Technical field

The present invention relates to a Mer Tyrosine Kinase (MerTK) ligand for use in adoptive T cell transfer cancer therapy in an individual.

Background

Cancer immunotherapy may be divided into three major categories: non-specific stimulation of the immune system, active immunization using cancer vaccines, and adoptive cell transfer immunotherapy.

Adoptive cell transfer (ACT) is based on transfer of immune cells with anti-tumor activity into cancer patients. Several cell types of the immune system are capable of cell killing, most notably T cells. More specifically, T cells are generally divided into CD4 and CD8 T cells. To this end, CD4 T cells are functionally plastic and may possess helper function for CD8 T cells, but may also differentiate into regulatory T cells which suppress CD8 T cell responses. CD8 T cells are less plastic and this cell type has as its main function to kill cells - be it infected cells (e.g., with virus) or cancer cells.

T cells used for ACT can be derived from tumor tissue (tumor infiltrating lymphocytes or TILs). TILs comprise relatively high frequencies of tumor specific T cells which can be expanded in vitro and transferred back to the patient in what is called“TIL based ACT” therapy. Blood T cells can also be used for harvest of cells. For treatment of cancer, tumor specific T cells are, however, scarce in blood, but T cells harvested from blood may be genetically engineered to express T cell receptors (TCRs) that recognize human leukocyte antigen (HLA)/peptide complexes expressed by cancer cells, or chimeric antigen receptors (CARs) which equip the cells with antibody recognition capacity, i.e., unleash the killing capacity based on expression of a molecule expressed on the cell surface. Several strategies based on the genetic engineering of T cells for exploitation of the capacity of this cell type to kill target cells upon ACT are in development. Some data have suggested that CD4 T cells may play a role in ACT. To this end, it has been shown that administered CD4 T cells specific for a mutated antigen could induce tumor regression in a cancer patient (Tran et al. 2014). However, whereas data on the efficacy of CD4 T cells are anecdotal, several lines of evidence suggest that CD8 T cells are the main cell types associated with clinical response in ACT therapy. In TIL based ACT in melanoma, CD8 frequency in administered TIL cultures is associated with clinical response (Andersen et al. 2016, Rosenberg et al. 2011 ). To this end, CD8 act as a co-stimulatory molecule to HLA class I which is expressed by all nucleated cells including cancer cells, whereas CD4 is a co-receptor for HLA class II molecules which is only expressed by antigen presenting cells. These data fit well with the fact that, although CD4 T cells are capable of killing target cells, the killing capacity of CD8 T cells is more efficient (June et al. 2015). Another characteristic of transferred cells, which is key to clinical efficacy, is persistence in the patient, i.e., memory or memory- stem cell features of the cells as opposed to terminally differentiated effector T cells.

Beyond conventional T cells expressing ab T cell receptors, several more rare sub-sets exist, e.g., gd T cells (expressing a gd T cell receptor), and invariant chain T cells which express a TCR with limited heterogeneity. In addition, NK T cells - expressing a T cell receptor along with more NK cell recognition and killing features - could potentially be exploited in ACT. However, the use of ACT based on the killing capacity of CD8 T cells is by far the most mature and clinically validated.

Mer Tyrosine Kinase (MerTK) is a member of the TAM family of receptor kinases which consists of Tyro3, Axl, and Mer. Best described ligands for TAM are are growth arrest- specific protein (Gas6) and Protein S (Prosl ). Gas6 is a ligand for all three TAMs whereas Prosl is a ligand for Tyro and MerTK only.

Cabezon et al. 2015 disclose expression of MerTK and Prosl in activated CD4 T cells and that addition of Prosl to activated CD4 T cell cultures induces proliferation and cytokine production.

Further development of adoptive cell therapy with e.g. autologous tumor infiltrating lymphocytes has the potential to markedly change the long-term prognosis of cancer patients and modifications of the original protocol that can improve its clinical efficacy are highly desirable. Summary

The present inventors have surprisingly found that CD8+ T lymphocytes express MerTK and Prosl and that stimulation of these cells with a MerTK ligand, such as Prosl , leads to increased proliferation and pro-inflammatory cytokine release. Thus, the present inventors propose an improved method for adoptive T cell transfer cancer therapy wherein T cells are stimulated by a MerTK ligand thereby increasing their tumor reactivity.

Particularly, the present disclosure relates to a Mer Tyrosine Kinase (MerTK) ligand for use in a method of adoptive cell transfer cancer therapy in an individual. The method may be performed by the following steps:

a. providing a MerTK ligand,

b. providing a population of T lymphocytes expressing MerTK, c. administering the MerTK ligand to the population of T lymphocytes in vitro, and

d. administering the T lymphocytes to the individual. Definitions

Mer Tyrosine Kinase (MerTK), also referred to as c-mer, Mer, MER, Proto-oncogene c-Mer, Receptor Tyrosine Kinase MerTK, Tyrosine-protein Kinase Mer, STK Kinase, RP38, or MGC133349, is a member of the TAM family of receptor tyrosine kinases, which also include AXL and TYR03 kinases. MerTK transduces signals from the extracellular space via activation by binding of ligands, most notably Gas-6, a soluble protein and Prosl . Three other TAM ligands have recently been described: Galectin-3, Tubby, and Tulp-1. CD8+ T lymphocytes kill target cells expressing the cognate antigenic peptide target. The term“CD8 T cells” or“CD8+ T lymphocytes” as used interchangeably herein refers to conventional CD8 positive Hla class I restricted T lymphocytes of the adaptive immune system. Memory T cells are a subset of T cells that have previously encountered and responded to their cognate antigen. These cells are long lived. The term TCM refers to T central memory cells.

Tumor infiltrating lymphocytes, abbreviated as TILs, are white blood cells that have left the bloodstream and migrated into a tumor.

Adoptive cell therapy, abbreviated ACT is herein used interchangeably with the term adoptive cell transfer. ACT involves the transfer of immune cells with antitumor activity into cancer patients. ACT is a treatment approach that usually involves the

identification, in vitro, of lymphocytes with antitumor activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Of particular interest for the disclosure is autologous ACT, i.e. adoptive cell therapy in which the immune cells with antitumor activity to be transferred into cancer patients originate from the patient itself.

Autologous: a situation in which the donor and recipient of e.g. lymphocytes are the same person.

Heterologous: a situation in which the donor and recipient of e.g. lymphocytes are not the same person.

Cytokines are small, secreted polypeptides from higher eukaryotes which are responsible for intercellular signal transduction and which affect the proliferation, division and functions of other cells. They are potent, pleiotropic polypeptides that, e.g. via corresponding receptors, act as local or systemic intercellular regulatory factors, and therefore play crucial roles in many biologic processes, such as immunity, inflammation, and hematopoiesis. Cytokines are produced by diverse cell types including fibroblasts, endothelial cells, epithelial cells, macrophages/monocytes, and lymphocytes.)

Interleukins are a group of cytokines important for the function of the immune system.

Pro-inflammatory refers to an agent capable of promoting inflammation. Pro- inflammatory cytokines include TNF-a, IFN-g and lnterleukin-2 (IL-2). Description of Drawings

Figure 1. MerTK receptor and ligand PROS1 are expressed by TCR-activated human CD8+ T cells.

Human CD8+ T cells were negatively isolated from peripheral blood mononuclear cells (PBMCs) and activated with aCD3/CD28 beads. (A) Representative histogram of (B) PROS1 surface expression (MFI) on unstimulated and aCD3/CD28 activated CD8+ T cells. Cells were harvested at different time points and analysed by flow cytometry (n=3). (C) RT-qPCR evaluated expression of PROS1 mRNA of 3-day aCD3/CD28 activated CD8+ T cells, normalized to unstimulated (n=3). (D) RT-qPCR evaluated expression of MerTK mRNA of 3-day aCD3/CD28 activated CD8+ T cells, normalised to unstimulated (n=3). (E) MerTK surface expression (MFI) on unstimulated and aCD3/CD28 activated CD8+ T cells, harvested on different time points for 4 days (n=3). (F) Representative histogram of (E). (G) MerTK protein expression (top) on day 3 of activation of PBMCs or CD8+ T cells, as analysed by western blot (representative of at least 3 independent experiments) b-actin (bottom) served as a loading control. Data are plotted as mean ± SEM. *p<0.05, **p<0.01 , ***p<0.001.

