Decorin-secreting chimeric antigen receptor immune cells

Abstract

The present invention relates to an immune cell expressing a chimeric antigen receptor (CAR) and a heterologous decorin polypeptide and the medical use thereof in treaing cancer. In particular, the specification teaches a CD19 CAR- expressing T cell that secretes decorin.

Description

[0001] Decorin-Secreting Chimeric Antigen Receptor Immune cells

[0002] Technical field

[0003] The present invention relates, in general terms, to Chimeric Antigen Receptor (CAR) immune cells. In particular, the specification teaches CAR immune cells that secrete decorin.

[0004] Background

[0005] The success of chimeric antigen receptor T cell (CAR-T) immunotherapy shows that it is possible to generate T cells to specifically kill tumor cells expressing the tumor antigens. Recent therapies that have been approved by the United States Food and Drug Administration (FDA) include, for example, tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta) and idecabtagene vicleucel (Abecma). Such therapies have been successful towards haematological malignancies in the clinic. However, the success of CAR- T therapy in solid tumors has been modest. One of the main factors hindering effective CAR- T therapy in solid tumors lies in immune suppression of the CAR-T cells by the tumor microenvironment. The immune suppressive microenvironment is especially crucial and unique to solid tumors limiting the anti-tumor capacity of the CAR-T cells.

[0006] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0007] Brief description of the drawings

[0008] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0009] Figure 1. Expression of CD19 CAR and secretion of recombinant Decorin from CD19- Decorin CAR-T cells. A) Schematic representation of CAR constructs. Gene encoding CD19-CAR (19CAR) and CD19-Decorin CAR (19CAR.D) were inserted at the multiple cloning site (MCS) of the pMSCV vector respectively. B) Expression of GFP and CD19- CAR in transduced-T cells from one representative donor (dot plot; left) and across two different donors (bar chart; right) after sorting for GFP-positive cells. Mock-transduced T cells were transduced with pMSCV vector without any construct. C) Secretion of Decorin from different transduced-T cell groups from one representative donor (left) and across 4 different donors (right). Transduced-T cells were either cultured alone or in co-culture with Daudi or Raji at an effector: target ratio of 1: 1 for 48 hours in serum-free media.

[0010] Figure 2: Specific cytolysis and long-term suppression of tumor cell growth by CD19- Decorin CAR-T cells in vitro. Cytotoxicity assays were conducted against (A) Daudi mCherry, (B) Raji mCherry for 160 hours and (C) CD19-expressing MCF-7 cells for 100 hours at 3 different E:T ratio (1: 1, 1:2 and 1:4). Mean ± SD of 3 technical repeats.

[0011] Figure 3: CAR-T cells suppressed growth of tumor aggregates in a 3-dimensional cytotoxicity assay against CD19-expressing MCF-7 mCherry cell line. A) Representative confocal images illustrate the infiltration of effector cells (green) into the collagen matrix containing target cells (blue) across 3 days. B) Fluorescent signals from CD19-expressing MCF-7 mCherry cells in the confocal images were analysed using Fiji to calculate the area occupied against the entire field imaged. Mean ± SD of 3 technical repeats.

[0012] Figure 4 Prolonged survival of mice treated with 19CAR.hDc T cells compared to PBS or Mock T cell treatments. Immune competent BALB / c mice were inoculated with A20 cells on Day 0. Mice were treated with PBS, Mock-transduced T cells, 19CAR T cells or 19CAR.hDc T cells (3 million cell per mouse) on Day 8, n= 5. Kaplan-Meier curve shows the survival of mice in each of the treatment groups. Mantel-cox log-rank test is used to assess significance, * p < 0.05.

[0013] Figure 5: Human 19CAR.D T cells could overcome the immune suppressive effects of TGF-β . A) Long-term cytotoxicity assay against MCF-7-CD19 mCherry in the presence of 5 ng / ml of TGF-β or equivalent concentration of citric acid as the solvent control. B) Bar graph shows the number of viable target cell at the 80-hour mark of the cytotoxicity assay. One-way ANOVA with Tukey’s multiple comparison test is used to assess significance. All data are shown as mean ± s.d, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. Figure 6: Induction of Treg differentiation in naive CD4+T cells by TGF-β . A) CD45.1+CAR-T cells and CD45.2+naive CD4+T cells were co-cultured at a ratio of 1:20 for five days in the presence of TGF-β, IL-2 and anti-CD3 and anti-CD28 antibodies to stimulate Treg differentiation. B) CAR-T cells and naive CD4+T cells were distinguished by their CD45.1 and CD45.2 expression out of the gated live CD45+population. Cells were further gated by CD4+and CD8+before analysis on the percentages of Tregs (CD25+FoxP3+) and phenotype of each cell population were performed.

[0014] Figure 7: Decorin-secreting CAR-T cells reduced TGF-P-induced Treg differentiation in both naive T cells and CAR-T cells. A) The percentage of CD45.1 and CD45.2 populations after five days of co-culture in the presence of TGF-β to stimulate Treg differentiation. B) Percentage of CD4+and CD8+populations in CD45.1+CAR-T cells when cultured alone or together with naive CD4+T cells. C-D) Representative contour plots (left) illustrate the percentage of CD25+FoxP3+Treg population gated on CD4+population. Bar graph (right) shows the percentage of Treg population in C) CD45.2+cells after co-culture and in D) CAR-T cells when cultured alone or together with naive CD4+T cells. One-way ANOVA with Tukey’s multiple comparison test is used to assess significance. All data are shown as mean ± s.d, n = 3, * p < 0.05, ** p < 0.01, **** p < 0.0001.

[0015] Figure 8: Decorin-secreting CAR-T cells were less exhausted and maintained high levels of cytokine expression after treatment with TGF-β . A) Exhausted CAR-T cells were determined as double positive for the expression of PD-1 and TIM-3, gated on CD8 (left) or CD4+(right). B) Expression of both IFN-y and TNF-a in CD8+(left) or CD4+(right) CAR-T cells. One-way ANOVA with Tukey’s multiple comparison test is used to assess significance. All data are shown as mean ± s.d, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 .

[0016] Figure 9: Treatment with a single dose of 19CAR.hDc T cells showed significantly better control of tumour growth in B16-CD19 melanoma model. A) Immune competent C57BL / 6 mice were injected with 0.5xl06CD19-cxprcssing B 16 tumour cells. Tumour cells were allowed to engraft for 10 days before mice given the respective treatments on Day 0, n=10. B) Rate of tumour growth in individual mice treated with PBS (left), 19CAR T cells (middle) or 19CAR.hDc T cells (right). C) Mean tumour size of mice in respective treatment groups. One-way ANOVA with Tukey’s multiple comparison test is used to assess significance. All data are shown as mean ± s.d, n = 10, ** p < 0.01, *** p < 0.001.

[0017] Figure 10: Decorin-secreting CAR-T cells reduced B16-CD19 tumour growth in syngeneic melanoma model. A) Immune competent C57BL / 6 mice were given subcutaneous injection of B16-CD19 cells. Tumour cells were allowed to engraft for 9 days before mice were given the respective treatments twice on Day 0 and Day 7. B) Size of tumours measured in mice treated with 19CAR or 19CAR.hDc T cells. Unpaired Student’s t-test is used to assess significance, * p < 0.05. All data are shown as mean ± s.d, n = 5.

[0018] Figure 11: Mice treated with 19CAR.hDc T cells had smaller tumours. A) Immune competent C57BL / 6 mice were given subcutaneous injection of CD45.2+B16-CD19 cells. Tumour cells were allowed to engraft for 9 days before mice were given the respective treatments of CD45.1+CAR-T cells twice on Day 0 and Day 7. B) Images of tumours harvested from the mice (top panel); scale bar: 10mm. Tumours were weighed and measured. Unpaired Student’s t-test is used to assess significance, * p < 0.05. All data are shown as mean ± s.d, n = 5.

[0019] Figure 12: CAR-T cells mainly localized to the tumour site. A) Representative dot plots (left) and bar graph (right) illustrate the prevalence of CD45.1+CAR-T cells in the tumour draining lymph node (LN), spleen or tumour of mice. Plots were gated on CD3+cells. Percentage of CD4+and CD8+B) CD45.1+CAR-T cells or C) CD45.2+host T cells detected in the LN, spleen or tumour. All data are shown as mean ± s.d, n = 5.

