TECHNICAL FIELD OF THE INVENTION

The present invention relates to a combination of adoptively transferred cells (adoptive cells) and at least one anti-PDLl/PDl antibody, wherein the adoptive cells comprises natural killer (NK) lymphocytes and T lymphocytes obtained by adoptive cellular therapy. In particular, the present invention relates to a combination as described above for use in the treatment or alleviation of cancer in a patient.

BACKGROUND OF THE INVENTION

Targeting the immune checkpoint receptors programmed cell-death protein 1 (PD1) and programmed cell-death 1 ligand 1 (PDL1) has revolutionized cancer therapy and become the backbone of cancer immunotherapy. Yet, across cancers, the clinical benefit is limited to approximately 13% of patients. The underlying reason is likely multifactorial including both redundant and/or compensatory immunosuppressive mechanisms as well as insufficient endogenous immunity. Absent or low endogenous anti-tumor immunity can in some cases be circumvented by adoptive cell transfer (ACT) of ex v/ o-expanded tumor infiltrating lymphocytes (TILs). Although many TILs are bystanders, they are assumed to contain an enriched source of tumor reactive T cells that can recognize neoantigens or cancer/testis antigens (CTAs) presented on MHC molecules. The immense potential of TIL-based therapy has been illustrated in melanoma and certain other types of solid cancers, but for unknown reasons it remains challenging to obtain cancer reactive TIL cultures from non-melanoma cancers. Furthermore, as the presence of TILs is a prerequisite for TIL expansion, TIL therapy can only be offered patients having "hot" and resectable tumors.

A novel strategy for obtaining cancer reactive lymphocytes directly from peripheral blood, known as autologous lymphoid effector cells specific against tumor (ALECSAT), was recently developed (WO 2008/081035). In this procedure antigen-presenting CD4 ⁺ T cells are induced to express and present various CTAs by epigenetic therapy. These cells are subsequently leveraged to expand CTA-reactive lymphocytes. ALECSAT was recently evaluated in patients with late-stage or newly diagnosed glioblastoma. Encouragingly, ALECSAT was well tolerated and a subset of patients displayed signs of anti-cancer activity, although overall survival was not improved. Here, the present inventors demonstrate a synergistic anti-cancer activity of the combined action of anti-PDLl and adoptive cells, such as ALECSAT II, in translational models of triple negative breast cancer.

SUMMARY OF THE INVENTION

Thus, an aspect of the present invention relates to a combination comprising adoptive cells and at least one anti-PDLl/PDl antibody for use in adoptive cellular therapeutic treatment or alleviation of cancer in a patient, wherein the adoptive cells comprises natural killer (NK) lymphocytes and T lymphocytes.

Another aspect of the present invention relates to the combination for the use of the present invention, wherein the adoptive cells are obtained by a process comprising, typically in vitro, the following steps: a. Providing a blood sample and separating isolated peripheral blood mononuclear cells (PBMCs) into a fraction enriched for lymphocytes and a fraction enriched for monocytes; b. Culturing a portion of the monocyte-enriched fraction under conditions facilitating maturation of dendritic cells; c. Co-culturing the matured dendritic cells obtained in step b) with a first portion of the lymphocyte-enriched fraction obtained in step a); d. Isolating proliferating lymphocytes from co-cultured cells in step c) and inducing expression of cancer/testis antigens by contacting the lymphocytes with an agent; e. Co-culturing the cancer/testis antigen-expressing cells obtained in step d) with a second portion of the lymphocyte-enriched fraction from step a) to stimulate proliferation of CD8 ⁺ and natural killer (NK) lymphocytes.

Yet another aspect of the present invention relates to a pharmaceutical composition comprising adoptive cells and at least one anti-PDLl/PDl antibody of the present invention as well as one or more pharmaceutically acceptable adjuvants. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the characterization of ALECSAT II cells. A) Representative flow cytometry analysis demonstrating that ALECSAT II preparations is exclusively composed of NK (CD56 ⁺ CD3 ) and T cells (CD3 ⁺ ) (left panel). The T cells further subdivide into CD4 ⁺ (middle panel), CD8 ⁺ (middle panel), and y6 T cells (right panel). B) Comparison of NK and T cell ratios in ALECSAT II cells and PBMCs. Mean ± SD of 8 PBMC and 6 ALECSAT II preparations is shown. C) Comparison of CD86 expression (a marker of NK activation) in N KAH cells (NK cells from ALECSAT II) and circulating NK cells. Mean ± SD of 8 PBMC and 6 ALECSAT II preparations is shown. D) Representative flow cytometry analysis of NK (CD3 ) CD56 ^br ' ^9ht/dim expression on NKAH cells and peripheral NK cells. E) Comparison of CD56 ^br ' ^9ht and CD56 ^dim on N KAH cells and peripheral NK cells. Mean ± SD of 8 PBMC and 6 ALECSAT II preparations is shown. F) Representative flow cytometry analysis demonstrating CD16 positivity of NKAH cells (CD56 ⁺ ). G) Representative flow cytometry analysis of NKAH cells demonstrating high expression of CD62L and NKG2D but not CCR7. H) Flow cytometry analysis showing that ALECSAT II cells express CD45RO (*) but not CD45RA («). I-J) Flow cytometry analysis showing that the majority of CD8’ (I) and CD8 ⁺ (J) TAII cells (T cells from ALECSAT II) do not express CCR7 but express high levels of CD62L and subsets of TAII cells also express CD27. Statistical difference was determined by the unpaired t-test method. *0.05 > P > 0.01, **0.01 > P > 0.001, ***0.001 > P. SSC: Side scatter.

Fig. 2 shows the characterization of the functional capacity of NKAH and TAII cells to detect and kill cancer cells in vitro. A) Cancer cell viability analysis (luminescence) following 24-hour coculturing with ALECSAT II cells at the indicated ratios, demonstrates concentrationdependent MDA-MB-231 cell killing using different blood donors (n=6). Data are shown with error bars representing mean ± SEM of three biological replicates. B) Potency comparison (LD ₅ o) of ALECSAT II preparations produced from the same donors (donors A, B and C) at different time points. C) Same setup as in A, but using MDA-MB-468 cells (n=6). D) Comparison of different cancer cell models sensitivity towards ALECSAT Il-mediated killing using the same experimental setup as in A. The results are categorized into donor origin. The results demonstrate that the MDA-MB-231 model is relatively resistant to ALECSAT II- mediated killing. Data are shown with error bars representing mean ± SEM of three biological replicates. E) Degranulation analysis (CD107a positivity) of ALECSAT II cells upon 4-5 hours culture alone (top), with PHA (middle), or with MDA-MB-231 cells (bottom), demonstrating degranulation of both N KAH (left), CD8 ⁺ (middle) and CD8- TAII cells (right). A representative of five independent experiments is shown. F) Flow cytometry analysis demonstrating expression of EGFR (left) and CD73 (right) on MDA-MB-231 cells. G) Same setup as in A but with addition of an anti-EGFR antibody (10 pg/mL) («) demonstrating enhanced killing capacity compared with ALECSAT II cells (#). Data are shown with error bars representing mean ± SEM of three biological replicates. H) Degranulation analysis of ALECSAT II cells after 4 hours co-culture with MDA-MB-231 cells alone (left) or supplemented with an anti- EGFR antibody (10 pg/mL, middle panel) or an anti-CD73 antibody (10 pg/mL, right panel) as determined by CD107a expression measured by flow cytometry. A representative of three biological replicates is shown. I) Same setup as in A but using purified immune cell fractions i.e. TAH (#) and NKAH («) cells. The bar plot is a potency comparison. A representative of seven independent experiments is shown. J) Same setup as in A, however, using y6 T cell depleted fractions («) and ALECSAT II cells (#). A representative of two biological replicates is shown. K-L) Same setup as in A but comparing untreated and zoledronic acid-treated cancer cell (MDA-MB-231 (K) or MDA-MB-468 (L)) sensitivity towards y6 T cell enriched fractions. Mean ± SEM of triplicate measurements is shown. Representative of three biological replicates is shown. M) Same setup as in D comparing the sensitivity of cancer models to ALECSAT II mediated killing with or without zoledronic acid-treatment. A representative of two biological replicates performed in triplicates is shown. Mean ± SD is shown. Statistical difference was determined by the one-way ANOVA method following Bonferoni's multiple correction testing (D and G) or by the unpaired t-test (I and M). *0.05 > P > 0.01, **0.01 > P > 0.001, ***0.001 > P. ET: effector cell/target cell ratio, zol: zoledronic acid.

