A method to determine simultaneously Anti-cancer activity and hematotoxicity of an antibody-drug conjugate

Abstract

The present invention refers to an in vitro method to determine simultaneously anti-cancer activity and hematotoxicity of an antibody-drug conjugate, comprising: (a) mixing a sample comprising cancer cells with a bone marrow sample; (b) adding an antibody-drug conjugate to the mixture of step (a); (c) determining anti-cancer activity of the antibody-drug conjugate by measuring cell depletion of tumor cells; and (d) determining hematotoxicity of the antibody-drug conjugate by measuring cell depletion of cells of the bone marrow sample.

Description

[0001] DESCRIPTION

[0002] A METHOD TO DETERMINE SIMULTANEOUSLY ANTI-CANCER ACTIVITY AND HEMATOTOXICITY OF AN ANTIBODY-DRUG CONJUGATE

[0003] TECHNICAL FIELD

[0004] The present invention relates to assays for determining the activity of antibody-drug conjugates against cancer cells and to assays for determining the hematotoxicity of the antibody-drug conjugates.

[0005] BACKGROUND ART

[0006] Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. ADCs are composed of an antibody linked to a biologically active anticancer payload, often a cytotoxic compound, or drug. ADCs combine the targeting properties of monoclonal antibodies with the cancer-killing capabilities of cytotoxic or other drugs, designed to discriminate between healthy and diseased tissue.

[0007] In ADCs, an anticancer drug is coupled to an antibody that targets a specific tumor antigen (or protein) found on tumor cells. Antibodies attach themselves to the antigens on the surface of cancerous cells. The biochemical reaction that occurs upon attaching triggers a signal in the tumor cell, which then absorbs, or internalizes, the antibody together with the linked payload. After the ADC is internalized, the payload may kill the cancer cell. Once the payload is free, it is distributed throughout the body reaching the bone marrow, where it may kill hematopoietic cells inducing hematotoxicity.

[0008] SUMMARY OF INVENTION

[0009] The invention is defined in the claims.

[0010] The present invention provides an in vitro method to determine simultaneously anti-cancer activity and hematotoxicity of an antibody-drug conjugate, comprising:

[0011] (a) mixing a sample comprising cancer cells with a bone marrow sample;

[0012] (b) adding an antibody-drug conjugate to the mixture of step (a);

[0013] (c) determining anti-cancer activity of the antibody-drug conjugate by measuring cell depletion of tumor cells; and

[0014] (d) determining hematotoxicity of the antibody-drug conjugate by measuring cell depletion of cells of the bone marrow sample. In step (a), two samples are mixed, the sample comprising cancer cells and the bone marrow sample. Preferably, both samples are from different subjects. Nevertheless, the two samples may be obtained from the same subject.

[0015] There are several types of Antibody-drug conjugates. Each type of ADC is tailored to address specific tumor characteristics and improve the selectivity, potency, and stability of the therapeutic agent. Several types of Antibody-drug conjugates are described below:

[0016] 1 . Traditional ADCs

[0017] Monoclonal Antibody (mAb)-based ADCs: These ADCs use a monoclonal antibody to target a specific tumor antigen, conjugated with a cytotoxic payload via a linker. Examples include trastuzumab emtansine (Kadcyla) targeting HER2 and brentuximab vedotin (Adcetris) targeting CD30.

[0018] 2. Bispecific and Multispecific ADCs

[0019] Bispecific ADCs: These use a bispecific antibody to recognize two distinct antigens, often one on the tumor cell and one on an immune cell, to improve targeting and efficacy. They can deliver cytotoxic agents to tumors while engaging the immune system.

[0020] Multispecific ADCs: These go a step further and target multiple antigens, helping increase selectivity and allowing ADCs to overcome heterogeneous antigen expression in tumors.