Figure 2. PROS1 positively regulates CD8+ T cell proliferation.

(A) Human CD8+ T cells were cultured in serum-free medium, stained with a proliferation dye (CTV, Cell Trace Violet), and activated for 3 days with aCD3/CD28 in the presence or absence of 50 nM PROS1. Proliferation was measured by flow cytometry. Representative histogram of triplicates of 1 donor. (B) Relative proliferation, with aCD3/CD28 activated CD8+ T cells set as 100, of activated CD8+ T cells cultured for 3 days in the presence or absence of 50 nM PROS1 (n=4). (C) IFN-g and (D) TNF-a production in culture supernatants (n=3). (E) Relative proliferation of CD8+ T cells activated with aCD3/CD28 beads for 5 days, treated with aPROS1 mAb (n=5). (F) Representative histogram of (E). Data are plotted as mean ± SEM. *p<0.05, **p<0.01 , ***p<0.001.

Figure 3. PROS1 impacts on human melanoma TIL expansion.

TILs from 4 metastatic melanoma patients were isolated, cultured and expanded according to the clinical Rapid Expansion Protocol (REP, with human serum-containing medium, 6000U/ml IL-2, and feeder cells) in the presence or absence of 50 nM PROS1 or aPROS1 mAb. Total live cells were counted and fold expansion was calculated on day 16 and 23 of culture, relative to day 0 (n=4). Data are plotted as mean ± SEM.

Figure 4. Domains in Gas6 and Prosl for TAM binding.

The figure represents a schematic representation of domains in Gas6 and Prosl . The domains predicted to be involved in TAM binding are the laminin G-like domains 1 and 2.

Figure 5. MERTK expression on T cell subsets.

(A) Gating strategy for CD8+ subset classification using CCR7 and CD45R0, gated on unstimulated CD8+CD3+ live cells. (B) Mean fluorescence intensity (MFI) of MERTK on CD8+ T cell subsets, as measured on day 3 of stimulation. CM = central-memory, EM = effector-memory, TEMRA = terminally differentiated EM cells. Data are plotted as mean ± SEM and statistical significance was determined with Student’s t tests or two- way ANOVA with Bonferroni’s multiple comparisons tests. *p<0.05, **p<0.01 ,

***p<0.001

Figure 6. MERTK is expressed by naturally occurring activated peripheral CD8+ T cells.

Human PBMCs were stimulated for 48 hours with peptides derived from CMV, EBV and influenza to track naturally occurring CD8+ T cell activation. (A) Representative dot plots of CD137 and MERTK co-expression on peptide-stimulated CD8+ T cells from 5 healthy human donors. (B) Gating strategy for resting (CD137-) and activated

(CD137+) T cells. (C) Representative histogram of MERTK on CD137- and CD137+ CD8+ T cells. (D) and (E) Percentage (D) and MFI (E) of MERTK-expressing CD8+ T cells in resting and activated peripheral CD8+ T cells as classified by CD137 expression (n=10). Data are plotted as mean ± SEM and statistical significance was determined with Student’s t tests (D,E), ***p<0.001.

Figure 7. CXCL10 and IL-7 produced by CD8+ T cells in presence or absence of Prosl .

Activated CD8+ T cells cultured for 3 days in the presence or absence of 50 nM PROS1. (A) CXCL10 and (B) IL-7 production in culture supernatants. Figure 8. MERTK acts as a co-stimulatory molecule on CD8+ T cells.

(A) siRNA-mediated knockdown (compared to control) of MERTK on three-day CD3/CD28-stimulated CD8+ T cells, followed for 24, 48 and 72h after siRNA knockdown as analyzed by MERTK protein expression via westernblot (representative of at least 3 independent experiments) b-actin (bottom) served as a loading control.

(B) Quantification of (A) using relative density compared to control (normalized with loading control). (C) Cytokine concentrations (IFN-g, IL-2, IL-7, IL-15) in supernatants of MERTK-knockdown and control CD8+ T cells re-stimulated overnight with aCD3/CD28, 48 hours after siRNA knockdown (n=4). (D) Human CD8+ T cells were cultured in serum-free medium, stained with a proliferation dye and activated for three days with aCD3/CD28 in the presence or absence of 250 nM MERTK-inhibitor UNC2025. Proliferation was measured by flow cytometry and relative proliferation was calculated compared to control (n=3). (E) % of live cells of CD8+ T cells activated with or without 200 nM MERTK-inhibitor UNC2025 (n=3). (F) IFN-g concentration in culture supernatants of activated CD8+ T cells stimulated with or without PROS1 or MERTK- inhibitor UNC2025 (n=3). Data are plotted as mean ± SEM and statistical

significancewas determined with Student’s t tests (C,D,F). *p<0.05, **p<0.01 ,

***p<0.001 .

Figure 9. PROS1 impacts on human melanoma TIL expansion

(A) Tumor infiltrating lymphocytes (TILs) from biopsies originating from four metastatic melanoma patients were cultured and expanded according to the‘young’ TIL protocol in the presence or absence of exogenous PROS1 or aPROS1 mAb. Fold expansion was calculated on day 16 and 23 of culture, relative to day 0 (n=4). (B) Phenotypic analysis on TILs was done on day 23 of expansion using CCR7 and CD45R0 as T cell subset markers (n=4). CM = central-memory, EM = effector-memory, TEMRA = terminally differentiated EM cells. (C) TAM receptor protein expression status of tumor cells from 3 metastatic melanoma patients. Actin was used a loading control. (D) Real- time in vitro cytolysis of autologous cancer cells from metastatic melanoma patient 3 after addition of antigen-selected autologous TILs (1 :10 or 1 :20 targeheffector ratio). (E) % Cytolysis 48 hours post TIL addition. Data are plotted as mean ± SEM and statistical significance was determined with two-way AN OVA with Bonferroni’s multiple comparisons tests (A). *p<0.05, **p<0.01 , ***p<0.001. Figure 10. Blocking PROS1 -MERTK axis decreases mitochondrial respiration in CD8+ T cells.

Bioenergetic properties of CD3/CD28-stimulated CD8+ T cells cultured for three days in the presence or absence of aPROS1. (A) Basal respiration was determined as initial resting consumption of oxygen. (B) ATP turnover was measured as decrease of oxygen consumption after addition of oligomycin. (C) Reserve respiratory capacity was measured as percentage of basal respiration, after addition of Carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone (FCCP). (D) Glycolytic capacity was measured after addition of oligomycin. (E) Raw levels of oxygen consumption. Cells were treated with either oligomycin or FCCP at stage A and antimycin A at stage B. (F) Whole-cells levels of ATP normalized to control. Data are plotted as mean ± SEM and statistical significance was determined with Student’s t tests (A,B,C,D,F). *p<0.05, **p<0.01 , ***p<0.001.

Figure 11. Blocking PROS1 -MERTK axis decreases mitochondrial respiration in CD8+ T cells.

Nanostring-measured IRF4 mRNA expression in three-day activated CD8+ T cells, analyzed as in Figure 8.

Figure 12. PROS1 impacts on anti-tumor tumor-infiltrating lymphocytes.

(A) Experimental setup of (B). (B) IFN-g concentrations in co-culture supernatants (n=3). Significance shown in comparison with 0 nM PROS1 condition. (C) Real-time in vitro cytolysis of autologous cancer cells from metastatic melanoma patient 3 after addition of antigen-selected autologous TILs (1 :10 target:effector ratio) and PROS1 titration from ranging from 0-100 nM PROS1. (D) % Cytolysis 12 hours post TIL addition.

Figure 13. PROS1 and MERTK co-expression on 3-day activated CD8+ T cells.

