Engineered immune cells

Abstract

The invention relates to an immune cell which comprises a nucleic acid sequence encoding an antigen binding molecule, and optionally an exogenous nucleic acid sequence encoding a cytokine. The invention also relates to such antigen binding molecules. Pharmaceutical compositions comprising the immune cell or antigen binding molecule of the invention, and their use in methods of treatment, are also provided.

Description

[0001] ENGINEERED IMMUNE CELLS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an immune cell which comprises a nucleic acid sequence encoding an antigen binding molecule, and optionally an exogenous nucleic acid sequence encoding a cytokine. The invention also relates to such antigen binding molecules. Pharmaceutical compositions comprising the immune cell or antigen binding molecule of the invention, and their use in methods of treatment, are also provided.

[0004] BACKGROUND TO THE INVENTION

[0005] Immune cells such as gd T cells can kill cancer via multiple innate and adaptive mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) against tumour antigens. Infiltration of gd T cell into tumours correlates with favourable clinical outcome.

[0006] B7 Homolog 3 (B7-H3) is a transmembrane protein that is expressed at a low level on normal tissues but is over-expressed in a variety of cancers, including non-small-cell lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma, ovarian cancer, and pancreatic cancer. B7-H3 plays a role in promoting tumour progression and metastasis, as well as immune escape. In addition, B7-H3 is expressed on tumour-associated vasculature and plays an important role in angiogenesis. B7-H3 therefore represents an attractive target for cancer immunotherapies.

[0007] To optimise their therapeutic potential, it would be desirable to engineer gd T cells to secrete molecules that enhance their anti-cancer effects, particularly against B7-H3 positive cancers.

[0008] SUMMARY OF THE INVENTION

[0009] The present inventors have identified novel antibodies that bind B7-H3 with high affinity, and mediate potent ADCC activity against cells expressing B7-H3.

[0010] ADCC activity is influenced by a number of different factors and cannot be reliably predicted. For example, high affinity binding may improve antibody-target stability. It may also have detrimental effects, such as promoting internalisation thereby decreasing FcyR engagement. ADCC activity is also considered to be epitope dependent. Similarly, off-target binding can sequester antibody or cause nonspecific effector activation, reducing on-target ADCC. Antigen density, dynamics and shedding, and other factors influencing antigen-antibody interactions contribute to the unpredictable nature of ADCC. The present inventors have surprisingly identified B7-H3 binders (for example, antibodies and scFv-Fc fusion proteins) that are effective mediators of ADCC.

[0011] Moreover, the present binders are effectively expressed by immune cells. Thus, the binders may be delivered to a subject via an immune cell engineered to express and secrete said binder. The immune cell may be capable of ADCC such that the secreted binder mediates ADCC by the cell from which it was expressed. Immune cells engineered to express said binders may therefore have increased cytotoxic activity. The inventors have further found that engineering immune cells to express a modified IL-15 cytokine results in increased cell purity and viability. Immune cells armoured with both binder and cytokine efficiently mediate B7-H3+ tumour cell killing and outperform pure anti-B7-H3 antibodies even at equimolar concentrations.

[0012] As the binders according to the present invention are effective mediators of ADCC, it is considered that they will outperform known anti-B7-H3 antibodies in the context of IL15Ra- IL15 fusion protein armoured immune cells.

[0013] Further, and without wishing to be bound by theory, it is considered that yb T cells expressing a combination of a B7H3 binder according to the present invention and modified IL-15 (e.g. I L15Ra-IL15 fusion protein) will demonstrate synergistic improvement, particularly in vivo, in cytotoxicity compared to y<5 T cells expressing either IL15Ra-IL15 fusion protein or B7H3 binder alone. For example, expression of the modified IL-15 cytokine by the immune cell engineered to express the binder according to the invention may improve the recruitment and / or persistence of said cells in vivo, resulting in a synergistic improvement in ADCC activity of the immune cell.

[0014] The invention provides an immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule; wherein the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0015] The invention provides an immune cell that is capable of antibody dependent cellular cytotoxicity (ADCC) and which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule; wherein the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0016] The immune cell may be capable of expressing and secreting the antigen binding molecule.

[0017] The immune cell may further comprise an exogenous nucleic acid sequence encoding a cytokine. The cytokine may be Interleukin-15 (IL-15). In some embodiments, the cytokine is a modified IL-15, preferably an I L-15Ra-IL-15 fusion protein. In some embodiments, the cytokine comprises an amino acid sequence according to SEQ ID NO: 99.

[0018] The cytokine may stimulate and / or activate the immune cell, and / or increase cell proliferation or expansion, and / or reduce cell apoptosis. For example, the cytokine may provide stimulatory or activatory signals to the immune cell.

[0019] The immune cell may further comprise an exogenous nucleic acid sequence encoding an expression marker. In some embodiments, the expression marker is CD19 or a truncated CD19.

[0020] In some embodiments, the immune cell is an NK cell.

[0021] In some embodiments, the immune cell is a gamma delta T cell. The gamma delta T cell may be a V51+ gamma delta T cell, a V52+ gamma delta T cell, or a V51- / V52- gamma delta T cell. In some embodiments, the gamma delta T cell is a V62+ gamma delta T cell, preferably a Vy9V<52 T cell.

[0022] In some embodiments, the immune cell does not express a chimeric antigen receptor (CAR).

[0023] The invention provides a pharmaceutical composition comprising an immune cell according to the invention, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0024] The invention further provides an antigen binding molecule comprising heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0025] The antigen binding molecule may comprise an scFv, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, an Fv, a dAb, an Fd, a dsFv, a ds-scFv, an scFv2, a bi-specific T-cell engager, a nanobody, a DARPin, an antibody mimetic, a diabody, a triabody, or a tetrabody.

[0026] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0027] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0028] In some embodiments, the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0029] In some embodiments, the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% identity thereto.

[0030] In some embodiments, the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 19 or a variant having at least 80% identity thereto.

[0031] In some embodiments, the antigen binding molecule is capable of binding to an Fc receptor.

[0032] The antigen binding molecule may comprise an Fc region or a modified Fc region. The antigen binding molecule may comprise an Fc region comprises an amino acid sequence according to SEQ ID NO: 94.

[0033] In other embodiments, the antigen binding molecule is not capable of binding to an Fc receptor. The antigen binding molecule may comprise a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 93.

[0034] In some embodiments, the antigen binding molecule is an antibody.

[0035] In some embodiments, the antigen binding molecule comprises an scFv. In some embodiments, the antigen binding molecule is an scFv-Fc fusion protein, optionally wherein the scFv-Fc fusion protein comprises an amino acid sequence according to SEQ ID NO: 91 or SEQ ID NO: 92. Without wishing to be bound by theory, it is considered that the scFv-Fc format of the antigen binding molecules of the present invention is advantageous over other Fc-containing formats due to advantageous transduction efficiency.

[0036] Suitably, transduction of yb T cells with a binder according to the present invention may be more efficient in scFv-Fc format as compared to IgG 1 format, for example due to the need to express only one polypeptide chain and subsequent formation of homodimer (as opposed to two polypeptide chains and formation of hetero-tetramer).

[0037] The invention provides one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention.

[0038] The invention provides a vector comprising the one or more nucleic acid sequence(s) according to the invention. The vector may further comprise a nucleic acid sequence encoding a cytokine, such as an I L-15Ra-l L-15 fusion protein, and / or a nucleic acid sequence encoding an expression marker, such as a truncated CD19.

[0039] The invention provides a cell comprising an antigen binding molecule according to the invention, or one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention. The cell may be capable of expressing an antigen binding molecule according to the invention. The cell may be an ADCC-incompetent cell.

[0040] The invention provides a method of making a cell according to the invention, comprising administering to the cell one or more one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention.

[0041] The invention provides a pharmaceutical composition comprising the antigen binding molecule according to the invention.

[0042] The invention provides a pharmaceutical composition comprising the immune cell or antigen binding molecule according to the invention, for use as a medicament. The pharmaceutical composition may be for use in the treatment of cancer.

[0043] The invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition.

[0044] The cancer may be selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T- cell lymphoma, and B-cell lymphoma.

[0045] The invention provides a method of manufacturing a population of immune cells, comprising: i. providing a population of immune cells; ii. incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL- 15; and iii. introducing into the immune cells a nucleic acid encoding an IL-15Ra-IL-15 fusion protein.

[0046] The I L-15Ra-l L-15 fusion protein may stimulate and / or activate the immune cells, and / or increase cell proliferation or expansion, and / or reduce cell apoptosis. The presence of an IL- 15Ra-IL-15 fusion protein may increase the purity and / or viability of the population of immune cells.

[0047] DESCRIPTION OF THE FIGURES

[0048] Figure 1. The selection of an optimal B7H3-targeting ADCC mAb, clone B7C18, for use in combination with y<5 T cell immunotherapy. Cytotoxicity assay for y<5 T cells co-cultured with a B7H3ne9or B7H3posSupT 1 cell line pre-coated with antibody (N = 3 biological replicates; mean ± SEM).

[0049] Figure 2. Clone B7C18 lgG1 vs SFP as ADCC drugs in the context of y<5 T cell immunotherapy, (a) Format of B7C18 lgG1 or scFv-Fv fusion protein, (b and c) Cytotoxicity assays for unmodified y<5 T cells co-cultured with B7H3+carcinoma cell lines pre-coated with B7C18 lgG1 (b) or B7C18 SFP (c) (N = 3 biological and 3 technical replicates; mean ± SEM). Inverted triangle = 10 nM, triangle = 1 nM, square = 0.1 nM, circle = 0 nM.

