and Cet conjugate, replacing the Cet antibody with the Rit antibody and using the same amount as used for ZA (1 mg/ml) or a tenth of this amount (0.1 mg/ml). The conjugates were analysed by MALDI and the presence of ZA-Rit ADC was demonstrated, with a different amount of ZA depending on the preparation, which demonstrates the possibility of modulating the amount of the drug ZA linked to the Rit antibody. Furthermore, the conjugate was used to demonstrate its surface reactivity with the CD20+ Raji and Karpas lines, its ability to inhibit the proliferation of the two Raji and Karpas lines compared with the antibody not conjugated with Rit and its ability to induce the specific expansion of V52 T+ cells present in the peripheral blood of healthy subjects. The experiments represented in figure 7 demonstrate that ZA-Rit ADC is effectively conjugated based on the differences in molecular weight that are revealed after MALDI-TOF analysis (7A), has a reactivity similar to that of non-conjugated native Rit (7B), inhibits the proliferation of both Raji and Karpas in a similar manner to Rit (7C) and induces the expansion of V52 T cells present in the peripheral blood of healthy donors (7D). These data indicate that ZA-Rit ADC, too, has properties of reactivity and of blocking the proliferation of CD20+ cancer cell lines and, furthermore, it induces the specific stimulation of V52 T cells as demonstrated for the ZA-Cet ADC antibody in the case of colon cancer. Overall, these data suggest that the conjugation of ZA ad monoclonal antibodies maintains their activity, while adding the possibility of precisely identifying the target cell, preserving the functional properties of the antibody and adding the activation of V52 antitumor T cells; this suggests a potential use of the antibody-ZA conjugates in solid and haematologic neoplasms.

References

1 Scott LJ. Brentuximab Vedotin: A Review in CD30-Positive Hodgkin Lymphoma. Drugs. 2017 Mar;77(4):435-445. doi: 10.1007/s40265-017-0705-

5.PMID: 28190142

2 Thomas A, Teicher BA, Hassan R. Antibody-drug conjugates for cancer therapy. Lancet Oncol. 2016 Jun;17(6):e254-e262. doi: 10.1016/S1470- 2045(16)30030-4. PMID: 27299281

3 Monteverde M, Milano G, Strola G, Maffi M, Lattanzio L, Vivenza D,

Tonissi F, Merlano M, Lo Nigro C. The relevance of ADCC for EGFR targeting: A review of the literature and a clin ical ly-appl icable method of assessment in patients. Crit Rev Oncol Hematol. 2015 Aug;95(2):179-90. doi:

10.1016/j.critrevonc.2015.02.014. Epub 2015 Mar 12.PMID: 25819749

4 Musso A, Catellani S, Canevali P, Tavella S, Vene R, Boero S, Pierri I, Gobbi M, Kunkl A, Ravetti JL, Zocchi MR, Poggi A. Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor V5 2 T lymphocytes in non-Hodgkin lymphomas. Haematologica. 2014 Jan;99(1 ):131 - 9. doi: 10.3324/haematol.2013.097311. Epub 2013 Oct 25.PMID: 24162786

5 Fuertes MB, Domaica Cl, Zwirner NW. Leveraging NKG2D Ligands in Immuno-Oncology. Front Immunol. 2021 Jul 29;12:713158. doi: 10.3389/fimmu.2021 .713158. eCollection 2021. PMID: 34394116

6. Ribot JC, Lopes N, Silva-Santos B. gammadelta T cells in tissue physiology and surveillance. Nat Rev Immunol. 2021 Apr;21 (4):221 -232. doi: 10.1038/S41577-020-00452-4. Epub 2020 Oct 14. PMID: 33057185

7. Li Y, Li G, Zhang J, Wu X, Chen X. The Dual Roles of Human gammadelta T Cells: Anti-Tumor or Tumor-Promoting. Front Immunol. 2021 Feb 16;11 :619954. doi: 10.3389/fimmu.2020.619954. eCollection 2020.PMID: 33664732