[0020] Figure 13: Mice treated with 19CAR.hDc T cells had lesser Treg cells at the tumour site. Representative dot plots (left) and bar graph (right) illustrate the percentage of CD25+FoxP3+CAR-T cells at the tumour. Plots were gated on CD4+cells. Unpaired Student’s t- test is used to assess significance, * p < 0.05. All data are shown as mean ± s.d, n = 5.

[0021] Figure 14: Mice treated with 19CAR.hDc T cells had substantially reduced naive T cells and seemingly increased percentage of effector T cells. A) Representative contour plots illustrate the categorization of memory subsets of T cells based on CD44 and CD62L expression. Plots were gated on CD8+cells of tumour samples. Bar graphs show the percentages of naive T cells, effector T cells (Tuff) and central memory T cells (TCM) of B) CD8+CAR-T cells and C) CD8 (upper panel) and CD4+(lower panel) of host T cells. Unpaired Student’s t-test is used to assess significance, * p < 0.05. All data are shown as mean ± s.d, n = 5.

[0022] Detailed description

[0023] Disclosed herein is an immune cell engineered to express a chimeric antigen receptor (CAR) and a heterologous decorin polypeptide.

[0024] To overcome immune suppression, the inventors have generated decorin-secreting CAR-T cells. Decorin is an extracellular matrix protein that binds to a wide range of signaling molecules, from growth factors to growth factor receptors, that mediate immune suppression and cancer progression. Decorin-secreting CAR-T cells have the capacity to modulate the immune suppressive tumor microenvironment and significantly improve the outcome of CAR-T cell therapy in solid tumors. CAR-T cells with the capacity to express and secrete recombinant decorin has the potential to sequester immune suppressive factors in the tumor microenvironment and may lead to improved therapeutic efficacy of CAR-T cell immunotherapy against solid tumors. The CAR-T cells may be engineered to constitutively produce and secrete decorin. Furthermore, the secreted decorin may be targeted to cancer cells.

[0025] The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificial (i.c., man-made) transmembrane protein expressed on a mammalian cell comprising at least an ectodomain, a transmembrane, and an endodomain. Optionally, the CAR protein includes a “spacer” which covalently links the ectodomain to the transmembrane domain. A spacer is often a polypeptide linking the ectodomain to the transmembrane domain via peptide bonds. The CAR is typically expressed on a mammalian lymphocyte. In some embodiments, the CAR is expressed on a mammalian cell such as a T-cell or a tumor infiltrating lymphocyte (TIL). A CAR expressed on a T-ccll is referred to herein as a “CAR T-ccll” or “CAR-T.” In some embodiments the CAR-T is a T helper cell, a cytotoxic T-cell, a natural killer T-cell, a memory T-ccll, a regulatory T-ccll, or a gamma delta T-ccll. When used clinically in, c.g. adoptive cell transfer, a CAR-T with antigen binding specificity to the patient's tumor is typically engineered to express on a native T-cell obtained from the patient. The engineered T-cell expressing the CAR is then infused back into the patient. The CAR-T is thus often an autologous CAR-T although allogeneic CAR-T are included within the scope of the invention. The ectodomain of a CAR comprises an antigen binding region, such as an antibody or antigen binding fragment thereof (e.g. scFv), that specifically binds under physiological conditions with a target antigen, such as a tumor specific antigen. Upon specific binding a biochemical chain of events (i.e., signal transduction) results in modulation of the immunological activity of the CAR-T. Thus, for example, upon specific binding by the antigen binding region of the CAR-T to its target antigen can lead to changes in the immunological activity of the T-cell activity as reflected by changes in cytotoxicity, proliferation or cytokine production. Signal transduction upon CAR-T activation is achieved in some embodiments by the CD3-zeta chain (“CD3-z”) which is involved in signal transduction in native mammalian T-cells. CAR-Ts can further comprises multiple signaling domains such as CD28, 41BB or 0X40, to further modulate immunomodulatory response of the T-cell. CD3-z comprises a conserved motif known as an immunoreceptor tyrosinebased activation motif (IT AM) which is involved in T-cell receptor signal transduction.

[0026] In one embodiment, the transmembrane domain is a transmembrane domain selected from the group consisting of a T cell receptor a chain, a T cell receptor 0 chain, a CD3 zeta chain, a CD28, a CD3c, a CD45, a CD4, a CD5, a CD8, a CD9, a CD16, a CD22, a CD33, a CD37, aCD64, a CD80, a CD86, a CD134, a CD137, an ICOS, a CD154, and a GITR. In one embodiment, the transmembrane domain is CDS.

[0027] In embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDl la / CD18), ICOS (CD278), and 4-1BB (CD137). In one embodiment, the costimulatory domain is a functional signaling domain obtained from 4-1BB (CD137).

[0028] In one embodiment, the activating domain comprises a CD3 zeta activating domain.

[0029] By “antigen-binding molecule” is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigenbinding activity. Representative antigen-binding molecules that are useful in the practice of the present invention include antibodies and their antigen-binding fragments. The term “antigen-binding molecule” includes antibodies and antigen-binding fragments of antibodies. The term “antibody”, as used herein, is understood to mean any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to, or interacts specifically with, the target antigen. The term “antibody” includes full-length immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (c.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR, VH or VH) and a heavy chain constant region. The heavy chain constant region typically comprises three domains - CHI, CH2 and CH3. Each light chain comprises a light chain variable region (which may be abbreviated as LCVR, VL, VK, VK or VL) and a light chain constant region. The light chain constant region will typically comprise one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, also referred to as framework regions (FR). Each VH and VL typically comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antigen-binding molecules described herein may be identical to the FR of germline sequences of the target species (i.e., the species to which the antigen-binding molecules or antigen-binding fragments thereof, as described herein, will be administered). In some embodiments, the FR may be naturally or artificially modified. Whilst it is generally desirable that each of the FR sequences arc identical to FR sequences derived from immunoglobulin molecules of the target species, including to minimize an immune response being raised against the binding molecule upon administration to a subject of the target species, in some embodiments, the antigen-binding molecule, or antigenbinding fragment thereof, may comprise one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in one or more FR from the target species.

[0030] An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called a, 5, e, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to a person skilled in the art.

[0031] As used herein, the term “complementarity determining regions” (CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which arc necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a “complementarity determining region” as defined for example by Kabat (i.e., about residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from a “hypervariable loop” (i.e., about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

[0032] An “antigen-binding site” refers to the site, i.e., one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site. An antigen-binding site of an antigen-binding molecule described herein typically binds specifically to an antigen and more particularly to an epitope of the antigen.

[0033] Tumor antigens arc proteins that arc produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. The selection of the antigen binding domain of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, BCMA, glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1 , MN-CAIX, human telomerase reverse transcriptase, RU 1 , RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostase, pro state- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2 / neu, surviving, telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-l, 1GF-H, 1GF-1 receptor and mesothelin.

[0034] In one embodiment, the CAR of the invention can be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding moiety that specifically binds to an antigen on a tumor cell. In one embodiment, the antigen-binding domain is capable of binding specifically to a tumor / cancer antigen, such as a cell-surface tumor / cancer antigen.

[0035] The type of tumor antigen referred to in the invention may also be a tumor- specific antigen (TSA) or a tumor- associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at extremely low levels on normal cells, but which are expressed at much higher levels on tumor cells.

[0036] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-l / MelanA (MART-1), glOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, 1GH- IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein -based antigens include TSP-180, MAGE-4, MAGE-5, MAGE- 6, RAGE, NY-ESO, pl85crbB2, pl80crbB- 3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta- Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4 (791Tgp72) alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\ I , CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein, Acyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0037] In an embodiment, the antigen is human CD 19. The present disclosure extends to antigen binding molecules that bind specifically to native CD19 (i.e., naturally-occurring CD19), as well as to valiants thereof. Such valiants may include CD 19 molecules that differ from a naturally-occurring (wild-type) molecule by one or more amino acid substitutions, deletions and I or insertions. Variant CD19 molecules of this type may be naturally-occurring or synthetic (e.g., recombinant) forms. It is to be understood, however, that in one embodiment, the antigen-binding molecules described herein bind specifically to a native form of CD19, whether of a human or non-human species.