[0050] Figure 3. B7C18 SFP was cloned into a y<5 T cell immunotherapy expression cassette, JF125. (a) Schematic of JF114 and JF125 expression cassettes, (b) y<5 T cell viability and purity of unmodified cells, or cells engineered with either JF114 or JF125 expression cassettes (N = at least 2 and up to 7 biological and at least 4 technical replicates per condition; mean; One-Way ANOVA and F-test). (c) Correlation between SFP in JF125 y<5 T cell supernatant and number of transduced (GFP+) y<5 T cells in culture (N = 5 biological and 2 technical replicates per condition; Linear Regression), (d) Cytotoxicity assay for unmodified (non-transduced, NTD), JF114 or JF125-modified yb T cells co-cultured with luciferase-expressing BT474 breast carcinoma cells at an effectortarget ratio of 2: 1 , in the presence of a range of zoledronic acid dilutions (N = 1 T cell donor with 3 technical replicates; mean ± SEM; Two-Way A NOVA).

[0051] Figure 4. B7C18 SFP-expressing JF125 yb T cell immunotherapy was efficacious against intratibial models of osteosarcoma and carcinoma, (a) Treatment schedule for mice engrafted with either a luciferase-expressing patient-derived model of osteosarcoma (PDX) or with a luciferase-expressing HS520 lung carcinoma cell line, (b) Efficacy of unmodified yb T cells, with and without zoledronic acid, JF114 yb T cells and JF125 yb T cells against osteosarcoma PDX (N > 6 animals per group; statistical comparisons done by Two-Way ANOVA comparisons of the individual animal linear regression slopes), (c) Efficacy of JF125 yb T cell, with and without zoledronic acid, against an intratibial model of HS520 lung carcinoma (N = 3 animals per group; Two-Way ANOVA).

[0052] Figure 5. B7C18 SFP-mediated yb T cell combination immunotherapeutic efficacy could be enhanced by mutating the SFP Fc region with a DLE mutation, (a) Schematic of JF25, JF162 (Fc-incompetent (Fcnuii) variant), and JF172 (Fc affinity-enhanced (FCDLE) variant) expression cassettes, (b) Flow cytometric assay of unmodified yb T cell cytotoxicity against MCF-7 and PC-3 carcinoma cell lines, following target co-culture either with plain media, media with exogenously-added B7C18 SFP Fcwr, NTD yb T cell supernatant, or supernatants from JF125, JF162 or JF172 yb T cells (N = 3 biological and 3 technical replicates), (c) Cytotoxicity of cryopreserved, thawed JF172 yb T cells co-cultured at a 1 :2 effectortarget ratio with isogenic luciferase-modified B7H3ne9(left column) or B7H3pos(right column) SupT1 lymphoma target cells (N = 2 biological replicates and 3 technical replicates; mean ± SEM; Two-Way ANOVA).

[0053] Figure 6. B7C18 SFP with an FCDLE in the context of a J F172 cassette did not increase yb T cell killing of non-malignant targets, (a) Schematic of JF125 and JF172 expression cassettes.

[0054] (b) Opsonisation of target cell evaluated by flow cytometry assay, (c) Flow cytometric cytotoxicity assay of unmodified yb T cells against supernatant pre-treated target cell (N = 3 biological replicates and 3 technical replicates; mean ± SEM).

[0055] Figure 7. B7C18 SFP I IL15Ra-IL15 fusion protein yb T cell immunotherapy is functional with a translationally-compatible membrane-bound marker gene, tCD19. (a) Schematic of JF172 and JF174 (comprising tCD19 construct) expression cassettes, (b) tCD19 expression of yb T cells at different multiplicities of infection (MOI) (representative data shown from one donor).

[0056] (c) yb T cell cytotoxicity against MCF-7 and PC-3 carcinoma cell lines pre-treated with either media only, T cell supernatant from NTD y<5 T cells, or y<5 T cell supernatant from JF172 or JF174 (N = 3 biological replicates and 3 technical replicates; mean ± SEM).

[0057] Figure 8. B7C18 SFP with an FCDLE outperforms antibodies enoblituzumab and B7C18. Cytotoxicity of unmodified Vd2 gd T cells co-cultured with B7H3+ tumour cells at a range of effectortarget ratios. Target cells were pre-treated with titred concentrations of enoblituzumab, B7C18 or supernatant from B7C18(DLE) secreting gd T cells (JF172 supe).

[0058] Figure 9. IL15 super-agonist (stlL15) armouring enhances y<5 T product quality.

[0059] Figure 10. (a) Cytotoxicity of unmodified Vd2 gdT cells co-cultured with B7H3+ PC-3 tumour cells at a range of effectortarget ratios. Targets were pre-treated with titred, equimolar concentrations of binder in supernatant from Vd2 cells secreting either chimeric B7C18 scFv- Fc fusion protein (JF174 supe and JF187 supe) or humanised B7C18 scFv-Fc fusion protein with DLE Fc mutation (JF189 supe). Supernatant from unmodified gdT cells was used as a control (non-transduced, NTD supe). (n=3). (b) Cytotoxicity of Vd2 cells engineered to express binder (n=9 across 3 donors).

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0062] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0063] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0064] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0065] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0066] All publications mentioned in the specification are herein incorporated by reference.

[0067] Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

[0068] Immune cell

[0069] The invention provides an immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule as defined herein.

[0070] The invention provides an immune cell that is capable of antibody dependent cellular cytotoxicity (ADCC) and which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule as defined herein. The immune cell may be any immune cell that is capable of ADCC. Immune cells capable of ADCC are known in the art.

[0071] ADCC is a well-known mechanism of adaptive, cell-mediated immunity. During ADCC, an immune effector cell actively lyses a target cell whose surface antigens have been bound by specific antibodies. Specifically, antibodies bind their cognate antigen on the surface of target cells. Fc receptors present on the surface of immune effector cells recognise the Fc region of the bound antibodies. Cross-linking of Fc receptors triggers the formation of a lytic synapse between the immune effector cell and the target cell, into which the immune effector cell degranulates lytic granules. Apoptosis of the target cell is therefore triggered. Immune effector cells capable of ADCC are known to include natural killer (NK) cells, macrophages, neutrophils and eosinophils, gd T cells are also capable of ADCC. The immune cell may be from any species, such as a human, dog, cat, mouse, rat, pig, sheep, cow, goat or horse. The immune cell is typically a human immune cell. The immune cell may be a canine, feline, murine, porcine, ovine, caprine, bovine or equine immune cell.

[0072] Preferably, the immune cell is not an ab T cell. Ab T cells are T cells that possess a T cell receptor (TCR) that comprises an alpha chain and a beta chain. They are usually activated in an MHC-dependent manner. ADCC has not been reported for ab T cells. Ab T cells may be referred to as “conventional” T cells.

[0073] The immune cell may be a gd T cell, gd T cells are T cells that have a gd T cell receptor (TCR) on their surface. That is, gd T cells possess a TCR that comprises a gamma chain and a delta chain. Therefore, gd T cells are structurally different from ab T cells, gd T cells are also functionally different from ab T cells. In particular, gd T cells are capable of ADCC. gd T cells are usually activated in an MHC-independent matter, gd T cells may be referred to as “unconventional” T cells.

[0074] Several subsets of gd T cell exist. For example, the gd T cell may be a Vb2+ gd T cell, a Vb1 + gd T cell, or a V61- / V62- gd T cell, for example a Vb3+, Vb4+, Vb5+, Vb6+, Vb7+, or Vb8+ T cell. Preferably, the gd T cell is a Vb2+ gd T cell.

[0075] The b-chain may pair with any suitable y-chain, for example, Vy2, Vy3, Vy4, Vy5, Vy8 or Vy9. The gd T cell may be a Vy9Vb2 T cell.

[0076] Vb2+ gd T cells, Vb l+ gd T cells, and Vb1-A / b2- T cells all have an excellent capacity for ADCC and exhibit good anti-tumour toxicity.

[0077] Methods for expanding gd T cells are known in the art. For instance, gd T cells may be expanded by culturing in the presence of IL-2 and zoledronic acid (Fisher J et al., 2015, Oncoimmunology, 5(1): el025194). gd T cells are thus readily available for use in the invention.

[0078] The immune cell may be a myeloid cell. Myeloid cells are cells that arise from a common myeloid progenitor cell, such as platelets, erythrocytes, mast cells, macrophages, basophils, neutrophils, and eosinophils. ADCC has been reported for macrophages, basophils, neutrophils, and eosinophils. Preferably, therefore, the myeloid cell is a macrophage, basophil, neutrophil or eosinophil. Methods for isolating and expanding myeloid cells are known in the art.

[0079] Natural killer (NK) cells are also capable of ADCC. The immune cell may be a NK cell. NK cells are a class of innate lymphocytes with roles in immunity against a variety of diseases. For example, NK cells have roles in detecting and controlling cancer, and in killing virally- infected cells. Methods for isolating and expanding NK cells are known in the art.

[0080] Preferably, the immune cell does not express a chimeric antigen receptor (CAR). Preferably, therefore, the immune cell is not a CAR T cell.

[0081] Cytokine

[0082] In some embodiments, the immune cell comprises an exogenous nucleic acid sequence encoding a cytokine. In some embodiments, the immune cell expresses the cytokine.

[0083] The cytokine may stimulate the immune cell, for example, the cytokine may promote expansion of the immune cell, and / or increase cytotoxicity and / or viability of the immune cell.

[0084] The cytokine may be soluble or membrane bound. In some embodiments, the immune cell secretes the cytokine.

[0085] The cytokine may be Interleukin-15, lnterleukin-2, Interleukin-18, Interleukin-12, Interleukin- 21.

[0086] In some embodiments, the cytokine is Interleukin-15.

[0087] IL-15 belongs to the four a-helix bundle family of cytokines. IL-15 has structural similarity to lnterleukin-2 (IL-2). IL-15 is crucial for the generation of multiple lymphocyte subsets (e.g. natural killer (NK), NK-T cells, and memory CD8 T cells).

[0088] An exemplary IL-15 amino acid sequence is provided by SEQ ID NO: 97:

[0089] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVE NLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0090] The cytokine may comprise or consist of a sequence according to SEQ ID NO: 97.