8 Herrmann T, Fichtner AS, Karunakaran MM. An Update on the Molecular Basis of Phosphoantigen Recognition by Vy9V52 T Cells. Cells. 2020 Jun 9;9(6):1433. doi: 10.3390/cells9061433.PMID: 32527033

9. Russell RG. Bisphosphonates: the first 40 years. Bone. 2011 Jul;49(1 ):2-19. doi: 10.1016/j.bone.2011.04.022. Epub 2011 May 1.PMID: 21555003

10. Dhillon S. Zoledronic Acid (ReclastQ, Aclasta()): A Review in Osteoporosis. Drugs. 2016 Nov;76(17): 1683-1697. doi: 10.1007/s40265-016-0662- 4.PMID: 27864686

11. Terpos E, Zamagni E, Lentzsch S, Drake MT, Garcia-Sanz R, Abildgaard N, Ntanasis-Stathopoulos I, Schjesvold F, de la Rubia J, Kyriakou C, Hillengass J, Zweegman S, Cavo M, Moreau P, San-Miguel J, Dimopoulos MA, Munshi N, Durie BGM, Raje N; . Treatment of multiple myeloma-related bone disease: recommendations from the Bone Working Group of the International Myeloma Working Group. Bone Working Group of the International Myeloma Working Group. Lancet Oncol. 2021 Mar;22(3):e119-e130. doi: 10.1016/S1470- 2045(20)30559-3. Epub 2021 Feb 2.PMID: 33545067

12. Shmeeda, H.; Amitay, Y.; Tzemach, D.; Gorin, J.; Gabizon, A. Liposome encapsulation of zoledronic acid results in major changes in tissue distribution and increase in toxicity. J. Control Release2013, 167, 265-275, doi: 10.1016/j.jconrel.2013.02.003.

13. Salzano, G.; Zappavigna, S.; Luce, A.; D’Onofrio, N.; Balestrieri, M.L.; Grimaldi, A.; Lusa, S.; Ingrosso, D.; Artuso, S.; Porru, M.; et al. Transferrin-Targeted Nanoparticles Containing Zoledronic Acid as a Potential Tool to Inhibit Glioblastoma Growth. J. Biomed. Nanotechnol.2016, 12, 811-830, doi:10.1166/jbn.2016.2214.

14. Porru, M.; Zappavigna, S.; Salzano, G.; Luce, A.; Stoppacciaro, A.; Balestrieri, M.L.; Artuso, S.; Lusa, S.; De Rosa, G.; Leonetti, C.; et al. Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid. Oncotarget2014, 5, 10446- 10459, doi: 10.18632/oncotarget.2182.

15. Kolmas, J.; Pajor, K.; Pajchel, L.; Przekora, A.; Ginalska, G.; Oledzka, E.; Sobczak, M. Fabrication and physicochemical characterization of porous composite microgranules with selenium oxyanions and risedronate sodium for potential applications in bone tumors. Int. J. Nanomedicine.2017, 12, 5633-5642, doi:10.2147/IJN.S140935.

16. Hodgins, N.O.; Al-Jamal, W.T.; Wang, J.T.-W.; Parente-Pereira, A.C.; Liu, M.; Maher, J.; Al-Jamal, K.T. In vitro potency, in vitro and in vivo efficacy of liposomal alendronate in combination with y5 T cell immunotherapy in mice. J. Control Release2016, 241 , 229-241 , doi: 10.1016/j.jconrel.2016.09.023.

17. Hodgins, N.O.; Wang, J.T.-W.; Al-Jamal, K.T. Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of y5 T cells for anticancer immunotherapy. Adv. Drug Deliv.

Rev.2017, 114, 143-160, doi:10.1016/j.addr.2017.07.003.