[0021] 3. Immunostimulatory ADCs

[0022] Immune-stimulatory payload ADCs: These combine an antibody with a payload that activates immune cells rather than directly killing the tumor cells. The immune-stimulatory payload could involve agents like toll-like receptor (TLR) agonists or STING agonists to trigger an immune response against the tumor.

[0023] 4. Targeted Protein Degraders (TPD)-based ADCs

[0024] PROTAC-ADCs: PROteolysis TArgeting Chimeras (PROTACs) use ADCs to deliver proteindegrading molecules specifically to tumor cells. These ADCs target and degrade specific proteins essential for tumor growth or survival, combining traditional ADC targeting with protein degradation.

[0025] 5. Radioimmunoconjugates

[0026] Radiolabeled ADCs: Instead of cytotoxic drugs, these ADCs carry a radioactive isotope as the payload. When the ADC binds to the tumor cell, it delivers radiation directly to the cell, potentially damaging DNA and inhibiting cell replication. Examples include Zevalin (ibritumomab tiuxetan) for non-Hodgkin lymphoma. 6. Antibody-siRNA Conjugates (ARCs) siRNA-based ADCs: These conjugates deliver small interfering RNA (siRNA) to knock down specific gene expression within cancer cells. The siRNA payload silences genes that are critical for tumor cell survival, making them an attractive alternative to traditional cytotoxic drugs.

[0027] 7. Photodynamic Therapy (PDT)-based ADCs

[0028] Photoactivated ADCs: These ADCs carry a photosensitive compound as the payload. Once the ADC binds to a tumor cell, it can be activated with light exposure, causing localized damage to the cell. This allows for high precision with minimal damage to surrounding tissues.

[0029] 8. Enzyme-Activated ADCs

[0030] Prodrug-based ADCs: In these ADCs, the payload is a prodrug that requires enzymatic activation within the tumor microenvironment. This approach increases specificity by ensuring the cytotoxic effect is confined to the tumor area.

[0031] 9. Self-lmmolative Linker ADCs

[0032] Self-Immolation Triggered ADCs: These ADCs employ self-immolative linkers that spontaneously release the drug once the linker is cleaved by tumor-specific conditions, allowing for precise control over drug release within the tumor.

[0033] 10. Polyclonal ADCs

[0034] Polyclonal Antibody ADCs: These ADCs use a mix of antibodies against multiple antigens to improve targeting in tumors with heterogeneous antigen expression. They are particularly useful in solid tumors, where a single antigen may not be consistently present.

[0035] 11 . Miniaturized ADCs (mADCs)

[0036] Miniaturized Antibody ADCs: These ADCs use smaller antibody fragments or scaffolds (e.g., singlechain variable fragments or nanobodies) that have improved tumor penetration and shorter half-lives, making them ideal for solid tumors.

[0037] In a preferred embodiment of the method of the invention, the sample comprising cancer cells is from a solid tumor or from a hematological cancer.

[0038] In a preferred embodiment of the method of the invention, the sample comprising cancer cells is from a subject having cancer.

[0039] In a preferred embodiment of the method of the invention, the sample comprising cancer cells is from a cancer cell line.

[0040] In a preferred embodiment of the method of the invention, the bone marrow sample is a whole bone marrow sample. Preferably, the bone marrow sample is a whole bone marrow sample, in which substantially the cellular and non-cellular components of the bone marrow are maintained and substantially maintains its structure. As an example, some bone marrow samples treatments include removal of complement proteins. Advantageously, whole bone marrow samples maintain the complement proteins, which can have an effect on the activity of antibody-drug conjugates to be determined.

[0041] In a preferred embodiment of the method of the invention, the bone marrow sample is from a healthy donor.

[0042] In a preferred embodiment of the method of the invention, the bone marrow sample comprises CD34+ progenitor cells, myeloid progenitor cells, erythroid progenitor cells, megakaryocytic progenitor cells, NK cells, and complement proteins.

[0043] In a preferred embodiment of the method of the invention, the bone marrow sample comprises NK cells, and complement proteins.