(A) Representative dot plots of PROS1 and MERTK co-staining on CD8+ T cells, activated for 3 days with aCD3/CD28 in the presence or absence of 5 mg/ml unconjugated Annexin V. (B) Percentage of PROS1 or MERTK negative, single- positive or double-positive CD8+ T cells (n=4). Data are plotted as mean

± SEM and statistical significance was determined with two-way ANOVA with

Bonferroni’s multiple comparisons tests (B). *p<0.05, **p<0.01. Figure 14. PROS1 consumption by cancer cells and T cells affects TIL-mediated killing.

aCD3/CD28-stimulated PBMCs (A, n=3) or cancer cell lines (B) were cultured for four to six days in the presence of 50 nM PROS1. Daily, samples from the culture medium were harvested and PROS1 concentration in culture medium was analyzed

Sequences

SEQ ID NO: 1 is the full length Prosl polypeptide having the following sequence:

MRVLGGRCGALLACLLLVLPVSEANFLSKQQASQVLVRKRRANSLLEETKQGNLERE

CIEELCNKEEAREVFENDPETDYFYPKYLVCLRSFQTGLFTAARQSTNAYPDLRSCV N AIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEFDINECKDPSNINGGC SQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICGTAVCKNIPGDFECEC PEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDGKKGFKLAQDQKSCEV VSVCLPLNLDTKYELLYLAEQFAGVVLYLKFRLPEISRFSAEFDFRTYDSEGVILYAESI DHSAWLLIALRGGKIEVQLKNEHTSKITTGGDVINNGLWNMVSVEELEHSISIKIAKEAV MDINKPGPLFKPENGLLETKVYFAGFPRKVESELIKPINPRLDGCIRSWNLMKQGASGI KEIIQEKQNKHCLVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVNVTLNIRPSTGTG VMLALVSGNNTVPFAVSLVDSTSEKSQDILLSVENTVIYRIQALSLCSDQQSHLEFRVN RNNLELSTPLKIETISHEDLQRQLAVLDKAMKAKVATYLGGLPDVPFSATPVNAFYNG

CMEVNINGVQLDLDEAISKHNDIRAHSCPSVWKKTKNS

SEQ ID NO: 2 is a fragment of Prosl and comprises an alternative predicted sequence of the LG-like binding domain 1 known to be involved in MerTK binding and

corresponds to amino acids 329-459 of SEQ ID NO: 1 :

FRTYDSEGVILYAESIDHSAWLLIALRGGKIEVQLKNEHTSKITTGGDVINNGLWNMVS

VEELEHSISIKIAKEAVMDINKPGPLFKPENGLLETKVYFAGFPRKVESELIKPINP RLDG

CIRSWNLMKQG

SEQ ID NO: 3 is a fragment of Prosl and comprises an alternative predicted sequence of the LG-like binding domain 2 known to be involved in MerTK binding and

corresponds to amino acids 514-647 of SEQ ID NO: 1 :

IRPSTGTGVMLALVSGNNTVPFAVSLVDSTSEKSQDILLSVENTVIYRIQALSLCSDQQ SHLEFRVNRNNLELSTPLKIETISHEDLQRQLAVLDKAMKAKVATYLGGLPDVPFSATP VNAFYNGCMEVNINGV

SEQ ID NO: 4 is fragment of Prosl and comprises a predicted sequence of the LG-like binding domain 1 as shown in fig 4 and corresponds to amino acids 299-475 of SEQ ID NO: 1 :

YLAEQFAGVVLYLKFRLPEISRFSAEFDFRTYDSEGVILYAESIDHSAWLLIALRGGKIE

VQLKNEHTSKITTGGDVINNGLWNMVSVEELEHSISIKIAKEAVMDINKPGPLFKPE NG

LLETKVYFAGFPRKVESELIKPINPRLDGCIRSWNLMKQGASGIKEIIQEKQNKHC SEQ ID NO: 5 is a fragment of Prosl and comprises a predicted sequence of the LG- like binding domain 2 as shown in fig. 4 and corresponds to amino acids 484-666 of SEQ ID NO: 1 :

YYPGSGIAQFHIDYNNVSSAEGWHVNVTLNIRPSTGTGVMLALVSGNNTVPFAVSLVD

STSEKSQDILLSVENTVIYRIQALSLCSDQQSHLEFRVNRNNLELSTPLKIETISHE DLQ

RQLAVLDKAMKAKVATYLGGLPDVPFSATPVNAFYNGCMEVNINGVQLDLDEAISKH

NDIRAHSC

SEQ ID NO: 6 is a fragment of Prosl and comprises a predicted sequence comprising the LG-like binding domains 1 and 2 as shown in fig. 4 and corresponds to amino acids 299-666 of SEQ ID NO: 1 :

LLYLAEQFAGVVLYLKFRLPEISRFSAEFDFRTYDSEGVILYAESIDHSAWLLIALRGGK I

EVQLKNEHTSKITTGGDVINNGLWNMVSVEELEHSISIKIAKEAVMDINKPGPLFKP EN

GLLETKVYFAGFPRKVESELIKPINPRLDGCIRSWNLMKQGASGIKEIIQEKQNKHC LV

TVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVNVTLNIRPSTGTGVMLALVSGNNTVP

FAVSLVDSTSEKSQDILLSVENTVIYRIQALSLCSDQQSHLEFRVNRNNLELSTPLK IETI

SHEDLQRQLAVLDKAMKAKVATYLGGLPDVPFSATPVNAFYNGCMEVNINGVQLDLD

EAISKHNDIRAHSC

SEQ ID NO: 7 is a fragment of Prosl and comprises a predicted sequence comprising the LG-like binding domains 1 and 2 and corresponds to amino acids 329-647 of SEQ ID NO: 1 :

FRTYDSEGVILYAESIDHSAWLLIALRGGKIEVQLKNEHTSKITTGGDVINNGLWNMVS

VEELEHSISIKIAKEAVMDINKPGPLFKPENGLLETKVYFAGFPRKVESELIKPINP RLDG

CIRSWNLMKQGASGIKEIIQEKQNKHCLVTVEKGSYYPGSGIAQFHIDYNNVSSAEG W

HVNVTLNIRPSTGTGVMLALVSGNNTVPFAVSLVDSTSEKSQDILLSVENTVIYRIQ ALS

LCSDQQSHLEFRVNRNNLELSTPLKIETISHEDLQRQLAVLDKAMKAKVATYLGGLP D

VPFSATPVNAFYNGCMEVNINGV

SEQ ID NO: 8 is fragment of Prosl and comprises amino acids predicted to be involved in binding to MerTK and corresponds to amino acids 42-666 of SEQ ID NO:1 :

ANSLLEETKGNLERECIEELCNKEEAREVFENDPETDYFYPKYLVCLRSFQTGLFTA A

RQSTNAYPDLRSCVNAIPDQCSPLPCNEDGYMSCKDGKASFTCTCKPGWQGEKCEF

DINECKDPSNINGGCSQICDNTPGSYHCSCKNGFVMLSNKKDCKDVDECSLKPSICG

TAVCKNIPGDFECECPEGYRYNLKSKSCEDIDECSENMCAQLCVNYPGGYTCYCDG

KKGFKLAQDQKSCEWSVCLLNLDTKYELLYLAEQFAGWLYLKFRLPEISRFSAEFDF

RTYDSEGVILYAESIDHSAWLLIALRGGKIEVQLKNEHTSKITTGGDVINNGLWNMV SV

EELEHSISIKIAKEAVMDINKPGPLFKPENGLLETKVYFAGFPRKVESELIKPINPR LDGC

IRSWNLMKQGASGIKEIIQEKQNKHCLVTVEKGSYYPGSGIAQFHIDYNNVSSAEGW H

VNVTLNIRPSTGTGVMLALVSGNNTVPFAVSLVDSTSEKSQDILLSVENTVIYRIQA LSL

CSDQQSHLEFRVNRNNLELSTPLKIETISHEDLQRQLAVLDKAMKAKVATYLGGLPD V

PFSATPVNAFYNGCMEVNINGVQLDLDEAISKHNDIRAHSC Detailed description

The present disclosure provides a method for improving the clinical efficacy of ACT by using T lymphocytes which have been stimulated with a MerTK ligand.

Hence, the present disclosure relates to a Mer Tyrosine Kinase (MerTK) ligand for use in a method of adoptive cell transfer cancer therapy in an individual comprising the consecutive steps of

a. providing a MerTK ligand,

b. providing a population of T lymphocytes expressing MerTK, c. administering the MerTK ligand to the population of T lymphocytes in vitro, and

d. administering the T lymphocytes to the individual.