18. Clancy, J. P., Dupont, L.,Konstan,M.W., Billings, J., Fustik,S. , Goss, C.H., etal.(2013).PhasellstudiesofnebulisedArikaceinCFpatientswith Pseudomonas aeruginosa infection. Thorax 68, 818-825. doi:10.1136/thoraxjnl-2012-202230

19. Bendas, G. (2001 ). Immunoliposomes: a promising approach to targeting cancer therapy. BioDrugs 15,215-224. doi: 10.2165/00063030- 200115040-00002

20.Puri,A.,Loomis,K.,Smith,B.,Lee,J.H.,Yavlovich,A.,Heldman,E., etal. (2009). Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit. Rev. Then Drug Carrier Syst. 26, 523-580. doi: 10.1615/CritRevTherDrugCamerSyst. v26. i6.10

21 .Ishida, T., lchihara,M., Wang,X., Yamamoto, K., Kimura, J. ,Majima, E., et al. (2006a). Injection of PEGylated liposomes in rats elicits PEG-specificIgM, which is responsible for rapid elimination of a second dose of PEGylated liposomes. J. Control. Release 112,15-25.doi:10.1016/j.jconrel.2006. 01.005

22. Ishida, T., Ichihara, M., Wang, X., and Kiwada,H. (2006b). Spleen plays an important role in the induction of accelerated blood clearance of PEGylated liposomes. J. Control. Release 115,243-250. doi: 10.1016/j.jconrel.2006.08.001 23. Ishida, T., Harada, M., Wang, X.Y., Ichihara, M., Irimura, K., and Kiwada, H. (2005). Accelerated blood clearance of PEGylated liposomes following preceding liposome injection: effects of lipid dose and PEG surface-density and chain length of the first-dose liposomes. J. Control. Release 105,305-317. doi: 10.1016/j.jconrel.2005.04.003

24. Ishida, T., Harashima,H., and Kiwada, H. (2001 ). Interactions of liposomes with cells invitro and in vivo: opsonins and receptors. Curr. Drug Metab. 2,397-409. doi: 10.2174/1389200013338306

25. Tinkle, S., McNeil, S.E.,Muhlebach,S.,Bawa,R., Borchard,G., Barenholz, Y.C., et al. (2014). Nanomedicines: addressing the scientific and regulatory gap. Ann. N.Y.Acad.Sci. 1313, 35-56. doi:10.1111/nyas.12403

26. Narang, A.S., Chang, R.K., and Hussain, M.A.(2013). Pharmaceutical development and regulatory considerations for nanoparticles and nanoparticulate drug delivery systems. J.Pharm.Sci. 102,3867-3882. doi: 10.1002/jps.23691

27. Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and Challenges of Liposome Assisted Drug Delivery. Front Pharmacol. 2015 Dec 1 ;6:286. doi: 10.3389/fphar.2015.00286. PMID: 26648870; PMCID: PMC4664963.

28. Wong SF. Cet: an epidermal growth factor receptor monoclonal antibody for the treatment of colorectal cancer. Clin Then 2005 Jun;27(6):684-94. doi: 10.1016/j.clinthera.2005.06.003. PMID: 16117976

29. Fasano M, Della Corte CM, Viscardi G, Di Liello R, Paragliola F, Sparano F, lacovino ML, Castrichino A, Doria F, Sica A, Morgillo F, Colella G, Tartaro G, Cappabianca S, Testa D, Motta G, Ciardiello F. Head and neck cancer: the role of anti-EGFR agents in the era of immunotherapy.Ther Adv Med Oncol. 2021 Mar 9; 13: 1758835920949418. doi: 10.1177/1758835920949418. eCollection 2021.30. Doggrell SA. Zoledronate once-yearly increases bone mineral densityimplications for osteoporosis. Expert Opin Pharmacother. 2002 Jul;3(7): 1007-9. doi: 10.1517/14656566.3.7.1007. PMID: 12083999

31 . Hedrich WD, Fandy TE, Ashour HM, Wang H, Hassan HE. Antibody- Drug Conjugates: Pharmacokinetic/Pharmacodynamic Modeling, Preclinical Characterization, Clinical Studies, and Lessons Learned. Clin Pharmacokinet. 2018 Jun;57(6):687-703. doi: 10.1007/s40262-017-0619-0.PMID: 29188435

32. Birrer MJ, Moore KN, Betella I, Bates RC. Antibody-Drug Conjugate- Based Therapeutics: State of the Science. J Natl Cancer Inst. 2019 Jun 1 ; 111 (6):538-549. doi: 10.1093/jnci/djz035.PMID: 30859213