[0038] The terns “antigen-binding fragment”, “antigen-binding portion”, “antigen-binding domain” and “antigen- binding site” are used interchangeably herein to refer to a part of an antigen-binding molecule that participates in antigen-binding. These terms include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0039] Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0040] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain- specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, one- armed antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression “antigen-binding fragment” as used herein.

[0041] An antigen- binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH- VH, VH-VL or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0042] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the present invention include: (i) Vn-Cnl; (ii) VL-CH2-CH3; and (xiv) VL-CL- In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigenbinding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalcnt association with one another and / or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)). A multispecific antigen-binding molecule will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0043] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antigen binding molecule to antigen. The variable domains of the heavy chain and light chain (Vn and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0044] The term “constant domains” or “constant region” as used herein denotes the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in binding of an antigen, but exhibits various immune effector functions.

[0045] In one embodiment, the antigen-binding molecule or antigen-binding fragment thereof is modified for compatibility with the target species. Thus, in an embodiment, the antigenbinding molecule or antigen-binding fragment thereof is humanized or felinized.

[0046] By “humanized” is meant that the antigen- binding molecule comprises an amino acid sequence that is compatible with humans, such that the amino acid sequence is unlikely to be seen as foreign by the immune system of a human subject. In an embodiment, the humanized antigen-binding molecule comprises one or more immunoglobulin framework regions derived from one or more human immunoglobulin molecules. In some embodiments, all of the framework regions of the humanized antigen-binding molecule will be derived from one or more human immunoglobulin molecules. The humanized antibody may optionally comprise an immunoglobulin heavy chain constant region derived from a human immunoglobulin molecule.

[0047] The phrase “specifically binds” or “specific binding” refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity to that determinant. This selection may be achieved by subtracting out antigen-binding molecules that cross-react with other molecules. A variety of immunoassay formats may be used to select antigen- binding molecules (e.g., immunoglobulins) such that they are specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual ( 1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods of determining binding affinity and specificity are also well known in the art (see, for example, Harlow and Lane, supra); Friefelder, “Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co. 1976)).

[0048] “Affinity” or “binding affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antigen-binding molecule) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair e.g., an antigen-binding molecule. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by common methods known in the art, including those described herein. A particular method for measuring affinity is Surface Plasmon Resonance (SPR).

[0049] The terms "polypeptide", "peptide", or "protein" are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acid residues are usually in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. The terms “heterologous polynucleotide,” “foreign polynucleotide” and “exogenous polynucleotide” are used interchangeably to refer to any nucleic acid (e.g., a gene sequence or regulatory sequence) which is introduced into the genome of an organism by experimental manipulations and may include gene sequences found in that organism so long as the introduced gene contains some modification (e.g., a point mutation, the presence of a endonuclease cleavage site, the presence of a loxP site, etc.) relative to the naturally- occurring gene(s).

[0050] The terms “heterologous polypeptide,” “foreign polypeptide” and “exogenous polypeptide” are used interchangeably to refer to any peptide or polypeptide which is encoded by a heterologous polynucleotide,” “foreign polynucleotide” and “exogenous polynucleotide,” as defined above.

[0051] As used herein, the term “modified antibody” includes synthetic forms of antibodies which are altered such that they are not naturally occurring, e.g., antibodies that comprise at least two heavy chain portions but not two complete heavy chains (such as domain deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and arc described, e.g., in U.S. Pat. No. 5,892,019. In addition, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen).

[0052] The antigen-binding molecule may comprise a) a heavy chain variable (VH) region comprising the VHCDR1 amino acid sequence of GVSLPDYGVS (SEQ ID NO: 1 ), the VHCDR2 amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 2) and the VHCDR3 amino acid sequence of HYYYGGSYAMDY (SEQ ID NO: 3); and b) a light chain variable (VL) region comprising the VLCDR1 amino acid sequence of RASQDISKYLN (SEQ ID NO: 4), the VLCDR2 amino acid sequence of HTSRLHS (SEQ ID NO: 5) and the VLCDR3 amino acid sequence of QQGNTLPYT (SEQ ID NO: 6).

[0053] The antigen-binding fragment may be an antibody or antigen-binding fragment thereof. The antibody or antigen binding fragment thereof may be a full-length antibody, a substantially intact antibody, a Fab fragment, a scFab, a Fab’, a single chain variable fragment (scFv) or a one-armed antibody.

[0054] In one embodiment, the antibody or antigen-binding molecule therefore is humanized.

[0055] The antigen-binding molecule may comprise: a) a VH region comprising an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to:

[0056] EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET TYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTATYYCAKHYYYGGSYAMDYW GQGTSVTVSS (SEQ ID NO: 7); and b) a VL region comprising an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to:

[0057] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 8).

[0058] The antigen-binding molecule may comprise CDR sequence from a BCMA CAR molecule described in WO2021091978A1. The antigen-binding molecule may comprise a) a heavy chain variable (VH) region comprising the VHCDR1 amino acid sequence of DYSIN (SEQ ID NO: 18), the VHCDR2 amino acid sequence of WINTETREPAYAYDFRG (SEQ ID NO: 19) and the VHCDR3 amino acid sequence of DYSYAMDY (SEQ ID NO: 20); and b) a light chain variable (VL) region comprising the VLCDR1 amino acid sequence of RASESVTILGSHLIH (SEQ ID NO: 21 ), the VLCDR2 amino acid sequence of LASNVQT (SEQ ID NO: 22) and the VLCDR3 amino acid sequence of LQSRTIPRT (SEQ ID NO: 23).

[0059] The term “sequence identity” as used herein refers to the extent that sequences are identical on a nuclcotidc-by-nuclcotidc basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0060] The antigen-binding molecule as defined herein may comprise one or more conservative amino acid substitutions.

[0061] A “conservative amino acid substitution” is to be understood as meaning a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as shown in the table "Amino Acid Classification" , below:

[0062] AMINO ACID SUB-CLASSIFICATION

[0063] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity.

[0064] Conservative substitutions are also shown in the table below (EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS'). Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants can be screened for their ability to bind specifically to an antigen using methods known to persons skilled in the art, including those methods described elsewhere herein.

[0065] EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS

[0066] Representative antigen-binding molecules contemplated by the present disclosure include full-length immunoglobulins and antigen-binding fragments, including recombinant antigen-binding molecules, which may be monovalent or multivalent, monospecific or multispecific.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody, a substantially intact antibody, a Fab fragment, scFab, Fab’, a single chain variable fragment (scFv) or a one-armed antibody.

[0068] In one embodiment, the antigen-binding molecule of the present invention is a monovalent antigen-binding molecule. Non-limiting monovalent antigen-binding molecules include: a Fab fragment consisting of VL, VH, CL and Cnl domains; a Fab’ fragment consisting of VL, VH, CL and Cnl domains, as well as a portion of a CH2 domain; an Fd fragment consisting of VH and CHI domains; an Fv fragment consisting of VL and VH domains of a single arm of an antibody; a single-chain antibody molecule (e.g., scFab and scFv); a single domain antibody (dAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and a one-armed antibody, such as described in US20080063641 (Genentech) or other monovalent antibody, e.g., such as described in W02007048037 (Amgen).

[0069] In one embodiment, a monovalent antigen-binding molecule comprises an Fv fragment. The Fv fragment is the smallest unit of an immunoglobulin molecule with function in antigenbinding activities. An antigen-binding molecule in scFv (single chain fragment variable) format consists of variable regions of heavy (VH) and light (VL) chains, which are joined together by a flexible peptide linker that can be easily expressed in functional form in an expression host such as E. coli and mammalian cells, allowing protein engineering to improve the properties of scFv such as increase of affinity and alteration of specificity (Ahmed et al., 2012. Clin Dev Immunol. 2012:980250). Representative examples of linker sequences are described in Section 4.5 infra. In the scFv construction, the order of the domains can be either Vn-linker-Vr or Vr-linker-Vn and both orientations can be applied.