In one embodiment the present disclosure relates to in vitro method of stimulating CD8+ T lymphocyte proliferation and/or cytokine release comprising the steps of

a. providing a MerTK ligand,

b. providing a population of CD8+ T lymphocytes expressing MerTK, c. administering the MerTK ligand to the population of CD8+ T lymphocytes,

d. culturing the CD8+ T lymphocytes.

In one embodiment the present disclosure relates to method for treatment of cancer in an individual comprising the consecutive steps of:

a. providing a MerTK ligand,

b. providing a population of T lymphocytes expressing MerTK, c. administering the MerTK ligand to the population of T lymphocytes in vitro, and

d. administering a therapeutically effective amount of the T lymphocytes to the individual.

In one embodiment, said method further comprises administering a therapeutically effective amount of a MerTK ligand, such as Prosl to said individual. The MerTK ligand maybe be administered once a week, for example twice a week, such as three times a week, for example four times a week, such as five times a week, for example six times a week, such as daily or for example monthly.

The MerTK ligand, such as Prosl , can e.g. be administered throughout treatment of said individual, i.e. until the tumour has regressed/decreased and/or until amelioration of symptoms and/or until said individual is cured.

The MerTK ligand may be administered prior to, simultaneously with or after the T lymphocytes to said individual.

The stimulation of T lymphocytes with the MerTK ligand is believed to stimulate tumor cell immunogenicity, thereby augmenting tumor reactivity of T lymphocytes. An increase in T lymphocyte reactivity will result in enhanced efficacy of adoptive cell therapy (ACT). ACT

ACT involves the transfer of lymphocytes with anti-tumor activity into cancer patients. ACT is a treatment approach that usually involves the identification, in vitro, of lymphocytes with anti-tumor activity, the in vitro expansion of these cells to large numbers and their infusion into the cancer-bearing host. Lymphocytes used for adoptive transfer can either be derived from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs), or from blood: genetically engineered to express anti- tumor T cell receptors (TCRs) or chimeric antigen receptors (CARs) as described previously by Rosenberg (Rosenberg et al., 2011 ); enriched with mixed lymphocyte tumor cell cultures (MLTCs) as described by Mazzarella (Mazzarella et al., 2012) or cloned using autologous antigen presenting cells and tumor derived peptides as described by Yee (Yee et al., 2002). The lymphocytes used for infusion can be isolated from a donor, or from the cancer-bearing host itself. ACT in which the lymphocytes originate from the cancer-bearing host to be infused is termed autologous ACT. ACT in which the lymphocytes originate from a donor to be infused is termed heterologous ACT.

According to the present disclosure, ACT may be performed by (i) obtaining lymphocytes from an individual, (ii) culturing said lymphocytes with a MerTK ligand, and (iii) administering the expanded lymphocytes to the individual. The lymphocytes are usually expanded prior to administration to the individual. In one embodiment, the lymphocytes are tumor-derived, i.e. they are TILs, and are isolated from the individual to be treated, i.e. autologous transfer.

In another embodiment, the cancer therapy comprises heterologous adoptive cell therapy of T lymphocytes.

MerTK ligand

The MerTK ligand as described herein is capable of activating MerTK signalling and of stimulating proliferation and/or cytokine release of T cells.

In one embodiment, the ligand that specifically binds to MerTK is selected from the group of Prosl , Gas6, Galectin-3, Tubby and Tulp-1 , or active fragments, variants or variants of fragments thereof.

In a preferred embodiment, the MerTK ligand is a Prosl polypeptide, which may be full length Prosl , an active variant of Prosl , a fragment of Pros 1 or a variant of a fragment of Prosl .

In one embodiment, the MerTK ligand is a Prosl polypeptide in the form of a fragment or variant of full length Prosl (SEQ ID NO: 1 ) comprising the amino acid residues which are involved in binding of Prosl to MerTK.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising an amino acid sequence according to SEQ ID NO: 2 and/or SEQ ID NO: 3. SEQ ID NO: 2 and SEQ ID NO: 3 are predicted sequences of LG-like domains 1 and 2 and have been predicted to be involved in Prosl binding to MerTK.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising an amino acid sequence according to SEQ ID NO: 4 and/or SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 are predicted sequences of LG-like domains 1 and 2 (as shown in fig. 4) and have been predicted to be involved in Prosl binding to MerTK.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising a variant of SEQ ID NO: 2 and/or SEQ ID NO: 3 having at least 80% sequence identity to said sequences, such as least 85% sequence identity to said sequences, for example at least 90% sequence identity to said sequences, such as at least 95% sequence identity to said sequences, for example at least 97% sequence identity to said sequences. In one embodiment the sequence identity is at least 90%.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising a variant of SEQ ID NO: 4 and/or SEQ ID NO: 5 having at least 80% sequence identity to said sequences, such as least 85% sequence identity to said sequences, for example at least 90% sequence identity to said sequences, such as at least 95% sequence identity to said sequences, for example at least 97% sequence identity to said sequences. In one embodiment the sequence identity is at least 90%.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising SEQ ID NO: 6 or a variant thereof having at least 80% sequence identity to said sequence, such as least 85% sequence identity to said sequence, for example at least 90% sequence identity to said sequence, such as at least 95% sequence identity to said sequence, for example at least 97% sequence identity to said sequence. In one embodiment the sequence identity is at least 90%.

In one embodiment the MerTK ligand is a Prosl polypeptide comprising SEQ ID NO: 7 or a variant thereof having at least 80% sequence identity to said sequence, such as least 85% sequence identity to said sequence, for example at least 90% sequence identity to said sequence, such as at least 95% sequence identity to said sequence, for example at least 97% sequence identity to said sequence. In one embodiment the sequence identity is at least 90%.

In one embodiment, the MerTK ligand is a Prosl polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , such as least 85% sequence identity to SEQ ID NO: 1 , for example at least 90% sequence identity to SEQ ID NO: 1 , such as at least 95% sequence identity to SEQ ID NO: 1 , for example at least 97% sequence identity to SEQ ID NO: 1 , or a fragment thereof comprising at least 150 amino acids, such as at least 200 amino acids, for example at least 250 amino acids, such as at least 300 amino acids, for example at least 350 amino acids, such as at least 400 amino acids, for example at least 450 amino acids, such as at least 500 amino acids, for example at least 550 amino acids, such as at least 600 amino acids, for example at least 650 amino acids.

In one embodiment, the MerTK ligand is a Prosl polypeptide comprising or consisting of SEQ ID NO: 8 or a fragment or variant thereof. SEQ ID NO: 1 comprises amino acid residues which are involved in binding of Prosl to MerTK. In one embodiment the Prosl polypeptide comprises SEQ ID NO: 8 or a variant thereof having at least 80% sequence identity to said sequence, such as least 85% sequence identity to said sequence, for example at least 90% sequence identity to said sequence, such as at least 95% sequence identity to said sequence, for example at least 97% sequence identity to said sequence. In one embodiment the sequence identity is at least 90%.

In one embodiment the MerTK ligand is a polypeptide, such as a Prosl polypeptide comprising less than 700 amino acids, such as less than 650 amino acids, for example less than 600 amino acids, such as less than 550 amino acids, for example less than 500 amino acids, such as less than 450 amino acids, for example less than 400 amino acids, such as less than 350 amino acids, for example less than 300 amino acids, such as less than 250 amino acids, for example less than 200 amino acids, such as less than 150 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Prosl polypeptide, comprising less than 500 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Prosl polypeptide, comprising less than 300 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Prosl polypeptide, comprising less than 150 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Prosl polypeptide comprising at least 100 amino acids, such as at least 150 amino acids, for example at least 200 amino acids, such as at least 250 amino acids, for example at least 300 amino acids, such as at least 350 amino acids, for example at least 400 amino acids, such as at least 450 amino acids, for example at least 500 amino acids, such as at least 550 amino acids, for example at least 600 amino acids, such as at least 650 amino acids. In one embodiment the MerTK ligand is a polypeptide, such as a Pros1 polypeptide comprising at least 100 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Pros1 polypeptide comprising at least 300 amino acids.

In one embodiment the MerTK ligand is a polypeptide, such as a Pros1 polypeptide comprising at least 500 amino acids.