Fig. 3 shows the anti-cancer effect of ALECSAT II cells in vivo by intravenous (i.v.) administration of ALECSAT II cells or by injecting ALECSAT II cells into the mammary fat pad (MFP). A) Growth curves of tumors from orthotopically transplanted MDA-MB-231 cells in suspension (susp) in NOG mice with or without administration of ALECSAT II cells injected i.v. and in hIL15 NOG mice with ALECSAT II cells injected i.v.. Data are shown as mean tumor volume ± SEM. B) At endpoint MDA-MB-231 tumors were excised and tumor mass measured for the mice described in A. Mean ± SD is shown. C) Growth curves of tumors arising from orthotopically transplanted MDA-MB-231 tumor pieces in NOG mice with or without subsequent MFP administration of ALECSAT II cells. Tumor size is presented as mean ± SEM. D) MDA-MB-231 tumors as in B were excised and tumor mass measured. Mean ± SD is shown. E) Representative images from formalin fixed paraffin embedded sections from tumors (top panel), livers (middle panel), and spleens (bottom panel) from orthotopically transplanted MDA-MB-231 cells in NOG mice with or without (first panel from left) administration of ALECSAT II cells injected i.v. (second panel from left) or into the MFP (fourth panel from left) and in hIL15 NOG mice with ALECSAT II cells injected i.v. (third panel from left) stained for CD3. Scale bar 250 pm. F) Same setup as in E but stained for both CD3 and Ki67. Scale bar 25 pm. White arrows indicate double positive cells. G) Same setup as in E but stained for PDL1. Scale bar 250 pm. Statistical difference was determined by the two-way ANOVA method following Bonferoni's multiple correction testing (A and C) or the Mann-Whitney test (B and D). *0.05 > P > 0.01, **0.01 > P > 0.001.

Fig. 4 shows the therapeutic effect of combining ALECSAT II therapy with anti-PDLl blockade. A) Growth of orthotopically transplanted MDA-MB-231 tumors in NOG mice MFP injected with either ALECSAT II (#) or ALECSAT II in combination with intraperitoneal- administered anti-PDLl antibody (&) or left untreated («). Tumor size is presented as mean ± SEM. B) Excised MDA-MB-231 tumors from A with tumor masses presented as mean ± SD. C) Immunohistochemistry (IHC) analysis of excised tumors from A, demonstrating comparable infiltration of CD8 ⁺ TAII cells (left panel) and tumor PDL1 expression (middle panel), but an enhanced level of PD1 expression (right panel) in mice treated with ALECSAT II in combination with anti-PDLl antibody as compared to mice treated with only ALECSAT II. Scale bar 100 pm. D) Same setup as in A but using a second donor (Donor 2) for ALECSAT II preparation. The control groups not receiving ALECSAT II cells were treated with an anti- PDLl antibody (a-PDLl («), TA+a-PDLl (+)). Furthermore, groups with in vitro purified TAII cells were included and administered with (TA + a-PDLl (+)) or without (TA (>)) an anti- PDLl antibody. E) Excised tumors from D presenting tumor mass as mean ± SD. F) IHC analysis of tumors from D demonstrating similar extent of CD8 ⁺ TAII cell infiltration across all groups (except the control group without TAII cells). Scale bar 100 pm. G) Same setup as in A and D but with a third donor (Donor 3) for the ALECSAT II preparation. H) Image of excised tumors from G. Scale bar: 5 mm. I) Tumor (left) and spleen (right) mass excised from mice from G at end-point. Presented as mean ± SD. Asterisks indicate significant differences in Students t-test (A) or Mann-Whitney-Wilcoxon test (E, I, J). * 0.05 > P > 0.01, ** 0.01 > P > 0.001, NS: non-significant.

Fig. 5 shows the effect of ALECSAT II therapy and PDLl-blockade on the formation of spontaneous and experimental metastasis in the lungs and liver. Quantification of spontaneous lung A) and liver B) metastasis following MFP transplantation of MDA-MB-231 tumor pieces in NOG mice treated with ALECSAT II, ALECSAT II and an anti-PDLl antibody or left untreated. Data presented as mean ± SD (Primary tumor expansion for this animal experiment is shown in Fig 4G (Donor 3)) C-D) Similar quantification as for A-B but using tissues from animal experiment presented in Fig 4D-E (Donor 2). E) IHC analysis with markup of metastasis in lung sections from (C) stained for pan-cytokeratin. Scale bar 5 mm. Quantification of experimental lung F) and liver G) metastasis following i.v. injection of MDA- MB-231 in hIL15 NOG mice treated with ALECSAT II, ALECSAT II and an anti-PDLl antibody or left untreated. Data presented as mean ± SD. H) IHC analysis with markup of lung metastasis in lung sections from (F) stained for pan-cytokeratin. Scale bar 5 mm. I) IHC analysis of size matched liver metastasis, demonstrating extensive TAII cell infiltration in resistant tumors. CKAE is used as a tumor stain. Asterisks indicate significant differences in Mann-Whitney-Wilcoxon test. * 0.05 > P > 0.01, ** 0.01 > P > 0.001.

Fig. 6 shows that ALECSAT II cells exert anti-tumor activity in autologous PDX models. Female hIL15 mice were transplanted orthotopically with IB01002 PDX tumor pieces followed by i.v. administration of autologous ALECSAT II cells (#) or ALECSAT II in combination with anti-PDLl antibody (&) or treated with an anti-PDLl antibody («). Tumor growth (presented as mean ± SEM) A) as well as size B) and mass (presented as mean ± SD) C) were evaluated at endpoint, demonstrating anti-cancer activity of ALECSAT II. D) IHC analysis of tumors treated with ALECSAT II (top) or ALECSAT II in combination with anti-PDLl antibody (bottom), demonstrating CD8 ⁺ T cell infiltration (middle panel) but no increase in PDL1 (right panel) and Cytokeratin (left panel) expression. E) Tumor growth and F) development of IB01003 PDX tumors in NOG mice treated with either an anti-PDLl antibody («), ALECSAT II (#) or ALECSAT II in combination with an anti-PDLl antibody (&). G) IHC analysis of spleen and tumor sections from mice from E stained for CD3. These results demonstrate that ALECSAT II cells were detectable in both spleen and tumor as late as day 150 upon administration when co-treated with anti-PDLl but not when administered alone. Female hIL15 mice were transplanted orthotopically with IB01004 PDX tumor pieces followed by i.v. administration of autologous ALECSAT II cells (#) or ALECSAT II in combination with anti- PDLl antibody (&) or treated with an anti-PDLl antibody («). Tumor growth (presented as mean ± SEM) H) as well as mass (presented as mean ± SD) I) were evaluated at endpoint, demonstrating anti-cancer activity of ALECSAT II.

Fig. 7. Microscopy pictures of cultures.

Panels A-D: Control MDA-MB-231 cultures; panels E-H : MDA-MB-231 cultured in the presence of ALECSAT cells. Panels A and E: No addition; panels B and F: addition of anti-PDLl antibodies; panels C and G: addition of dendritic cells; panels D and H : addition of anti- PD-L1 antibodies and dendritic cells.

See Example 7 for details.

Fig. 8. Bar graphs showing values of Normalized Cell Index in cultures of MDA-MB-231 cells in the presence of ALECSAT cells and the effect of addition of anti-PD-Ll and/or dendritic cells.

A: Values recorded before addition of dendritic cells

B: Values recorded 72 hours after addition of dendritic cells.

See Example 7 for details. The present invention will now be described in more detail in the following.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

Adoptive cellular therapy

Adoptive cellular therapy, also known as cellular immunotherapy, is a personalized treatment strategy that involves the isolation of a patient's own immune cells followed by their ex vivo expansion and reinfusion. Sometimes the immune cells are genetically engineered via gene therapy to enhance their cancer-fighting capabilities, e.g. chimeric antigen receptor (CAR) T cells. The majority of adoptive cellular therapy strategies utilize T cells isolated from tumor or peripheral blood but may also utilize other immune cell subsets such as natural killer cells. The cells generated by adoptive cellular therapy are termed adoptive cells herein.