[0070] In some embodiments, the linker sequences used in scFvs are multimers of the pentapeptide GGGGS (SEQ ID NO: 9) (or G4S or Gly4Ser). Those include the 15-mer (G4S)3 (Huston et al., 1988. Proc Natl Acad Sci USA. 85(16), 5879-83), the 18-mer GGSSRSSSSGGGGSGGGG (SEQ ID NO: 10) (Andris-Widhopf ct al., “Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.” Cold Spring Harbor Protocols, 2011(9)) and the 20-mer (G4S)4 (Schaefer et al., “Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly.” In: Antibody Engineering, R. Kontermann and S. Diibel, Springer Verlag, Heidelberg, Germany (2010) pp. 21-44). Many other sequences have been proposed, including sequences with added functionalities, e.g., an epitope tag or an encoding sequence containing a Cre-Lox recombination site or sequences improving scFv properties, often in the context of particular antibody sequences.

[0071] Cloning of the scFv is usually done by a two-step overlapping PCR (also known as Splicing by Overlap Extension or SOE-PCR), as described (Schaefer et al., 2010, supra). The Vu and VL domains are first amplified and gel-purified and secondarily assembled in a single step of assembly PCR. The linker is generated either by overlapping of the two inner primers or by adding a linker primer whose sequence covers the entire linker or more (three-fragment assembly PCR).

[0072] In one embodiment, the heterologous decorin polypeptide is fused to a homing (or targeting) molecule or peptide. The homing molecule or peptide may comprise a secretory signal peptide of CARSKNKDC (SEQ ID NO: 1 1). The homing molecule or peptide may be one that is described in WO2016172515A1, which is entirely incorporated herein by reference. The homing molecule or peptide may be positioned downstream of the decorin polypeptide. The heterologous decorin polypeptide may be a mammalian decorin polypeptide that is fused to a homing (or targeting) molecule or peptide. In one embodiment, the heterologous decorin polypeptide is a human decorin polypeptide that is fused to a homing (or targeting) molecule or peptide. The term "homing molecule" as used herein, means any molecule that selectively homes in vivo to specified target sites, such as cells or tissues, in preference to normal or other nontarget sites, cells, or tissues. Similarly, the term "homing peptide" or "homing peptidomimetic" means a peptide that selectively homes in vivo to specified target sites, such as cells or tissues, in preference to normal or other non-target sites, cells, or tissues. It is understood that a homing molecule that selectively homes in vivo to, for example, tumors can home to all tumors or can exhibit preferential homing to one or a subset of tumor types.

[0073] By "selectively homes" it is meant that in vivo, the homing molecule binds preferentially to the target as compared to non-target. For example, the homing molecule can bind preferentially to certain molecules, proteins, cells, tissues, etc. as compared to other molecules, proteins, cells, tissues, etc. For example, the homing molecule can bind preferentially to tumor vasculature or one or more tumors as compared to non-tumoral tissue. Such a homing molecule can selectively home, for example, to tumors. Selective homing to, for example, certain molecules, proteins, cells, tissues, etc. generally is characterized by at least a two-fold greater localization the molecules, proteins, cells, tissues, etc. (or other target), as compared to other certain molecules, proteins, cells, tissues, etc. A homing molecule can be characterized by, for example, 5-fold, 10-fold, 20-fold or more preferential localization to the target as compared to one or more non-targets. For example, a homing molecule can be characterized by, for example, 5-fold, 10-fold, 20-fold or more preferential localization to tumor vasculature as compared to vasculature of several or many tissue types of non-tumoral tissue, or as compared to vasculature of most or all non-tumoral tissue. As another example, a homing molecule can be characterized by, for example, 5-fold, 10-fold, 20-fold or more preferential localization to tumors as compared to several or many tissue types of non-tumoral tissue, or as compared to-most or all non-tumoral tissue. Thus, it is understood that, in some cases, a homing molecule homes, in part, to one or more normal organs in addition to homing to the target tissue. Selective homing can also be referred to as targeting. The molecules, proteins, cells, tissues, etc. that arc targeted by homing molecules can be referred to as targeted molecules, proteins, cells, tissues, etc.

[0074] In one embodiment, the heterologous decorin polypeptide comprises an amino acid sequence having at least 70% (including at least 71% to 99% and all integer percentages therebetween) sequence identity to: MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPF RCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALIL

[0075] VNNKTSKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKV

[0076] TFNGLNQMIVIELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTEL HLDGNK1SRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKL

[0077] TRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQY WEIQPSTFRCVYVRSAIQLGNYK (SEQ ID NO: 12) or a fragment thereof.

[0078] In one embodiment, the heterologous decorin polypeptide comprises an amino acid sequence having at least 70% (including at least 71 % to 99% and all integer percentages therebetween) sequence identity to:

[0079] MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGPVCPF RCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALIL VNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKV

[0080] TFNGLNQMIVIELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTEL HLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKL

[0081] TRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQY WEIQPSTFRCVYVRSAIQLGNYKGSEFCARSKNKDCVDLVPRGSS (SEQ ID NO: 13) or a fragment thereof.

[0082] In one embodiment, the transmembrane (and hinge) domain comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence of:

[0083] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYC (SEQ ID NO: 14).

[0084] In one embodiment, the co-stimulation signaling region comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence of:

[0085] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 15).

[0086] In one embodiment, the CD3f signaling domain comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence of:

[0087] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSE1GMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (SEQ ID NO: 16). The immune cell may comprise a nucleic acid molecule encoding a CAR and a heterologous decorin polypeptide. The CAR and the heterologous decorin polypeptide may be expressed as a fusion protein. The CAR and the heterologous decorin polypeptide may be expressed as a fusion protein that is separated by a 2A self-cleaving peptide. The nucleic acid molecule may further encoded a truncated EGFR.

[0088] The immune cell may comprise an expression construct comprising a nucleic acid molecule encoding a CAR and a heterologous decorin polypeptide.

[0089] The nucleic acid molecule may comprise a nucleic acid sequence having at least 90% sequence identity to:

[0090] CGCGGATCCGGCTTCCACCATGGCCCTGCCCGTCACCGCCCTGCTGCTGCCCCTGGCTCTGC TGCTGCACGCTGCTAGACCCGACATTCAGATGACTCAGACAACAAGCTCCCTGTCCGCCTC TCTGGGCGACAGGGTGACCATCTCTTGCCGCGCCAGCCAGGATATCTCCAAGTATCTGAAC TGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTATCACACATCTCGGCTGC ACAGCGGCGTGCCTTCCAGATTCAGCGGCTCCGGCTCTGGCACCGACTACTCTCTGACAAT CAGCAACCTGGAGCAGGAGGATATCGCCACCTATTTCTGCCAGCAGGGCAATACCCTGCC ATACACATTTGGCGGCGGCACCAAGCTGGAGATCACCGGAGGAGGAGGAAGCGGAGGAG GAGGATCCGGCGGCGGCGGCTCTGAGGTGAAGCTGCAGGAGTCCGGACCTGGCCTGGTGG CACCAAGCCAGTCCCTGTCTGTGACCTGTACAGTGTCCGGCGTGTCTCTGCCCGACTACGG CGTGTCTTGGATCCGGCAGCCCCCTAGAAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGG CAGCGAGACAACATACTATAATTCCGCCCTGAAGTCTAGGCTGACCATCATCAAGGATAAC AGCAAGTCCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGATACAGCCATCTAC TATTGCGCCAAGCACTACTATTACGGCGGCAGCTATGCCATGGACTACTGGGGCCAGGGCA CCAGCGTGACAGTGTCTAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCA CCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCG CAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGG GACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAG AAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAG ATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGT TCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTG AGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC CCTTCACATGCAGGCCCTGCCCCCTCGCGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAG CAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGAAGGCCACTATCATCCTCCTTCTGC TTGCACAAGTTTCCTGGGCTGGACCGTTTCAACAGAGAGGCTTATTTGACTTTATGCTAGA AGATGAGGCTTCTGGGATAGGCCCAGAAGTTCCTGATGACCGCGACTTCGAGCCCTCCCTA GGCCCAGTGTGCCCCTTCCGCTGTCAATGCCATCTTCGAGTGGTCCAGTGTTCTGATTTGGG TCTGGACAAAGTGCCAAAGGATCTTCCCCCTGACACAACTCTGCTAGACCTGCAAAACAAC AAAATAACCGAAATCAAAGATGGAGACTTTAAGAACCTGAAGAACCTTCACGCATTGATT CTTGTCAACAATAAAATTAGCAAAGTTAGTCCTGGAGCATTTACACCTTTGGTGAAGTTGG AACGACTTTATCTGTCCAAGAATCAGCTGAAGGAATTGCCAGAAAAAATGCCCAAAACTC TTCAGGAGCTGCGTGCCCATGAGAATGAGATCACCAAAGTGCGAAAAGTTACTTTCAATG GACTGAACCAGATGATTGTCATAGAACTGGGCACCAATCCGCTGAAGAGCTCAGGAATTG AAAATGGGGCTTTCCAGGGAATGAAGAAGCTCTCCTACATCCGCATTGCTGATACCAATAT CACCAGCATTCCTCAAGGTCTTCCTCCTTCCCTTACGGAATTACATCTTGATGGCAACAAAA TCAGCAGAGTTGATGCAGCTAGCCTGAAAGGACTGAATAATTTGGCTAAGTTGGGATTGA GTTTCAACAGCATCTCTGCTGTTGACAATGGCTCTCTGGCCAACACGCCTCATCTGAGGGA GCTTCACTTGGACAACAACAAGCTTACCAGAGTACCTGGTGGGCTGGCAGAGCATAAGTA CATCCAGGTTGTCTACCTTCATAACAACAATATCTCTGTAGTTGGATCAAGTGACTTCTGCC CACCTGGACACAACACCAAAAAGGCTTCTTATTCGGGTGTGAGTCTTTTCAGCAACCCGGT CCAGTACTGGGAGATACAGCCATCCACCTTCAGATGTGTCTACGTGCGCTCTGCCATTCAA CTCGGAAACTATAAGGGTAGCGAATTTTGTGCACGTAGCAAGAACAAAGATTGCGTTGAT CTGGTGCCGCGTGGTAGCAGTCATCACCACCATCACCATTGACGCGGATCCGCG (SEQ ID NO: 17).