In one embodiment, the MerTK ligand is a Gas6 polypeptide, which may be full length Gas6, an active variant of Gas6, a fragment of Gas6 or a variant of a fragment of Gas6.

The MerTK ligand is provided in an amount capable of activating MerTK.

In one embodiment, the MerTK ligand is provided in the amount between about 10nM to about 1000nM, for example about 10nM to about 900 nM, such as about 10nM to about 800nM, for example about 10nM to about 700nM, such as about 10nM to about 600nM, for example about 10nM to about 500nM, such as about 10nM to about 400nM, for example about 10nM to about 300nM, such as about 10nM to about 200nM, for example about 10nM to about 100nM, for example about 50nM.

In one embodiment, the MerTK ligand is provided in the amount between about 10nM to about 100nM, for example about 10nM to about 90nM, such as about 10nM to about 80nM, for example about 10nM to about 70nM, such as about 10nM to about 60nM, for example about 10nM to about 50nM, such as about 10nM to about 40nM, for example about 10nM to about 30nM, such as about 10nM to about 20nM.

In one embodiment, the MerTK ligand promotes the proliferation and cytokine production by CD8+ T cells. The ability of CD8 T cells to proliferate and produce cytokines plays an important role in the capacity of such T cells to control tumor progression.

In one embodiment, the MerTK ligand promotes the secretion of pro-inflammatory cytokines by CD8+ T cells. In yet another embodiment, the MerTK ligand promotes the secretion of interleukins by CD8+ T cells. In yet another embodiment, the MerTK ligand promotes the secretion of interferons such as IFNy by CD8+ T cells. In yet another embodiment, the MerTK ligand promotes the secretion of TNFa by CD8+ T cells. In yet another embodiment, the MerTK ligand promotes the secretion of IL-2 by CD8+ T cells.

In one embodiment, the MerTK ligand upregulates IRF4 expression. The term upregulates used herein is to be understood as an increase in the quantity of a cellular component, such as RNA or protein in response to the said MerTK ligand stimulation.

In one embodiment, provided herein is an antibody capable of stimulating MerTK signalling for use as a MerTK ligand. Antibodies that specifically bind to human Mer Tyrosine Kinase (MerTK) capable of stimulating MerTK signalling are described in, e.g., WO2016106221 , which is incorporated herein by reference in its entirety.

Lymphocytes

TILs may be obtained from the stroma of resected tumors. Tumor samples are obtained from patients and a single cell suspension is obtained. The single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACS (TM) Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Alternatively, T cells may be grown from tumor pieces by IL-2 and MerTK ligand with or without activating CD3 antibody

Prior to administration of the MerTK ligand, the population of T lymphocytes is stimulated by an agent capable of stimulating MerTK expression. In one embodiment, the agent is selected from the group consisting of activating CD3 antibody, activating CD28 antibody, IL-2 and combinations thereof.

In one embodiment, the population of T lymphocytes comprises or consists essentially of CD8+ T lymphocytes. In another embodiment, the CD8+ T cells comprise effector T lymphocytes expressing CD8. The T lymphocytes are usually conventional CD8 positive Hla class I restricted T cells of the adaptive immune system, expressing variant ab T cell receptors. In a preferred embodiment the population of T lymphocytes does not comprise or consist essentially of cells selected from the group of NKT cells, CD4+ T lymphocytes and T regulatory lymphocytes.

In one embodiment, the population of lymphocytes comprises memory T cells. In one embodiment, the population of lymphocytes comprises effector T cells and memory T cells.

In one embodiment, the T lymphocytes used in the adoptive T cell cancer therapy according to the present disclosure are TILs. In one embodiment, the population of T lymphocytes comprises or consists essentially of TILs.

Culturing of lymphocytes to expand the number of lymphocytes, including tumor- infiltrating lymphocytes, such as T cells can be accomplished by any of a number of methods as are known in the art. For example, T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2), IL-7, IL-15 and IL-21 , with IL-2 being preferred.

T cells for use according to the ACT methods described herein can also be obtained from blood, lymphoid tissue (e.g. spleen) or from a tumor (e.g. tumor infiltrating lymphocytes). In one embodiment, T cells for use according to the ACT methods described herein are produced from PBMCs that have a histocompatible phenotype to the subject with cancer to be treated. These PBMCs are thus heterologous to the subject with cancer to be treated. In related embodiments, the PBMCs are autologous to the subject with cancer to be treated.

In one embodiment of the present disclosure the T cells are genetically engineered to express T cell receptors (TCRs) that recognize human leukocyte antigen (HLA)/peptide complexes expressed by cancer cells or chimeric antigen receptors (CARs) of the individual to be treated.

I one embodiment the T lymphocytes have been cultured for between about 14 to about 40 days prior to administration to the individual. In one embodiment, the T lymphocytes have been cultured for about 19 to about 35 days prior to administration to the individual. In other embodiments of the disclosure, the administered lymphocytes have been cultured for less than 14 days and in yet other embodiments, the lymphocytes have been cultured for longer than 40 days prior to administration to the individual.

The lymphocytes can be administered by any suitable route known in the art.

Preferably, the lymphocytes are administered as an intra-arterial or intravenous infusion, which may last about 30 to about 60 minutes. Other examples of routes of administration include intraperitoneal, intrathecal and intralymphatic.

Any therapeutically effective amount of lymphocytes can be administered. In one embodiment, about 1 x 10 ⁴ lymphocytes to about 1 x 10 ¹⁰ lymphocytes are

administered, such as about 1 x 10 ⁵ lymphocytes to about 1 x 10 ⁹ lymphocytes, for example about 1 x 10 ⁵ lymphocytes to about 1 x 10 ⁸ lymphocytes, such as about 1 x 10 ⁵ lymphocytes to about 1 x 10 ⁷ lymphocytes, for example about 1 x 10 ⁵ lymphocytes to about 1 x 10 ⁶ lymphocytes, such as about 1 x 10 ⁵ lymphocytes to about 9 x 10 ⁵ lymphocytes, for example about 1 x 10 ⁵ lymphocytes to about 8 x 10 ⁵ lymphocytes, such as about 1 x 10 ⁵ lymphocytes to about 7 x 10 ⁵ lymphocytes, for example about 1 x 10 ⁵ lymphocytes to about 6 x 10 ⁵ lymphocytes, such as about 1 x 10 ⁵ lymphocytes to about 5 x 10 ⁵ lymphocytes, for example about 1 x 10 ⁵ lymphocytes to about 4 x 10 ⁵ lymphocytes, such as about 1 x 10 ⁵ lymphocytes to about 3 x 10 ⁵ lymphocytes, for example about 2 x 10 ⁵ lymphocytes

The T lymphocytes may be administered one or more times to the individual.

Inhibition of Prosl was shown to increase the expression of the genes selected from the group consisting of LTA, TNFSRF9, IL-2, and IFN-g. Inhibition of Prosl was shown to decrease the expression of the genes selected from the group consisting of IL4R, DUSP4, CD99, ITGAL and CCL5.

Cancer

The cancer to be treated by the methods of the present disclosure can be any cancer that is likely to benefit from ACT, including any of acute lymphocytic leukaemia, acute myeloid leukaemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukaemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureter cancer, urinary bladder cancer, and digestive tract cancer such as, e.g., oesophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumor, cancer of the oral cavity, colorectal cancer, and hepatobiliary cancer.

The cancer can be a recurrent cancer.

In one embodiment the cancer is a solid cancer.

In one embodiment the cancer is selected from melanoma, ovarian, breast and colorectal cancer.

In one embodiment the cancer is a cancer of the hematopoietic or lymphoid system.

In one embodiment the cancer is melanoma, such as cutaneous metastatic melanoma.

Individual

The individual to be treated according to the methods described herein may be any subject likely to benefit from ACT.

The individual is usually a mammal, preferably a human. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order

Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids

(humans and apes).

Constitutive active MerTK

In an alternative embodiment, the CD8+ T cells used in ACT are not stimulated with a MerTK ligand but rather express a constitutive active MerTK, thus allowing for constitutive MerTK signalling without ligand stimulation. CD8 T cells can e.g. be isolated from tumor tissue, i.e. TILs, or CD8 T cells from blood samples can be rendered tumor specific by genetic modification e.g. by forced introduction, e.g., by viral transduction with a vector expressing a receptor that recognize cancer cells, e.g., a T cell receptor (TCR), or a chimeric antigen receptor (CAR).