35. Abdollahpour-Alitappeh M, Lotfinia M, Gharibi T, Mardaneh J, Farhadihosseinabadi B, Larki P, Faghfourian B, Sepehr KS, Abbaszadeh-Goudarzi K, Abbaszadeh-Goudarzi G, Johari B, Zali MR, Bagheri N. Antibody-drug conjugates (ADCs) for cancer therapy: Strategies, challenges, and successes. J Cell Physiol. 2019 May;234(5):5628-5642. doi: 10.1002/jcp.27419. Epub 2018 Nov 27. PMID: 30478951

33. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 2009 May;157(2):220-33. doi: 10.1111/j.1476-5381 .2009.00190.x. PMID: 19459844

34. Chanier T, Chames P. Nanobody Engineering: Toward Next Generation Immunotherapies and Immunoimaging of Cancer. Antibodies (Basel). 2019 Jan 21 ;8(1 ):13. doi: 10.3390/antib8010013.PMID: 31544819

35. Seki T, Fang J, Maeda H. Enhanced delivery of macromolecular antitumor drugs to tumors by nitroglycerin application. Cancer Sci. 2009 Dec; 100(12):2426-30. doi: 10.1111/j.1349-7006.2009.01323.x. Epub 2009 Aug 25.PMID: 1979308

36. Maeda T, Miyazono Y, Ito K, Hamada K, Sekine S, Horie T. Oxidative stress and enhanced paracellular permeability in the small intestine of methotrexate- treated rats. Cancer Chemother Pharmacol. 2010 May;65(6):1117-23. doi: 10.1007/s00280-009-1119-1 . Epub 2009 Sep 16. PMID: 19756603

37. Ghosh SS, Philip M. Kao, Ann W. McCue, and Hugh L. Chappelle Bioconjugate Chemistry 1990 1 (1 ), 71 -76 DOI: 10.1021 /bc00001 a009

38. Shabarova ZA, Ivanovskaya MG, Isaguliants MG. DNA-like duplexes with repetitions: efficient template-guided polycondensation of decadeoxyribonucleotide imidazolide. FEBS Lett. 1983 Apr 18;154(2):288-92. doi: 10.1016/0014-5793(83)80168-9. PMID: 6832371.

39. Shabarova ZA. Synthetic nucleotide-peptides. Prog Nucleic Acid Res Mol Biol. 1970;10:145-82. doi: 10.1016/s0079-6603(08)60564-4. PMID: 4910303

40. Itumoh EJ, Data S, Leitao EM. Opening up the Toolbox: Synthesis and Mechanisms of Phosphoram idates. Molecules. 2020 Aug 13;25(16):3684. doi: 10.3390/molecules25163684. PMID: 32823507

41. Fujii M, Shimokawa M, Date S, et al. A Colorectal Tumor Organoid Library Demonstrates Progressive Loss of Niche Factor Requirements during Tumorigenesis. Cell Stem Cell. 2016; 18: 827-38. doi:

10.1016/j.stem.2016.04.003. Epub 2016 May 19

42. Jauhiainen, M., Monkkonen, H., Raikkonen, J., Monkkonen, J., Auriola, S. Analysis of endogenous ATP analogs and mevalonate pathway mebolites in cancer cell cultures using liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Analyt Technol. Biomed Life Sci.2009;877(27):2967-2975. doi: 10.1016/j.jchromb.2009.07.010.

43. Zocchi, M.R.; Costa, D.; Vene, R.; Tosetti, F.; Ferrari, N.; Minghelli, S.; Benelli, R.; Scabini, S.; Romairone, E; Catellani, S.; Profumo, A.; Poggi, A. Zoledronate can induce colorectal cancer microenvironment expressing BTN3A1 to stimulate effector yb T cells with anti-tumor activity. Oncoimmunol. 2017;6(3):e1278099.doi: 10.1080/2162402X.2016.1278099.