[0044] In a preferred embodiment of the method of the invention, the mixture formed in step (b) is incubated from 4 hours up to 7 days.

[0045] In a preferred embodiment of the method of the invention, the mixture formed in step (b) is incubated with 3D culture constructs mimicking the microenvironment architecture of solid tumors. Preferably, said 3D culture constructs are selected from the group consisting of: spheroids, organoids, extracellular matrix gels, synthetic scaffolds, rotary cell culture systems, culture plastics, tumor explants, and tumoroids.

[0046] In a preferred embodiment of the method of the invention, the anti-cancer activity of the antibodydrug conjugate is determined by flow cytometry.

[0047] In a preferred embodiment of the method of the invention, the hematotoxicity of the antibody-drug conjugate is determined by flow cytometry.

[0048] ADCs bind to tumor cells in tumor tissues (e.g. HER2 in breast tumors), and eventually the toxin is released traveling to the bone marrow where it can kill myeloid progenitors generating hematotoxicity. We mix the tumor cells with the bone marrow sample. This artifact represents an extreme case of ADC targeting tumor cells right next to myeloid progenitors, rather than at a distance tissue in the body. By removing the distance between tumor and progenitors, we simplify the system, and we represent the worst-case scenario where maximal toxin can be exposed to progenitors. But it allows us to study activity and hematotoxicity in the same assay, and we can study these two processes.

[0049] Activity of different ADCs in 5 different doses was tested, against tumor cell lines expressing or not the ADC target, Her2. The tested cell lines were the breast cancer cell line, Her2 positive SKBR3 and the Her2 negative cell line MCF-7 (expressing low levels of Her2), both commercially available at ATCC. Besides dose-response tumor depletion, the effects of ADC candidates were also evaluated on healthy cells, as T-lymphocytes (T Cells), B-lymphocytes (B Cells), and CD34 positive cells, derived from Normal Bone Marrow (NBM) samples, in the same assay, by mixing donor-derived cells with tumor cells.

[0050] This ADCs in vitro testing combined with hematotoxicity assay, include target expression by tumor cells, in this case, solid tumor cell lines expressing Her2. The tumor cells are mixed with the NBM cells and this mix is incubated with the ADCs candidates, in the presence of donor’s plasma and red blood cells for 72 hours.

[0051] Once the incubation time is finished, the tumor and NBM-derived cells are recovered from the plates and stained with flow-cytometry antibodies. The flow-based readout includes tumor depletion curves, and off-target toxicity in healthy CD34 positive cells, T Cells and B Cells. A scheme of experimental design is shown in Figure 1.

[0052] The advantages of co-incubating bone marrow samples together with target tumor cells are as follows: (i) the bone marrow contain autologous plasma and cells that could improve the Complement-dependent cytotoxicity (CDC) mediated by the antibody of the ADCs; (ii) the presence of healthy effector cells, as NK Cells, could allow the Antibody-dependent cellular cytotoxicity (ADCC) evaluation of the antibody of the ADC; (iii) the presence of proteases in bone marrow plasma could cleave the payload, unmasking possible untargeted toxicity; and (iv) the reduced tumor cell lines proliferation, due to nutrients competition with healthy cells could also impair tumor depletion by payloads dependent of cell proliferation, reducing their activity compared with conventional assays with tumor cells alone.

[0053] In a preferred embodiment of the method of the invention, cell depletion of cells of the bone marrow sample includes measuring depletion of CD34+ progenitor cells, cell depletion of myeloid progenitor cells (responsible, for example, for clinical anaemia, neutropenia, lymphopenia, or thrombocytopenia), cell depletion of cells of myeloid origin responsible for toxic adverse effects, cell depletion of cells of lymphoid origin, cell depletion of erythroid progenitor cells, or cell depletion of megakaryocytic progenitor cells.