[0091] The immune cell may comprise an exogenous nucleic acid sequence encoding a cytokine comprising or consisting of an amino acid sequence according to SEQ ID NO: 97, or a variant having at least 80% (such as 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0092] The IL- 15 receptor (IL-15R) is a type I cytokine receptor. The IL- 15 receptor is composed of the IL-2RP and yc subunits, along with a unique ligand-specific subunit, IL-15Ra, which bears homology to IL-2Ra. These receptor proteins feature protein-binding motifs known as "sushi domains" (for example, amino acids 31-95 of SEQ ID NO: 98 or SEQ ID NO: 101).

[0093] An exemplary IL-15Ra amino acid sequence is provided by SEQ ID NO: 98:

[0094] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLS PSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWE LTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAME ALPVTWGTSSRDEDLENCSHHL

[0095] Sushi domain of IL15Ra (SEQ ID NO: 101):

[0096] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLK CIR

[0097] In some embodiments, the immune cell comprises an exogenous nucleic acid sequence encoding a modified IL-15 protein. In some embodiments, the immune cell expresses the modified IL-15 protein.

[0098] In some embodiments, the modified IL-15 comprises an IL-15 fusion protein.

[0099] A “fusion protein” may refer to a protein created through the joining of two or more polynucleotides providing coding sequences that originally coded for separate proteins. Translation of this fusion polynucleotide results in a single polypeptide with functional properties derived from each of the original proteins.

[0100] The modified IL-15 may comprise a sushi domain of IL-15Ra. In some embodiments, the IL- 15Ra-IL-15 fusion protein may comprise an IL-15Ra sushi domain fused to an IL-15 protein, optionally by a flexible linker.

[0101] In some embodiments, the modified IL-15 comprises an amino acid sequence according to SEQ ID NO: 99.

[0102] MYRMQLLSCIALSLALVTNSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL TECVLNKATNVAHWTTPSLKCIRGGSGGSGGSGGSGGSGGNWVNVISDLKKIEDLIQSMHI DATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 99).

[0103] The modified IL-15 may comprise a secretory sequence, such as an IL2 secretory sequence as shown in SEQ ID NO: 102:

[0104] MYRMQLLSCIALSLALVTNS Suitable flexible linkers are known in the art. The flexibly linker may comprise an amino acid sequence according to SEQ ID NO: 103:

[0105] GGSGGSGGSGGSGGSGG

[0106] The immune cell may comprise an exogenous nucleic acid sequence encoding a cytokine having an amino acid sequence according to SEQ ID NO: 99, or a variant having at least 80% (such as 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0107] Expression marker

[0108] In some embodiments, the immune cell comprises an exogenous nucleic acid sequence encoding an expression marker. In some embodiments, the immune cell expresses the expression marker.

[0109] A nucleic acid sequence encoding an expression marker may be referred to as a “marker gene”, which may be a gene used to determine if a nucleic acid sequence has been successfully inserted into a host cell, for example integrated into a host cell genome. The marker gene may encode an “expression marker” that facilitates screening of a cell in order to determine whether a nucleic acid sequence has been introduced into said cell.

[0110] For example, the marker gene may encode a fluorescent protein, such as a green fluorescent protein (GFP).

[0111] The expression marker may be a membrane-bound protein.

[0112] In some embodiments, the expression marker is CD19. In some embodiments, the expression marker is a truncated CD19. For example, as set out in SEQ ID NO: 100.

[0113] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPF LKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGEL FRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSL NQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDM WVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAY LIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF

[0114] Suitable methods for screening a cell are known in the art and may include, for example microscopy, immunohistochemistry, flow cytometry or fluorescence-activated cell sorting (FACS). B7H3

[0115] An antigen binding molecule according to the invention specifically binds to B7 Homolog 3 (B7-H3).

[0116] In some embodiments, B7-H3 is considered the antigen of the antigen binding molecule according to the invention.

[0117] B7-H3 (also known as CD276), is a member of the B7 / CD28 immunoglobulin superfamily. B7- H3 is a single-pass transmembrane protein, existing in two isoforms. The 2lg form of B7-H3 contains a single pair of IgV-like and IgC-like immunoglobulin domains, a transmembrane region, and a cytoplasmic tail. The dominantly expressed form of human 4lgB7-H3 contains tandemly duplicated VC domains with four Ig-like domains.

[0118] Exemplary human B7-H3 sequences are provided in SEQ ID NO: 85 and 86.

[0119] 4lg-B7-H3 (SEQ ID NO: 85)

[0120] MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSL

[0121] AQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSF

[0122] TCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDG

[0123] QGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRS

[0124] PTGAVEVQVPEDPWALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQ

[0125] GSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPS

[0126] MTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHS

[0127] VLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAF

[0128] VCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA

[0129] 2lg-B7-H3 (SEQ ID NO: 86)

[0130] MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSL AQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSF TCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDG QGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQP MTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPL KHSDSKEDDGQEIA

[0131] The antigen binding molecule may "specifically bind" to an epitope or antigenic molecule, which means that the antigen binding molecule interacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the foregoing to an epitope or antigenic molecule than alternative substances, including unrelated proteins. In specific embodiments, "specifically binds" means, for instance, that an antigen binding molecule binds to a protein with a KD of approximately 0.1 mM or less, but more usually, less than about 1 pM. In specific embodiments, "specifically binds" means that an antigen binding molecule binds to a protein at times with a KD of approximately 0.1 pM or less, and at other times, with a KD of approximately 0.01 pM or less.

[0132] Suitable assays and techniques for measuring / quantifying binding activity of an antigen binding molecule may include, but are not limited to, ELISA, surface plasmon resonance (SPR), bio-layer interferometry (BLI), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.

[0133] Antigen binding molecule

[0134] The invention provides an immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule as defined herein. The immune cell may express and secrete the antigen binding molecule.

[0135] The invention provides an immune cell that is capable of antibody dependent cellular cytotoxicity (ADCC) and which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule as defined herein. The immune cell may express and secrete the antigen binding molecule.

[0136] The invention also provides an antigen binding molecule comprising heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0137] The term “antigen binding molecule” may refer to an antibody or antigen binding-fragment thereof.

[0138] The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof. The term “antibody” or “antigen-binding fragment thereof” includes monoclonal antibodies and fragments or derivatives of antibodies, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, e.g., scFvs and antigen binding antibody fragments such as Fab and Fab' fragments and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein.

[0139] Within an antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, and each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.

[0140] The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0141] A CDR refers to one of three hypervariable regions (H1 , H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH p-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL p- sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., 1977, J. Biol. Chem. 252:6609-6616; Kabat, 1978, Adv. Prot. Chem. 32:1-75). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved p-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917). Alternatively, IMGT or EU numbering may be used. These terminologies are well recognized in the art. The positions of CDRs within a canonical antibody variable domain have been determined by comparison of numerous structures (Al-Lazikani et al., 1997, J. Mol. Biol. 25 273:927-948; Morea et al., 2000, Methods 20:267-279). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable domain numbering scheme (Al-Lazikani et al., supra). Such nomenclature is similarly well known to those skilled in the art.

[0142] Suitably, the antibody or antigen binding fragment may be defined by the presence of HCDRs and LCDRs determined according to CDR numbering schemes which are known in the art. For example, the CDRs may be defined according to the IMGT, Chothia and / or Kabat numbering schemes. CDRs defined according to each of the IMGT, Chothia and Kabat numbering schemes are described herein.

[0143] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0144] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 3, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7.

[0145] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the IMGT numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 4, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 7.

[0146] Table 1. CDR sequences according to IMGT numbering scheme

[0147] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21 or SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23 or SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25 or SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27 or SEQ ID NO: 28, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0148] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0149] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 21 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 25, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0150] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0151] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 23, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 28, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0152] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Kabat numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 20, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 22, iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 24, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 26, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 27, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 29.

[0153] Table 2. CDR sequences according to Kabat numbering scheme

[0154] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32 or

[0155] SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34 or SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36 or SEQ ID NO: 37, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38; optionally, wherein one or more of the HCDRs or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0156] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0157] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 34, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0158] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38. In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 32, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 37, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0159] In some embodiments, the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3 according to the Chothia numbering scheme, wherein: i. HCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 30, ii. HCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 31 , iii. HCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 33, iv. LCDR1 comprises or consists of an amino acid sequence according to SEQ ID NO: 35, v. LCDR2 comprises or consists of an amino acid sequence according to SEQ ID NO: 36, and vi. LCDR3 comprises or consists of an amino acid sequence according to SEQ ID NO: 38.

[0160] Table 3. CDR sequences according to Chothia numbering scheme

[0161] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations. In some embodiments, HCDR1 may comprise one, two or three amino acid mutations. In some embodiments, HCDR2 may comprise one, two or three amino acid mutations. In some embodiments, may comprise one, two or three amino acid mutations. In some embodiments, LCDR1 may comprise one, two or three amino acid mutations. In some embodiments, LCDR2 may comprise one, two or three amino acid mutations. In some embodiments, LCDR3 may comprise one, two or three amino acid mutations.

[0162] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the antigen binding molecule from binding to B7-H3. In other words, an antigen binding molecule comprising a mutation in one or more CDRs described herein may suitably maintain the capacity (e.g. affinity) to bind to B7-H3. In some embodiments, the mutation suitably maintains the same capacity (e.g. affinity) to bind to B7-H3 as the parent antigen binding molecule. The term “parent” in this context refers to an antigen binding molecule without the mutation in question.

[0163] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto.

[0164] In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0165] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto.

[0166] In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto

[0167] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0168] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto.

[0169] In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto

[0170] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0171] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto

[0172] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 15, or a variant having at least 80% identity thereto.

[0173] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto.

[0174] In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto

[0175] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0176] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto.

[0177] In some embodiments, the antigen binding molecule comprises a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto

[0178] In some embodiments, the antigen binding molecule comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising or consisting of an amino acid sequence according to SEQ ID NO: 19, or a variant having at least 80% identity thereto.