Cancer/testis antigen (CTA)

Cancer/testis antigens (CTAs) are a group of proteins and their expression is, in general, restricted to male germ cells. However, these antigens are often re-expressed in cancer and classified as tumor antigens. CTAs are expressed by a broad spectrum of cancers and includes antigens such as MAGE-A (including MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4), BAGE, GAGE-1, NY-ESO-1, PRAME, CT83 and SSX2.

Dendritic cell (DC)

Dendritic cells (DCs) are antigen-presenting cells of the mammalian immune system. Their main function is to process antigen material and present it on the cell surface to T cells. In this way, DCs act as messengers between the innate and the adaptive immune systems. The term "mature dendritic cell" are used to denote DCs, which, in contrast to immature DCs, have a high potential for T cell activation. In the present invention, the mature DCs are obtained by plating and culturing adhering monocytes, treating them with interleukin- (IL) 4 (and/or IL-13) and GM-CSF to differentiate the monocytes into immature dendritic cells and subsequently treating the immature DCs with TNF-o, IL- and prostaglandin E2. These mature DCs are not loaded with an antigen, as would be the base for mature DCs isolated from lymphoid tissue.

Natural killer (NK) cell

Natural killer (NK) cells are a type of cytotoxic lymphocyte that belong to the group of innate lymphoid cells. NK cells can detect and kill virus-infected cells and other intracellular pathogens as well as cancer cells. NK cells are unique compared to cytotoxic T cells in that they have the ability to detect and kill stressed cells in the absence of antibodies or MHC molecules.

Peripheral blood mononuclear cell (PBMC) denotes any peripheral blood cell having a round nucleus. PBMCs consist of lymphocytes (T cells, B cells, NK cells) and monocytes (can differentiate into macrophages and dendritic cells). Erythrocytes and platelets have no nuclei and granulocytes have multi-lobed nuclei, thus, these cells are not classified as PBMCs.

Programmed cell-death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein on the cell surface of T and B cells that has a role in regulating immune responses. Hence, PD1 is an immune checkpoint. PD1 has two ligands, PDL1 and PDL2, and if PD1 interacts with either one of its ligands, immune responses are negatively regulated by inducing apoptosis in antigen-specific T cells in lymph nodes or reducing apoptosis in regulatory T cells. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells since cancer cells can express PDL1 or PDL2. PD1 inhibitors (neutralizing antibodies targeting PD1) have been developed to unleash the immune system to attack tumors and thereby treating or alleviating certain types of cancer. Programmed cell-death 1 ligand 1 (PDL1)

Programmed cell-death 1 ligand 1 (PDL1) also known as cluster of differentiation 274 (CD274) and B7 homolog 1 (B7-H1) is a ligand for Programmed cell-death protein 1 (PD1). Neutralizing antibodies targeting PDL1 has been developed to treat cancers, wherein the tumor or immune cells express PDL1.

T lymphocytes

T lymphocytes or T cells (the terms are used interchangeably herein) play a central role in the adaptive immune response. T cells can be categorized into two main subtypes: CD4 ⁺ and CD8 ⁺ T cells. CD4 ⁺ T cells refer to T cells of the T helper subtype, which assist other lymphocytes e.g. the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are known as CD4 ⁺ T cells as they express the CD4 ⁺ glycoprotein on the cell surface and become activated when the CD4 molecule interacts with a peptide antigen presented by MHC class II molecules on antigen- presenting cells. CD8 ⁺ T cells, also known as cytotoxic T cells, are cytotoxic and destroy virus-infected and cancer cells. These cells are defined by their expression of the CD8 glycoprotein on their cell surface and recognize their targets by binding to peptides associated with MHC class I molecules present on the surface of all nucleated cells. Another subtype of T lymphocytes mentioned herein is the y6 T cells, which possess a y6 T cell receptor (TCR) rather than the op TCR present on the cell surface of the majority of other T cells. y6 T cells are not MHC-restricted and seem to be able to recognize whole proteins rather than reguiring peptides to be presented by MHC molecules on antigen-presenting cells.

The invention will now be described in further details in the following.

As mentioned above, the present inventors have surprisingly demonstrated a synergistic anticancer activity of the combined action of anti-PDLl and adoptive cells, such as ALECSAT II, in translational models of triple negative breast cancer.

Thus, one aspect of the present invention relates to a combination or composition comprising adoptive cells (adoptively transferred cells) and at least one anti-PDLl/PDl antibody for use in the treatment or alleviation of cancer in a patient, wherein the adoptive cells comprises natural killer (NK) lymphocytes and T lymphocytes obtained by adoptive cellular therapy. In one embodiment of the present invention, the adoptive cells further comprises NK T lymphocytes.

In another embodiment of the present invention, the adoptive cellular therapy comprises stimulation of proliferation of lymphocytes using dendritic cells and/or chemical treatment, optionally followed by induction of the proliferating cells to express tumor antigens.

In yet another embodiment of the present invention, the T lymphocytes is a mixture of T lymphocytes with different phenotypes including CD8 ⁺ T lymphocytes, CD4 ⁺ T lymphocytes, CD4 /CD8" T lymphocytes, NK T lymphocytes, and y6 T lymphocytes.

In one embodiment of the present invention, the adoptive cells are obtained by a process comprising the following, typically in vitro, steps: a. Providing a blood sample and separating isolated peripheral blood mononuclear cells (PBMCs) into a fraction enriched for lymphocytes and a fraction enriched for monocytes; b. Culturing a portion of the monocyte-enriched fraction under conditions facilitating maturation of dendritic cells; c. Co-culturing the matured dendritic cells obtained in step b) with a first portion of the lymphocyte-enriched fraction obtained in step a); d. Isolating proliferating lymphocytes from co-cultured cells in step c) and inducing expression of cancer/testis antigens by contacting the lymphocytes with an agent; e. Co-culturing the cancer/testis antigen-expressing cells obtained in step d) with a second portion of the lymphocyte-enriched fraction from step a) to stimulate proliferation of CD8 ⁺ and natural killer (NK) lymphocytes.

In another embodiment of the present invention, the adoptive cells are obtained by a process comprising the following, typically in vitro, steps: a. Providing a blood sample and isolating peripheral blood mononuclear cells (PBMCs); b. Culturing the PBMCs in medium containing interferon (IFN) -y; c. Culturing the PBMCs from step (b) in medium containing anti-CD3 antibody and interleukin (IL) -2; d. Transferring the cells from step (c) to fresh medium containing IL-2.

In yet another embodiment of the combination for the use of the present inventnion, the adoptive cells are obtained by a process comprising the following steps:

Bl) isolating a sample of blood cells from a subject, wherein the sample is enriched for lymphocytes; B2) culturing a fraction of the sample under conditions that stimulate proliferation of CD4 ⁺ lymphocytes and increase the CD4 ⁺ /CD8 ⁺ ratio compared to the lymphocytes obtained from step b-1; B3) contacting the proliferating T lymphocytes with an agent that induces expression of cancer/testis antigens followed by a period of culture that results in said expression of cancer/testis antigens; B4) separating the cancer/testis antigen expressing T lymphocytes from the agent capable of activating T lymphocytes followed by mixing the cancer/testis antigen expressing lymphocytes with a second fraction of the sample from step a; and B5) subsequently culturing the lymphocyte mixture from step b-4 to stimulate proliferation of CD8+ and NK lymphocytes, wherein step B5) comprises addition of an agent capable of activating T lymphocytes via binding to CD3 and/or CD28.

Also, in any one of the embodiments of the combination for the use of the invention disclosed herein, the adoptive cellular therapy further comprises administration of dendritic cells, preferably mature dendritic cells, and in particular mature autologous dendritic cells to the patient.

The preferred adoptive T-cell therapy of the present invention entails administration of ALECSAT cells. For details about the production and characteristics of these, reference is made to the section headed "Preparation of ALECSAT cells", where several protocols are presented. The most recent of these protocols include, instead of using dendritic cells, the addition of an agent capable of activating T lymphocytes via binding to CD3 and/or CD28 in the step of culturing lymphocytes prior to the step of inducing cancer/testis antigens and/or in the step of co-culture between cancer/testis antigen expressing T lymphocytes with lymphocytes. As disclosed in WO 2022/269019, the addition in these steps of CD3 and/or CD28 binders provides for improvements in term of production time and/or in terms of the number of generated cells. The agent capable of activating T lymphocytes via binding to CD3 and/or CD28 preferably comprises antibodies, antibody fragments or antibody analogues which bind either CD3 or CD28, and in a preferred embodiment, the agent capable of activating T lymphocytes via binding to CD3 and/or CD28 comprises antibodies, antibody fragments or antibody analogues which bind CD3 and comprises antibodies, antibody fragments or antibody analogues which bind CD28. Such antibodies, antibody fragments or antibody analogues are typically linked to a solid or semi-solid phase, or to a polymer, such as dextran. A particularly important embodiment entails that the solid or semi-solid phase is constituted by separable beads, such as paramagnetic or superparamagnetic beads.