Such tumor specific T cells can be further manipulated to express a constitutively active form of MerTK, e.g. as described previously by Kasikara et al., e.g, by viral transduction, in turn leading to T cells having improved cytokine production and proliferation and ultimately improved effect in ACT.

Thus, in one embodiment the present disclosure provides a CD8+ T lymphocyte genetically modified to express a constitutive active MerTK.

The CD8+ T lymphocyte genetically modified to express a constitutive active MerTK may in turn be used in a method of ACT cancer therapy. The present disclosure is further described by the following examples that should not be construed as limiting the scope of the disclosure.

Examples

Materials and Methods Clinical specimens, peripheral blood cells, and cancer cell lines

All procedures were approved by the Scientific Ethics Committee for the Capital Region of Denmark. Written informed consent was obtained from the patients before inclusion according to the Declaration of Helsinki. Cell lines were cultured in RPMI 1640 (Gibco) + 2.5% FCS (R10). PBMCs from healthy donor buffy coats were isolated by gradient centrifugation, and used immediately or cryopreserved for later use.

Biopsies from patients with stage III or IV melanoma (MM) was collected from 2006- 2013, and used for expansion of tumor infiltrating lymphocytes (TIL). Biopsy material was cut into small fragments (1-2 mm2) and kept overnight (ON) in a humidified 37°C CO ₂ incubator. The following day, tumor fragments and cells were resuspended, washed twice in 1xPBS (Lonza) by centrifugation for 5 minutes at 1500 RPM and cryopreserved in fetal bovine serum (FBS, Gibco) + 10% DMSO (Sigma Aldrich). All biopsies originated from lymph nodes LN metastases removed under palliative surgery. Antibodies and reagents

The following fluorochrome-labelled anti-human antibodies were used to stain for various populations: anti-CD3 (clone UCHT1 ), anti-CD8 (RPA-T8), anti-CD45RO (UCHL-1 , all BD Biosciences), anti-CD4 (SK3), anti-CCR7 (G043H7, all Biolegend) anti-PROS1 (PS7, Santa Cruz Biotechnologies), anti-MerTK (125518), anti-Tyro3 (96201 ) and anti-Axl (108724, all R&D Systems).

Expansion of tumor infiltrating lymphocytes (TIL) in the presence ofProsl and mAb Prosl

Cell suspensions were thawn and placed in 24 well-culture plates (Nunc, Roskilde, Denmark) together with 2 ml of culture medium (90% RPMI 1640 (Invitrogen), 10% heat inactivated Human AB serum (Sigma-Albricht, USA), IL-2 6000 lU/ml (6000 lU/ml IL-2, Proleukin, Novartis), penicillin, streptomycin and fungi zone (Bristol-Myers Squibb, Lyngby, Denmark). Cells were split into 2-3 wells when cell concentration in 1 well exceeded 1.5 x 106 cells/ml. Expansions were set up in culture triplicates plus Prosl (50 nM PROS1 (Hematologic Technologies)), plus mAb Prosl (10 mg/ml, clone PS7, Santa Cruz Biotechnologies) , and control expansions. Outgrowth of T cells measured by counting of live CD3 positive T cells, and fold expansion was calculated.

Flow cytometry and cell sorting

Flow cytometry analysis was performed on a FACS Canto™ II, or LSR (BD

Biosciences, San Jose CA, USA), and cell sorting was performed on FACS Aria™ (BD Biosciences, San Jose CA, USA). Multicolor flow cytometry analyses were performed to characterize expression of surface markers.

ELISA

ELISA was used to study levels of IFN-g and TNF-a (ELISA Ready-Set-Go kits,

Thermo Fisher Scientific) in CD3/CD28 bead stimulated T cell cultures, upon addition of prosl or aProsl mAb. Similarly, CD3/CD28 bead stimulated T cell cultures were studied upon siRNA gene knock down. Culture supernatants were tested at different dilutions following the manufacturer’s protocol.

T-cell sorting and stimulation

Human CD8+ T cells were isolated from human PBMCs by negative selection using MagniSort™ Human CD8 T cell Enrichment Kit, according to the manufacturer’s instructions (Thermo Fisher Scientific). Purified total CD8 T cells, or for some experiments total PBMCs, were cultured in the presence (1 :1 ratio) or absence of anti- human CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) in X-VIVO 15 medium (Lonza), supplemented with 5% human serum and 50 U/ml hlL-2 for 1 to 7 days. To evaluate PROS1 and TAM receptor expression, cells were harvested daily (for surface expression) or after 3 days (for RT-qPCR). Alternatively, cells were cultured in serum free medium in the presence or absence of 50 nM PROS1 (Hematologic Technologies) and cells were harvested for analysis after 3 days of activation. For PROS1 blocking experiments, cells were cultured in medium with 5% human serum for 3-5 days prior to analysis, in the presence or absence of anti-PROS1 (clone PS7, Santa Cruz

Biotechnologies).

Western blotting

Cells were lysed using Pierce RIPA Lysis and Extraction buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Following protein content quantification by BCA assay, equal amounts were loaded on pre-cast 4-12% Bolt Bis-Tris Plus gels and run according to manufacturer’s instructions. Subsequently, samples were loaded on gels as described above. Proteins were transferred to nitrocellulose membranes using the Bolt system (Invitrogen/Thermo) and membranes were subsequently blocked for 1 hour at room temperature with 5% BSA (Sigma) in TBS-T (Tris-buffered saline with 0.1 % Tween, Sigma). Membranes were incubated with primary antibodies (rabbit anti-human MERTK (D21 F11 , Cell Signalling), at 4°C overnight in TBS-T with 5% BSA and reprobed with mouse anti-human actin (C4, SCBT)). After several washing steps and incubation with HRP-linked secondary antibodies (Cell Signalling), proteins were detected using SuperSignal West ECL Kit and BioRad Analyzer. Quantification of signal was done using Fiji ImageJ (v.1.49).

Gene expression by Quantitative RT-PCR

At the indicated time points, T cells were harvested and RNA isolation was done using the NucleoSpin® RNA kit (Cat# 740955.50, MACHEREY-NAGEL, Düren, Germany) kit according to manufacturer’s instructions. RNA was reverse-transcribed to cDNA using Superscript® VI LO™ cDNA Synthesis Kit (Invitrogen, Thermo Fisher Scientific, Cat# 11754-050). qPCR was performed in Agilent AriaMX System using the Brilliant III Ultra- Fast QPCR Master Mix (Agilent). Amplified products were checked by dissociation curves and expression was normalized to housekeeping genes. siRNA gene knockdown

A set of three Stealth siRNA duplexes for targeted silencing of human MERTK were obtained from Invitrogen. For control experiments three siRNAs with scrambled sequences possessing similar GC content (Invitrogen) were used. Following magnetic bead removal, three-day stimulated CD8+ T cells were transfected with MERTK or Mock siRNA with the ECM830 square wave electroporation system (BTX) using electroporation parameters as previously described. Knockdown on protein level was confirmed for every individual experiment.

In vitro killing assay

The tumor-specific killing ability of TILs was assessed with a novel impedance-based cytotoxicity assay. Briefly, antigen-specific TILs were thawed and rested in IL-2 free media (RPMI 1640 supplemented with 10% human serum, penicillin and streptomycin) for 72 hours. Autologous tumor cells were seeded on E-plate 96 plates (ACEA Biosciences Inc) which were loaded onto RTCA SP real-time cell analyzer (ACEA Biosciences Inc). After 24 hours, TILs were added with or without 50 nM PROS1.