[0054] In a preferred embodiment of the method of the invention, cell depletion of steps (c) or (d) is measured by a parameter selected from the group consisting of: number or percentage of cells killed relative to control samples without the antibody-drug conjugate using as reference a control sample at initial conditions, a post incubation control sample, or both, to take into account the spontaneous cell death, cell depletion at a single dose, cell depletion at multiple doses to generate a doseresponse curve, that enables calculation of Area Under the Curve (AUC), EC50, and Emax parameters.

[0055] In a preferred embodiment of the method of the invention, the parameters of cell depletion of steps (c) or (d) are analyzed across tumor cells from multiple patients, either multiple cell lines or multiple patient samples, and multiple bone marrow from multiple donors. More preferably, said analysis can estimate interpatient variability and the confidence intervals associated to the different parameters. Yet more preferably, analysis of a sufficient number of samples enables the use of mixed effects populational models that converge, to quantitate the population behaviour.

[0056] In a preferred embodiment, the method of the invention further comprises determining an ex vivo therapeutic index.

[0057] As used herein, “ex vivo therapeutic index” (also called therapeutic ratio) refers to a quantitative measurement of the relative efficacy vs safety of a drug. It is a comparison of the amount of a therapeutic agent that causes toxicity to the amount that causes the therapeutic effect. Said ex vivo therapeutic index can be calculated by: (i) comparing anti-cancer activity of the antibody-drug conjugate to hematototoxicity of the antibody-drug conjugate; (ii) determining the ratio AUC(anti- cancer activity of the antibody-drug conjugate) / AUC(hematototoxicity of the antibody-drug conjugate) or the inverse ratio; (iii) determining the ratio EC50(anti-cancer activity of the antibody-drug conjugate) / EC50(hematototoxicity of the antibody-drug conjugate) or the inverse ratio; or (iv) determining the ratio Emax(anti-cancer activity of the antibody-drug conjugate) / Emax(hematototoxicity of the antibody-drug conjugate) or the inverse ratio.

[0058] In a preferred embodiment, the method of the invention further comprises using the results obtained, either alone or combined with other data from different assays or sources, in statistical methods or in Artificial Intelligence methods, aimed to discover behaviour patterns with a potential clinical or pharmacological application.

[0059] In a preferred embodiment of the method of the invention, the antibody-drug conjugate comprises an antibody selected from the group consisting of trastuzumab, daratumumab, gemtuzumab, brentuximab, inotuzumab, moxetuzumab, polatuzumab, enfortumab, sacituzumab, disitamab, loncastuximab, tisotumab, and mirvetuximab.

[0060] In a preferred embodiment of the method of the invention, the antibody-drug conjugate comprises a drug linked to an antibody by a linker. Preferably, said drug is selected from the group consisting of: a cytotoxic compound, an antiproliferative compound, and a degrader.

[0061] As used herein, the term “degrader” refers to molecules responsible for targeted protein degradation. In particular, it refers to molecules that reprogram the protein homeostasis system to destroy target proteins.

[0062] In a preferred embodiment of the method of the invention, the antibody-drug conjugate comprises a drug selected from the group consisting of: calicheamicin, monomethyl auristatin E, DM1 , PE38, DXD, SN-38, PBD SG3199, and DM4.

[0063] In a preferred embodiment of the method of the invention, the antibody-drug conjugate comprises a linker selected from the group consisting of: hydrazone, mc-VC-PABC, SMCC, tetrapeptide, CL2A, and sulfo-SPDB. In a preferred embodiment of the method of the invention, the antibody-drug conjugate is selected from the group consisting of: gemtuzumab ozogamicin (commercial name Mylotarg, targeting CD33), brentuximab vedotin (commercial name Adcetris, targeting CD30), trastuzumab emtansine (commercial name Kadcyla, targeting HER2), inotuzumab ozogamicin (commercial name Besponsa, targeting CD22), moxetumomab pasudotox (commercial name Lumoxiti, targeting CD22), polatuzumab vedotin (commercial name Polivy, targeting CD79b), enfortumab vedotin (commercial name Padcev, targeting Nectin-4), trastuzumab deruxtecan (commercial name Enhertu, targeting HER2), sacituzumab govitecan (commercial name Trodelvy, targeting TROP2), disitamab vedotin (commercial name Aidixi, targeting HER2), loncastuximab tesirine (commercial name Zynlonta, targeting CD19), tisotumab vedotin (commercial name Tivdak, targeting TF), and mirvetuximab soravtansine (commercial name Elshere, targeting FRa).