[0091] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system, Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0092] The nucleic acid sequence may allow for the expression of CAR and decorin under one promoter, with the CAR targeted for surface expression while allowing decorin to be cleaved with the sclf-clcavagc peptide and be targeted for secretion.

[0093] The term “polynucleotide” or “nucleic acid” are used interchangeably herein to refer to a polymer of nucleotides, which can be mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

[0094] By “vector” is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or virus, into which a nucleic acid sequence may be inserted or cloned. A vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a lineal' or closed circular plasmid, an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

[0095] The term “construct” refers to a recombinant genetic molecule including one or more isolated nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin arc assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that arc not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present invention will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct, such as, for example, a target nucleic acid sequence or a modulator nucleic acid sequence. Such elements may include control elements or regulatory sequences such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1 , 2, and 3. ,T. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.

[0096] By “control element”, “control sequence”, "regulatory sequence" and the like, as used herein, mean a nucleic acid sequence (c.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell. The control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cis-acting sequence such as an operator sequence and a ribosome binding site. Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.

[0097] A "constitutive" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0098] An "inducible" promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0099] Disclosed herein is an expression construct comprising a nucleic acid molecule encoding a CAR and a heterologous decorin polypeptide.

[0100] Disclosed herein is a vector comprising the expression construct as defined herein.

[0101] The vector may be a viral vector, such as a retrovirus. In one embodiment, the viral vector is Murine stem cell virus (MSCV).

[0102] In one embodiment, there is provided a method of preparing an immune cell as defined herein. The method may comprising contacting an immune cell with a vector as defined herein to generated an immune cell as defined herein.

[0103] Disclosed herein is an immune cell as defined herein for use as a medicament.

[0104] Disclosed herein is a method of treating cancer in a subject, wherein the method comprises administering an immune cell as defined herein to the subject.

[0105] Disclosed herein is the use of an immune cell as defined herein in the manufacture of a medicament for treating cancer.

[0106] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. By “non-metastatic” is meant a cancer that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. The term "metastatic cancer" refers to cancer that has spread or is capable of spreading from one part of the body to another. Generally, a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer, and occasionally a Stage III cancer. A metastatic cancer, on the other hand, is usually a stage IV cancer.

[0107] The term "cancer" includes but is not limited to, breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular- melanoma), testicular cancer, oral cancer, pharyngeal cancer or a combination thereof.

[0108] In one embodiment, the cancer cell is a solid or haematological cancer cell.

[0109] The term “solid cancer” may refer to one or more of breast cancer, large intestinal cancer, lung cancer, small cell lung cancer, gastric (stomach) cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and / or neck cancer, cutaneous or intraocular melanoma, uterine sarcoma, ovarian cancer, rectal or colorectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vulval cancer, squamous cell carcinoma, vaginal carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue tumor, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, glioma, astrocytoma, glioblastoma multiforme, primary CNS lymphoma, bone marrow tumor, brain stem nerve gliomas, pituitary adenoma, uveal melanoma (also known as intraocular melanoma), testicular cancer, oral cancer, pharyngeal cancer, sarcomas or a combination thereof.

[0110] The term “haematological cancer’ may refer to one or more of leukemia, lymphoma. Chronic Myeloproliferative Disorders, Langerhans Cell Histiocytosis, Multiple Myeloma / Plasma Cell Neoplasm, Myelodysplasia Syndromes, Myelodysplastic / Myeloproliferative Neoplasms or a combination thereof. In some embodiments, leukemia is any one or more of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Hairy Cell Leukemia (HCL) or a combination thereof. In some embodiments, lymphoma is any one or more of AIDS-Related Lymphoma, Cutaneous T- Cell Lymphoma, Hodgkin Lymphoma, Mycosis Fungoides, Non-Hodgkin Lymphoma, Primary Central Nervous System Lymphoma, Sezary Syndrome, T-Cell Lymphoma, Cutaneous, Waldenstrom Macroglobulinemia, B cell lymphoma or a combination thereof.

[0111] The cancer may be a cancer associated with the undesired expression of a cancer antigen.

[0112] The term “treating" as used herein may refer to (1) delaying the appearance of one or more symptoms of the condition; (2) inhibiting the development of the condition or one or more symptoms of the condition; (3) relieving the condition, i.e.. causing regression of the condition or at least one or more symptoms of the condition; and / or (4) causing a decrease in the severity of the condition or of one or more symptoms of the condition.

[0113] The terms “subject”, “patient”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such as from the genus Macaca (e.g., cynomolgus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In another embodiment, the subject is a human subject.

[0114] The methods as disclosed herein may comprises the administration of a “therapeutically effective amount” of an agent to a subject. As used herein the term "therapeutically effective amount” includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.

[0115] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0116] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0117] Throughout this specification and the statements which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0118] Throughout this specification and the statements which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of' will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0119] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0120] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0122] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0123] EXAMPLES

[0124] Methods

[0125] Plasmid vectors & constructs

[0126] The retroviral transfer plasmid pMIG II and packaging plasmid pEQ-Pam3(-E) was a gift from Dario Vignali, and the envelop plasmid pRDF was a gift from Dario Campana. To produce the 19CAR construct, gene encoding aCD19-41BB-CD3^ CAR was synthesized and inserted into the multiple cloning site of pMIG 11 (flanked by EcoRI and Xhol restriction sites) by GenScript. To produce the gene construct for 19CAR.D, Decorin-CARSKNKDC (SEQ ID NO: 11) was linked to the 3’ end of aCD19-41BB-CD3g CAR, separated by a P2A self-cleaving peptide1. The production of Decorin-CARSKNKDC (SEQ ID NO: 11) was previously described in the work by Jarvinen and Ruoslahti2.

[0127] Expansion of human T cells

[0128] Peripheral blood samples were obtained from discarded anonymized by-products of platelet donations from healthy adult donors at the Health Science Authority Blood Bank, Singapore. Ficoll density centrifugation was performed to obtain the fraction containing the peripheral blood mononuclear cells (PBMCs). To expand for T cells, PBMCs were seeded into 12-well plate at a concentration of IxlO6cells / ml. Wells were precoated with 10 ug / ml of Ultra- LEAF purified anti-human CD3 antibody (HlT3a; Biolegend) and 2.5 ug / ml Ultra-LEAF purified anti-human CD28 antibody (CD28.2; Biolegend). Cells were cultured in conditioned RPMI supplemented with lOOIU / ml of IL-2 every two days.