Anti-CD3 and CD28 monoclonal antibodies are available, both for therapeutic applications and for in vitro application: Examples of therapeutic grade antibodies in this groupare muromonab-CD3, otelixizumab, teplizumab and visilizumab, all anti-CD3, and therelizumab (anti-CD28). Also, "cocktails" of murine anti-CD3 and anti-CD28 antibodies are available as well as bispecific anti-CD3/anti-CD28 antibodies.

The adoptive cellular therapeutic treatment disclosed herein entails administration of an immune checkpoint inhibitor drug (also termed simply a checkpoint inhibitor). Particularly preferred checkpoint inhibitor drugs in this context are inhibitors of PD-1 and/or PD-L1, cf. below.

Hence, an effective amount of a PD-1 inhibitor or a PD-L1 inhibitor is administered. The cotreatment man take place prior to and/or concurrent with and/or after the treatment with the cells administered.

The PD-1 and PD-L1 inhibitors can in this interesting embodiment be any of the following approved or experimental inhibitors:

Approved PD-1 inhibitors

Pembrolizumab (formerly MK-3475 or lambrolizumab, Keytruda), approved for the treatment of melanoma, metastatic non-small cell lung cancer and head and neck squamous cell carcinoma.

Nivolumab (Opdivo), approved for the treatment of melanoma, squamous cell lung cancer, renal cell carcinoma, and Hodgkin's lymphoma. Cemiplimab (Libtayo), approved for the treatment of cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation.

Dostarlimab (Jemperli), approved for treatment of mismatch repair deficient (dMMR) recurrent or advanced endometrial cancer by the FDA in April of 2021. [13] On August 17, 2021, the FDA granted accelerated approval for the treatment of mismatch repair deficient (dMMR) recurrent or advanced solid tumours.

PD-1 inhibitors currently being investigated

JTX-4014 by Jounce Therapeutics, which is currently undergoing a Phase I clinical trial;

Spartalizumab (PDR001) developed to treat both solid tumours and lymphomas;

Camrelizumab (SHR1210), which has been conditionally approved for treatment of Hodgkin's lymphoma.

Sintilimab (IBI308) developed to treat non-small cell lung cancer (NSCLC).

Tislelizumab (BGB-A317), which is developed for treatment of solid tumors and hematologic cancers.

Toripalimab (JS 001).

INCMGA00012 (MGA012).

AMP-224.

AMP-514 (MEDI0680).

Approved PD-L1 inhibitors

Atezolizumab (Tecentriq), which is approved for treatment of urothelial carcinoma and non- small cell lung cancer.

Avelumab (Bavencio), which is approved for the treatment of metastatic merkel-cell carcinoma

Durvalumab (Imfinzi), which is approved for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer after chemoradiation.

PD-L1 inhibitors currently being investigated

KN035; CK-301 for treatment of NSCLC;

AUNP12 (a 29-mer peptide);

CA-170 for treatment of mesothelioma patients; and

BMS-986189.

In preferred embodiments, an anti-PDLl antibody is administered. The anti-PDLl antibody preferably has a heavy chain according to SEQ ID NO: 1 or a sequence having at least 80% to SEQ ID NO: 1 and a light chain according to SEQ ID NO:2 or a sequence having at least 80% to SEQ ID NO:2 (Atezolizumab), a heavy chain according to SEQ ID NO:3 or a sequence having at least 80% to SEQ ID NO:3 and a light chain according to SEQ ID NO:4 or a sequence having at least 80% to SEQ ID NO:4 (Avelumab) or a heavy chain according to SEQ ID NO: 5 or a sequence having at least 80% to SEQ ID NO: 5 and a light chain according to SEQ ID NO:6 or a sequence having at least 80% to SEQ ID NO:7 (Durvalumab).

More preferably, 9, the anti-PDLl antibody has a heavy chain protein sequence according to SEQ ID NO: 1 or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 94% sequence identity to SEQ ID NO: 1 and wherein the anti-PDLl antibody has the light chain protein sequence according to SEQ ID NO:2 or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 94% sequence identity to SEQ ID NO:2.

In another preferred embodiment, the adoptive cellular therapy is combined with the administration to the patient of at least one anti-PDl antibody. Preferably, the anti-PDl antibody has a heavy chain according to SEQ ID NO:7 or a sequence having at least 80% to SEQ ID NO:7 and a light chain according to SEQ ID NO:8 or a sequence having at least 80% to SEQ ID NO:8 (Nivolumab), a heavy chain according to SEQ ID NO:9 or a sequence having at least 80% to SEQ ID NO:9 and a light chain according to SEQ ID NO: 10 or a sequence having at least 80% to SEQ ID NO: 10 (Pembrolizumab), a heavy chain according to SEQ ID NO: 11 or a sequence having at least 80% to SEQ ID NO: 11 and a light chain according to SEQ ID NO: 12 or a sequence having at least 80% to SEQ ID NO: 12 (Cemiplimab) or a heavy chain according to SEQ ID NO: 13 or a sequence having at least 80% to SEQ ID NO: 13 and a light chain according to SEQ ID NO: 14 or a sequence having at least 80% to SEQ ID NO: 14 (Dostarlimab).

All of the above-mentioned sequence identities can be higher than the at least 80% indicated : at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, and at least 95% are preferred sequence identities for each of SEQ ID NO: 1-14 - and in all cases amino acid residues that substitute an amino acid residue in any of SEQ ID Nos: 1-14 will preferably be a proteogenic amino acid residues, i.e. one of the 22 amino acid that are encoded by DNA in living organisms.

In a further embodiment of the present invention, the cancer is selected from the list consisting of carcinoma, adenocarcinoma, sarcoma (including liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma), glioma (in particular glioblastoma), neuroblastoma, medullablastoma, malignant melanoma, neurofibrosarcoma, choriocarcinoma, myeloma, and leukemia. Examples of cancers are hence lung cancer, colon cancer, gastric cancer, esophagus cancer, pancreatic cancer, liver cancer, head and neck cancer, ovarian cancer, gynecological cancer, prostate cancer, urological cancer, kidney cancer, thyroid cancer, brain cancer, basal cell carcinoma, squamous cell carcinoma, and blood cancer, preferably prostate cancer or breast cancer, most preferably triple-negative breast cancer.

In an additional embodiment of the present invention, the combination is administered to a subject by parenteral administration, such as intravenous, intraarterial, intratumoral or intralymphatic administration, preferably intravenous, intraarterial or intratumoral administration, more preferably intravenous or intratumoral administration, most preferably intravenous administration. The combination of adoptive cells and at least one anti- PDL1/PD1 antibody may be administered concomitantly or separately by administering the adaptive cells followed by administration of at least one anti-PDLl/PDl antibody or vice versa by administering at least one anti-PDLl/PDl antibody followed by administration of the adaptive cells.

In yet another embodiment of the present invention, the patient receives at least 2, at least 3, or at least 4 administrations.

A second aspect of the present invention relates to a pharmaceutical composition comprising adoptive cells and at least one anti-PDLl/PDl antibody according to the present invention as well as one or more pharmaceutically acceptable adjuvants.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. The invention will now be described in further details in the following non-limiting examples.

PREAMBLE TO THE EXAMPLES

Materials and methods

Cancer cell culture

Human MDA-MB-231 (triple-negative breast cancer (TNBC)), MDA-MB-468 (TNBC), and M4A4-LM3-2 (LM3, melanoma) were obtained from American Type Culture collection (ATCC) and grown in Dulbecco's modified Eagle medium (DMEM) AQmedia (Sigma-Aldrich, D0819), supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (P/S). T47D and Daudi cells were gift from Anne Lykkesfeldt and Maria Ormhoj, respectively. Daudi cells were grown in RPMI

Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10 % FBS, 1% P/S. T47D cells were grown in similar media supplemented with 6 ng/mL insulin.