[0064] BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1. Scheme of experimental design combining anti-cancer activity of ADCs assay with hematotoxicity assay, which includes target expression by tumor cells. The tumor cells are mixed with the Normal Bone Marrow (NBM) cells and the mix is incubated with the ADCs candidates, in the presence of donor’s plasma and red blood cells for 72 hours. Once the incubation time is finished, the tumor and NBM-derived cells are recovered from the plates and stained with flow-cytometry antibodies. The flow-based readout includes tumor depletion curves, and off-target toxicity in healthy CD34 positive cells, T Cells and B Cells.

[0066] Figure 2. Plate design of an assay mixing a cell line with different Her2-ADC compounds.

[0067] Figure 3. Plate designs of assays mixing a cell line with different Her2-ADC compounds.

[0068] Figure 4. Her2 expression was evaluated in cell lines SKBR3 and MCF-7 and in Normal Bone Marrow (NBM) cells. SKBR3 cell line presented higher Her2 expression compared with MCF-7 cell line. NBM cells were negative for Her2 expression. FMO: Fluorescence minus one (FMO) flow cytometry control.

[0069] Figure 5. Cell lines SKBR3 and MCF-7 were incubated with antibody drug conjugates for 72 hours. Tumor live cells were counted, and the values were normalized according to the basal counts (counts of live tumor cells in ineffective compound doses). The SKBR3 cell line, Her2 positive, presented dose-dependent cell depletion. The MCF-7 cell line, Her2 negative, did not present sensibility to ADCs.

[0070] Figure 6. Antibody drug conjugates were incubated for 72 hours with SKBR3 cells alone or with SKBR3 cells and cells derived from Normal Bone Marrow (NBM) sample.

[0071] Figure 7. Antibody drug conjugates were incubated for 72 hours with MCF-7 cells alone or with MCF- 7 cells and cells derived from Normal Bone Marrow (NBM) sample. Figure 8. Results comparing the SKBR3 and MCF-7 cell lines tumor depletion.

[0072] Figure 9. CD34+ cells were counted and the counts were normalized according to values from basal conditions. CD34+ cells, when incubated with sensitive tumor cells (SKBR3), presented a dosedependent depletion. When incubated with non-sensitive tumor cells (MCF-7), CD34+ cells kept stable in culture.

[0073] Figure 10. Cell depletion of SKBR3 tumor cells and of CD34+ cells.

[0074] Figure 11 . Cell depletion of MCF-7 tumor cells and of CD34+ cells.

[0075] Figure 12. Antibody drug conjugates were incubated for 72 hours with SKBR3 cells and healthy T cells. Antibody drug conjugates were incubated for 72 hours with MCF-7 cells and healthy T cells.

[0076] Figure 13. Antibody drug conjugates were incubated for 72 hours with SKBR3 cells and healthy B cells. Antibody drug conjugates were incubated for 72 hours with MCF-7 cells and healthy B cells.

[0077] DESCRIPTION OF EMBODIMENTS

[0078] Example 1. Materials and Methods

[0079] A. Samples:

[0080] • Target Cells: PKH67 labelled

[0081] - SKBR3 as Her2 positive cell line.

[0082] - MCF-7 as Her2 negative cell line.

[0083] • Hematotoxicity Evaluation: CD34+ cells population, T and B Cells from a healthy donor NBM.

