Use of eggs grafted with tumor cells to study the anticancer efficacy of immunotherapy in the absence of immune effector cells other than the immune effector cells of the grafted eggs

Abstract

The present invention relates to the use of a developing egg model grafted with tumor cells to investigate anticancer efficacy or screen immunotherapeutic molecules in the absence of immune effector cells other than the immune effector cells of the grafted egg.

Description

Technology Field

[0001] The present invention relates to a personalized medicine for prescribing the most promising immunotherapy in terms of effectiveness to cancer patients, particularly in the field of immuno-oncology. Background Technology

[0002] Despite existing therapeutic solutions for cancer treatment (surgery, radiation therapy, chemotherapy, and targeted therapy), some malignant tumors remained incurable until a mechanism was discovered through which the immune system could act on the tumor.

[0003] Due to these advancements in the aforementioned fields, a new therapeutic approach called immunotherapy has been developed, which specifically attacks tumor cells by reactivating or stimulating the immune system.

[0004] One of the concerns in immunotherapy is to develop treatments that rely on the genetic profile of the tumor and the presence of specific biomarkers (PDL-1 type) rather than on the given type of cancer. Therefore, these treatments consider the patient's profile and their tumor, marking the first step toward personalized medicine.

[0005] Since 2010, various mechanisms of action have been considered and have yielded promising results:

[0006] - The use of monoclonal antibodies, regardless of whether they are associated with cytotoxic molecules,

[0007] - The use of immune checkpoint inhibitors (checkpoints in pathways specifically activated or inhibited in cancer mechanisms) to activate or inhibit certain mechanisms related to the developing immune system, such as the use of CTLA-4 inhibitors (ipilimumab),

[0008] - Stimulation of the immune system to fight tumor cells more effectively, specifically through non-targeted immunotherapy, prophylactic or curative cancer vaccines,

[0009] - The use of adoptive cell therapy, such as CAR-T cells (Chimeric Antigen Receptor T Lymphocytes), which aims to fight tumors with promising results by modifying the patient's cells in vitro and then re-injecting them into the patient.

[0010] Most immunotherapies require the presence of effective immune cells, which limits the feasibility of testing these molecules in vitro. Consequently, the development phase is rapidly shifting from primarily mice to animal models.

[0011] Mouse models with spontaneously developing tumors lack the genetic complexity present in patient tumors, which can suppress or, conversely, amplify arbitrary therapeutic effects. Consequently, this makes it difficult to extrapolate the results to humans.

[0012] The first in vivo models used for xenografts were immunodeficient mice that promote tumor development not attacked by the host immune system. Subsequently, these mouse models were 'humanized' by creating transgenic models through the expression of human genes (knock-in) or by grafting human hematopoietic cells into immunodeficient mice. However, these models have several disadvantages, such as the time required to develop the models (taking several months to obtain random results) or a faster rate of tumor development than in humans, and this development does not involve chronic inflammation in the tumor environment as it does in humans. The problem to be solved

[0013] Considering these difficulties and the associated costs and time required to conduct investigations in humanized mouse models, there is a need to develop other simpler, faster, and more reliable models to develop and validate the efficacy of novel immunotherapies. means of solving the problem

[0014] The present invention relates to the use of an embryonic bird egg model grafted with tumor cells, particularly at the level of the chorioallantoic membrane (CAM), to evaluate the anticancer activity of one or more immunotherapeutic molecule(s), wherein the model excludes the presence of immune effector cells other than the immune effector cells of the grafted egg.

[0015] Preferably, the immunotherapeutic molecule is selected from adoptive cell therapies, e.g., CAR-T, vaccines, bispecific antibodies, immune checkpoint inhibitors, e.g., anti-PD1, or anti-PDL1, or anti-CTLA-4 antibodies.

[0016] In particular, when isolating tumor cells from cancer patient samples, testing various immunotherapeutic molecules allows determining which molecule is most promising in terms of cancer treatment efficacy in that patient.

[0017] Within the scope of use of this embryonic egg, it is also possible to determine or even quantify the toxicity of immunotherapeutic molecule(s) tested in both the tumor developed from grafted tumor cells and the entire embryo.

[0018] The present invention also

[0019] - A step of grafting tumor cells at the level of CAM in a previously incubated avian embryo that has been incubated to a developmental stage corresponding to the formation of chorionic alluvial membrane (CAM) and equivalent to at least 8 days of development in a chicken embryo,

[0020] - A step of administering immunotherapeutic molecule(s) to the developing egg at least 12 hours after grafting,

[0021] - A step of investigating the effect of the administered immunotherapeutic molecule(s) on the tumorigenesis of a tumor developed in a grafted embryo.

[0022] including,

[0023] And implementing in the absence of effector immune cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0024] The present invention relates to a method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) characterized by [specific feature].

[0025] The present invention also

[0026] - A step of grafting tumor cells at the level of CAM in an avian embryo previously incubated to a developmental stage corresponding to the formation of chorionic alluvial membrane (CAM) and equivalent to at least 8 days of development in a chicken embryo,

[0027] - A step of administering candidate immunotherapeutic molecule(s) to a developing egg at least 12 hours after grafting,

[0028] - A step of investigating the effect of the immunotherapeutic molecule(s) thus administered on the tumorigenesis of a tumor developed in a grafted embryo.

[0029] including,

[0030] This relates to a screening method for immunotherapeutic molecules having anticancer activity, and

[0031] And the above method is implemented in the absence of effector immune cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0032] Finally, the present invention relates to a method for monitoring cancer patients or animals, and

[0033] - A step of preparing a first avian embryo as described above using tumor cells from the patient or animal at time T1, and investigating the tumor formation of a tumor developing in the first avian embryo,

[0034] - A step of preparing a second avian embryo as described above using tumor cells from a sample of the same patient or animal at time T2, and investigating the tumor formation of a tumor developing in the second avian embryo,

[0035] - A step of comparing tumor formation of tumors developed in the first and second avian embryos.