[0179] Table 4. VH / VL sequences

[0180] It will be understood that a VH and / or VL having a percentage identity to the SEQ ID NO of any VH and / or VL defined herein may have an equivalent function to the VH and / or VL having the sequence set forth in the SEQ I D NO defined herein and may suitably maintain the capacity to bind to B7-H3, e.g. the same capacity as the VH and / or VL domain having the sequence set forth in the SEQ ID NO defined herein.

[0181] In some embodiments, the VH has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0182] In some embodiments, the VL has at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0183] In some embodiments, the antigen binding molecule comprises one or more immunoglobulin constant domains. In some embodiments, the immunoglobulin constant domains comprise a constant light chain domain (CL). In some embodiments, the immunoglobulin constant domains comprise a constant heavy 1 (CH1) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 3 (CH3) domain. In some embodiments, the immunoglobulin constant domains comprise a constant heavy 2 (CH2) domain. In some embodiments, the immunoglobulin constant domains comprise a CH2 and a CH3 domain. In some embodiments, the CH2 and CH3 domains are considered to be an Fc (fragment crystallisable) region. In some embodiments, the immunoglobulin constant domains comprise a CL, CH1 , CH2 and CH3 domain.

[0184] In some embodiments, the antigen binding molecule comprises an Fc region.

[0185] In some embodiments, the antigen binding molecule comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 39 or SEQ ID NO: 40.

[0186] In some embodiments, the antigen binding molecule, e.g. via the Fc region, binds to one or more or all of the Fc receptors. The Fc receptors may comprise one or more or all of FcyRI (CD64), FcyRlla (CD32A), FcyRllb (CD32B), FcyRIII (CD16), C1q and FcRn. In some embodiments, the antigen binding molecule, e.g. via the Fc region, binds to FcyRI. It will be understood that the Fc region may interact with Fc receptors presented on the surface of a cell and / or may interact with proteins of the complement system. The Fc receptors may be Fc gamma receptors, e.g. FcyRI. The proteins of the complement system may include C1q.

[0187] In some embodiments, the Fc region of the antigen binding molecule is modified to increase or improve one or more Fc receptor binding and / or functionalities, for example ADCC. Such mutations and / or modifications are well known to those skilled in the art.

[0188] In other embodiments the Fc region of the antigen binding molecule is silenced to reduce, negate or abolish one or more Fc receptor binding and / or functionalities. In some embodiments, the Fc region of the antigen binding molecule is modified to negate one or more Fc receptor functionalities. In some embodiments, the Fc region of the antigen binding molecule is silenced in respect of one or more or all of FcyRI (CD64), FcyRI la (CD32A), FcyRllb (CD32B), FcyRIII (CD16) and C1q functionality. Thus, in some embodiments, the Fc region of the antigen binding molecule is a modified Fc region. Such silencing mutations and / or modifications are well known to those skilled in the art.

[0189] In some embodiments, the antigen binding molecule comprises a modified Fc region.

[0190] In some embodiments, the binding of the modified Fc region to FcyRI may be reduced compared to a wild-type Fc region.

[0191] In some such embodiments, the Fc region of the antigen binding molecule comprises a silencing modification selected from the LALA mutation and the LALA-dCTK mutation, as defined herein.

[0192] In some embodiments, the antigen binding molecule comprises an Fc region comprising an amino acid sequence according to SEQ ID NO: 41 , SEQ ID NO: 42 or SEQ ID NO: 93.

[0193] In some embodiments, the binding of the modified Fc region to an Fc receptor may be increased compared to a wild-type Fc region. In some embodiments, the functionality of the modified Fc region may be increased compared to a wild-type Fc region. For example, the modified Fc may have increased ADCC activity as compared to a wild-type Fc region.

[0194] In some such embodiments, the Fc region of the antigen binding molecule comprises an affinity enhancing modification, such as the DLE mutation as defined herein.

[0195] In some embodiments, the antigen binding molecule an Fc region comprising an amino acid sequence according to SEQ ID NO: 94. Table 5. Fc and constant region sequences

[0196] In one embodiment, the antigen binding molecule according to the invention is an antibody.

[0197] The antibody may comprise a heavy chain(s) and a light chain(s). The term “heavy chain” refers to a large protein subunit of an immunoglobulin. Heavy chains can be of any immunoglobulin isotype (for example IgG, IgE, IgM, IgD, IgA or IgY), subtype (for example lgG1 , lgG2, lgG2a, lgG2b, lgG2c, lgG3, lgG-4, lgA1 or lgA2) or allotype. The term “light chain” refers to a small protein subunit of an immunoglobulin. Light chains can be of any type (for example kappa or lambda), subtype or allotype. Antibodies described herein include polyclonal and monoclonal antibodies and include IgA such as I gA 1 or I gA2, IgG such as IgG 1 , lgG2, lgG3, or lgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an lgG1 antibody, more particularly an lgG1 , kappa or lgG1 , lambda isotype (i.e. lgG1 , K, A), an lgG2a antibody (e.g. lgG2a, K, A), an lgG2b antibody (e.g. lgG2b, K, A), an lgG3 antibody (e.g. lgG3, K, A) or an lgG4 antibody (e.g. lgG4, K, A). In preferred embodiments the antibody is an I gG 1 , preferably I gG 1 , lambda. The antibody may be of any species (for example human, monkey, camel, llama, goat, sheep, rabbit, mouse, rat, mouse, hamster or chicken) or it may be a hybrid derived from more than one species. It may be naturally occurring or it may be non-naturally occurring (i.e. an isolated antibody). The antibody may be created by genetic engineering (for example a chimeric antibody, humanised antibody, camelised antibody, intrabody, bispecific antibody).

[0198] In one embodiment the antigen binding molecule is a monoclonal antibody. In one embodiment the antigen binding molecule is a humanised antibody. In one embodiment the antigen binding molecule is a chimeric antibody.

[0199] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a variant having at least 80% identity thereto.

[0200] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 44, or a variant having at least 80% identity thereto.

[0201] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 43, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 44, or a variant having at least 80% identity thereto.

[0202] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 45, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 46, or a variant having at least 80% identity thereto.

[0203] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 45, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 46, or a variant having at least 80% identity thereto.

[0204] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47, or a variant having at least 80% identity thereto.

[0205] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 48, or a variant having at least 80% identity thereto.

[0206] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 47, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 48, or a variant having at least 80% identity thereto.

[0207] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 49, or a variant having at least 80% identity thereto.

[0208] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 50, or a variant having at least 80% identity thereto.

[0209] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 49, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 50, or a variant having at least 80% identity thereto.

[0210] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 55, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 56, or a variant having at least 80% identity thereto.

[0211] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 55, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 56, or a variant having at least 80% identity thereto.

[0212] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 57, or a variant having at least 80% identity thereto.

[0213] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 58, or a variant having at least 80% identity thereto.

[0214] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 57, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 58, or a variant having at least 80% identity thereto.

[0215] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 59, or a variant having at least 80% identity thereto.

[0216] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 60, or a variant having at least 80% identity thereto.

[0217] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 59, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 60, or a variant having at least 80% identity thereto.

[0218] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 61 , or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 62, or a variant having at least 80% identity thereto.

[0219] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 61 , or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 62, or a variant having at least 80% identity thereto.

[0220] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 63, or a variant having at least 80% identity thereto.

[0221] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 64, or a variant having at least 80% identity thereto.

[0222] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 63, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 64, or a variant having at least 80% identity thereto.

[0223] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 65, or a variant having at least 80% identity thereto.

[0224] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 66, or a variant having at least 80% identity thereto.

[0225] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 65, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 66, or a variant having at least 80% identity thereto.

[0226] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 67, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 68, or a variant having at least 80% identity thereto.

[0227] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 67, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 68, or a variant having at least 80% identity thereto.

[0228] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 69, or a variant having at least 80% identity thereto.

[0229] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 70, or a variant having at least 80% identity thereto.

[0230] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 69, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 70, or a variant having at least 80% identity thereto.

[0231] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 71 , or a variant having at least 80% identity thereto.

[0232] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 72, or a variant having at least 80% identity thereto.

[0233] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 71 , or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 72, or a variant having at least 80% identity thereto.

[0234] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 73, or a variant having at least 80% identity thereto. In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 74, or a variant having at least 80% identity thereto.

[0235] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 73, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 74, or a variant having at least 80% identity thereto.

[0236] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 75, or a variant having at least 80% identity thereto.

[0237] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 76, or a variant having at least 80% identity thereto.

[0238] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 75, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 76, or a variant having at least 80% identity thereto.

[0239] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 77, or a variant having at least 80% identity thereto.

[0240] In some embodiments, the antigen binding molecule comprises a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 78, or a variant having at least 80% identity thereto.

[0241] In some embodiments, the antigen binding molecule comprises a light chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 77, or a variant having at least 80% identity thereto, and a heavy chain comprising or consisting of an amino acid sequence according to SEQ ID NO: 78, or a variant having at least 80% identity thereto.

[0242] Table 6. Full-length heavy and light chains

[0243] It will be understood that a heavy chain and / or light chain having a percentage identity to the SEQ ID NO of any heavy chain and / or light chain defined herein may have an equivalent function to the heavy chain and / or light chain having the sequence set forth in the SEQ ID NO defined herein and may suitably maintain the capacity to bind to B7-H3, e.g. the same capacity as the heavy chain and / or light chain having the sequence set forth in the SEQ ID NO defined herein.

[0244] In some embodiments, the heavy chain has at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0245] In some embodiments, the light chain has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in the SEQ ID NO defined herein.

[0246] The antigen binding molecule may comprise an scFv, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, an Fv, a dAb, an Fd, a dsFv, a ds-scFv, an scFv2, a bi-specific T-cell engager, a nanobody, a DARPin, an antibody mimetic, a diabody, a triabody, a tetrabody, or a polypeptide ligand for a receptor expressed on the surface of a cell that is targeted by the immune cell.