44.Varesano, S.; Zocchi, M.R.; Poggi, A. Zoledronate triggers V52 T cells to destroy and kill spheroids of colon carcinoma: quantitative image analysis of three-dimensional cultures. Front Immunol.2018;9:998. doi: 10.3389/fimmu.2018.00998. eCollection 2018. Erratum in: Front Immunol. 2018. 9:1343.

45. Di Mascolo, D.; Varesano, S.; Benelli, R.; Mollica, H.; Salis, A.; Zocchi, M.R.; Decuzzi, P.; Poggi, A. Nanoformulated Zoledronic Acid Boosts the V52 T Cell Immunotherapeutic Potential in Colorectal Cancer. Cancers (Basel) 2019, 12, doi: 10.3390/cancers12010104.

46. Jandke A, Melandri D, Monin L, Ushakov DS, Laing AG, Vantourout P, East P, Nitta T, Narita T, Takayanagi H, Feederle R, Hayday A. Butyrophilin-like proteins display combinatorial diversity in selecting and maintaining signature intraepithelial yb T cell compartments. Nat Commun. 2020 Jul 28;11 (1 ):3769. doi: 10.1038/S41467-020-17557-y. PMID: 32724083

47. Wesch D, Marx S, Kabelitz D. Comparative analysis of alpha beta and gamma delta T cell activation by Mycobacterium tuberculosis and isopentenyl pyrophosphate. Eur J Immunol. 1997 Apr;27(4):952-6. doi:

10.1002/eji.1830270422. PMID: 9130649

48. Morita CT, Beckman EM, Bukowski JF, Tanaka Y, Band H, Bloom BR, Golan DE, Brenner MB. Direct presentation of nonpeptide prenyl pyrophosphate antigens to human gamma delta T cells. Immunity. 1995 Oct;3(4):495-507. doi: 10.1016/1074-7613(95)90178-7. PMID: 7584140

49. Vavassori S, Kumar A, Wan GS, Ramanjaneyulu GS, Cavallari M, El Daker S, Beddoe T, Theodossis A, Williams NK, Gostick E, Price DA, Soudamini DU, Voon KK, Olivo M, Rossjohn J, Mori L, De Libero G. Butyrophilin 3A1 binds phosphorylated antigens and stimulates human y5 T cells. Nat Immunol. 2013;14(9):908-16. doi: 10.1038/ni.2665. PMID: 23872678.

50. Poggi A, Zocchi MR. y5 T Lymphocytes as a First Line of Immune Defense: Old and New Ways of Antigen Recognition and Implications for Cancer Immunotherapy. Front Immunol. 2014 Nov 11 ;5:575. doi: 10.3389/fimmu.2014.00575. PMID: 25426121 ; PMCID: PMC4226920

51. Bonneville, M., O'Brien, R. & Bom, W. y5 T cell effector functions: a blend of innate programming and acquired plasticity. Nat Rev Immunol 10, 467-478 (2010). doi: 10.1038/nri2781 .

52. Ayuso JM, Truttschel R, Gong MM, Humayun M, Virumbrales-Munoz M, Vitek R, Felder M, Gillies SD, Sondel P, Wisinski KB, Patankar M, Beebe DJ, Skala MC. Evaluating natural killer cell cytotoxicity against solid tumors using a microfluidic model. Oncoimmunology. 2018 Dec 12;8(3): 1553477. doi: 10.1080/2162402X.2018.1553477. PMID: 30723584; PMCID: PMC6350694.55. Chen T, Berenson J, Vescio R, Swift R, Gilchick A, Goodin S, LoRusso P, Ma P, Ravera C, Deckert F, Schran H, Seaman J, Skerjanec A. Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases. J Clin Pharmacol. 2002 Nov;42(11 ): 1228-36. doi: 10.1177/009127002762491316. PMID: 12412821.

53. Le Louedec F, Alix-Panabieres C, Lafont T, Allal BC, Garrel R, Digue L, Guigay J, Cupissol D, Delord JP, Lallemant B, Alfonsi M, Aubry K, Mazel M, Becher F, Perriard F, Chatelut E, Thomas F. Cet pharmacokinetic/pharmacodynamics relationships in advanced head and neck carcinoma patients. Br J Clin Pharmacol. 2019 Jun;85(6):1357-1366. doi: 10.1111/bcp.13907. Epub 2019 Apr 13. PMID: 30811063; PMCID: PMC6533440.