[0036] Includes,

[0037] The above method is implemented in the absence of immune effector cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0038] The present invention excludes the presence of immune effector cells other than immune effector cells in avian embryos grafted with tumor cells.

[0039] The use and method according to the present invention shall never include the presence or addition of immune effector cells other than the immune effector cells of the developing egg grafted with tumor cells. Brief explanation of the drawing

[0040] Figure 1 shows a model of a developing egg in which a tumor cell deposition zone and major tissues are present. Figure 2 shows an example of an investigation timeline from cell grafting to sample collection. Figure 3 shows the effect of atezolizumab (anti-PD-L1 Tecentriq) treatment on tumors originating from MDA-MB-231 cells. Figure 4 shows the effect of pembrolizumab (anti-PD1 Keytruda) treatment on tumors originating from (A) MDA-MB-231 cells or (B) SU-DHL-4 cells. Figure 5 shows the effect of RMP1-14 (anti-PD1) treatment on tumors originating from SU-DHL-4 cells. Figure 6 shows the effect of nivolumab (anti-PD1 Opdivo) treatment on tumors originating from MDA-MB-231 cells. Figure 7 shows the effect of pembrolizumab (anti-PD1 Keytruda) treatment on metastasis in the downstream CAM after grafting MDA-MB-231 cells. Figure 8 shows the relative amounts of CD3 (A) and CD4 (B) expression (compared to a negative control) in tumors obtained from SU-DHL-4 cells with or without treatment with atezolizumab (anti-PD-L1 Tecentriq). Figure 9 shows the relative expression levels of CD3 (A), CD45 (B), CD56 (C), and CD8 (D) in tumors obtained from SU-DHL-4 cells with or without treatment with pembrolizumab (anti-PD-1 Keytruda) (compared to a negative control). Figure 10 shows the relative amount of CD3 expression (compared to a negative control) in tumors obtained from MDA-MB-231 cells with or without treatment with nivolumab (anti-PD1 Opdivo). Figure 11 shows different immune cell populations (CD4+ T lymphocytes, CD8+ T lymphocytes, and monocytes) detected by flow cytometry in peripheral blood mononuclear cells of chicken embryos at E16. Figure 12 shows the increase in the cytotoxic effect of chicken T lymphocytes on human tumor cells H460 after treating T lymphocytes with pembrolizumab (anti-PD-1 Keytruda®). Specific details for implementing the invention

[0041] The present invention relates to the use of grafted avian embryos with tumor cells, particularly at the level of CAM, to evaluate the anticancer activity of one or more immunotherapeutic molecule(s), wherein the model excludes immune effector cells other than the immune effector cells of the grafted egg. Preferably, the immunotherapeutic molecule is selected from adoptive cell therapies, e.g., CAR-T, vaccines, bispecific antibodies, immune checkpoint inhibitors, e.g., anti-PD1, or anti-PDL1, or anti-CTLA-4 antibodies, and much more preferably from adoptive cell therapies, e.g., CAR-T, bispecific antibodies, and immune checkpoint inhibitors, e.g., anti-PD1, or anti-PDL1, or anti-CTLA-4 antibodies. Advantageously, the immunotherapeutic molecule is selected from immune checkpoint inhibitors, e.g., anti-PD1, or anti-PDL1, or anti-CTLA-4 antibodies.

[0042] Eggs, particularly chicken models, in which tumors are grafted at the level of the chorionic villi (CAM) are already widely used to test the efficacy and toxicity of various types of anticancer agents, such as chemotherapy, peptides, or nanoparticles. However, this has never been used to test the efficacy of immunotherapeutic anticancer molecules that rely on the activation, or more precisely, the reactivation, of the cancer patient's own immune system.

[0043] The inventors have demonstrated that, surprisingly, this model is very different from humans and that, although many authors have considered that the chicken immune system is immature and may be unable to induce any immune response, it can be used in the same way to test the efficacy of immunotherapeutic molecules using only the grafted egg immune system. Therefore, the use of this model according to the present invention is implemented in the absence of and without the addition of immune effector cells other than those of the grafted embryo. Thus, this implementation relies solely on the immune system of the grafted egg. This implementation of the model has several advantages over existing models, as follows:

[0044] - Cost (cost of eggs compared to mice and maintenance of the animal house for a few weeks or months);

[0045] - The presence of a complete immune system that does not require the presence or addition of effector immune cells other than the immune effector cells of the developed grafted egg.

[0046] Furthermore, since this is an embryonic model, the immune system is still developing. Nevertheless, the maturation of this immune system is sufficient to verify its efficacy, as it is activated by therapeutic immune compounds for a few hours after grafting.

[0047] Preferably, the developing egg according to the present invention is gallipformes ( Galliformes ) or strutioniformes( Struthioniformes It is an egg of a bird of the order ) In particular, it is particularly preferable that the egg be an egg of a purebred bird, especially a chicken, quail, turkey, pheasant, peacock, guinea fowl, or other farm bird. It may also be an ostrich egg. Advantageously, the developing egg according to the present invention is an egg (gallus galus( Gallus gallus ))am.

[0048] Within the scope of the present invention, the term “developing egg” refers to a fertilized bird egg in which an embryo can develop under suitable conditions, particularly in an incubator at a temperature of 37°C to 38°C. Under these conditions, the incubation time required for the egg to hatch is 21 days for chickens.

[0049] The developmental stages reported herein are defined as a function of the incubation time after fertilization of the egg, specifically as defined above, under appropriate conditions.