[0247] In some embodiments, the antigen binding molecule according to the invention comprises an scFv. The scFv may comprise the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a short linker peptide.

[0248] In some embodiments, the antigen binding molecule comprises an scFv comprising or consisting of an amino acid sequence according to SEQ ID NO: 83, or a variant having at least 80% identity thereto.

[0249] In some embodiments, the antigen binding molecule comprises an scFv comprising or consisting of an amino acid sequence according to SEQ ID NO: 84, or a variant having at least 80% identity thereto.

[0250] In some embodiments, the scFv has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO 83 or 84 defined herein.

[0251] The antigen binding molecule may be a scFv-Fc fusion protein (SFP).

[0252] The scFv may comprise the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, connected with a short linker peptide, fused to an Fc region. In some embodiments, the antigen binding molecule comprises an scFv-Fc comprising or consisting of an amino acid sequence according to SEQ ID NO: 91 or SEQ ID NO: 92, or a variant having at least 80% identity thereto.

[0253] In some embodiments, the scFv-Fc has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO: 91 or 92 defined herein.

[0254] In preferred embodiments, the antigen binding molecule comprises an amino acid sequence according to SEQ ID NO: 92, or a variant having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto.

[0255] Table 7. ScFv (with Chothia and Kabat CDR annotation) and ScFv-Fc sequences.

[0256] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences. Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0257] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0258] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1): 187-8).

[0259] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0260] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0261] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0262] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally- occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0263] Polynucleotide

[0264] The present invention provides one or more nucleic acid sequence(s) encoding the antigen binding molecule according to the invention. In other words, the present invention provides one or more nucleic acid sequence(s) capable of expressing the antigen binding molecule according to the invention.

[0265] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. The nucleic acid sequence(s) may be RNA or DNA sequences, or a mixture of RNA and DNA sequences. In an embodiment, the nucleic acid sequence(s) are one or more DNA sequences, such as cDNA sequences. In an embodiment, the nucleic acid sequence is a DNA sequence, such as a cDNA sequence. In an embodiment, the nucleic acid sequence(s) are RNA sequences, such as mRNA sequences. In an embodiment, the nucleic acid sequence is an RNA sequence, such as an mRNA sequence.

[0266] The nucleic acid sequence(s) may be single-stranded or may be double-stranded. The nucleic acid sequence(s) may be, for example, genomic, recombinant, mRNA or cDNA. The nucleic acid sequence(s) may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance in vivo activity and / or stability.

[0267] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These variations in nucleic acid sequences are encompassed by the present invention. Therefore, multiple nucleic acid sequence(s) are envisaged, each of which may be different, but which still encode a binding molecule or antibody according to the present invention. It is known in the art how to design and produce such nucleic acid sequences.

[0268] In some embodiments, the nucleic acid sequence(s) may be codon optimised for production in the host cell of choice. In some embodiments, the nucleic acid sequence(s) may be operably linked to further sequence(s) such as control sequence(s), e.g. promoter sequence(s), enhancer sequence(s), polyadenylation signal sequence(s) and / or other regulatory sequence(s), which control transcription and / or translation. The nucleic acid sequence(s) may be in the form of one or more expression cassettes. The nucleic acid sequences may be suitable for expression in prokaryotic cells or in eukaryotic cells, such as mammalian cells. Any promoter may be used, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0269] Exemplary nucleic acid sequences (encoding an scFV according to the invention) are provided in SEQ ID NOs: 95 and 96.

[0270] Vector

[0271] The present invention provides a vector comprising the one or more nucleic acid sequence(s) of the invention.

[0272] Accordingly, the vector may comprise a polynucleotide comprising a nucleic acid sequence or sequences encoding the antigen binding molecule according to the invention.

[0273] The vector may further comprise a nucleic acid sequence encoding a cytokine, such as an IL- 15Ra-IL-15 fusion protein as defined herein.

[0274] The vector may further comprise a nucleic acid sequence encoding an expression marker. The expression marker may be a truncated CD19 as defined herein.

[0275] The vector may be used to introduce nucleic acid sequence(s) into a cell so that the cell expresses and / or produces the antigen binding molecule according to the invention and / or the cytokine and / or expression marker. As used herein, the term “vector” may be considered interchangeable with the term “expression vector” and “expression construct”. The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[0276] In some embodiments, the vector may be an expression cassette or construct.

[0277] In some embodiments, the nucleic acid sequences encoding the antigen binding molecule, cytokine and / or expression marker may be included on the same expression construct, for example separated by a sequence encoding a self-cleaving peptide.

[0278] The vector according to the invention may be any agent capable of delivering nucleic acid sequence(s) according to the invention to a cell and / or expressing nucleic acid sequence(s) according to the invention in a cell. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0279] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector. In some embodiments, the vector is a lentiviral vector.

[0280] The vector may be capable of transfecting or transducing a cell.

[0281] Cell

[0282] The invention provides a cell comprising an antigen binding molecule according to the invention.

[0283] The invention provides a cell comprising one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention. The invention provides a cell comprising a vector according to the invention.

[0284] The cell may be any cell capable of expressing an antigen binding molecule according to the invention. The cell may be capable of secreting the antigen binding molecule according to the invention.

[0285] Expression cell lines are known in the art and include, for example, human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells. In preferred embodiments, the cell is not an expression cell line. Preferably, the cell is not a human embryonic kidney cell.

[0286] The cell may be an immune cell, such as a T cell.

[0287] Thus, the invention provides an immune cell comprising an antigen binding molecule according to the invention. The invention provides an immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention. The immune cell may be capable of expressing the antigen binding molecule according to the invention. The immune cell may be capable of secreting the antigen binding molecule according to the invention.

[0288] As used herein the term “immune cells” refers to any cell forming part of the immune system and which may play a role in protecting the body from for example, infectious disease, foreign entities and cancers. The term may refer to white blood cells (also known as leukocytes) and includes granulocytes (such as basophils, eosinophils, neutrophils, and mast cells), lymphocytes (such as T cells, B cells, and NK cells) and monocytes (such as macrophages and dendritic cells).

[0289] In some embodiments, the immune cell may be a T cell (such as an ab T cell or gd T cell), a myeloid cell (such as a platelet, erythrocyte, mast cell, macrophage, basophil, neutrophil, and eosinophil), or an NK cell, as defined herein.

[0290] The cell may be an isolated cell.

[0291] The cell, preferably an immune cell, may be administered to a subject, for example in order to deliver an antigen binding molecule according to the invention to a subject.

[0292] In some embodiments, the immune cell is ADCC-incompetent. In these embodiments, the ADCC-incompetent cell may express and secrete the antigen binding molecule thereby inducing or enhancing ADCC activity of an ADCC-competent cell.

[0293] In preferred embodiments, the immune cell is capable of ADCC. In these embodiments, the ADCC-competent cell may express and secrete the antigen binding molecule thereby inducing or enhancing ADCC activity of itself and / or other ADCC-competent cells.

[0294] Thus, the invention provides a method of producing an immune cell, optionally wherein the immune cell is capable of ADCC, which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention. The method may comprise: i. obtaining or providing an immune cell; and ii. introducing into said cell one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention.

[0295] The method may comprise introducing into said cell an exogenous nucleic acid sequence encoding a cytokine.

[0296] The method may comprise introducing into said cell an exogenous nucleic acid sequence encoding an expression marker.

[0297] The nucleic acid sequences may be introduced into the cell by transduction, for example transduction by lentiviral vector.

[0298] The method may further comprise stimulating the immune cell with, such as by culturing or incubating the immune cell in the presence of, zoledronic acid and / or IL-2 and / or IL-15. The immune cell may be stimulated before, during and / or after introducing into the cell the nucleic acid sequences.

[0299] Thus, in some embodiments, the method comprises the steps of: i. obtaining or providing an immune cell; ii. culturing or incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL-15; and iii. introducing into said cell one or more nucleic acid sequence(s) encoding an antigen binding molecule according to the invention; wherein steps ii) and iii) are performed in any order or simultaneously.

[0300] The invention provides a method of producing a population of immune cells, for example gd T cells, the method comprising: i. obtaining or providing a population of immune cells; ii. culturing or incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL-15; and iii. introducing into the immune cells a nucleic acid encoding an IL-15Ra-IL-15 fusion protein; wherein steps ii) and iii) are performed in any order or simultaneously.

[0301] The method may further comprise introducing into said cell a nucleic acid sequence encoding an antigen binding molecule according to the invention, and / or an exogenous nucleic acid sequence encoding an expression marker.

[0302] The IL-15Ra-IL-15 fusion protein may comprise an amino acid sequence according to SEQ ID NO: 99, or a variant having at least 80% (such as 85%, 90%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0303] A population of immune cells, for example gd T cells, engineered to express an IL-15Ra-IL- 15 fusion protein may have may have increased purity (e.g. a higher proportion of gd T cells) and / or viability as compared to immune cells that do not express an I L-15Ra-IL-15 fusion protein.

[0304] Thus, in some embodiments, the invention provides a method for improving the purity of a population of immune cells, comprising: i. providing a population of immune cells; ii. incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL-15; and iii. introducing into the immune cells a nucleic acid encoding an I L-15Ra-IL-15 fusion protein.

[0305] The population of immune cells may comprise about 60-100%, about 70-100%, about 80- 100% gd T cells. For example, about 60-100%, about 70-100%, about 80-100% V52+ gd T cells.

[0306] The population of immune cells may comprise about 60-100%, about 70-100%, about 80- 100% viable gd T cell. For example, about 60-100%, about 70-100%, about 80-100% viable V52+ gd T cells.

[0307] Suitable assays for determining cell purity and / or viability are known in the art and may include flow cytometry or FACS analysis, for example in combination with an appropriate antibody or dye.