Transcriptomic analysis of CD8+ T cells

Sorted CD8+ T cells from three healthy donors were cultured in the presence or absence of aCD3/CD28 beads or 10 mg/ml aPROS1 for 3 days. Subsequently, cells were incubated with Brefeldin A for four hours. Final input for transcriptomic analysis was 1x10 ⁵ viable cells per condition. RNA and protein samples were split and processed separately. Transcriptomic analysis was performed using the nCounter Vantage 3D RNA:Protein Immune Cell Signaling Panel. Samples were subsequently processed in the fully automated nCounter Prepstation (NanoString Technologies) and analyzed in the nCounter Digital Analyzer (NanoString Technologies). The nSolver4 software (NanoString Technologies) was used for data normalization and differential gene expression analyses. The significance of differential gene expression between paired groups was estimated using a mixed module significance testing with the algorithm included in the nCounter Advanced Analysis. In this module, a negative binomical mixture model for low expression probes or a simplified negative binomial model for high expression probes was used. Differential expression is indicated as the log2 fold change in gene or protein expression and the obtained p-values were adjusted for multiple testing by the Benjamini and Hochberg method (BH. p-value) to control the false discovery rate. Differentially expressed genes and proteins were depicted as volcano plot using R/RStudio v1.0.44.

Measurements of bioenergetics

The bioenergetics from CD8+ T cells were measured in the presence or absence of co- stimulatory MERTK signalling in real-time using an XF-96 Extracellular Flux Analyzer. aCD3/CD28 stimulated CD8+ T cells were grown in the presence or absence of 50 nM PROS1 (serum free medium) or aPROS1 (10 mg/ml, serum-containing medium) for three days prior to use. Cells were resuspended in Seahorse assay media (Seahorse Bioscience, Agilent), supplemented with 1 mM pyruvate, 2 mM glutamine, adjusted to pH 7.4, and subsequently seeded in a Seahorse 96-well plate using Cell-Tak adherent. Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured where after wells were treated with 1 mM oligomycin and 10 mM 2-deoxy-D- glucose to measure ATP turnover and glycolytic capacity from the changes in OCR and ECAR respectively, or with 0.4 pM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) to determine reserve respiratory capacity from change in OCR. All wells received a final treatment with 2mM Antimycin A.

Determination of ATP content

Whole-cell levels of ATP were measured in three-day aCD3/CD28-stimulated CD8+ T cells grown in the presence or absence of 50 nM PROS1 or aPROS1 (10 mg/ml) using a luciferase-based assay (ViaLight MDA Plus Detection Kit, Lonza). Luminescence was quantified in a MicroBeta2 Scintillation Counter (Perkin Elmer).

Expansion of TILs

Expansion conditions included culture medium in the presence or absence of 50 nM PROS1 or 10 mg/ml aPROS1. Outgrowth of‘young’ TILs was measured by manual, unblinded counting of live cells and fold expansion was calculated.

TILs designated for in vitro killing assays were isolated and expanded in vitro from metastatic melanoma lesions with a two-step process as described previously (52). Expanded TILs with high specificity for the HLA-A2 restricted MART-1 /MelanA peptide analogue ELAGIGILTV (>90% specific with peptide-MHC multimer staining) were obtained through electronic sorting of relevant CD8+‘young’ TILs, using peptide-MHC multimers. TILs were subsequently subjected to the rapid expansion protocol, as previously described.

T cell stimulation

For PtdSer-blocking experiments, cells were cultured in medium with 5% human serum with aPROS1 (10 mg/ml) or unconjugated Annexin V (5 mg/ml), respectively.

In vitro killing assay and co-cultures

For co-culture experiments, high TAM receptor-expressing MDA-MB-231 cell line was cultured in serum-free X-VIVO medium for 1 week prior to co-culture. Subsequently, MDA-MB-231 cells were plated in a flat-bottom 96-well plate and left to adhere for approximately 4 hours. Sorted allogenic non-reactive CD8+ T cells and aCD3/CD28 beads were added in a 1 :10 tumor cell:T cell ratio. A PROS1 titration was added in the range of 0-100 nM PROS1. After 4 days of co-culture, supernatants were harvested and analyzed by ELISA. Example 1. MerTK receptor and ligand PROS1 are expressed by TCR-activated human CD8+ T cells.

Human CD8+ T cells were negatively isolated from PBMCs and activated with aCD3/CD28 beads. PROS1 and MerTK surface expression was investigated on unstimulated and aCD3/CD28 activated CD8+ T cells by different methods. Cells were harvested at different time points and analysed by flow cytometry. Moreover, expression of PROS1 mRNA and MerTK mRNA of 3-day aCD3/CD28 activated CD8+

T cells was evaluated by RT-qPCR. MerTK protein expression was analysed on day 3 of activation of PBMCs or CD8+ T cells, as analysed by western blot. We show that activated CD8 T cells express MerTK as well as Prosl . The results are shown in fig. 1.

Example 2. PROS1 positively regulates CD8+ T cell proliferation.

Human CD8+ T cells were cultured in serum-free medium, stained with a proliferation dye (CTV, Cell Trace Violet), and activated for 3 days with aCD3/CD28 in the presence or absence of 50 nM PROS1. Proliferation was measured by flow cytometry and IFN-g as well as TNF-a production was measured in culture supernatants.

Addition of recombinant Prosl improved functionality of CD8 T cells upon activation and promoted proliferation of CD8 T, whereas it had no impact on non-activated T cells. Similarly, addition of Prosl to CD8 T cells upon stimulation induced production of more cytokine, specifically TNF-a and IFN-g. Conversely, blocking of Prosl significantly inhibited proliferation and cytokine release. The results are shown in fig. 2.

Example 3. PROS1 impacts on human melanoma TIL expansion.

Our data suggests that TAM signalling in CD8 T cells represent an important co- stimulatory immune checkpoint. Since both cancer cells and activated T cells express ligands for Prosl , we hypothesize that the TAM axis plays a role in suppressing T cell functionality upon interaction with cancer cells: We propose that cancer cells through upregulated expression of TAMs compete for stimulatory Prosl secreted by T cells, by which cancer cells acquire a dual advantage; prevent the stimulatory TAM signalling in the T cell, and at the same time augment intrinsic oncogenic TAM signalling.

Importantly, this strategy for T cell suppression would potentially inflict on all T cells interacting with cancer cells in the microenvironment, not just T cells expressing individual inhibitory immune check point molecules, e.g., PD-1.

We have conducted an experiment in which we compared the outgrowth of T cells from tumors when we added prosl , blocked prosl and controlled out-growth of T cells from the same tumor suspensions.

TILs from 4 metastatic melanoma patients were isolated, cultured and expanded according to the clinical Rapid Expansion Protocol in the presence or absence of 50 nM PROS1 or aPROS1 mAb. Total live cells were counted and fold expansion was calculated on day 16 and 23 of culture, relative to day 0. Tumor biopsy material was digested and homogenized and equal“tumor soups” were put in culture in normal media, media plus Prosl , and media with blocking of Prosl (aPros). Outgrowth of T cells was given as fold expansion, showing a clear trend towards limited outgrowth when prosl was blocked, and possibly a positive impact when Prosl was added.

The data show that blocking of prosl decreases the out-growth of T cells from biopsy material, supporting the notion of the co-stimulatory signal in T cell proliferation in tissues. The results are shown in fig. 3.

Example 4. MERTK expression by T cell subsets.

To assess if MERTK expression was limited to a certain CD8+ T cell subsets, MERTK expression was analysed on three-day activated CD8+ T cells which were co-stained with subset markers CCR7 and CD45R0. MERTK expression was significantly higher expressed on TCM CD8+ T cells. The results are shown in fig. 5.

Example 5. PROS1 co-stimulation on CD8+ T cells acts via MERTK

To confirm that MERTK upregulation was not due to persistent stimulation by

CD3/CD28, human PBMCs were activated with a pool of 23 peptides derived from cytomegalovirus, Epstein-Barr virus and influenza. Using CD137, recently TCR- activated, naturally occurring, CD8+ T cells can be accurately identified . Only recently activated CD137-positive CD8+ T cells expressed MERTK, in levels similar to

CD3/CD28-activation. The results are shown in fig. 6. Additionally, we found that resting or activated CD8+ T cells expressed only low levels of TYRO3 and do not express AXL (data not shown).

Example 6. PROS1 induces CD8+ T cell cytokine production.

Human CD8+ T cells were cultured in serum-free medium and activated for 3 days with aCD3/CD28 in the presence or absence of 50 nM PROS1. Chemokine CXCL10 and memory IL-7 production was measured in culture supernatants.