[0050] "Grafting at the level of a CAM" is intended to specify administration by addition or injection onto a CAM, whether it is an upper CAM or a lower CAM.

[0051] The embryonic model according to the present invention has cells derived from two different organisms or xenografts: cells from a "host" or "recipient" bird and tumor cells grafted into an egg from a human or animal organism of a different species from that of the "recipient" bird. Particularly preferably, the tumor cells grafted into the avian embryonic egg are human cells. These grafted cells will then develop in the embryo by forming one or more solid tumors and / or by migrating from the egg.

[0052] According to the present invention, grafting tumor cells is performed in the absence of immune effector cells other than the immune effector cells of the developing egg, and the use of the egg once grafted excludes the presence and addition of immune effector cells other than the immune effector cells of the grafted egg.

[0053] By definition, "grafting at the level of CAM" is performed once CAM is formed, under normal and standard growth conditions, at a stage corresponding to at least 8 days of development in chickens. If the bird used is a chicken, this stage corresponds to at least 8 days of development. The number of developmental days may vary by species, and grafting may occur after varying developmental periods. For example, at least 8 days of development in chickens corresponds to at least 6.5 days of development in quails.

[0054] It is understood that the grafted embryo used according to the present invention is not intended to hatch and is therefore not intended to produce an adult organism. This It is used only as an animal model during the period of investigating the effects of immunotherapeutic molecule(s), and is not used until hatching corresponding to 21 days of development in chickens. In any case, the avian embryo according to the present invention will be sacrificed in accordance with ethical rules in effect before hatching and after the grafted tumor cells have developed one or more tumors in the egg.

[0055] The grafted tumor cells may be tumor cells of different types of cancer, but may also be derived from a tumor sample from a cancer patient, for example from a tumor biopsy of the patient, or from any other biological sample containing tumor cells from the patient, provided that effector immune cells have been removed, that is, only tumor cells have been isolated from the biological sample.

[0056] Within the scope of the present invention, effector immune cells will not be added when grafting a biological sample from a tumor cell line or cancer patient from which effector immune cells have been removed, when using an egg model, or when implementing the method according to the present invention.

[0057] According to one embodiment of the present invention, tumor cells obtained from a sample of a patient or animal suffering from cancer are circulating tumor cells (CTCs) that have been purified before being grafted into a developing egg. Such purification can be achieved by any method known to those skilled in the art. In particular, various methods were described by Zheyu Shen et al., 2017. So-called "negative" enrichment can be achieved when the objective is to capture non-target lymphocytes and elute CTCs. A number of different methods are specifically described by the literature [Zheyu Shen et al., 2017]. These make it possible to achieve so-called "negative" enrichment when the objective is to capture non-target lymphocytes and elute CTCs, or so-called "positive" enrichment when the objective is to capture CTCs and elute non-target cells from a sample. In particular, one can mention those described by Han Wei Hou et al. in 2013, or again by Laget S et al. in 2017 (when these are circulating tumor cells (CTCs)), those described by Petit Vincent et al. in 2013 (when these are tumor cells isolated from xenografts derived from patient cells (patient-derived xenografts or PDX)), and finally those described by DeBord Logan C et al. in 2018.

[0058] Preferably, the patient is a human individual. In this case, the sample is a xenograft or PDX (patient-derived xenograft) derived from the patient's tumor.

[0059] Tumor cells grafted into the embryonic egg may be from lung cancer, prostate cancer, breast cancer, melanoma, kidney cancer, and any other cancer that may benefit from immunotherapy treatment.

[0060] Advantageously, the developing egg model used according to the present invention is an egg in which a tumor, preferably a human cell, is grafted at the level of CAM. Preferably, the use of the grafted egg model excludes the presence of human immune effector cells.

[0061] "Immune effector cells" refers to lymphocytes, particularly T, B, and NK lymphocytes, macrophages, and dendritic cells.

[0062] Within the scope of the present invention, the terms tumor and cancer are used interchangeably and with the same meaning to define the proliferation of malignant cells. The same applies to the use of the terms anti-tumor and anticancer.

[0063] The term “immunotherapeutic molecule” is intended to designate any compound or product capable of activating an immune response or restoring the action developed by the patient’s immune system against the patient’s tumor. These immunotherapeutic molecules target the function of controlling the immune system that has been blocked by the tumor. Such compounds may be antibodies, specifically monoclonal antibodies, ajuvants, chemical molecules, etc. In the developing eggs used according to the present invention, the immunotherapeutic molecule may stimulate the immune response of the “host” or “recipient” bird against the cancer developing from the tumor cells grafted in this case. Among the immunotherapeutic molecules, particularly adoptive cell therapies, e.g., CAR-T, vaccines, bispecific antibodies, immune checkpoint inhibitors, e.g., anti-PD1, or anti-PDL1, or anti-CTLA-4 antibodies may be mentioned.

[0064] Especially, and When tumor cells are derived from a cancer patient sample, testing various immunotherapeutic molecules allows for the selection of the most promising immunotherapeutic molecule for the treatment of the patient's tumor. Accordingly, according to one preferred embodiment of the present invention, a avian embryo grafted with tumor cells is used to determine which of the different immunotherapeutic molecules has the best anticancer activity.

[0065] Bird eggs grafted with tumor cells can also be used to test the anticancer efficacy of immunotherapeutic molecule combinations by comparing the effects obtained with each molecule tested independently according to the present invention.

[0066] Within the scope of use of this embryo, it is also possible to determine or even quantify the toxicity of immunotherapy(s) tested on both the tumor developed from the grafted tumor cells and the entire embryo. Accordingly, another object of the present invention relates to the use of avian embryos grafted with tumor cells to quantify the toxicity of immunotherapy molecule(s) to the tumor and / or the entire embryo.