[0308] The population of immune cells may be isolated from a subject or from a sample from a subject. The sample may be a peripheral blood sample, a cord blood sample, a tumour, a stem cell precursor, a tumor biopsy, a tissue, or a lymph. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a tissue sample, such as skin or gut. Peripheral blood mononuclear cells can be collected from a subject, for example, with an apheresis machine, including the Ficoll-Paque™ PLUS (GE Healthcare) system, or another suitable device / system. Immune cells may be purified from the collected sample with, for example, flow cytometry techniques.

[0309] The step of obtaining a sample of immune cells may be performed prior to the steps of the present method.

[0310] The population of immune cells may be peripheral blood mononuclear cells (PBMCs).

[0311] The cells may be allogeneic. The cells may be autologous.

[0312] The duration of the incubation period may be from 1 day to about 30 days, from 1 day to about 10 days, from 1 day to about 9 days, from 1 day to about 8 days, from 1 day to about 7 days, from 1 day to about 6 days, from 1 day to about 5 days, from 1 day to about 4 days, from about 2 days to about 10 days, from about 2 days to about 8 days, from about 2 days to about 7 days, about 3 days to about 7 days, or about 4 to about 6 days, from about 7 days to about 30 days, from about 10 days to about 25 days. The duration of the incubation period may be about 25 days.

[0313] In some embodiments, the cells are stimulated with zoledronic acid.

[0314] In some embodiments, the cells are stimulated with zoledronic acid and IL-2.

[0315] In some embodiments, the cells are stimulated with zoledronic acid and IL-5.

[0316] In some embodiments, the cells are stimulated with zoledronic acid, IL-2 and IL-15.

[0317] The concentration of zoledronic acid, during the culture or incubation step may be from about 0.1 pM to about 500 pM, from about 0.1 pM to about 400 pM, from about 0.1 pM to about 300 pM, from about 0.1 pM to about 200 pM, from about 0.1 pM to about 100 pM, from about 0.5 pM to about 100 pM, from about 1 pM to about 500 pM, from about 1 pM to about 400 pM, from about 1 pM to about 300 pM, from about 1 pM to about 200 pM, from about 1 pM to about 100 pM, from about 1 pM to about 90 pM, from about 1 pM to about 80 pM, from about 1 pM to about 70 pM, from about 1 pM to about 60 pM, from about 1 pM to about 50 pM, from about 1 pM to about 40 pM, from about 1 pM to about 30 pM, from about 1 pM to about 20 pM, from about 1 pM to about 15 pM, from about 1 pM to about 10 pM, from about 1 pM to about 9 pM, from about 1 pM to about 8 pM, from about 1 pM to about 7 pM, from about 1 pM to about 6 pM, from about 1 pM to about 5 pM, from about 2 pM to about 5 pM, from about 3 pM to about 5 pM, from about 2 pM to about 5 pM, from about 2 pM to about 10 pM, from about 3 pM to about 8 pM, or from about 4 pM to about 6 pM.

[0318] In a preferred embodiment, the concentration of zoledronic acid is about 5 pM.

[0319] The concentration of IL-2 during the culture or incubation step may be from about 10 lll / ml to about 1000 lU / ml, from about 10 lll / ml to about 500 lU / ml, from about 10 lll / ml to about 400 lU / ml, from about 10 lll / ml to about 300 lU / ml, from about 10 lll / ml to about 200 lU / ml, from about 10 lll / ml to about 150 lU / ml, from about 10 lll / ml to about 100 lU / ml, from about 20 lll / ml to about 100 lU / ml, from about 30 lll / ml to about 100 lU / ml, from about 40 lll / ml to about 100 lU / ml, from about 50 lll / ml to about 100 lU / ml, from about 75 lll / ml to about 125 lU / ml, from about 20 lll / ml to about 80 lU / ml, from about 25 lll / ml to about 100 lU / ml, or from about 50 lll / ml to about 150 lU / ml.

[0320] In a preferred embodiment, the concentration of IL-2 is about 100 lU / ml.

[0321] The concentration of IL-15 during the culture or incubation step may be from about 10 ng / ml to about 1 pg / ml, from about 10 ng / ml to about 500 ng / ml, from about 10 ng / ml to about 400 ng / ml, from about 10 ng / ml to about 300 ng / ml, from about 10 ng / ml to about 200 ng / ml, from about 10 ng / ml to about 150 ng / ml, from about 10 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 100 ng / ml, from about 30 ng / ml to about 100 ng / ml, from about 40 ng / ml to about 100 ng / ml, from about 50 ng / ml to about 100 ng / ml, from about 60 ng / ml to about 100 ng / ml, from about 20 ng / ml to about 80 ng / ml, from about 30 ng / ml to about 60 ng / ml, or from about 50 ng / ml to about 120 ng / ml.

[0322] In a preferred embodiment, the concentration of IL-15 is about 70 ng / ml.

[0323] Throughout the incubation period the culture medium may be removed and / or refreshed, or supplemented with a feed medium, such as a feed medium containing cytokines and / or other suitable agents.

[0324] Pharmaceutical composition

[0325] The invention provides a pharmaceutical composition comprising an antigen binding molecule according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0326] The invention provides a pharmaceutical composition comprising an immune cell according to the invention, together with a pharmaceutically acceptable carrier, diluent or excipient. The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile.

[0327] The pharmaceutical composition may be formulated to be suitable for administration to a patient in order to prevent and / or treat disease. Pharmaceutical compositions can be formulated for administration by different routes, for example, for oral, parenteral, topical, inhalative, intravenous, intramuscular, rectal, sublingual, transdermal, subcutaneous, intratumoral application routes, according to their chemical and physical properties.

[0328] Method of treatment

[0329] The invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antigen binding molecule according to the invention. The invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immune cell according to the invention. The invention provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to the invention. In some embodiments, the disease is cancer.

[0330] The invention provides an antigen binding molecule according to the invention for use as a medicament. The invention provides an immune cell according to the invention for use as a medicament. The invention provides a pharmaceutical composition according to the invention for use as a medicament.

[0331] The invention provides an antigen binding molecule according to the invention for use in a method of treatment and / or diagnosis. The invention provides an immune cell according to the invention for use in a method of treatment and / or diagnosis. The invention provides a pharmaceutical composition according to the invention for use in a method of treatment and / or diagnosis. In some embodiments, the antigen binding molecule, immune cell or pharmaceutical composition according to the invention is for use in a method of treating cancer.

[0332] The invention provides for the use of an antigen binding molecule according to the invention for the manufacture of a medicament. The invention provides for the use of an immune cell according to the invention for the manufacture of a medicament. The invention provides for the use of pharmaceutical composition according to the invention for the manufacture of a medicament.

[0333] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a bisphosphonate, such as zoledronic acid or risedronic acid.

[0334] In some embodiments, the zoledronic acid is administered at a dose of about 4 mg. In some embodiments, the zoledronic acid is administered at a dose of about 4 mg every 28 days.

[0335] In some embodiments, the risedronic acid is administered at a dose of about 30 mg. In some embodiments, the risedronic acid is administered daily at a dose of about 30 mg.

[0336] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterised by unregulated cell growth.

[0337] “Tumour” refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

[0338] The term “subject” may refer to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0339] An "effective amount" as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0340] The term "therapeutically effective amount" refers to an amount of drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumour size; inhibit (i.e., slow to some extent and in a certain embodiment, stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and in a certain embodiment, stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with cancer.

[0341] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0342] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumour size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumour metastasis; inhibition or an absence of tumour growth; relief of one or more symptoms associated with specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumourigenicity, tumourigenic frequency, or tumourigenic capacity, of a tumour; reduction in the number or frequency of cancer stem cells in a tumour; differentiation of tumourigenic cells to a non-tumourigenic state; or some combination of effects.

[0343] The cancer may express B7-H3. In some embodiments, the expression of B7-H3 is increased compared to the expression of B7-H3 by the same non-cancerous tissue or cells.

[0344] An increase may refer to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression is only found in a diseased tissue, while expression in a corresponding healthy tissue is repressed. According to the invention, diseases associated with cells expressing B7-H3 include cancer diseases. Furthermore, according to the invention, cancer diseases preferably are those wherein the cancer cells express B7-H3.

[0345] The cancer may be melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and B- cell lymphoma. In some embodiments, the cancer is selected from prostate cancer, colorectal cancer, lung cancer and breast cancer.

[0346] The invention will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0347] EXAMPLES

[0348] Methods

[0349] Cell lines

[0350] The following human cell lines were from the American Type Culture Collection (ATCC): SUP- T1 (T cell Lymphoblastic Lymphoma), BT474 (breast adenocarcinoma), MCF-& (breast adenocarcinoma), PC-3 (prostatic carcinoma bone metastasis), 22Rv1 (prostate carcinoma), H520 (lung squamous cell carcinoma). SUP-T1-B7H3 were a kind gift from Prof. John Anderson (University College London). Patient-derived osteosarcoma cells designated PDOS25 corresponds to patient-derived xenograft line PDX_OS#25-C which were a kind gift from Dr. Katia Scotlandi (Istituto Ortopedico Rizzoli, Bologna) and outgrown in-vitro.

[0351] Cell culture conditions

[0352] Cell lines were maintained in a base medium of RPMI-1640 (Sigma Aldrich). Patient derived osteosarcoma lines were maintained in a base medium of IMDM (Sigma Aldrich). All base media were supplemented with 2mM L-Glutamine (Sigma-Aldrich) and 10% (v / v) heat inactivated fetal bovine serum (FBS, Thermo Fisher Scientific).