[0084] B. Tumor cells membrane labelling:

[0085] - Once expanded the tumor cell lines, according to ATCCs instructions, detach cells from flasks.

[0086] - Count live cells and stain the cell membrane using PKH67 Green Fluorescent Cell Linker Midi Kit for General Cell Membrane Labeling (Sigma-Aldrich, ref. MIDI67-1 KT), following vendor’s instructions.

[0087] - Confirm tumor cells labelling and viability by flow cytometry.

[0088] - For tumor depletion evaluation, calculate 10 thousand PKH67-labelled tumor cells per condition (ADC candidate / dose), and reserved. - For tumor depletion evaluation combined with hematotoxicity assay, calculate 60 thousand labelled cells per condition (ADC candidate I dose) from each cell line and reserve in RPMI- 1640 medium, supplemented with 20% fetal bovine serum (FBS), and antibiotics (Pen / Strep 1x).

[0089] C. Normal Bone Marrow cells identification:

[0090] - NBM samples were received according to local ethical committee approval and patient consent. Only samples containing enough cells CD34 positive were considered for the assays.

[0091] - Check NBM cell populations and viability by flow-cytometry, using the antibodies panel described in Table 1.

[0092] - For tumor depletion evaluation combined with hematotoxicity assay, calculate approximately 1 thousand live CD34 positive cells per condition (ADC candidate I dose), plus other cell populations, and reserve in RPMI-1640 medium, supplemented with 20% fetal bovine serum (FBS), and antibiotics (Pen / Strep 1x).

[0093] Table 1

[0094] D. ADCs treatment plates:

[0095] Her2-ADC compounds are described in Table 2A. The compounds were diluted in PBS according to the desired concentrations, and dispensed into the treatment plates using a Labcyte Echo 550 acoustic dispenser (Labcyte, Inc. Sunnyvale, CA). The plates were sealed with a PlateLoc plate sealer (Agilent Technologies, Santa Clara, CA) and stored refrigerated until the moment of use. The concentrations of the compound used in the assay are described in Table 2B. The plates designs are described in Figure 2 and Figure 3.

[0096] Table 2A

[0097] Table 2B E. Cells mixing and plating:

[0098] - Plate PKH67-labelled tumor cells in control wells (non-treated wells).

[0099] - Plate 10 thousand PKH67-labelled tumor cells per well, in the 96 wells plates designed for tumor depletion assay (without hematotoxicity evaluation), containing different ADCs candidates and doses. - Mix labelled tumor cells with NBM-derived cells for tumor depletion assay combined with hematotoxicity assay, respecting desired target cells numbers: 60 thousand tumor cells plus 1 thousand CD34 positive cells (containing other NBM-derived cell populations) per condition.

[0100] - Plate the cells mix in the 96 wells plates designed for tumor depletion assay plus hematotoxicity evaluation, containing different ADCs candidates and doses.

[0101] Each condition (ADC candidate / dose) for tumor depletion assay plus hematotoxicity evaluation may be plated in a pool-well of 6 wells, to avoid excess of cell confluence. Later those 6 wells may be processed together during flow-based analysis. F. Baseline cells evaluation:

[0102] Immediately after plating cells, take 6 wells of each PHK67-labelled tumor cell line, and 6 wells from each mix of tumor cells + BMB cells for a flow-based baseline evaluation.

[0103] - Process those wells to detach tumor cells from the plates. Lysate red blood cells derived from NBM, and stain the cells, using the antibodies panel described in Table 3.

[0104] Incubate cells for 15 minutes, at room temperature, protected from light.

[0105] - Wash cells, centrifuge the plates and discard the supernatant.

[0106] - Resuspend cells in 150 pL of binding buffer and acquire cells in Attune Omnicyt Cytometer.

[0107] Table 3

[0108] G. Assay incubation:

[0109] Incubate the co-culture at 37°C, in 5% CO2, in humid atmosphere, for 72 hours.