[0067] In one preferred embodiment, the use according to the present invention described above is carried out with grafted avian eggs that have been previously incubated to a developmental stage corresponding to CAM formation and corresponding to at least 9 days or much more preferably 9.5 days of development of a chicken.

[0068] The present invention also

[0069] - A step of grafting tumor cells at the level of CAM in avian embryos previously incubated to a developmental stage corresponding to at least 8 days in chickens at the time of graft, corresponding to the formation of chorionic alluvial membrane (CAM),

[0070] - A step of administering immunotherapeutic molecule(s) to the developing egg at least 12 hours after grafting,

[0071] - A step of investigating the effect of the immunotherapeutic molecule(s) thus administered on the tumorigenesis of a tumor developed in a grafted embryo.

[0072] including,

[0073] And implementing in the absence of effector immune cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0074] The present invention relates to a method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) characterized by [specific feature].

[0075] A person skilled in the art in the relevant technical field will know how to determine the time for grafting tumor cells depending on the species of bird used, that is, the minimum number of days of incubation or development of a developing egg that reaches the formation of CAM and a developmental stage equivalent to at least 8 days of development in chickens. For example, in chickens, grafting can be performed from day 8 of development, and in quail from day 6.5 of development.

[0076] According to one preferred embodiment, the developing egg was incubated before grafting to a developmental stage corresponding to the formation of CAM and at least 9 days or much more preferably at least 9.5 days in a chicken.

[0077] Incubation is carried out under appropriate conditions, that is, conditions that enable the normal development of the developing egg, particularly at a temperature of 37°C to 39°C, preferably 38°C, or even 38.5°C.

[0078] The grafting of tumor cells can be performed at any location above or below the CAM, preferably at the level of the upper CAM. Any method widely known to those skilled in the art may be used for this grafting, and in particular, it is possible to use the grafting technique referenced in the literature [Crespo P. & Casar B, 2016].

[0079] According to one specific embodiment, the amount of grafted tumor cells is about 10 cells to about 5.10 6 It is a range of dog cells.

[0080] According to one preferred embodiment, the tumor cells used were frozen before grafting onto the embryonic egg, for tumor cells isolated from a cell line or from a cancer patient or animal sample.

[0081] In particular, when grafted tumor cells are derived from samples of patients or animals with cancer, testing several immunotherapeutic molecules allows for the selection of the most promising one for treating the tumors of these patients or animals. Accordingly, according to one preferred embodiment of the present invention, a method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) allows for the determination of the immunotherapeutic molecule exhibiting the best anticancer activity among the different molecules tested.

[0082] The method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) according to the present invention also enables testing the anticancer efficacy of a combination of immunotherapeutic molecule(s) in relation to the respective effects of the immunotherapeutic molecule(s) tested independently.

[0083] The step of administering immunotherapeutic molecule(s) to a developing egg may be performed in different ways by techniques widely known to those skilled in the art. Administration may be performed particularly by addition or injection at the level of CAM, by injection into a tumor, or by injection into the embryonic or extra-embryonic structure of the egg.

[0084] The administration of the immunotherapeutic molecule(s) is performed at least 12 hours after grafting the tumor cells, preferably at least 24 hours after grafting, or much more preferably at least 48 hours after grafting, i.e., 1 to 2 days after grafting. The immunotherapeutic molecule(s) may be administered according to different patterns in terms of duration, but may also be administered until the last day of egg incubation in terms of the frequency of administration, such as every 2 days, or daily, or twice a day, or as a single injection. These choices will be determined by the immunotherapeutic molecule being administered.

[0085] According to one preferred embodiment, a method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) according to the present invention further comprises, before studying the effect on tumorigenesis, administering the immunotherapeutic molecule(s) to the grafted embryo and then incubating the grafted embryo for at least one hour. Advantageously, the incubation is performed for at least four days and at most twelve days, corresponding to an embryonic development stage of at most 21 days and, advantageously, 18 days of development.

[0086] According to one specific embodiment, a method for evaluating the anticancer activity of one or more immunotherapeutic molecule(s) according to the present invention further comprises collecting a tumor developing from a grafted tumor cell after administration of the administered immunotherapeutic molecule(s), and particularly at the end of the incubation of said developing egg by microdissection.

[0087] Accordingly, investigations into the effects of immunotherapeutic molecule(s) on tumorigenesis may employ several complementary approaches, particularly after collecting tumors developed from grafted embryos. This may include analyzing parameters such as tumor growth, metastatic invasion, angiogenesis, neo-angiogenesis, inflammation and / or tumor immune infiltration, and toxicity.

[0088] Accordingly, the tumor may be subjected to analysis to measure and / or analyze these different parameters, such as tumor weight and / or volume to investigate tumor growth, expression of different specific markers to investigate metastatic invasion such as amplification of Alu sequences by quantitative PCR for human metastasis, number of blood vessels in the tumor for angiogenesis and neovascularization, quantification of interleukin for inflammation to evaluate tumor immune invasion and / or quantification of markers such as CD3, CD8, CD4, CD45 and CD56 particularly by rtQPCR, body weight, and histological analysis to evaluate toxicity to the tumor.

[0089] Investigations into metastatic invasion can be performed in easily accessible lower CAMs, but may also be performed in any target organ within the embryo, depending on known data regarding the type of cancer and associated metastasis phenomena.

[0090] Inflammation and / or tumor immune infiltration can be specifically investigated by analyzing the expression of various markers such as CD3 (membrane marker for T lymphocytes), CD4 (membrane marker for regulatory T lymphocytes, monocytes, and macrophages), CD8 (marker for cytotoxic T lymphocytes), CD45 (membrane marker for leukocytes), and CD56 (marker for NK cells). Oligonucleotide pairs specific to these markers can be developed to avoid cross-breeding between species.