Flow cytometry

Cells were blocked by human Fc block (BD, 564220) and stained with Pacific blue CD3 (SK7, Biolegend), FITC CD4 (SK3, Biolegend), PE CD8 (SKI, Biolegend), Alexa Fluor 647 CD27 (M- T271, Biolegend), Alexa Fluor 647 CD28 (CD28.2, Biolegend), FITC CD56 (TULY56, eBioscience), Alexa Fluor 647 CD56 (NCAM, Biolegend), Alexa Fluor 647 CD62L (DREG-56, Biolegend), Alexa Fluor 647 CD86 (IT2.2, Biolegend), FITC TCR y6 (Bl, BD), Alexa Fluor 647 NKG2D (1D11, Biolegend), FITC CD107a (H4A3, Biolegend), Alexa Fluor 647 CD107a (H4A3, Biolegend), Alexa Fluor 647 PD1 (MIH4, BD), Alexa Fluor 647 PDL1 (2340D, R&D systems), Alexa Fluor 647 CD73 (AD2, Abeam), PE CD16 (3G8, BD). Cells incubated with anti-EGFR (Cetuximab, Merck) were subsequently stained by an Alexa Fluor 488-labeled goat antihuman IgG (Life Technologies, A11013). Damaged cells were excluded by TO-PRO-3 staining (ThermoFischer, T3605) or LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFischer, L10119).

Preparation of ALECSAT cells

The present invention is based in part on the previously developed a method of adoptive immunotherapy of cancer, which relies on generation of cytotoxic lymphocytes specifically targeting a broad spectrum of cancer-testis antigens, which constitutes a group of shared tumour antigens appearing in tumour cells as a result of genome wide DNA de-methylation (Kirkin et al., 2018; WO 2008/081035). This existing procedure consists of four steps:

1) Generation of monocyte-derived mature dendritic cells;

2) Induction of proliferation of predominantly CD4+ cells after co-culture of the mature dendritic cells and peripheral blood lymphocytes (PBLs);

3) Treatment of the activated CD4-enriched lymphocytes with a DNA de-methylating agent leading to induction of expression of a broad spectrum of cancer-testis antigens (CTA); and

4) Generation of cytotoxic lymphocytes (via an "immunization/expansion step") after coculture of DNA de-methylated activated CD4 ⁺ enriched lymphocytes with unstimulated PBLs.

This method is universal and can be applied to treatment of many types of malignancies. In the present application, this process is generally termed the ALECSAT (Autologous Lymphoid Effector Cells Specific Against Tumor cells) process, more specifically the "ALECSAT-1" or "ALECSAT I" process.

A later developed improvement of the method originally disclosed in WO 2008/081035 is disclosed in WO 2020/208054; here a portion of the mature dendritic cells are used as feeder cells in the co-culture step (step 4), thus at at a very early stage during the co-culture of DNA de-methylated activated CD4 ⁺ enriched lymphocytes with unstimulated PBLs- This leads to a pronounced acceleration of the process in general and to an increase in the final number of effector cells. This improved process is termed "ALECSAT-2" or "ALECSAT II" herein.

ALECSAT I and II cells can thus be prepared according to protocols, which are described in detail in WO 2008/081035 and WO 2020/208054; in the latter, the protocol for ALECSAT I cell production is provided in Example 1 as the "standard protocol", and the protocol for ALECSAT II cell production is provided in Example 2 as the protocol where dendritic cells are added during the immunization step.

Both ALECSAT I and II cells require the addition of dendritic cells in the early stages of above-described step 2. However, recently an alternative ALECSAT process (ALECSAT III) was disclosed in WO 2022/269019; here, the production time is reduced because no mature dendritic cells need to be prepared prior to culture of the PBLs. Instead the PBLs are cultured in the presence of an agent (typically a bead-coupled antibody) that binds CD3 and/or CD28. Later in the process, in the immunization/expansion step, the same type of agent is also present.

Finally, a different version of the ALECSAT I and II protocols (both requiring dendritic cells in step 2) is also provided in WO 2022/259019; here, the immunization/expansion step in each protocol is carried out by addition of an agent (typically a bead-coupled antibody) that binds CD3 and/or CD28, i.e. as is also done in the ALECSAT III process. These versions of the ALECSAT I and II processes are termed ALECSAT 1.2 and 2.2 herein. of mature dendritic cells

If relevant for preparing ALECSAT cells using the ALECSAT I or II processes, such dendritic cells can be prepared as described in Example 1 in WO 2022/259019, cf. the process described prior to day 6;

Dav 0

Buffy coats (about 60 ml) are diluted with 60 ml of Ca and Mg free Dulbecco's Phosphate Buffered Saline (DPBS, Product No. BE17-512F, Cambrex, Belgium), and approximately 30 ml are layered on 15 ml of Lymphoprep® (Product No. 1053980, AXIS-SHIELD PoC AS, Norway) in four 50 ml tubes. After the first centrifugation at 200 G, 20 min, 20°C, 15-20 ml of the upper layer of plasma (so-called platelet rich plasma, PRP) are collected to a separate tube, and used for the preparation of serum. For this, CaCI ₂ is added to a concentration of 25 mM, and after mixing, the plasma is transferred to a T225 flask (Nunc, Denmark), and placed in a CO ₂ -incubator. The flask is left in the CO ₂ -incubator until the next day. Centrifugation of tubes with Lymphoprep® is continued at 460 G, 20 min, 20°C. After termination of centrifugation, mononuclear cells are collected from the interface between Lymphoprep® and plasma to tubes with 25 ml of cold DPBS-EDTA (Cambrex) and washed three times with cold DPBS-EDTA by centrifugation, first at 300 G, then two times at 250 G, each time for 12 min at 4°C. After the last wash, cells are re-suspended in 30 ml of cold Ca and Mg free DPBS, and counted using a Moxi counter.

The concentration of monocytes is determined by gating the corresponding peaks of cells. Generation of dendritic cells (DCs) is performed in T225 tissue culture flasks pretreated with 30 ml of 5% human AB serum in RPMI 1640. After removal of pretreatment medium, 30 ml of a cell suspension containing 4-5x l0 ⁷ monocytes in AIM-V medium are added. After 30 min of incubation at 37°C, non-adherent lymphocytes are collected, adherent monocytes rinsed twice with pre-warmed RPMI 1640 medium and further cultured in 30 ml of AIM-V medium. The collected lymphocytes are frozen in several aliquots of 25-30x 10® cells.

Dav 1

GM-CSF and IL-4 (both from Gentaur, Belgium, or CellGenix, Germany) are added to the flask with monocytes to final concentrations of 100 ng/ml and 25 ng/ml, respectively.

The T225 flask with the clotted plasma is transferred to a refrigerator and placed in an inclined position, with the clotted plasma down, and after 15-30 minutes, serum transferred to a 50 ml tube, and transferred to a -20°C freezer.

Dav 2

A tube with the frozen serum is transferred to the refrigerator (4°C).

Dav 3

GM-CSF and IL-4 (both from Gentaur, Belgium, or CellGenix, Germany) are added to the flask with monocytes to final concentrations of 100 ng/ml and 25 ng/ml, respectively.

Tubes with the thawed serum are centrifuged at 2000 G, 15 min, 20°C, and the supernatant is transferred to a new 50 ml tube. This serum (termed "later plasma- derived serum") is stored at 4°C. Dav 4

IL-ip, IL-6, TNF-a (all from Gentaur), and PGE2 (Sigma) are added to final concentrations of 10 ng/ml, 1000 lU/ml, 10 ng/ml and 0.2 pg/ml, respectively, in 10 ml of AIM-V medium.

Dav 6 - start co-culture of dendritic cells and lymphocytes.

Non-adherent dendritic cells are harvested, counted and stored for further use.

Isolation of immune cells

T and NK cells were purified using the Dynabeads Untouched Human T cells (Invitrogen, 11344D) and the EasySep Human CD56 Positive Selection Kit II (Stem cell, 17855), according to manufacturer's instructions, respectively. y6 T cells were depleted and enriched using the Anti-TCRy/6 MicroBead Kit human (Miltenyi Biotec, 130-050-701) according to the manufacturer's instructions.