[0110] H. Post-incubation assay processing:

[0111] - Centrifuge assay plates and discard supernatant

[0112] - Wash the wells with PBS, centrifuge the plate again, and discard supernatant.

[0113] - Add Trypsin 0.25% (30 pL / well) to detach adherent tumor cells, shake the plate and check cells under microscope.

[0114] Inactivate with 50 pL of medium containing 10% of FBS.

[0115] - Transfer the whole content of wells to a new conic bottom plate.

[0116] - Centrifuge the plate and discard the supernatant.

[0117] - Perform red blood cells lysis and wash cells with PBS.

[0118] - Centrifuge the plate and discard the supernatant, if red blood cells are still visible, repeat the lysis.

[0119] - Stain the cells with flow cytometer panel described in the Table 3.

[0120] Incubate cells for 15 minutes, at room temperature, protected from light. Wash cells, centrifuge the plates and discard the supernatant.

[0121] - Resuspend cells in 150 pL of binding buffer and acquire cells in Attune Omnicyt Cytometer.

[0122] Example 2. Results

[0123] Her2 expression was evaluated in cell lines at baseline using PE-conjugated anti-Her2 antibody. As expected, SKBR3 cell line presented higher Her2 expression compared with MCF-7 cell line. NBM cells were negative for Her2 expression (Figure 4).

[0124] Once the cells were incubated for 72 hours in the presence of ADC candidates, the cells were recovered from testing plates and stained with flow antibodies. The tumor live cells were counted in each experimental condition (ADC candidate and dose) and the values were normalized according to the basal counts (counts of live tumor cells in ineffective compound doses). The SKBR3 cell line, Her2 positive, presented dose-dependent cell depletion: VTX009-HER2-MDC = Normal = Stable > Control. The MCF-7 cell line, Her2 negative, did not present sensibility to ADCs (right): VTX009- HER2-MDC > Normal = Stable = Control (Figure 5).

[0125] The same analysis of live tumor cells was performed when SKBR3 cells were co-incubated with cells derived from NBM sample. The NBM co-incubation assay protects target tumor cells from ADC activity, except for Trastuzumab Control (red), that presented increased depletion activity: VTX009- HER2-MDC = Normal = Stable > Control. No CDC or ADCC effects observed. The lower activity could be explained by lower tumor cells proliferation rate in presence of NBM-derived cells, as the SKBR3 cell line, when plated “alone” in the wells increased 7.2-fold in 72 hours, but when coincubated with NBM increased 4.4-fold (Figure 6).

[0126] The same analysis of live tumor cells was performed when MCF-7 cells were co-incubated with cells derived from NBM sample. MCF-7 cell line did not present sensitivity to compounds when incubated alone or combined with NBM. No ADC-mediated activity, no free payload-med iated activity* and no CDC or ADCC-mediated activity were observed (Figure 7). *MCF-7 cell line proliferation rate was similar when incubated alone, or in presence of NBM.

[0127] Another way to represent tumor depletion results comparing the sensitive (SKBR3) and non-sensitive (MCF-7) cell lines tumor depletion is represented in Figure 8. MCF-7 cell line did not present sensitivity to compounds when incubated combined with NBM, except for VTX009-HER2-MDC, where a tendency of depletion activity was observed.

[0128] Regarding hematotoxicity evaluation, the CD34 positive cells were counted in each experimental condition (ADC candidate and dose), the counts were normalized according to values from basal conditions. CD34+ cells, when incubated with sensitive tumor cells (SKBR3), presented a dosedependent depletion: Normal > Stable > Control > VTX009-HER2-MDC. When incubated with nonsensitive tumor cells (MCF-7), CD34+ cells kept stable in culture (Figure 9). The ADCs depletion effects on SKBR3 tumor cells, and healthy CD34 positive cells were represented together in Figure 10. The four different ADC candidates were able to deplete sensitive tumor cells (Her2 positive SKBR3 cell line), and for 3 of them a marked off-target toxicity on CD34 positive cells was observed. One ADC candidate showed a very low toxicity for CD34 positive cells, demonstrating its higher selectivity.