[0091] By extension, it is also possible to monitor inflammation and infiltration of immune system cells at the metastatic site.

[0092] The analysis of the combination of all these factors, which are widely known to those skilled in the art, enables the determination of the anticancer efficacy of immunotherapeutic molecule(s) administered to embryos. These parameters are essential parts of decision trees used by clinicians to determine the treatment regimen to adopt, particularly in cancer patients.

[0093] Within the scope of all methods according to the present invention, including investigating the effect of immunotherapeutic molecule(s) on tumor formation, anticancer activity is preferably evaluated by comparing the tumor formation of a tumor collected after administration of the immunotherapeutic molecule(s) in an embryo that has been grafted once with that of a tumor collected from another embryo of the same bird that was previously grafted with the same tumor cells according to the same method and was not administered the immunotherapeutic molecule. Similarly, when the effect of multiple immunotherapeutic molecules is investigated, anticancer activity will be preferentially evaluated by comparing the tumor formation of a tumor collected after administration of the immunotherapeutic molecule in an embryo that has been grafted once with that of a tumor collected from one or more other embryo(s) of the same bird that were previously grafted with the same tumor cells according to the same method but to which each immunotherapeutic molecule was individually administered.

[0094] Advantageously, within the scope of the method for evaluating anticancer activity according to the present invention, the anticancer activity of one or more immune checkpoint inhibitors is evaluated, preferably the anticancer activity of an anti-PD1 antibody or an anti-PDL1 antibody.

[0095] The present invention also relates to a method for screening immunotherapeutic molecules for in vivo cancer treatment. Accordingly, according to another aspect, the present invention

[0096] - A step of grafting tumor cells at the level of CAM in avian embryos previously incubated to a developmental stage corresponding to the formation of chorionic alluvial membrane (CAM) and equivalent to at least 8 days of development in chickens,

[0097] - A step of administering candidate immunotherapeutic molecule(s) to a developing egg at least 12 hours after grafting,

[0098] - A step of investigating the effect of the immunotherapeutic molecule(s) thus administered on the tumorigenesis of a tumor developed in a grafted embryo.

[0099] The present invention relates to a method for screening immunotherapeutic molecules having anticancer activity, including,

[0100] The above method is implemented in the absence of effector immune cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0101] "Candidate immunotherapeutic molecule" refers to a chemical or biological immunotherapeutic molecule as defined above, which may possess antitumor / anticancer activity and is potentially effective, in particular, for treating types of cancer developed from tumor cells grafted into embryonic eggs.

[0102] The screening method according to the present invention enables determining whether a candidate therapeutic agent has anticancer activity and whether it has anti-metastasis activity.

[0103] According to another aspect, the present invention also relates to a method for monitoring cancer patients or animals, and

[0104] - A step of preparing a first avian embryo as described above using tumor cells from the patient or animal at time T1, and investigating the tumor formation of a tumor developing in the first avian embryo,

[0105] - A step of preparing a second avian embryo as described above using tumor cells from a sample of the same patient or animal at time T2, and investigating the tumor formation of a tumor developing in the second avian embryo,

[0106] - A step of comparing tumor formation of tumors developed in the first and second avian embryos.

[0107] Includes,

[0108] The above method is implemented in the absence of immune effector cells other than the immune effector cells of the grafted egg, and without adding said cells.

[0109] All the aforementioned preferences and details regarding the method for evaluating the anticancer activity of immunotherapeutic molecules are applied to the monitoring and screening method according to the present invention.

[0110] Examples

[0111] The present invention is exemplified by the use of hens developed with tumors of human origin on the chorionic villi (CAM) to verify the efficacy of antibodies against two membrane proteins that play a significant role in the interaction between different immunotherapeutic molecules, particularly the immune system and tumors: PD-1 and PDL-1, in oncology.

[0112] PD-1 (or PDC1 in the case of programmed apoptosis 1) is a membrane protein expressed on the surface of activated T lymphocytes. Its binding to its ligand, PDL-1 (programmed apoptosis ligand 1), which is present on the surface of tumor cells, causes the inactivation (inhibition of proliferation and cytokine secretion) of T lymphocytes in relation to tumor cells.

[0113] Immunocheckpoint inhibitors were developed with the goal of blocking lymphocytes and removing obstacles to prevent them from attacking tumors. Therefore, anti-PD1 or anti-PD-L1 antibodies must reactivate the immune system's attack on the tumor.

[0114] Materials and Methods

[0115] Embryo, incubation conditions, and graft

[0116] Opening an embryo on the chorionic allantium (CAM) and grafting tumor cells is already known and has been widely documented for many years (Crespo P. & Casar B., 2016). A schematic diagram of an embryo with a grafting site of tumor cells on the upper CAM is shown in Fig. 1.

[0117] Only the specific conditions used in the verification study are listed here:

[0118] - Modified hen eggs were incubated in a supine position at 37°C and 40% humidity for 9.5 days.

[0119] - In the development of E9.5, the egg was opened while maintaining the integrity of the CAM. After grafting the filament onto the CAM, the egg was incubated again for 24 hours at 37.5°C and 40% humidity.

[0120] - For each group (control group and treatment group), results were provided for at least 9 eggs (eggs surviving at E18).

[0121] cell line

[0122] Standard lymphoma (SU-DHL-4), breast adenocarcinoma (MDA-MB-231), or glioblastoma (U87) cell lines were grafted in the amounts and conditions listed in Table 1.

[0123]

[0124] Immunotherapy molecules

[0125] In ovo-grafted tumors were treated with anti-human PD1 or anti-PDL1 antibodies in four cases (E10.5; E12.5; E14.5; E16.5), and 100 μl of antibody (anti-PD1 or anti-PDL1) was used at different concentrations (detailed in the figure below).