Co-culture studies

Cancer cells (5xl0 ³ ) were suspended in AIM V media (Gibco, 12055-083) supplemented with 2% human serum and seeded in a white 96-well plate and allowed to attach for 2 h. Immune effector cells were subsequently added to the wells and incubated at 37°C for 24 h. After incubation, cancer viability was assessed by addition of D-luciferin (3 mg/ml in PBS) and luminescence was immediately measured using a Victor3 Multilabel Plate Reader. In some assays cancer cells were grown for 48 hours with 10 pM zoledronic acid (Fresenius Kabi) before the addition of effector cells. Cancer cell viability was calculated as: Viability = (sample - background)/(Cancer cells only - background)xl00%

Deqranulation assays

Cancer cells (lxlO ⁵ ) were suspended in growth media (AIM V media supplemented with 5% human serum). Effector cells (3xl0 ⁵ ) were subsequently added together with growth media supplemented with 2 pg/mL Phytohemagglutinin-L (Merck, 11249738001), GolgiStop (BD, 554724), or growth media supplemented with GolgiStop and anti-CD107a. After 5-hour incubation, cells were harvested, stained and analyzed by flow cytometry. Immunohistochemistry (IHC)

Tissue sections from the formalin-fixed and paraffin embedded tissue were cut, deparaffinized, and rehydrated prior to antigen retrieval by boiling in either Tris EGTA buffer (10 mM Tris and 0.5 mM EGTA, pH 9) for 15 min (for programmed death ligand 1 (PDL1 staining)), or in Cell Conditioning 1 buffer (Ventana Medical Systems, Oro Valley, AZ, USA) for 32 min (for CD3 and CD8 stainings), for 48 min (ki67 and CD3 double stainings) or incubated with Protease 3 (Ventana Medical systems) at 36°C for 4 min followed by 32 min of Cell Conditioning 1 buffer at 95°C (for pan-cytokeratin and PD1 stainings). Sections were incubated with anti-CD3 (2GV6; Ventana Medical systems) for 8 min at 36°C, anti-CD8 (1 : 100, M7103; DAKO, Glostrup, Denmark) for 32 min at 36°C, anti-PDLl (1 :500, EPR19759; Abeam pic., Cambridge, UK) for 1 hour at room temperature, anti-PDl (Abeam, AB52587) for 16 min at 36°C, or anti-pancytokeratin (1 :30, KL1; AbD Serotec, Hercules, CA, USA) for 1 hour at room temperature. Double stainings were initially with anti-Ki67 (Roche, 790-4286) at 36°C for 12 min, and subsequently stained with anti-CD3 (Roche, 790-4341) at 36°C for 12 min. Primary antibody binding was detected with either OptiView DAB IHC detection kit (760-700; Ventana Medical systems; CD3, CD8, pan-cytokeratin) or Envision FLEX DAB (DAKO; PDL1) as chromogen. Double stainings were detected by OptiView DAB IHC detection kit and ultraView Universal Alkaline phosphatase Red Detection Kit (760-501, Ventanam Medical systems). All sections were counterstained with hematoxylin. Hematoxylin and eosin staining was performed by routine stainings. Slides were scanned using a NANOZOOMER 2.0- HT Whole Slide Imager (Hamamatsu, San Diego, CA, USA).

In vivo experiments

All animal experiments were performed at the animal core facility at the University of Southern Denmark. Mice were housed under pathogen-free conditions with ad libitum food and water. The lig ht/dark cycle was 12 h lig ht/dark, with lights turned on from 6 a.m. to 6 p.m. Housing temperature was 21 ± 1°C and relative humidity 40-60%. Sample size was guided by previous experience and pre-liminary data. No animals were excluded from analysis. No randomization was performed as treatment was given before tumor size could be reliably determined. Investigators performing the experiments were not blinded. Mice were acclimatized for 2 weeks before initiation of experiments. Generation of patient derived xenograft (POX') models

Female NOG (NOD.Cg-PrkdcSCIDII2rgtmlSug/JicTac, Taconic) mice were anesthetized, and the fourth mammary fat pad was surgically exposed and injected with 50 pL extracellular matrix (ECM) gel (Merck, E1270-5). The mammary fat pad was subsequently opened, and a tumor piece (approximately 8 mm3) were implanted in the ECM. The mammary fat pad and skin was subsequently closed by internal and outer stitches, respectively.

Comparison between injection routes

Female NOG (n = 5) or hIL15 NOG (NOD.Cg-Prkdcscid II2rgtmlSug Tg(CMV-IL2/IL15)l- lJic/JicTac, Taconic) (n = 6) were injected with 1x106 MDA-MB-231 cells into the fourth mammary fatpad. Three days later 5x106 ALECSAT II cells were injected intravenously (i.v.). In parallel, separate mice were surgically transplanted with MDA-MB-231 tumor pieces into the mammary fat pat using extracellular matrix (ECM) gel (Merck, E1270-5) with (n = 3) or without (n=3) ALECSAT II cells.

Primary tumor growth, spontaneous metastasis and survival

Female NOG mice were transplanted with MDA-MB-231 tumor pieces into ECM gel with or without 5x106 ALECSAT II cells. Anti-PDLl (200 pg Atezolizumab) was administered intraperitoneally on day 0, 3 and weekly until termination. Mice were sacrificed as tumors reached 1.2 cm in diameter. For survival analysis, animals were considered dead as tumors reached a volume of 200 mm3.

Experimental metastasis

Female hIL15 NOG mice were injected i.v. with 1x106 MDA-MB-231 cells. Seven days later mice were injected with 5x106 ALECSAT II cells. Anti-PDLl (200 pg Atezolizumab) was administered intraperitoneally on day 1 and 3 followed by weekly injections until termination.

Quantification of metastasis

The NDP.view 2.3.14 software (Hamamatsu) annotation tool was used to markup full section and tumor area in lung and liver, respectively. Metastatic load was calculated as tumor area I tissue area xl00%. EXAMPLE 1

Aim of study

Because most studies suggest a dose-dependent effect of ACT cancer therapy, the inventors aimed at optimizing the expansion protocol for generation of ALECSAT cells. The aim of the present example was therefore to characterize these output cells (ALECSAT II) using flow cytometry.

Results

ALECSAT II cells constitute a mixture of NK cells, CD8 ⁺ T cells, CD4 ⁺ T cells and y6 T cells (Fig 1A). While cell numbers were greatly enhanced by the expansion protocol, the ratio of NK cells and T cells remained stable, albeit with a slight increase in the CD8 ⁺ /CD4 ⁺ T cell ratio (P<0.05) (Fig IB). To get a better appreciation of the individual cell types, the ALECSAT II cells were analyzed by flow cytometry. Compared to circulating NK cells, ALECSAT II NK (N KAH) cells expressed high level of CD86 - a marker of NK activation (Fig 1C). NK cells are conventionally subdivided into cytotoxic (CD56 ^dim CD16 ⁺ ) and regulatory (CD56 ^br ' ^9ht CD16 ). To our surprise N KAH cells were predominantly CD56 ^br ' ^9ht CD16 ⁺ (Fig 1D-F). Furthermore, whereas CD56 ^br ' ^9ht NK cells usually express CCR7, CD62L and NKG2D, NKAH cells express CD62L, NKG2D, but not CCR7 (Fig 1G), suggesting that these cells represent an intermediary previously undescribed phenotype.

The anti-cancer efficacy of ACT is believed to partly depend on low level of differentiation of T cells as well as expression of the co-stimulatory receptor CD27. The inventors therefore stained cells for CD45RA, CD45RO, CCR7, CD62L and CD27. Almost all ALECSAT II cells were positive for CD45RO and negative for CD45RA, indicating that they become activated during culture (Fig 1H). To our surprise the majority of both CD8 ⁺ and CD8" ALECSAT II T (TAII) cells were negative for CCR7, positive for CD62L, and partly positive for CD27 (Fig 1I-J). This expression pattern suggests that not only NKAH cells, but also TAII cells acquire a unique phenotype during culture. Conclusion

The ALECSAT II cells constitute a mixture of NK cells, CD8 ⁺ T cells, CD4 ⁺ T cells and y5 T cells. The characterization of these cells revealed that both NKAH and TAII acquire a unique phenotype during culture.

EXAMPLE 2

NKAH and TAII cells detect and eradicate cancer cells

Aim of study

The aim of this study was to characterize the functional capacity of NKAH and TAII cells to detect and kill cancer cells.