[0129] The ADCs depletion effects on MCF-7 tumor cells, and healthy CD34 positive cells were represented together in Figure 11. None of four different ADC candidates were able to deplete non-sensitive tumor cells (Her2 negative MCF-7 cell line). When no tumor depletion was caused by ADCs, none (or very low) toxicity for CD34 positive cells was observed, demonstrating that the ADC payloads are not been released nonspecifically in the assay medium.

[0130] The ADCs toxic effects on healthy T lymphocytes were evaluated by counting T Cells in each experimental condition and normalizing the values according to values from basal conditions. T cells counts upon ADCs incubation (72h) were not affected, when NBM sample was incubated with sensitive and non-sensitive tumor cells (Figure 12). The same analysis was performed in B Cells, with similar results (Figure 13).

Claims

CLAIMS1 . An in vitro method to determine simultaneously anti-cancer activity and hematotoxicity of an antibody-drug conjugate, comprising:(a) mixing a sample comprising cancer cells with a bone marrow sample;(b) adding an antibody-drug conjugate to the mixture of step (a);(c) determining anti-cancer activity of the antibody-drug conjugate by measuring cell depletion of tumor cells; and(d) determining hematotoxicity of the antibody-drug conjugate by measuring cell depletion of cells of the bone marrow sample.

2. The method according to claim 1 , wherein the sample comprising cancer cells is from a solid tumor or from a hematological cancer.

3. The method according to claim 1 or 2, wherein the sample comprising cancer cells is from a subject having cancer.

4. The method according to claim 1 or 2, wherein the sample comprising cancer cells is from a cancer cell line.

5. The method according to any one of claims 1 to 4, wherein the bone marrow sample is a whole bone marrow sample.

6. The method according to any one of claims 1 to 5, wherein the bone marrow sample is from a healthy donor.

7. The method according to any one of claims 1 to 6, wherein the bone marrow sample comprises CD34+ progenitor cells, myeloid progenitor cells, erythroid progenitor cells, megakaryocytic progenitor cells, NK cells, and complement proteins.

8. The method according to any one of claims 1 to 7, wherein the bone marrow sample comprises NK cells, and complement proteins.

9. The method according to any one of claims 1 to 8, wherein the mixture formed in step (b) is incubated from 4 hours up to 7 days.

10. The method according to any one of claims 1 to 9, wherein the mixture formed in step (b) is incubated with 3D culture constructs mimicking the microenvironment architecture of solid tumors.

11. The method according to claim 10, wherein said 3D culture constructs are selected from the group consisting of: spheroids, organoids, extracellular matrix gels, synthetic scaffolds, rotary cell culture systems, culture plastics, tumor explants, and tumoroids.

12. The method according to any one of claims 1 to 11 , wherein the anti-cancer activity of the antibody-drug conjugate is determined by flow cytometry.

13. The method according to any one of claims 1 to 12, wherein the hematotoxicity of the antibodydrug conjugate is determined by flow cytometry.

14. The method according to any one of claims 1 to 13, wherein cell depletion of steps (c) or (d) is measured by a parameter selected from the group consisting of: number or percentage of cells killed relative to control samples without the antibody-drug conjugate, cell depletion at a single dose, and cell depletion at multiple doses.

15. The method according to any one of claims 1 to 14, further comprising determining an ex vivo therapeutic index.

16. The method according to any one of claims 1 to 15, wherein the antibody-drug conjugate comprises a drug linked to an antibody by a linker.

17. The method according to any one of claims 1 to 16, wherein the antibody-drug conjugate comprises a drug selected from the group consisting of: a cytotoxic compound, an antiproliferative compound, and a degrader.

Images