[0126] The anti-PD1 and anti-PDL-1 antibodies tested are presented in Table 2.

[0127]

[0128] Atezolizumab (anti-PD-L1 Tecentriq) and pembrolizumab (anti-PD-1 Keytruda) are two monoclonal antibodies already prescribed in humans.

[0129] Atezolizumab (anti-PD-L1 Tecentriq) is the first anti-PD-L1 drug approved by the FDA in humans. It is used for metastatic non-small cell lung cancer. It is also used for urothelial carcinoma. Pembrolizumab (anti-PD-1 Keytruda) is an anti-PD-1 drug prescribed for many cancers (melanoma, lung, Hodgkin's lymphoma, prostate, bladder, breast, etc.) that is an anti-PD-1 drug but binds to a different site on the PD-1 membrane protein, and is a commercial competitor to nivolumab (Opdivo) (Fessas, P.; Semin Oncol. 2017).

[0130] Tumor collection and analysis

[0131] At E18, the eggs were opened, the tumors were collected, fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), and washed to remove CAM fragments surrounding the tumors, and weighed using a precision balance. For metastasis analysis, fragments of the lower CAM (opposite the graft site) may be collected and frozen. This will be used for whole-genome DNA extraction. Detection of human cells in these samples was performed by qPCR using primers specific to human Alu sequences (multi-copy sequences widely conserved in humans) (Zijlstra, A. et al., 2012).

[0132] Figure 2 shows the investigation timeline from cell graft to sample collection.

[0133] Results were obtained from various cancer models, demonstrating the potential of this type of treatment in many models.

[0134] result

[0135] 1. Efficacy of 4 Immunotherapy Methods Against Tumor Growth

[0136] Efficacy of atezolizumab (anti-PDL-1 Tecentriq) against MDA-MB-231 cells

[0137] After grafting the cells, the tumors were treated four times (E10.5; E12.5; E14.5; E16.5) with 100 μl of atezolizumab (anti-PDL-1 Tecentriq) at a dose of 4 μg / kg per gram (E10.5; E12.5; E14.5; E16.5).

[0138] The results of the tumor weight analysis initiated in MDA-MB-231 cells after atezolizumab administration were pooled in Table 3 (SD: standard deviation; SEM: standard error of the mean) and attached to Figure 3.

[0139]

[0140] Efficacy of Pembrolizumab (anti-PD-1 Keytruda) against MDA-MB-231 cells and SU-DHL-4 cells

[0141] After grafting the cells, the tumors were treated with 100 μl of pembrolizumab (anti-PD-1 Keytruda) four times (E10.5; E12.5; E14.5; E16.5).

[0142] The results of tumor weight analysis initiated in MDA-MB-231 cells or SU-DHL-4 cells after pembrolizumab administration were pooled in Tables 4 and 5 (SD: standard deviation; SEM: standard error of the mean) and attached to Figure 4.

[0143]

[0144]

[0145] Efficacy of murine anti-PD-1 RMP1-14 against SU-DHL-4 cells

[0146] After grafting the cells, the tumors were treated four times (E10.5; E12.5; E14.5; E16.5) with 100 μl of RMP1-14 (murine anti-PDL-1) at a dose of 166 μg / kg per gram.

[0147] The results of the tumor weight analysis initiated in SU-DHL-4 cells after RMP1-14 administration are shown in Table 6 (SD: standard deviation; SEM: standard error of the mean) and the attached Figure 5.

[0148]

[0149] Efficacy of nivolumab (anti-PD-1 opdivo) against MDA-MB-231 cells

[0150] After grafting the cells, the tumors were treated with 100 μl of nivolumab (anti-PD-1 Opdivo) four times (E10.5; E12.5; E14.5; E16.5).

[0151] The results of the tumor weight analysis initiated in MDA-MB-231 cells after nivolumab administration were pooled in Table 7 (SD: standard deviation; SEM: standard error of the mean) as well as in the attached Figure 6.

[0152]

[0153] 2. Monitoring of metastatic invasion

[0154] In addition to tumor weight, metastatic invasion within the developing egg can be monitored after administering immunotherapy.

[0155] This analysis was performed on genomic DNA extracted from any embryonic tissue (MagJET Genomic DNA kit; ThermoScientific; Ref. K2721) by qPCR (Bio-Rad; SsoAdvanced Univ SYBR Green Supermix Ref. 1725274) using oligonucleotides typically specific to human Alu sequences (multi-copy sequences of the human genome) (Zijlstra, A. et al., 2012).

[0156] After grafting MDA-MB-231 cells, and administering pembrolizumab (anti-PD-1 Keytruda) as described above, analysis was performed on genomic DNA extracted from a fragment of the sub-CAM, which is an easily available tissue.

[0157] The results are pooled in the attached Fig. 7.

[0158] 3. Analysis of tumor infiltration by immune system cells

[0159] In addition to the observed efficacy against tumor metastasis, the action of immunotherapy can be analyzed by measuring the infiltration of immune cells from the embryo into the tumor tissue in the presence or absence of immunotherapy.

[0160] As described above, avian-type CD3 (membrane marker for T lymphocytes), CD4 (membrane marker for regulatory T lymphocytes, monocytes, and macrophages), CD45 (membrane marker for leukocytes), CD8 (cytotoxic T lymphocyte marker) and CD56 (NK cell marker) markers were analyzed by qPCR in tumors originating from SU-DHL-4 cells, either after or without administration of anti-PD-1 pembrolizumab or nivolumab, or anti-PD-L1 atezolizumab.