54. Tan AR, Moore DF, Hidalgo M, Doroshow JH, Poplin EA, Goodin S, Mauro D, Rubin EH. Pharmacokinetics of Cet after administration of escalating single dosing and weekly fixed dosing in patients with solid tumors. Clin Cancer Res. 2006 Nov 1 ;12(21 ):6517-22. doi: 10.1158/1078-0432. CCR-06-0705. Epub 2006 Oct 25. PMID: 17065274 55. Azad N, Dasari A, Arcaroli J, Taylor GE, Laheru DA, Carducci MA, McManus M, Quackenbush K, Wright J J, Hidalgo M, Diaz LA Jr, Donehower RC, Zhao M, Rudek MA, Messersmith WA. Phase I pharmacokinetic and pharmacodynamic study of Cet, irinotecan and sorafenib in advanced colorectal cancer. Invest New Drugs. 2013 Apr;31 (2):345-54. doi: 10.1007/s10637-012-9820- z. Epub 2012 May 22. PMID: 22615057; PMCID: PMC3674958.

56. Bournazos S, Ravetch JV. Fcgamma Receptor Function and the Design of Vaccination Strategies. Immunity. 2017 Aug 15;47(2):224-233. doi: 10.1016/j.immuni.2017.07.009. PMID: 28813656

57. Taylor RP, Lindorfer MA. Fcgamma-receptor-mediated trogocytosis impacts mAb-based therapies: historical precedence and recent developments. Blood. 2015 Jan 29;125(5):762-6. doi: 10.1182/blood-2014-10-569244. Epub 2014 Dec 10. PMID: 25498911

58. Hepburn AL, Mason JC, Davies KA. Expression of Fcgamma and complement receptors on peripheral blood monocytes in systemic lupus erythematosus and rheumatoid arthritis. Rheumatology (Oxford). 2004 May;43(5):547-54. doi: 10.1093/rheumatology/keh112. Epub 2004 Jan 27.PMID: 14747618

59. Bournazos S, Ravetch JV. Diversification of IgG effector functions. Int Immunol. 2017 Jul 1 ;29(7):303-310. doi: 10.1093/intimm/dxx025. PMID: 28472280

60. Fridman, W., Pages, F., Sautes-Fridman, C. et al. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12, 298- 306 (2012). doi: 10.1038/nrc3245.

61. Church SE, Galon J. Tumor Microenvironment and Immunotherapy: The Whole Picture Is Better Than a Glimpse. Immunity. 2015 Oct 20;43(4):631 -3. doi: 10.1016/j.immuni.2015.10.004. PMID: 26488814.

62. Xie, YH., Chen, YX. & Fang, JY. Comprehensive review of targeted therapy for colorectal cancer. Sig Transduct Target Ther 5, 22 (2020). doi: 10.1038/S41392-020-0116-z

63. Costa D, Vene R, Benelli R, Romairone E, Scabini S, Catellani S, Rebesco B, Mastracci L, Grillo F, Minghelli S, Loiacono F, Zocchi MR, Poggi A. Targeting the Epidermal Growth Factor Receptor Can Counteract the Inhibition of Natural Killer Cell Function Exerted by Colorectal Tumor-Associated Fibroblasts. Front Immunol. 2018 May 29; 9:1150. doi: 10.3389/fimmu.2018.01150. eCollection 2018.

64.Tokumaru Y, Matsuhashi N, Takahashi T, Tanahashi T, Matsui S, Imai H, Tanaka Y, Yamaguchi K, Yoshida K. Efficacy of combination therapy with zoledronic acid and Cet for unresectable rectal cancer with bone metastases: A case report. Mol Clin Oncol. 2019 Jun;10(6):571 -574. doi: 10.3892/mco.2019.1836. Epub 2019 Apr 3.PMID: 31031973

65. Kato J, Futamura M, Kanematsu M, Gaowa S, Mori R, Tanahashi T, Matsuhashi N, Yoshida K. Combination therapy with zoledronic acid and Cet effectively suppresses growth of colorectal cancer cells regardless of KRAS status.