[0129] Retroviral transduction of cells

[0130] The viral packaging cell line, HEK 293T cells, were seeded into 100x20 mm cell culture dishes at a concentration of IxlO6cells for every 10 ml of conditioned DMEM 18 hours prior to the transfection of the cells. Cell were transfected with the pMIG II transfer vector containing the respective genetic constructs, the pEQ-Pam3(-E) packaging vector and pRDF envelop vector at a ratio of 3.5 : 3.5 : 3.0 pg. The procedure was done using calcium phosphate transfection kit (Sigma- Aldrich), according to the manufacturer’s protocol. Culture media in the HEK 293T cultures were replaced with conditioned RPMI 24 hours after the transfection.

[0131] Viral supernatant in the HEK 293T cell culture dishes were harve ted and fresh conditioned RPMI was added back to the cultures. The viral supernatant was filtered through a 0.2 pm filter (Satorius) and 3 ml was dispensed into tubes pre-coated with RetroNectin (TaKaRa). Tubes were centrifuged three times for 2600 rpm for 20 minutes every 2 hours. Viral supernatant in the tubes were then discarded and IxlO6expanded T cells or cell lines to be transduced were seeded in 2 ml of their respective conditioned media with the necessary supplements. Transduction was repeated 12 hours after the initial harvesting of the viral supernatant. Cells in the tubes were centrifuged at 1200 rpm for 5 minutes, and the old supernatant was discarded while the second harvest was filtered and added in. Transductions were repeated for a total of 6 times to maximize transduction efficiency.

[0132] Flow Cytometry

[0133] To prepare cells for flow cytometry analysis, a maximum of IxlO6cells were resuspended in 100 pl of phosphate- buffered saline (PBS) before the addition of the respective antibodies. Cells were stained for 10 minutes at room temperature in the dark, before washing twice and analysis using the LSRFortessa X-20 flow cytometer.

[0134] Collection and quantification of secreted Decorin by ELISA

[0135] To detect the expression of Decorin by transduced cells, 0.5xl06transduced cells were seeded with or without the respective target cells Daudi and Raji at a 1: 1 ratio. Cells were cultured for 48 hours in 2 ml of serum-free media to prevent background levels of Decorin from being measured. Cell culture supernatant was then harvested, centrifuged at 1200 rpm for 5 minutes followed by 12 000 rpm for 15 minutes in 4 °C to remove all cells and debris. Harvested supernatants were stored at -80 °C till further downstream application.

[0136] The levels of secreted Decorin in the harvested supernatant was measured using the Human Decorin DuoSet ELISA kit (R&D Systems) according to the manufacturer’s instructions. Fluorescence readings were obtained with the Sunrise plate reader (Tecan).

[0137] Long-term cytotoxicity assays

[0138] Effector cells and mCherry-expressing target cell lines were seeded at various E:T ratio into 96-well flat bottom plate. Adherent target cell lines were seeded 6 hours prior to allow for attachment onto the well. IL-2 was supplemented at 200 lU / ml every 3 days with minimal disruption to cells in the wells. The assay was incubated in the Incucyte S3 Live-Cell Analysis System (Sartorius) and imaged every 4 hours. 3 -dimensional cytotoxicity assays

[0139] To form spherical tumor aggregate, mCherry-expressing MCF-7 CD19 cells were seeded in custom-made laser-cut petri dishes coated with 0.2% Pluronic F127 to prevent cells from attaching to the plate. The plate was incubated for 2 days for aggregate formation.

[0140] Tumor aggregates were subsequently filtered through cell strainers to harvest for aggregates that were between 40 to 100 pm in diameter. Harvested aggregates were resuspended in 2.5 mg / ml of collagen solution at pH 7.4 and seeded into the gel channel of cell culture microfluidic chips (AIM Biotech) and incubated overnight.

[0141] Microfluidic chips were imaged using Opera Phenix High Content Screen System (PerkinElmer) before the addition of effector cells on one side of the media port. Microfluidic chips were imaged daily to monitor the growth of tumor aggregates.

[0142] EXAMPLE 1

[0143] Expression of CD 19 CAR and recombinant Decorin in transduced T cells

[0144] A construct containing the conventional CD19-41 BB-CD3^ and Decorin-CARSKNKDC (SEQ ID NO: 11) recombinant fusion protein separated by 2A self-cleaving peptides (termed 19CAR.D; Figure 1A) was developed. To determine that the constructs could be expressed in T cells, expanded human T cells were transduced with GFP only (Mock; negative control), conventional anti-CD19-CAR (termed 19CAR; positive control) or with 19CAR.D. Transduced T cells that were GFP-positive were sorted for and flow cytometry analysis showed that CAR was expressed in similar percentages across both CAR-T cell groups (Figure IB).

[0145] To determine that the recombinant Decorin could be expressed and secreted by CD19- Dccorin CAR-T cells, CAR-T cells were cultured in scrum-free media for 48 hours followed by detection of Decorin levels in the supernatant by ELISA. Serum-free media was used to avoid background levels of Decorin present in the serum. Decorin was secreted by CD 19- Decorin CAR-T cells and not mock-transduced T cells or CD 19 CAR-T cells (Figure 1C). Across four different donors, Decorin secretion was significantly higher when target cells (Daudi or Raji) were added to the culture at 1: 1 effector to target (E:T) ratio.

[0146] Example 2

[0147] Expression of recombinant Decorin did not negatively affect cytotoxic capacity of CAR-T cells

[0148] Long-term cytotoxicity assays against several CD19-expressing cell lines were performed to ensure that the CD19 CAR expressed on CAR-T cells were functional and to investigate whether the expression of recombinant Decorin would augment the cytotoxic capacity of CAR-T cells. Against mCherry-expressing target cells Daudi and Raji, both groups of CAR- T cells completely eradicated and suppressed the growth of tumor cell lines after 160 hours of culture across different E:T ratios, while tumor cells continued to grow when mock- transduced T cells were present (Figure 2A-B). Similarly, when co-cultured with CD19 and mCherry-expressing MCF-7, tumor growth was suppressed when either group of CAR-T cells from two independent donor was present (Figure 2C). Importantly, across the various E:T ratio and target cell lines, both CD19 CAR-T cells and CD19-Decorin CAR-T cells were observed to elicit comparable levels of cytotoxicity. Together, the results showed that both groups of CAR-T cells demonstrated comparable cytotoxic capacity and that the expression of recombinant Decorin did not adversely impact CAR-T cell function when immune suppressive factors were absent.

[0149] Example 3

[0150] CD19-Decorin CAR-T cell displayed similar cytotoxic capacity in 3-dimensional killing assay

[0151] The effects of recombinant Decorin secreted by CAR-T cells act on the complex and dynamic network between the tumor cells and its microenvironment. It would be difficult to model and replicate those interactions in the 2-dimcnsional assays performed conventionally, and a 3-dimensional (3D) system would be required to recapitulate this interaction. To ensure that CAR-T cells could effectively infiltrate into the tumor site and eliminate the tumor cells in a 3D environment, cytotoxic capacity of CAR-T cells were tested against MCF-7 CD 19 mCherry tumor aggregates embedded in collagen matrix and seeded in cell culture microfluidic chips. Both groups of CAR-T cells elicited comparable levels of cytotoxicity against the tumor aggregates after three days of culture (Figure 3A-B). Notably, CAR-T cells were able to suppress the growth of the tumor aggregates compared to mock- transduced T cells, where an increase in tumor area occupied by the aggregates was observed.

[0152] Example 4

[0153] Mouse CD19 CAR-T cell expressing human Decorin displayed superior anti-tumor efficacy

[0154] To fully elucidate the therapeutic benefits of Decorin-secreting CAR-T cells, an immune competent syngeneic mouse model was used. Murine B cell lymphoma A20 was established in BALB / c mice via intravenous infusion (i.v.). followed by treatment with PBS or the respective CAR-T cell groups via i.v. All mice treated with PBS, mock-transduced T cells or mouse CD 19 CAR-T cells succumbed to their tumour burden (Figure 4). Notably, mice treated with CD19-Decorin CAR-T was observed to have prolonged survival, with one out of five mice observed to be tumor- free after 100 days.