Results

ALECSAT II preparations from six healthy, HLA class I A2 ⁺ donors consistently displayed cancer cell kill in a concentration dependent manner of A2 ⁺ cancer cell lines (Fig 2A). To investigate whether the potency of the ALECSAT preparations was donor-dependent, the inventors generated multiple preparations from an additional set of donors and found that the potency variation was both inter- and intra-donor-dependent (Fig 2B). Killing of the HLA class I A2" MDA-MB-468 cells occurred in a concentration-dependent manner (Fig 2C). This was also observed in additional three HLA class I A2- cancer models of different origin (T47D, Daudi, LM3), suggesting a non-T cell mediated kill mechanism. Despite being the only cancer model having the potential to be recognized and destroyed by both T and NK cells, MDA-MB- 231 were significantly more resistant to ALECSAT Il-mediated killing compared to the other cancer cell models (Fig 2D, P < 0.001). To identify the immune cell type responsible for the killing, the inventors performed degranulation assays and found, as expected, that a substantial part of NKAH cells, but also TAII cells from both the CD8+ and CD8- compartments degranulated upon co-culturing with cancer cells as determined by CD107a membrane positivity (Fig 2E). As the majority of NKAH cells express CD16 (Fig IF), the inventors proceeded by evaluating whether the activity of NKAH cells could be increased by addition of a cancer targeting antibody. To that end, the inventors exploited that MDA-MB-231 cells express both EGFR and CD73 (Fig 2F). As expected, addition of the anti-EGFR antibody to the co-culture significantly enhanced the kill potency of ALECSAT II cells (Fig 2G). In agreement, CD107a assays showed significantly enhanced degranulation of N KAH cells (Fig 2H). To further confirm that the killing was mediated by both NKAH and TAII cells, the inventors separated each cell type, and found that both fractions were able to mediate cancer cell killing, although N KAH cells were significantly more effective in this assay (Fig 21, P < 0.05). As a subset of TAII cells are of the y6 T cell lineage, the inventors evaluated whether depleting these cells would alter the overall potency of ALECSAT II preparations and found that it did not (Fig 2J). Nevertheless, ALECSAT II y6 T cells were functional as MDA-MB-231 and MDA-MB-468 cells, pre-treated with zoledronic acid and was detected and killed in a concentration dependent manner by y6 T cells isolated from ALECSAT II preparations (Fig 2K-L). As y6 T cells accounts for a minor part of TAII cells, the inventors evaluated whether their killing-contribution would be detectable in complete cultures. Indeed, the potency of ALECSAT II towards MDA-MB-231, MDA-MB-468 and T47D was significantly increased (Fig 2M, P < 0.001).

Conclusion

Taken together, these data demonstrate that both NKAH and TAII cells can detect and eradicate cancer cells.

EXAMPLE 3

The anti-cancer activity of ALECSAT II cells in vivo

Aim of study

The aim of the present study was to investigate the anti-cancer effect of ALECSAT II cells in vivo by intravenous (i.v.) administration of ALECSAT II cells or by injecting ALECSAT II cells into the mammary fat pad (MFP).

Results

Secondary lymphoid organs, common-gamma-chain (y _c ) cytokine support (IL2, IL7, IL15, IL17 and IL21), and in particular IL15, support the activity of ACT therapy in preclinical models. Since secondary lymphoid organs are greatly compromised in NOG mice, which allow xenografting of human cancer and immune cells, the inventors initially investigated whether ALECSAT II cells would elicit anti-cancer responses when injected into tumor challenged NOG and hIL15 NOG mice. The inventors found significant inhibition of tumor growth of ALECSAT II cells in hIL15 NOG mice but not in NOG mice (Fig 3A-B, P < 0.05). The inventors hypothesized that the lack of response was due to delayed activation of ALECSAT II cells when administered intravenous (i.v.). Indeed, transplantation of a tumor piece into the mammary fat pad (MFP) and subsequent injection of ALECSAT II cells into the MFP resulted in modest but significant anti-tumor activity (Fig 3C P<0.05). Immunohistochemistry (IHC) confirmed enhanced level of TAII cells in tumors of hIL15 NOG mice and NOG mice when injected into the MFP as compared to NOG mice when administered i.v. (Fig 3E). Although injection directly into the MFP were required for survival of ALECSAT II cells in the absence of IL15, TAII cells were subsequently able to leave and survive outside the MFP, as TAII cells were detectable in the spleen and liver (Fig 3E). Importantly, TAII cells did not only survive but was found to proliferate in both tumor, liver, and spleen (Fig 3F). MDA-MB-231 tumors constitutively express PDL1, but the expression level was increased in ALECSAT II treated tumors (Fig 3G), suggesting that ALECSAT II cells stimulate enhanced PDL1 expression - a hallmark of adaptive resistance.

Conclusion

Taken together, these data demonstrate that ALECSAT II cells survive and expand in vivo resulting in anti-cancer activity. In addition, the administration of ALECSAT II cells stimulate enhanced PDL1 expression in tumor tissue.

EXAMPLE 4

Combined ALECSAT and anti-PDLl therapy inhibits triple-negative breast cancer (TNBC) growth

Aim of study

The enhanced cancer expression of PDL1 in ALECSAT II treated tumors (Fig 3G) prompted the inventors to investigate whether anti-PDLl therapy would support the therapeutic activity of ALECSAT II. Results

MDA-MB-231 tumor pieces were embedded with or without ALECSAT II (from an HLA-A2 ⁺ donor, donor 1) into the MFP of NOG mice and treated weekly with anti-PDLl starting on day 0. As expected, a modest growth reduction was observed following ALECSAT II monotherapy, but the anti-cancer activity was greatly enhanced when combined with anti-PDLl therapy (Fig 4A-B, P < 0.001). Despite the therapeutic benefit, there was no difference between CD8 ⁺ TAII cell infiltration or tumor PDL1 expression, whereas PD1 expression was greatly enhanced in mice receiving combination therapy (Fig 4C). Next, the inventors designed an experiment to 1) rule out that the observed tumor growth inhibition was caused by anti-PDLl alone, and 2) demonstrate that the effect was caused by TAII cells. To those ends, the inventors repeated the prior experiment (with a new HLA-A2 ⁺ donor, donor 2, but treated the control group not receiving ALECSAT II cells with anti-PDLl and included groups with in vitro purified TAII cells (54 vs 96 % purity). In accordance with prior experiments, ALECSAT II and TAII cells did not exert strong anti-cancer activity (Fig. 4D-E). In contrast, anti-PDLl in combination with purified TAII cells or ALECSAT II demonstrated significant tumor growth inhibition (Fig. 4D-E, P < 0.01). Importantly, there was no significant difference in the effect of ALECSAT II + anti- PDLl compared to TAII + anti-PDLl, demonstrating that TAII cells are the dominant effector population. The more modest anti-cancer activity of donor 2 compared to donor 1 was also reflected in lower levels of CD8+ TAII cell tumor infiltration (Fig 4C+4F). Considering the varied activity of ALECSAT II from donor 1 and 2, we repeated the analysis with a third HLA- A2 ⁺ donor, donor 3, which like the MDA-MB-231 cells, express HLA class I A*02:01, B*40 :02, and C*02:02, thereby allowing ALECSAT II cells to recognize antigens presented on all major types of HLA class I molecules. Again, ALECSAT II exerted modest, if any, anti-tumor activity alone. Consistent with our previous studies the activity was greatly enhanced by addition of anti-PDLl therapy (Fig 4G-I, P < 0.01) resulting in complete tumor eradication in 3/5 mice (Fig 4H-I).

Conclusion

The data demonstrate that the anti-tumor activity of ALECSAT II is greatly enhanced by addition of anti-PDLl. EXAMPLE 5

ALECSAT II suppress spontaneous and experimental metastasis formation

Aim of study

The inventors have previously demonstrated that MDA-MB-231 cells form extensive lung and liver metastasis in the presence of human leukocytes. Because ALECSAT II cells appeared to perform full body immune surveillance (Fig 3), the aim of this study was to investigate whether ALECSAT II therapy would inhibit formation of spontaneous metastasis to the lungs and liver.