[0161] Tissues were collected and total RNA was extracted (MagJET RNA Kit; Thermoscientific; Ref. K2731). From total RNA, cDNA was synthesized by specific pre-amplification for each desired biomarker (PrimePCR, Pre-Amp Assay, Probe Chicken; Bio-Rad; Ref. 10041596) (iScript Explore RT and PreAmp Kit; Bio-Rad; Ref. 12004856). Subsequently, quantitative PCR was performed using oligonucleotides specific to each biomarker (PrimePCR Assay FAM, Chicken; Bio-Rad; Ref. 12001961).

[0162] The results are pooled in Fig. 8 (anti-PDL-1 atezolizumab), Fig. 9 (anti-PD-1 pembrolizumab), and Fig. 10 (anti-PD-1 nivolumab) and show the infiltration of immune cells in chickens possessing CD3, CD4, CD45, CD8, and CD56 regardless of whether they received immunotherapy treatment with atezolizumab (anti-PDL-1), pembrolizumab (anti-PD-1), or nivolumab (anti-PD-1).

[0163] 4. Characterization of chicken embryonic immune cells and their response to immunotherapy

[0164] Characterization of immune cells from peripheral blood by flow cytometry

[0165] Chicken peripheral blood was collected at E16. After purification by Ficoll-Paque® density gradient centrifugation (Sigma-GE17-1440-02), peripheral blood mononuclear cells (PBMCs) were labeled with anti-chicken-CD45-FITC (Thermo Fisher-MA5-28679), anti-chicken-CD3-Pacific Blue® (CliniSciences 8200-26), anti-chicken-CD8 alpha-PE (Thermo Fisher-MA5-28726), anti-chicken-CD4-PE (Thermo Fisher-MA5-28686), and anti-chicken KUL01-PE (Thermo Fisher-MA5-28828) (which identify chicken monocytes and phagocytes). Different immune cell populations were detected by flow cytometry (BD FACSCanto™ II).

[0166] The results are pooled in Figure 11, and various immune cell populations (CD8+ T cells, CD4+ T cells, and monocytes) were detected in the peripheral blood of chicken embryos, which indicates that the immune system of chicken embryos is functional.

[0167] Verification of the cytotoxicity of chicken lymphocytes against human H460 tumor cells in the presence of pembrolizumab (Keytruda) in vitro

[0168] As described above, peripheral blood mononuclear cells (PBMCs) from blood collected at E16 were activated with phytohemagglutinin (PHA, Sigma-11249738001, 5 μg / ml) for 72 hours. Subsequently, to prevent interaction with PD-L1 expressed by tumor cells, pembrolizumab (Keytruda®, 5 μg / ml) was added to T lymphocytes maintained in culture for 12 hours to block PD-1 molecules. Pembrolizumab-treated and untreated T lymphocytes were then co-cultured with human H460 (lung) tumor cells at different ratios of T lymphocytes (effector cells = E) to tumor cells (target lymphocytes = C): E / C = 10:1; E / C = 20:1; and E / C = 40:1 (Fig. 12). PD-1 blockade on chicken T lymphocytes by pembrolizumab was confirmed by measuring the cytotoxicity of T lymphocytes against tumor cells, as revealed by the in vitro MTT cytotoxicity assay (Sigma-CGD1-1KT).

[0169] The results are pooled in Figure 12 and demonstrate an increase in the cytotoxic effect of chicken T lymphocytes on human H460 tumor cells after treatment with pembrolizumab.

[0170] Greater tumor cell viability was detected when tumor cells were incubated with pembrolizumab-treated T lymphocytes compared to when tumor cells were incubated with T lymphocytes not treated with pembrolizumab. This difference indicates an increase in T lymphocyte cytotoxicity, which indicates effective blockade of PD-1 on chicken T lymphocytes by pembrolizumab.

[0171] References

[0172] Crespo P. & Casar B.; Bio-protocol, Vol. 6, Iss 20, Oct 20, 2016; Chick Embryo Chorioallantoic Membrane as an in vivo Model to Study Metastasis

[0173] Han Wei Hou et al. Scientific Reports 3, Article number: 1259 (2013)

[0174] Laget S et al. PloS one, 2017 Jan 6;12(1):e0169427.

[0175] Petit Vincent et al. Lab Invest. 2013 May;93(5):611-21.

[0176] DeBord Logan C et al. Am J Cancer Res. 2018 Aug 1;8(8):1642-1660.

[0177] Zijlstra, A. et al. A Quantitative Analysis of Rate-limiting Steps in the Metastatic Cascade Using Human-specific Real-Time Polymerase Chain Reaction. Cancer Res. 62, 7083-7092 (2012)

[0178] Fessas, P.; Semin Oncol. 2017 Fessas, P. A molecular and preclinical comparison of the PD-1-targeted T lymphocyte checkpoint inhibitors nivolumab and pembrolizumab;.Semin Oncol. 2017 Apr; 44(2): 136-140. PMID: 28923212).