Int J Cancer. 2016 Mar 15; 138(6): 1516-27. doi: 10.1002/ijc.29881 . Epub 2015 Oct 23

Claims

1 ) Conjugate of a bisphosphonate compound with a monoclonal antibody having at least one free amino group, wherein said amino group of the monoclonal antibody is linked to at least one phosphonate group of the bisphosphonate compound.

2) Conjugate according to claim 1 , wherein the bisphosphonate compound is an amino bisphosphonate compound, preferably an amino bisphosphonate compound without a free NH2 group.

3) Conjugate according to claim 2, wherein the amino bisphosphonate compound is selected from the group consisting of zoledronate, risedronate, ibadronate, preferably zoledronate.

4) Conjugate according to any one of claims 1 -3, wherein the monoclonal antibody is an antitumor monoclonal antibody.

5) Conjugate according to claim 4, wherein the antitumor monoclonal antibody is selected from the group consisting of cetuximab, Rituximab, anti-Her2, anti-CD38 or anti-BCMA.

6) Conjugate according to any one of claims 1 -5, wherein the amino bisphosphonate is zoledronate and the antitumor monoclonal antibody is cetuximab, said zoledronate and cetuximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of cetuximab.

7) Conjugate according to any one of claims 1 -5, wherein the amino bisphosphonate is zoledronate and the monoclonal antibody is Rituximab, said zoledronate and Rituximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of Rituximab.

8) Pharmaceutical composition comprising or consisting of a conjugate as defined in any one of claims 1 -7, as an active ingredient, together with one or more excipients and/or adjuvants.

9) Conjugate as defined in any one of claims 1 -7 or pharmaceutical composition as defined in claim 8, for use as a medicament.

10) Conjugate as defined in any one of claims 1 -7 or pharmaceutical composition as defined in claim 8, for use in the treatment of tumor.

11 ) Process for the preparation of a conjugate as defined in any one of claims 1 -5, comprising the following steps: a) reacting the bisphosphonate compound with a suitable reagent to activate at least one phosphonate group of said bisphosphonate compound for reaction with a free amino group of a monoclonal antibody; b) reacting the bisphosphonate compound obtained in step a) with a monoclonal antibody having a free amino group in order to obtain the conjugate.

12) Process according to claim 11 , wherein step a) is carried out by reacting said bisphosphonate compound with 1 -ethyl-3-3-dimethylaminopropyl carbodiimide and imidazole to obtain an O-acylisourea intermediate of the bisphosphonate compound followed by of a phosphorim idazolide of the bisphosphonate compound.

13) Process according to any one of claims 11 -12, wherein both step a) and step b) are carried out in a medium comprising imidazole, preferably at pH 6±0.2.

14) Process according to any one of claims 11 -13, wherein the bisphosphonate compound is an amino bisphosphonate compound, preferably an amino bisphosphonate compound without a free NH2 group.

15) Process according to claim 14, wherein the amino bisphosphonate compound is selected from the group consisting of zoledronate, risedronate, ibadronate, preferably zoledronate.

16) Process according to any one of claims 11 -15, wherein the monoclonal antibody is an antitumor monoclonal antibody.

17) Process according to claim 16, wherein the antitumor monoclonal antibody is selected from the group consisting of cetuximab, Rituximab, anti-Her2, anti-CD38 or anti-BCMA.

18) Process according to any one of claims 11 -17, wherein the amino bisphosphonate is zoledronate and the antitumor monoclonal antibody is cetuximab, said zoledronate and cetuximab being linked together by a phosphoramide bond between at least one phosphonate group of zoledronate and at least one free amino group of cetuximab.

19) Process according to any one of claims 11 -17, wherein the amino bisphosphonate is zoledronate and the monoclonal antibody is Rituximab, said zoledronate and Rituximab being linked together by a phosphoramide bond between a phosphonate group of zoledronate and at least one free amino group of Rituximab.