[0155] Taken together, this study has generated recombinant Decorin- secreting CD19 CAR-T cells and has shown that both the CAR and Decorin could be expressed and secreted respectively. Levels of secreted recombinant Decorin was observed to increase when target cells were present. When comparing the cytotoxic capacity of CD19-Decorin CAR-T cells with that of conventional CD19-CAR-T cells used in the clinic, similar levels of cytotoxicity was elicited by both groups of CAR-T cells in a proof-of-principle long-term killing assay without any immune suppressive pressures. This implies that the secretion of recombinant Decorin would not impair the cytotoxic function of CAR-T cells. Both groups of CAR-T cells also suppressed CD19-expressing MCF-7 tumor aggregate growth in a 3-D killing assay. Since the main function of Decorin is to sequester immune suppressive factors in the tumor microenvironment, immune suppressive components such as TGF- are added to the collagen matrix in future experiments to mimic the tumor microenvironment. It is hypothesized that the recombinant Decorin secreted by CD19-Decorin CAR-T cells should sequester those added suppressive components and reduce the levels of immune suppression, leading to enhanced cytotoxicity from CD19-Decorin CAR-T cells compared to the conventional CD 19 CAR-T cells. Finally, only treatment with mouse CD19-Decorin CAR-T cells showed improved survival in vivo in a murine lymphoma model, demonstrating the potential therapeutic benefits of Decorin-secreting CAR-T cells. Mouse CD19-Decorin CAR-T will be tested in different animal models to further demonstrate its therapeutic efficacy and applicability for the treatment of solid tumors.

[0156] EXAMPLE 5

[0157] Human 19CAR.D T cells are resistant to the immune suppression mediated by TGF-P in vitro

[0158] The immune suppressive cytokine TGF-β is known to suppress the cytotoxicity of CAR-T cells. Therefore, it is important to determine whether the Decorin secreted by 19CAR.D T cells could sequester TGF-β and alleviate the immune suppression induced by TGF-β on CAR-T cells. To address this question, the cytotoxic capacity of CAR-T cells was investigated in a long-term killing assay against MCF-7-CD19 mCherry in the presence of TGF-β. The addition of TGF-β has no effect on the growth of MCF-7-CD19 mCherry cancer cells, as no significant difference was observed in the number of viable cells when MCF-7- CD19 mCherry was cultured alone with or without TGF-β (Figure 5).

[0159] Under the suppressive effects of TGF-β, the cytotoxic capacity of 19CAR.D T cells was unaffected and was superior compared to that of 19CAR T cells (Figure 5A). Under all three E:T ratios, 19CAR.D T cells were able to control and suppress the growth of MCF-7-CD19 mCherry' cells throughout the co-culture even in the presence of TGF-β. The conventional 19CAR T cells, however, could not efficiently suppress the growth of the cancer cells at the lowest E:T ratio of 1:8 when TGF-β was present (Figure 5A). At the end of the co-culture with 19CAR T cells in the presence of TGF-β, the number of viable target cells was also significantly higher than that of 19CAR.D T cells in all E:T ratios (Figure 5B). Likewise, an approximated 17% and 25% increase (E:T ratio of 1:4 and 1:8 respectively) in the number of viable target cells was observed in the 19CAR T cell co-cultures when TGF-β was added (Figure 5B). However, no significant difference in the number of viable target cells was observed in 19CAR.D co-cultures with or without TGF-β throughout all three E:T ratios (Figure 5B). These results indicate that the cytotoxic capacity of 19CAR T cells was suppressed by TGF-β while 19CAR.D T cells were able to overcome the immune suppression derived from TGF-β.

[0160] EXAMPLE 6

[0161] Murine 19CAR.hDc T cells reduced TGF-P-induced Treg differentiation in vitro TGF-β is responsible for the polarization of T cells towards the Treg population, which further contributes to the immune suppressive pressures in the TME. Therefore, it is important to determine whether the Decorin secreted by 19CAR.D T cells could reduce the differentiation of naive T cells into Tregs induced by TGF-β. Naive CD4+T cells were cocultured with the respective groups of transduced CAR-T cells in the presence of TGF-P to induce differentiation into Tregs. To distinguish the naive CD4+T cells from CAR-T cells, naive CD4+T cells were isolated from CD45.2 C57BL / 6 mice while CAR-T cells were generated from CD45.1 C57BL / 6 mice (Figure 6A). The efficiency of TGF-P-induced Treg differentiation is determined by the percentages of Treg (CD25+FoxP3+) in the CD45.1+CAR-T cell population and CD45.2+naive T cell population after five days of co-culture (Figure 6B).

[0162] A significantly lower percentage of CD45.2+T cells (initially naive CD4+T cells) was observed in co-culture with 19CAR.hDc T cells compared to 19CAR T cells (Figure 7A). The CAR-T cell population was made up of majority of CD8+in all groups of the transduced T cells (Figure 7B). The co-culture also did not affect the composition of CD4+and CD8+populations in the CAR-T cells, as comparable percentages was observed between cocultures with naive CD4+T cells and CAR-T cells alone (Figure 7B).

[0163] Decorin-secreting CAR-T cells had the capacity to reduce TGF-β induced Treg differentiation in naive CD4+T cells. The percentage of Treg in CD45.2+cells was almost halved in co-cultures with 19CAR.hDc compared to 19CAR T cells (Figure 7C). When the naive CD4+T cells were cultured alone, approximately 47% of the cells were converted into Tregs after five days of culture with TGF-P, implying that the induction of Treg differentiation in naive CD4+T cells were successful (Figure 7C).

[0164] When the CAR-T cells were cultured alone in the presence of TGF-P, the percentage of Tregs was also lower by approximately 33% in 19CAR.hDc T cells compared to 19CAR T cells (Figure 7D). The percentage of Tregs in both groups of CAR-T cells increased slightly by an equal magnitude when co-cultured with CD45.2+naive CD4+T cells. However, 19CAR.hDc T cells still had a lower percentage of Treg population compared to 19CAR T cells (Figure 7D). Collectively, these data suggests that 19CAR.hDc T cells were able to alleviate the effects of TGF-β in differentiating naive T cells and CAR-T cells towards a Treg phenotype.

[0165] The CD8+population of 19CAR.hDc T cells were less exhausted after treatment with TGF- P or co-culture with induced Tregs from naive CD4+T cells. When treated with TGF-P and cultured alone, the percentage of PD-1+TIM-3+cells in the CD8+population of 19CAR.hDc T cells was significantly lower than that of 19CAR T cells (Figure 8A). The same trend was also observed when the CAR-T cells were co-cultured with induced Tregs from naive CD4+T cells (Figure 8A). No significant difference in the percentage of exhausted CD4+population of both groups of CAR-T cells was observed (Figure 8A).

[0166] Both CD8+and CD4+populations of 19C AR.hDc T cells were able to maintain higher levels of cytokine expression after treatment with TGF-β or co-culture with induced Tregs from naive CD4+T cells, when compared to Mock T cells or 19CAR T cells. Importantly, when treated with TGF-β and cultured alone, the percentage of CD8+19CAR.hDc T cells expressing both IFN-y and TNF-a was significantly higher than that of 19CAR T cells (Figure 8B). The same trend was also observed when the CAR-T cells were co-cultured with induced Tregs from naive CD4+T cells (Figure 8B). Likewise, a significantly higher percentage of CD4+19C AR.hDc T cells was observed to express both cytokines when compared to that of 19CAR T cells regardless of culture conditions (Figure 8B).

[0167] EXAMPLE 7

[0168] Decorin-secreting CAR-T cells are better at controlling tumour growth in syngeneic melanoma tumour model

[0169] The capacity of 19CAR.hDc T cells to eradicate tumours in vivo were further examined in a solid tumour setting using a syngeneic melanoma model. Briefly, mice were given subcutaneous injections of CD19-expressing B 16 (B 16-CD19) cells. Tumours were allowed to engraft, and mice were treated with either PBS or a high dose of 19CAR or 19CAR.hDc T cells (Figure 9A).