[0353] Flow cytometry

[0354] Antibodies were diluted in phosphate buffered saline (PBS) using the manufacturers’ recommended volumes per test. Cells were washed in PBS prior to staining (1500 RPM for 5 minutes) and resuspended in a minimum volume of 100 pl antibody master-mix. They were incubated for 30m at 5°C and then washed twice in PBS (as above) prior to analysis. Where samples were fixed for analysis at a later time, 50 pl fixation buffer (Biolegend) was added to each tube. Flow cytometric analysis was performed using an LSRII or Symphony A5 flow cytometer (BD Biosciences). Data were collected using BD FACSDiva V8.0.1 and analyzed using FlowJo 10.8.1. Compensation was calculated on the basis of OneComp eBeads (Thermo Fisher Scientific) or MACS Comp anti-REA Bead Kit (Miltenyi Biotec) stained with single-color antibodies. Where absolute cell counts were required, Precision Count Beads (BioLegend) were added to tubes prior to sample acquisition. Quantification ofanti-B7H3 scFv-protein concentration in gdT supernatant

[0355] Standard sandwich ELISA technique was used. Nickel coated plates were pre-coated with His-tagged recombinant anti-B7H3. Supernatant from transduced gdT cells was added. Following incubation and washing, HRP-conjugated anti human Fc antibody was used as the detection reagent.

[0356] Vy9V52 T cell expansion

[0357] PBMC isolation is described below. For specific Vy9V<52 y<5 T cell expansion, PBMCs were cultured in RPMI-1640 medium supplemented with l-glutamine and 10% FBS (v / v) (Gibco). Vy9V<52 y<5 T cell expansion was stimulated using 5 iM zoledronic acid (Actavis) and IL-2 (100 lU / ml; Aldesleukin, Novartis) or recombinant human IL-15 (70ng / mL; Miltenyi Biotec). Unless otherwise stated, IL-2 or IL-15 was replenished every 2 to 3 days by removing half of the media from the well and replacing with fresh media containing IL-2 or IL-15 to achieve final concentrations of 100 lU / ml and 70ng / ml, respectively.

[0358] Lentiviral transduction of y5 T cells

[0359] PBMC were plated in tissue-culture treated plates at 1 to 2x106cells per cm2and stimulated with zoledronic acid and IL-2 or IL-15 as described above. Following 48h of stimulation, pretitered lentiviral vector was added directly to the expansion wells at a multiplicity of infection (MOI) of 2 to 4. No transduction enhancers were used for lentiviral transduction. Transduction efficiency was checked at day 5 after transduction by flow cytometry and cells were typically harvested on day 12 unless otherwise stated. Where transduction efficiency for lentiviral transductions is stated, it refers to the percentage of live Vy9V<52 T cells expressing the eGFP or tCD19 marker gene. No further purification steps were applied to the cells following expansion and transduction.

[0360] In vitro cytotoxicity assays

[0361] Effector cells were typically co-cultured for 18h with target cells at an E:T ratio of 1 :1 unless otherwise stated in the figure legends. E:T ratio was determined using the total number of lymphocytes present in the y<5 T cell expansion and was not adjusted for Vy9V<52 purity or transduction efficiency. Target cells were pre-labelled with Cell-Trace Violet (Invitrogen) using the manufacturer’s standard instructions to differentiate between target and effector cells. Flow cytometry was used to count live target cells and to assess target cell death within gated tumor cells using viability dyes. If relative cell numbers are expressed, then they are normalized to a monoculture of that cell type cultured in the same plate and with the same starting number. To assess bystander cytotoxicity, cell-free culture supernatants from engineered or unmodified-yb T cells were added to co-cultures of targets and allogeneic bystander effector cells (unmodified-yb T cells, NK cells, macrophages or neutrophils). Following co-culture, cells were harvested and analyzed by flow cytometry as above.

[0362] For some cytotoxicity assays, tumor cell lines were stably transduced to express firefly luciferase and used as target cells. Target cells were seeded overnight in 96-well black tissue culture plates and then co-cultured for a further 18 hours (unless otherwise stated) with effector cells. D-Luciferin (Cayman Chemical) was added to the cells to a final concentration of 150pg / mL and after 10mins, luminescence was read using a SpectraMax i3x plate reader (Molecular Devices).

[0363] Mouse experiments

[0364] All murine studies were approved by the UK home office (PP5675666). 6-week-old NOD.Cg- PrkdcscidH2rgtm1vw / SzJ (NSG) mice were purchased from Charles River Laboratories and housed in individually ventilated cages with a maximum of 5 mice per cage. Treatment group and mouse sex assignment was done using randomization, and pre-treatment bioluminescence used to ensure there was no statistically significant difference in mean tumor burden between groups at baseline.

[0365] 0.5x106cells from patient derived osteosarcoma line PDOS25 or lung cancer cell line H520 expressing firefly luciferase were orthotopically injected into the right tibia of NSG mice. Intratibial injection was performed under anesthesia. The leg to be engrafted was shaved and cleaned with Hibiscrub™ containing 4% chlorhexidine gluconate and the animal’s leg positioned in a bent position to facilitate access to the knee joint. A 27G needle was inserted under the patella into the anterior intercondylar area of the distal tibia and the bone penetrated using a careful drilling motion. Once the tibial cortex was penetrated the 27G needle was removed and 10 pl cell suspension injected using a 29G needle. Tumor engraftment and growth was monitored by bioluminescence. Once engraftment was confirmed, treatment for all groups in an experiment was deemed to have commenced when T cells were administered. Some mice received 1 107cells from engineered or unmodified yb T cell preparations by tail vein injection; cell dose was not modified for transduction efficiency or T cell content. On day 7 following treatment start, mice received either 3pg zoledronic acid or no further treatment. Interim blood samples were collected from some animals at days 7 and 14 following T cell injection. Results

[0366] Selection of B7H3-targeting mAb for use in combination with yd T cell immunotherapy

[0367] To determine the antibody-dependent cellular cytotoxicity (ADCC)-mediating capacity and antigen specificity of a library of new B7H3-targeting mAbs, four mAb clones were expressed in I gG 1 format with a human Fc (mAbs 1-3 and novel clone B7C18). These mAbs were then used to coat an isogenic SupT 1 lymphoma target cell line pair: SupT 1 cells which are naturally B7H3ne9, or an engineered SupT 1 cell line which is B7H3pos. SupT 1 targets were coated with the mAbs at 1 pg / mL of each antibody. These targets were then washed and incubated overnight with y<5 T cells. Following overnight co-culture, tumour killing was assessed by flow cytometric counting of viable tumour cells left in culture. Clone B7C18 was progressed for further evaluation, as it exhibited the highest ADCC against B7H3posSupT 1 targets, but no cytotoxicity against B7H3ne9targets (Figure 1). mAbs 1-3 correspond to anti-B7H3 antibodies TE9, TC6, and BH6 derived from a previous library selection (WO 2024 / 009075 A1).

[0368] B7C18 binds B7-H3 with high affinity

[0369] The binding kinetics of B7C18 versus known anti-B7-H3 antibodies was compared. B7C18 in IgG or scFv-Fc format bound with a KD of 0.14 and 0.7 nM, respectively (Table 8).

[0370] Table 8. Binding of anti-B7-H3 antibodies

[0371] B7C18 lgG1 vs SFP as ADCC drugs in the context of yd T cell immunotherapy.

[0372] Clone B7C18 mAb was synthesized either as a full human lgG1 or as an scFv-Fc fusion protein (SFP) for use in antibody-dependent cellular cytotoxicity (ADCC) assays with y<5 T cells (Figure 2A). Unmodified y<5 T cells were co-cultured for 4h with a range of B7H3+carcinoma cell lines that were pre-coated with various concentrations of anti-B7H3 clone B7C18 lgG1 or B7C18 SFP. Cytotoxicity was measured using a flow cytometric tally of dead tumour cells in the assay. Clone B7C18 lgG1 mediated y<5 T cell ADCC against a range of solid tumour targets, as did B7C18 SFP, albeit with different kinetics. Binder in lgG1 format mediated more potent ADCC at lower concentrations than binder in SFP format. (Figure 2b, c).

[0373] Cloning of B7C 18 SFP into a y5 T cell immunotherapy expression cassette

[0374] To evaluate the performance of clone B7C18 SFP in the context of genetically-modified y<5 T cell immunotherapy, y<5 T cells were engineered with two expression cassettes. One that expressed a GFP marker gene and a I L15Ra-IL15 fusion protein (construct ID: JF114) or a GFP marker gene, a IL15Ra-IL15 fusion protein and a B7C18 SFP (construct ID: JF125) (Figure 3a). An IL15Ra-IL15 fusion protein was included in the expression cassette as it increased y<5 T cell immunotherapy product batch viability and purity, as well as substantially decreased the batch-to-batch variability between different production runs (Figure 3b and Figure 9). The presence of SFP in JF125 y<5 T cell supernatant was measured using ELISA, and was found to correlate positively with the number of transduced (GFP+) y<5 T cells in culture (Figure 3c). Unmodified (non-transduced, NTD), JF114 or JF125-modified y<5 T cells were cocultured overnight with luciferase-expressing BT474 breast carcinoma cells at an effectortarget ratio of 2:1 , in the presence of a range of zoledronic acid dilutions. JF125 y6 T cells showed enhanced overnight killing against BT474 cells compared to JF114 y<5 T cells, in a manner that was enhanced by combination with zoledronic acid (ZOL) (Figure 3d). Cytotoxicity was measured using a luminescence-based assay, where residual living tumour cell burden was measured using luciferin co-culture.

[0375] Efficacy of B7C 18 SFP-expressing JF125 y5 T cell immunotherapy against osteosarcoma and carcinoma

[0376] To evaluate the in vivo performance of y<5 T cell immunotherapy against bone-resident cancer, NSG mice were engrafted either with a luciferase-expressing patient-derived model of osteosarcoma (PDX) or with a luciferase-expressing HS520 lung carcinoma cell line. Once tumour engraftment was confirmed via I VIS, each animal was treated was treated with a single dose of 10e6 JF125 y<5 T cells. This treatment was followed in some animals by an intraperitoneal injection of zoledronic acid (ZOL) (Figure 4a). Therapeutic efficacy against osteosarcoma PDX was compared between unmodified y<5 T cells, with and without zoledronic acid, JF114 y6 T cells and JF125 y<5 T cells. Zoledronic acid on its own did not enhance y<5 T cell immunotherapeutic efficacy, in contrast to modification with an I L15Ra-l L15 fusion protein (JF114 cassette). The most efficacious therapy, which controlled or cured the tumour burden in all animals examined, was IL15Ra-IL15 fusion protein and B7C18 SFP-modified y<5 T cells (JF125 cassette) (Figure 4b). JF125 y<5 T cell immunotherapeutic efficacy was similarly evaluated against an intratibial model of HS520 lung carcinoma. JF125 y<5 T cells displayed a degree of tumour control, in a manner that was enhanced by combination with intraperitoneal zoledronic acid (ZOL) (Figure 4c).