[0179] Zheyu Shen et al, Chem Soc Rev, 2017 Apr 18;46(8): 2038-2056. Current Detection Technologies of Circulating Tumour Cells

Claims

Claim 1 A composition comprising an embryonic bird egg model grafted with tumor cells at the level of the chorioallantoic membrane (CAM) for use in evaluating the anticancer activity of one or more immune checkpoint inhibitors, wherein the model excludes the presence of immune effector cells other than immune effector cells of the grafted egg, and wherein the immune checkpoint inhibitor is selected from anti-PD1, anti-PDL1, and anti-CTLA-4 antibodies. Claim 2 A composition according to claim 1, also to be used for selecting the immune checkpoint inhibitor having the most promising anticancer activity for the treatment of a tumor developed from tumor cells grafted into an egg cell from different immune checkpoint inhibitors tested. Claim 3 A composition comprising a avian developing egg model grafted with tumor cells at the level of the chorionic villi (CAM) for use in determining or even quantifying the toxicity of one or more immune checkpoint inhibitors on a tumor developed from grafted tumor cells, an entire embryo, or both, wherein the model excludes the presence of any immune effector cells other than the immune effector cells of the grafted egg, and the immune checkpoint inhibitor is selected from anti-PD1, anti-PDL1, and anti-CTLA-4 antibodies. Claim 4 A method for evaluating the anticancer activity of one or more immune checkpoint inhibitors, characterized by comprising: a step of grafting tumor cells at the level of CAM in a previously incubated avian embryo that corresponds to the formation of chorionic alluvial membrane (CAM) and corresponds to a developmental stage equivalent to at least 8 days of development in a chicken embryo; a step of administering an immune checkpoint inhibitor to the embryo at least 12 hours after grafting; and a step of investigating the effect of the immune checkpoint inhibitor so administered on the tumorigenesis of a tumor developed in the grafted embryo, wherein the method is implemented in the absence of effector immune cells other than the effector immune cells of the grafted egg and without the addition of effector immune cells other than the effector immune cells of the grafted egg, and wherein the immune checkpoint inhibitor is selected from anti-PD1, anti-PDL1, and anti-CTLA-4 antibodies. Claim 5 A method according to claim 4, further comprising the step of administering an immune checkpoint inhibitor into a grafted embryo before investigating the effect on tumor formation, and then incubating the grafted embryo for at least one hour. Claim 6 A method according to claim 5, further comprising the step of collecting a tumor developed from a tumor cell grafted at the end of the incubation of the embryo after administration of an immune checkpoint inhibitor. Claim 7 A method characterized in that, in any one of claims 4 to 6, investigating tumor formation includes measuring various parameters. Claim 8 A method according to claim 7, wherein the parameter is selected from tumor growth; metastatic invasion; angiogenesis; neo-angiogenesis; inflammation, tumor immune infiltration, or both; and toxicity to the tumor. Claim 9 A method according to any one of claims 4 to 6, characterized in that the anticancer activity is evaluated by comparing the tumor formation of a tumor collected after administering an immune checkpoint inhibitor to a developing egg that has been grafted once with the same tumor cells according to the same method and the tumor formation of a tumor collected from the same developing egg that has been previously grafted with the same tumor cells and has not been administered an immune checkpoint inhibitor. Claim 10 A method according to claim 9, characterized in that investigating tumor formation includes measuring various parameters. Claim 11 A method according to claim 10, wherein the parameter is selected from tumor growth; metastatic invasion; angiogenesis; neovascularization; inflammation, tumor immune infiltration, or both; and toxicity to the tumor. Claim 12 A method according to any one of claims 4 to 6, characterized in that the tumor cells grafted into the avian embryo are derived from a sample of a patient or animal with cancer. Claim 13 A method according to claim 12, characterized in that investigating tumor formation includes measuring various parameters. Claim 14 A method according to claim 13, wherein the parameter is selected from tumor growth; metastatic invasion; angiogenesis; neovascularization; inflammation, tumor immune infiltration, or both; and toxicity to the tumor. Claim 15 A method according to claim 12, characterized in that anticancer activity is evaluated by comparing the tumor formation of a tumor collected after administering an immune checkpoint inhibitor to a developing egg that has been grafted once with the same tumor cells according to the same method and the tumor formation of a tumor collected from the same developing egg that has been previously grafted with the same tumor cells and has not been administered an immune checkpoint inhibitor. Claim 16 A method for screening an immune checkpoint inhibitor having anticancer activity, comprising: - a step of grafting tumor cells at the level of CAM in an avian embryo previously incubated to a developmental stage corresponding to the formation of chorionic alluvial membrane (CAM) and equivalent to at least 8 days of development in chickens; - a step of administering a candidate immune checkpoint inhibitor to the embryo at least 12 hours after grafting; and - a step of investigating the effect of the immune checkpoint inhibitor so administered on the tumorigenesis of a tumor developed in the grafted embryo, wherein the method is implemented in the absence of effector immune cells other than the effector immune cells of the grafted egg and without the addition of effector immune cells other than the effector immune cells of the grafted egg, and wherein the immune checkpoint inhibitor is selected from anti-PD1, anti-PDL1, and anti-CTLA-4 antibodies. Claim 17 A method according to claim 16, characterized by further including the step of administering an immune checkpoint inhibitor into a grafted embryo before investigating the effect on tumor formation, and then incubating the grafted embryo for at least one hour. Claim 18 A method according to claim 17, further comprising the step of collecting a tumor developed from a tumor cell grafted at the end of the incubation of the embryo after administration of an immune checkpoint inhibitor. Claim 19 A method according to any one of claims 16 to 18, wherein the anticancer activity of a candidate immune checkpoint inhibitor is evaluated by comparing the tumor formation of a tumor collected after administering the immune checkpoint inhibitor to a developing egg that has been grafted once with the tumor formation of a tumor collected from the same developing egg that has been previously grafted with the same tumor cells according to the same method and has not been administered the immune checkpoint inhibitor. Claim 20 A method according to any one of claims 16 to 18, characterized in that the tumor cells grafted into the avian embryo are derived from a sample of a patient or animal with cancer. Claim 21 A method according to claim 20, characterized in that the anticancer activity of a candidate immune checkpoint inhibitor is evaluated by comparing the tumor formation of a tumor collected after administering the immune checkpoint inhibitor to a grafted embryo with the tumor formation of a tumor collected from the same avian embryo that was previously grafted with the same tumor cells according to the same method and was not administered the immune checkpoint inhibitor. Claim 22 delete

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