[0170] Treatment with 19CAR T cells did little in suppressing the growth of B 16-CD19 tumours in mice compared to those that received the PBS treatment. Mice treated with 19CAR.hDc T cells, however, showed significantly controlled tumour growth compared to those treated with PBS or 19CAR T cells (Figure 9B-C). From the 10 mice that received treatment, four of the mice that was treated with PBS and two that was treated with 19CAR T cells had tumours larger than 1500 mm3by Day 14 and had to be euthanized, while none of the 19C AR.hDc T cell-treated mice had tumours larger than 1500 mm3(Figure 9B). The tumour size of 19CAR.hDc T cell-treated mice was almost halved that of the remaining 19CAR T cell-treated mice (Figure 9C).

[0171] The melanoma model was repeated with lower doses of CAR-T cells but with increased number of treatments. Briefly, the mice were treated twice with 3 x 106of CAR-T cells and monitored for tumour growth (Figure 10A). Mice treated with 19CAR.hDc T cells showed significantly reduced tumour growth compared to 19C AR T cells by Day 12 (Figure 10B).

[0172] EXAMPLE 8

[0173] The effect of 19CAR.hDc treatment on host immune cells and TME

[0174] The effectiveness of 19CAR.hDc T cells on modulating the immune suppressive TME and potential effects on the host immune system were determined using the B16-CD19 melanoma model. Briefly, B 16-CD19 melanoma was first established in immune competent mice before two infusions of CAR-T cells were given (Figure 1 1 A). The mice were sacrificed on Day 12 post initial treatment and the tumour draining lymph nodes, spleen and tumours were harvested for analysis by flow cytometry. Although, no significant difference was observed in the weight of the tumours between the two treatment groups, the tumours of mice treated with 19C AR.hDc T cells were significantly smaller compared to those treated with 19CAR T cells (Figure 1 IB).

[0175] Analysis of the tumour draining lymph node, spleen and tumour samples revealed that CAR- T cells mainly infiltrated to the tumours, as shown by a distinct CD45.1+population representative of the CAR-T cells (Figure 12A). The percentages of CAR-T cells in the tumour draining lymph nodes and spleens were less than 0.2% (Figure 12A). The percentages of tumour-infiltrating 19CAR and 19CAR.hDc T cells were also comparable (Figure 12A). Further analysis showed that both 19CAR and 19CAR.hDc T cells were mainly made up of the CD8+population, and the CD8-to-CD4 composition between the two groups were comparable (Figure 12B). Similarly, 19CAR.hDc T cell treatment did not significantly affect the CD8 / CD4 ratio of host T cells (Figure 12C).

[0176] Mice treated with 19CAR.hDc T cells exhibited lower percentage of immune suppressive Treg cells in the TME derived from B 16-CD19 subcutaneous model. This is evident from the significantly lower percentage of CD25+FoxP3+Tregs cells detected in the tumour sites of mice treated with 19CAR.hDc T cells compared to those treated with 19CAR T cells (Figure 13).

[0177] Further analysis on the CAR-T cell and host T cell memory phenotype was performed to understand how treatment with 19CAR.hDc could contribute to the better therapeutic outcome observed in B16-CD19 tumour-bearing mice. Briefly, the markers CD44 and CD62L were used to categorize the T cells into the different subsets, namely naive T cells (CD44" CD62L+), effector T cells (CD44+CD62L"; Tuff) and central memory T cells (CD44+CD62L+; TCM; Figure 14A). Interestingly, tumour-infiltrating CD8+19CAR.hDc cells were mostly of the Tuff population, although no significant difference was observed when compared to 19CAR T cells (Figure 14B). Naive T cell population of 19CAR.hDc T cells was drastically lower than that of 19CAR T cells, while the percentage of TCM subset was similar between both groups (Figure 14B).

[0178] The memory phenotypes of host T cells in the tumour-bearing mice were largely similar to that of the CAR-T cells. Mice treated with 19CAR.hDc T cells had significantly lowered naive CD8+and CD4+T cell populations in the tumour draining lymph node, spleen and tumour compared to those treated with 19CAR T cells (Figure 14C). Likewise, both CD8+and CD4+Tuff populations at the tumour site of 19CAR.hDc T cell-treated mice seemed to be higher than those of 19CAR T cell, although no significant difference was observed (Figure 10C). The percentage of host TCM population were also comparable regardless of the treatment administered (Figure 14C). References

[0179] 1. Kim, J. H. et al. High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines , Zebrafish and Mice. PLoS One 6, 1-8 (2011).

[0180] 2. Jarvinen, T. A. H. & Ruoslahti, E. Target-seeking anti fibrotic compound enhances wound healing and suppresses scar formation in mice. Proc. Natl. Acad. Sci. 107, 21671-21676 (2010).

Claims

CLAIMS1 . An immune cell engineered to express a chimeric antigen receptor polypeptide and a heterologous decorin polypeptide.

2. The immune cell of claim 1, wherein the heterologous decorin polypeptide is fused to a secretory signal peptide.

3. The immune cell of claim 2, wherein the secretory signal peptide comprises an amino acid sequence of CARSKNKDC (SEQ ID NO: 11).

4. The immune cell of any one of claims 1 to 3, wherein the heterologous decorin polypeptide comprises an amino acid sequence having at least 70% sequence identity to:MKATIILLLLAQVSWAGPFQQRGLFDFMLEDEASGIGPEVPDDRDFEPSLGP VCPFRCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKN LKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQEL RAHENEITKVRKVTFNGLNQMTVIELGTNPLKSSGIENGAFQGMKKLSYIRI ADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAV DNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSS DFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK (SEQ ID NO: 12).

5. The immune cell of any one of claims 1 to 4, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, a co-stimulation signaling region, a CD3^ signaling domain.

6. The immune cell of claim 5, wherein the chimeric antigen receptor comprises a CD8 transmembrane domain.

7. The immune cell of claim 5 or 6, wherein the chimeric antigen receptor comprises a 4-1-BB co-stimulation signaling region.

8. The immune cell of any one of claims 1 to 7, wherein the antigen-binding domain is capable of binding specifically to a cancer antigen.

9. The immune cell of any one of claims 1 to 8, wherein the antigen-binding domain is capable of binding specifically to CD 19.

10. The immune cell of any one of claims 1 to 9, wherein the antigen-binding domain comprises a) a heavy chain variable (VH) region comprising the VHCDR1 amino acid sequence of GVSLPDYGVS (SEQ ID NO: 1), the VHCDR2 amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: 2) and the VHCDR3 amino acid sequence of HYYYGGSYAMDY (SEQ ID NO: 3); and b) a light chain variable (VL) region comprising the VLCDR1 amino acid sequence of RASQDISKYLN (SEQ ID NO: 4), the VLCDR2 amino acid sequence of HTSRLHS (SEQ ID NO: 5) and the VLCDR3 amino acid sequence of QQGNTLPYT (SEQ ID NO: 6).

11. The immune cell of claim 10, wherein the antigen-binding domain comprises a) a VH region comprising an amino acid sequence having at least 70% sequence identity to:EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSS (SEQ ID NO: 7); and b) a VL region comprising an amino acid sequence having at least 70% sequence identity to:DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTS RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EFT (SEQ ID NO: 8).

12. The immune cell of any one of claims 1 to 11, wherein the immune cell comprises a nucleic acid molecule encoding a CAR and a heterologous decorin polypeptide.

13. The immune cell of any one of claims 1 to 12, wherein the immune cell is a T cell or an NK cell.

14. An expression construct comprising a nucleic acid molecule encoding a CAR and a heterologous decorin polypeptide.

15. A vector comprising the expression construct of claim 14.

16. The immune cell of any one of claims 1 to 13 for use as a medicament.

17. A method of treating cancer in a subject, wherein the method comprises administering an immune cell of any one of claims 1 to 13 to the subject.

18. The method of claim 17, wherein the cancer is a solid cancer or a haematological cancer.

19. The method of claim 17 or 18, wherein the cancer is one that is associated with an undesired expression of a cancer antigen.

20. Use of an immune cell of any one of claims 1 to 13 in the manufacture of a medicament for treating cancer in a subject.

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