Addition of recombinant Prosl improved functionality of CD8 T cells upon activation and induced production of more cytokine, specifically Chemokine CXCL10 and memory IL-7 production. Conversely, blocking of Prosl significantly inhibited cytokine release. The results are shown in fig. 7.

Example 7. PROS1 co-stimulation on CD8+ T cells acts via MERTK.

To verify if the functional changes related to PROS1 were due to signaling through MERTK, MERTK signaling in activated CD8+ T cells was inhibited. We established a siRNA-mediated knockdown of MERTK. As our earlier results have shown that resting T cells do not express MERTK, CD8+ T cells were activated for three days prior to siRNA electroporation. We confirmed that siRNA knockdown resulted in a 70% reduction in MERTK protein levels compared to control. When re-activated, MERTK- knockdown cells produced less IFN-g. Moreover, IL-7, but not IL-15, secretion was significantly decreased. We verified these results using UNC2025, a MERTK-inhibitor which is currently in development for the treatment of leukemia. MERTK inhibition significantly decreased aCD3/CD28-mediated CD8+ T cell proliferation, while no decrease in cell viability was seen. Correspondingly, MERTK inhibition could reverse the positive effects of PROS1 on IFN-g secretion. The results are shown in fig. 8.

Example 8. Expansion of TILs.

We studied if PROS1-signaling had an impact on the primary expansion of‘young’ TILs from metastatic melanoma patients. Treatment of TILs with aPROS1 mAb during the outgrowth phase led to a significant decrease in fold expansion rate. Although PROS1- blocked conditions had a reduced total number of cells, analysis of T cell subsets revealed that no specific subset was depleted. Finally, we studied if joint TAM receptor expression on TILs and cancer cells could affect anti-tumor immunity. For this preliminary study, we screened cancer cells from three metastatic melanoma patients for TAM receptor expression. Next, rapidly expanded (‘REP’) antigen-selected TILs from the highest MERTK-expressing patient were co-cultured with their autologous TAM receptor-expressing tumor cells. Using xCELLigence technology, we could quantitatively follow real-time in vitro tumor-cell killing by autologous TILs. PROS1 alone had no effect on cancer cell growth. Strikingly, PROS1 -supplementation increased TIL-mediated killing, with the PROS1 -mediated increase in cytolysis the most pronounced 48 hours post TIL-addition. The results are shown in fig. 9.

Example 9. PROS1 -MERTK signaling influences CD8+ T cell metabolism

The metabolism of activated CD8+ T cells in the presence or absence of PROS1- MERTK signalling was investigated. Bioenergetic phenotypes are shown to be strongly predictive for CD8+ T cell differentiation into the various memory subsets. Strikingly, the basal respiration rate of PROS1 -blocked cells was significantly decreased to 35% of control-stimulated cells. Accordingly, the ATP turnover of PROS1 -blocked cells was reduced to 31 %. Finally, the reserve respiratory capacity (SRC) of PROS1 -blocked cells was significantly decreased. This contrasts with activated CD8+ T cells supplemented with PROS1 , where no significant changes were found. For both PROS1-blocked and PROS1 -supplemented cells no significant change of glycolytic reserve capacity was discovered. To test whether this effect was due to a lack of overall energy, the whole cell content of ATP was measured, which increased by 140% in PROS1 -blocked CD8+ T cells. This demonstrates that the decreased activity of oxidative phosphorylation and mitochondrial respiration in PROS1 -blocked cells was not a result of starvation of ATP. Taken together, these results indicate that when PROS1-MERTK signaling is absent in activated CD8+ T cells, the mitochondrial respiration capacity, necessary for long-lived T cell memory cells, is significantly decreased. The results are shown in fig. 10. Example 10. PROS1 -MERTK signaling in CD8+ T cells is associated with changes in gene expression

To investigate the intracellular effects of this hitherto unknown PROS1-MERTK axis on CD8+ T cells, the transcriptome of three-day aCD3/CD28-activated CD8+ T cells in the presence or absence of aPROS1 mAb was analyzed. Most differentially upregulated genes in PROS1-blocked cells versus control were LTA, TNFSRF9, IL-2, and IFN-g (Table 1 ). The most differentially downregulated genes were IL4R, DUSP4, CD99, ITGAL and CCL5 (Table 2). These results, along with the observation that activation- associated MERTK expression was more pronounced on T central memory cells demonstrates that PROS1 -MERTK signaling could influence differentiation of“long- lived” memory cells. Table 1. Upregulated genes in PROS1 -blocked cells versus control

LTA

TNFRSF9

IL2

I L7R

TRAF4

TNF

I FNg-protein

SLAM F7

TCF7

FCER1G

Table 2. Downregulated genes in PROS1 -blocked cells versus control

IL4R

CD99

DUSP4

CCL5

ITGAL

TIG IT Example 11. PROS1 -MERTK signaling influences CD8+ T cell metabolism

The gene encoding the transcription factor IRF4 was downregulated in PROS1-blocked cells versus control (Fig. 1 1 ). IRF4 has previously been correlated with metabolic programming of CD8+ T cells where it induces a metabolic shift, essential for antigen- specific responses and effector differentiation and function

Example 12. PROS1 -MERTK signaling affects melanoma TIL outgrowth and functionality

As cancer cell lines express higher levels of TAM receptors, their PROS1 consumption was higher than that of activated T cells (Fig. 14A-B). We aimed to study the possibility of ligand competition in a T cell/cancer cell co-culture using activated CD8+ T cells and the MDA-MB-231 cancer cell line (expressing TAM receptors at high levels) (Fig. 12A- B). Interestingly, scarcity of PROS1 in low concentrations resulted in an increased inhibition by cancer cells on CD8+ T cell activation. Correspondingly, once an excess of PROS1 was present, this PROS1-competive inhibitory effect was abrogated.

PROS1 -supplementation increased TIL-mediated killing in stepwise increments dependent on PROS1 levels (Fig. 12D). Cytokine secretion by T cells was inhibited at low levels of PROS1 as shown in the co-culture experiments, supposedly due to ligand competition (Fig. 12B).

Example 13. Human CD8+ T cells express ligand PROS1 and TAM receptor MERTK upon activation

PROS1 surface staining was party reversible by blockage of PtdSer (Figure 13).

Furthermore, activated CD8+ T cells significantly increased TAM receptor MERTK surface expression from day two onwards, only on PROS1 -positive cells.

References

Andersen, R., M. Donia, E. Ellebaek, T. Holz Borch, P. Kongsted, T. Z. Iversen, L. Rosenkrantz Holmich, H. Westergren Hendel, O. Met, M. H. Andersen, P. thor Straten, and I. M. Svane. 2016. Long-lasting complete responses in patients with metastatic melanoma after adoptive cell therapy with tumor-infiltrating lymphocytes and an attenuated IL-2 regimen. Clinical Cancer Research 22: 3734-3745.

Cabezon et al. 2015. MERTK as negative regulator of human T cell activation. J.

Leukocyte Biology. 97(4): 751-760.

June, C. H. 2015. Serial Killers and Mass Murderers: Engineered T Cells Are up to the Task. Cancer Immunol. Res. 3: 470-472.

Kasikara, C., S. Kumar, S. Kimani, W. I. Tsou, K. Geng, V. Davra, G. Sriram, C. Devoe, K. Q. Nguyen, A. Antes, A. Krantz, G. Rymarczyk, A. Wilczynski, C. Empig, B. D.

Freimark, M. Gray, K. Schlunegger, J.

Rosenberg, S. A., J. C. Yang, R. M. Sherry, U. S. Kammula, M. S. Hughes, G. Q.

Phan, D. E. Citrin, N. P. Restifo, P. F. Robbins, J. R. Wunderlich, K. E. Morton, C. M. Laurencot, S. M. Steinberg, D. E. White, and M. E. Dudley. 201 1. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin. Cancer Res. 17: 4550-4557.

Tran, E., S. Turcotte, A. Gros, P. F. Robbins, Y. C. Lu, M. E. Dudley, J. R. Wunderlich, R. P. Somerville, K. Hogan, C. S. Hinrichs, M. R. Parkhurst, J. C. Yang, and S. A.

Rosenberg. 2014. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 344: 641-645.