Results

As expected, anti-PDLl treated and untreated mice presented with extensive lung and liver metastases (Fig 5A-E). Remarkably, despite having very limited impact on primary tumor growth, ALECSAT II as single agent therapy exerted significant protection against metastasis formation in both lung (Fig 5A+C, P < 0.01) and liver (Fig 5B+D, P < 0.05). The protective effect was retained by purified TAII cells (Fig 5C-D). Although the protective effect appeared to be amplified upon addition of anti-PDLl therapy, it did not reach statistical significance (Fig 5E, P = 0.06). To rule out that the effect was due to differences in primary tumor size or related to local effects in the MFP, the inventors evaluated the efficacy of ALECSAT II on established experimental metastases. In these experiments, hIL15 NOG mice were challenged with an i.v. injection of MDA-MB-231 cells. Seven days later mice were treated with ALECSAT II or combined ALECSAT II and anti-PDLl. A strong anti-metastatic effect of ALECSAT II alone was observed that were significantly augmented by addition of anti-PDLl (Fig 5F-H, P < 0.05). In fact, whereas lung metastases were detectable in all ALECSAT II treated mice, cancer cells were undetectable in the lungs of mice receiving the combined ALECSAT II and anti-PDLl therapy (Fig 5F+H). Nevertheless, solitary liver metastasis was detectable in 2/6 mice treated with the ALECSAT II and anti-PDLl combination (Fig 51). Compared to untreated liver metastases these appeared less dense and extensively infiltrated by TAII cells. Conclusion

Taken together, these data demonstrate that ALECSAT II cells can detect and eradicate both spontaneous and experimental metastasis, and that the anti-metastatic effect is significantly augmented by addition of anti-PDLl therapy.

EXAMPLE 6

ALECSAT II display improved survival in a completely autologous system

Aim of study

As mentioned above the ALECSAT II donors used were all HLA class I A2 ⁺ to ensure proper interaction with the MDA-MB-231 cells. Because it was not possible to obtain a perfect match, the inventors cannot rule out that part of the observed responses was due to allogenicity rather than tumor antigen expression. The aim of the present study was therefore to examine whether ALECSAT II cells can in fact detect and kill autologous cancer cells.

Results

To demonstrate that ALECSAT II cells can detect and kill autologous cancer cells, the inventors created PDX models from patients with metastatic triple-negative breast cancer (TNBC). The PDL1 status of PDX IB01002 and IB01003 was 2% and <1%, respectively. IB01002 PDX tumors were initially transplanted into the MFP and autologous ALECSAT II cells were subsequently administered i.v. in hIL15 mice after 7 days. As expected, ALECSAT II exerted significant inhibition of tumor expansion. Adding anti-PDLl therapy did not further enhance the anti-cancer effect (Fig. 6A-C, P < 0.05). Encouragingly, ALECSAT CD8 ⁺ T cells infiltrated PDX tumors, but it did not stimulate tumor PDL1 expression (Fig. 6D). Next, the inventors co-implanted IB01003 PDX and autologous ALECSAT II cells into the MFP of NOG mice. Due to limited tumor material at the time of ALECSAT II delivery, group sizes were limited to 3-4 mice per group, which were insufficient to reach statistical significance. Nevertheless, the inventors observed heterogeneous tumor development with a tendency towards delayed development in mice treated with ALECSAT II and a-PDLl (Fig 6E). The inventors therefore repeated the experiment and monitored tumor development. As expected, combined ALECSAT II and anti-PDLl conferred a statistically survival benefit compared to either monotherapy (Fig 6F, P < 0.01). To the inventors' surprise ALECSAT II cells were detectable in both spleen and tumor as late as day 150 upon administration when co-treated with anti-PDLl (Fig 6G) but not when administered as mono therapy. Finally, the inventors repeated the analysis from A) with a third TNBC PDX model IB01004. In this model ALECSAT II did not reduce tumor growth compared to anti-PDLl treated, whereas the combination of ALECSAT and anti-PDLl significantly reduced tumor growth (Fig 6H-I).

Conclusion

Taken together, these data demonstrate that ALECSAT II cells home to cancer tissue and exert anti-cancer activity in autologous systems both when administered i.v. or directly into the MFP. It further suggests that tumors with very low tumor PDL1 positivity can benefit from combined ALECSAT II and anti-PDLl therapy. Finally, it suggests that blocking PDL1 improve survival of ALECSAT II cells.

EXAMPLE 7

In vitro experiments with ALECSAT cells, a PD-L1 inhibitor, and dendritic cells

It has previously been demonstrated that the effect of different types of immunotherapies including those employing PD-1/PD-L1 targeting critically depends on the presence of dendritic cells inside the tumour and involves "crosstalk" between lymphocytes and dendritic cells (Spranger S et al. 2017; Garris CS et al. 2018).

In order to demonstrate that antitumor activity of ALECSAT cells used alone or in the presence of anti-PD-Ll antibody can also be modulated by dendritic cells, we first performed an experiment with unseparated ALECSAT II cells prepared as described in detail in Example 2 in WO 2020/208054, where dendritic cells were added to the culture at day 15. MDA-MB- 231 breast cancer cells were seeded into the wells of 24-well plates and after overnight growth the ALECSAT cells were added. After 24 hours of co-incubation, significant but incomplete lysis of MDA-MB-231 cells in wells with the addition of ALECSAT cells was noticed. At this time point half of the medium (1 ml) was carefully removed, and one ml fresh medium with or without addition of mature dendritic cells (10 ⁵ cells, prepared according to the method described above and kept frozen) or/and anti-PD-Ll antibody (Atezolizumab (MedChemExpress), final concentration 10 pg/ml) was added to the wells according to the following scheme:

After additional 72 hours of incubation, cultures were inspected under the microscope and photographed. Control cultures of tumor cells (without addition of ALECSAT cells) reached full confluency, and no visible effects of addition of PD-L1 or/and dendritic cells can be seen (Fig. 7, panels A-D). The culture where ALECSAT cells were added also contained confluent tumor cells, indicating the fact that ALECSAT alone ceases to work after about 24-48 hours, and the surviving tumor cells continue to grow reaching the confluency to this date (Fig. 7, panel E). Addition of anti-PD-Ll antibody has no visible effect on the re-growth of tumor cells (Fig. 7, panel F). In contrast, addition of dendritic cells significantly increased the anti-tumor activity of ALECSAT cells, leading to disappearance of adherent tumor cells from the major part of the well, leaving only single areas of adherent tumor cells (one of them is shown in Fig. 7, panel G). Surprisingly, addition of a combination of dendritic cells and anti-PD-Ll antibody induced complete disappearance of the adherent tumor cells (Fig. 7, panel H).

The experiment has been repeated with cells from another healthy donor, and with inclusion of both cells produced according to the method described in detail in Example 2 in WO 2020/208054 where dendritic cells are added to the culture at day 15 (/.e. "ALECSAT-2 cells") and with cells produced according to the novel ALECSAT 3 protocol, cf. above.

The experiment was done using iCELLigence equipment to detect impedance of the adherent tumor cells (as described in Example 2 in 2022/259019). Tumour cells were seeded into one E-plate, and lymphocytes (100 x 10 ³ per well) were added after 22 hours in the absence or presence of anti-PD-Ll antibody (final concentration 10 pg/ml). After 24 hours, 0.2 ml of culture medium was removed from all wells, and dendritic cells were added to all wells in the amount of 20 x 10 ³ per wells in 0.2 ml of medium.

The impedance was constantly recorded during the following 72 hours. The values of the normalized Cell Index for two time points (before addition of dendritic cells and 72 hours after addition of dendritic cells) was used to obtain the bar graphs (Fig. 8A and Fig. 8B). The data presented in Fig. 8A indicates that addition of an anti-PD-Ll antibody has no effect on activity of ALECSAT- 2 cells, but slightly increases the activity of ALECSAT-3 cells. Addition of dendritic cells increased the activity of both ALECSAT-2 and ALECSAT-3 cells. On the other hand, addition of both dendritic cells and anti-PD-Ll antibody leads to practically complete disappearance of adherent tumor cells in the group of ALECSAT-2 cells, and to significant decrease in number of the remaining adherent tumor cells in the group of ALECSAT-3 cells. This data supports the results obtained in the previous experiment and clearly indicates that combination of dendritic cells with anti-PD-Ll antibody produces maximal antitumor effect, seen both for lymphocytes prepared by ALECSAT-2 and ALECSAT-3 protocol.

In summary, this example demonstrates for the first time that the in vitro anti tumor effect of ALECSAT cells can be significantly increased by combination with mature dendritic cells, and further increased by addition of anti-PD-Ll antibody. REFERENCES

• WO 2008/081035

• WO 2020/208054

• WO 2022/269019

BIOLOGICAL SEQUENCES Table 1 : Overview of sequences used