[0377] Enhancing immunotherapeutic efficacy of B7C18 SFP-mediated y5 T cell

[0378] To evaluate the contribution of the Fc portion of the SFP the y<5 T cell ADCC in the context of synthetic immunotherapy, two additional expression cassettes were created: (i) JF162, which mutated the JF125 B7C18 SFP Fc from a wild type (WT) sequence to an Fc-incompetent (Fcnuii) variant, and (ii) JF172, which mutated the JF125 B7C18 SFP Fc from a wild type (WT) sequence to an affinity-enhanced variant, with Fc mutations Ser293Asp I Ala330Leu I lle332Glu (known as ‘DLE’) (FCDLE) (Figure 5a). Using a flow cytometric assay, unmodified y<5 T cell cytotoxicity was evaluated against killing-refractory MCF-7 and PC-3 carcinoma cell lines, following target co-culture either with plain media, media with exogenously-added B7C18 SFP Fcwr, non-transduced y<5 T cell supernatant, or supernatants from JF125, JF162 or JF172 y<5 T cells. The supernatants used in this assay were normalised to contain 0.75 nM of SFP each. Only JF172 y<5 T cell-derived supernatant mediated significant cytotoxicity against refractory tumour targets, suggesting that the DLE Fc mutation can overcome tumour resistance to y<5 T cell ADCC (Figure 5b). The immunotherapeutic efficacy of y<5 T cell products was also compared using cryopreserved JF172 y6 T cells. Thawed JF172 y6 T cells were co-cultured at a 1 :2 effectortarget ratio with isogenic luciferase-modified SupT1 lymphoma target cells, which are naturally B7H3ne9, or with an engineered SupT1 cell line which is B7H3pos. Tumour killing was measured by tallying the remaining luminescence in cell culture using luciferin co-culture. Thawed JF172 y6 T cells displayed efficient and antigenspecific cytotoxicity against SupT 1 lymphoma targets after both first and second challenge with tumour (Figure 5c). y5 T cell killing of non-malignant targets

[0379] JF125 and JF172-modified y<5 T cells were compared in terms of their ability to kill B7H3+target cells: in vitro-killing refractory lung cancer cell line HS520 or healthy human umbilical vein endothelial cells (HUVEC) (Figure 6a). Both constructs were first evaluated by flow cytometry for their ability to opsonize the target cells (co-culture with T cell supernatant, followed by anti-Fc staining of the target cells), whereby similar opsonisation was achieved by both constructs and on both target cell types. (Figure 6b). Target cell killing was then evaluated by a 4h flow cytometric cytotoxicity assay with supernatant pre-treated target cells and unmodified y<5 T cells as the ADCC effector cells. While only JF172 y<5 T cell supernatant mediated HS520 target cell killing, none of the culture conditions resulted in the cytotoxic killing of HLIVEC targets, indicating that y<5 T cells do not perform ADCC against opsonized but non- malignant target cells (Figure 6c).

[0380] Use of a translationally-compatible membrane-bound marker gene

[0381] To evaluate whether DLE-enhanced B7C18 scFv Fc fusion protein (SFP) I IL15Ra-IL15 fusion protein y<5 T cell immunotherapy retains functionality when combined with a translationally- compatible marker gene, truncated CD19 (tCD19), the JF172 expression cassette was modified to remove GFP and replace it with a tCD19 construct (creating the JF174 expression cassette) (Figure 7a). y<5 T cells expressed tCD19 robustly and in a manner that was dependent on the multiplicity of infection (MOI) of lentivirus used (Figure 7b). y<5 T cell cytotoxicity was then evaluated against in vitro refractory target MCF-7 and PC-3 carcinoma cell lines. Unmodified y<5 T cell ADCC against tumour cells was compared between targets pre-treated with either media only, T cell supernatant from unmodified y<5 T cells, or y<5 T cell supernatant from JF172 or JF174 cultures normalised for SFP content. Both JF172 and JF174 supernatants mediated potent and equivalent cytotoxicity against refractory carcinoma target cells (Figure 7c). y5 T cell immunotherapy compared to antibody

[0382] Unmodified Vd2 gd T cells were co-cultured with B7H3+ SupT1 tumour cells at a range of effectortarget ratios. Target cells were pre-treated with titred concentrations of either enoblituzumab (Fc enhanced MGA271), B7C18 (wild type Fc), or supernatant from B7C18(DLE) secreting gd T cells, where the opsonin had been titred beforehand. In the latter case, supernatant from unmodified gd T cells was used as a control. Treatment with opsonin secreted from gd T cell outperformed both pure enoblituzumab and B7C18, even at equimolar binder concentrations (Figure 8).

[0383] Humanisation of binder improves cytotoxicity

[0384] Unmodified Vd2 gd T cells were co-cultured with B7H3+ PC-3 tumour cells at a range of effectortarget ratios. Targets were pre-treated with titred, equimolar concentrations of supernatant from Vd2 cells secreting either chimeric B7C18 scFv-Fc fusion protein (JF174, as above, or JF187 [as for JF174 but including an extra glycine-serine-glycine between the C terminus of the Fc portion and the P2A self-cleaving peptide, which enables the stl L15 to be cleaved from the Fc]) or humanised B7C18 scFv-Fc fusion protein (JF189), where the opsonin had been titred beforehand. Each construct had the structure: scFv-Fc-stlL15-tCD19 (tCD19 serves as a marker gene), and each Fc contained the DLE Fc mutation. Supernatant from non-transduced cells was used a control. In a separate experiment, Vd2 cells secreting either chimeric (JF174 and JF187) or humanised (JF189) B7C18 scFv-Fc fusion protein were co-cultured with B7H3+ tumour cells at a range of effectortarget ratios. Non-transduced cells were used as a control.

[0385] Humanised binders secreted by gd T cells outperform chimeric binders secreted by gd T cells in gd T ADCC assays in both indirect (i.e. killing by unmodified gd T cells; Figure 10a).

[0386] Prophetic Example 1

[0387] The performance of the present B7H3 binder in the context of genetically modified yb T cell immunotherapy is further evaluated in T cells engineered to express constructs selected from the following: a) Unmodified cells b) T cells expressing an IL15Ra-IL15 fusion protein c) T cells expressing an I L15Ra-l L15 fusion protein and B7H3 binder according to the invention d) T cells expressing B7H3 binder according to the invention e) T cells expressing an I L15Ra-l L15 fusion protein and comparative B7H3 binder

[0388] (e.g. enoblitzumab) f) T cells expressing comparative B7H3 binder (e.g. enoblitzumab)

[0389] Read-outs such as y6 T cell viability and purity, and cytotoxicity (in the presence and absence of zoledronic acid) are assessed. Illustrative models include the BT474 breast carcinoma cell line (as per Figure 3) and intratibial models of osteosarcoma and carcinoma (as per Figure 4).

[0390] Prophetic Example 2

[0391] Transduction efficiency of B7H3 binder in scFv-Fc and lgG1 format is compared in immune cells.

[0392] NUMBERED EMBODIMENTS

[0393] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs.

[0394] 1. An immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule; wherein the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences. An immune cell that is capable of antibody dependent cellular cytotoxicity (ADCC) and which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule; wherein the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences. The immune cell according to paragraph 1 or paragraph 2, wherein the immune cell comprises an exogenous nucleic acid sequence encoding a cytokine. The immune cell according to paragraph 3, wherein the cytokine is Interleukin-15 (IL-15). The immune cell according to paragraph 3 or paragraph 4, wherein the cytokine is a modified IL-15, preferably an I L-15Ra-l L-15 fusion protein. The immune cell according to paragraph 5, wherein the modified IL-15 comprises an amino acid sequence according to SEQ ID NO: 99. 7. The immune cell according to any preceding paragraph, wherein the immune cell comprises an exogenous nucleic acid sequence encoding an expression marker.

[0395] 8. The immune cell according to paragraph 7, wherein the expression marker is CD19, preferably a truncated CD19.

[0396] 9. The immune cell according to any preceding paragraph, wherein the immune cell is an NK cell.

[0397] 10. The immune cell according to any one of paragraphs 1-8, wherein the immune cell is a T cell.

[0398] 11 . The immune cell according to any of paragraphs 1-8 and 10, wherein the immune cell is a gamma delta T cell.

[0399] 12. The immune cell according to paragraph 11 , wherein the gamma delta T cell is a V51 + gamma delta T cell, a V52+ gamma delta T cell, or a V51- / V52- gamma delta T cell.

[0400] 13. The immune cell according to paragraph 12, wherein the gamma delta T cell is a V52+ gamma delta T cell, preferably a Vy9V<52 T cell.

[0401] 14. The immune cell according to any preceding paragraph, wherein the immune cell does not express a chimeric antigen receptor (CAR).

[0402] 15. The immune cell according to any preceding paragraph, wherein the antigen binding molecule comprises an scFv, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, an Fv, a dAb, an Fd, a dsFv, a ds-scFv, an scFv2, a bi-specific T-cell engager, a nanobody, a DARPin, an antibody mimetic, a diabody, a triabody, or a tetrabody.

[0403] 16. The immune cell according to any preceding paragraph, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0404] 17. The immune cell according to any one of paragraphs 1 to 15, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0405] 18. The immune cell according to any one of paragraphs 1 to 15, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0406] 19. The immune cell according to any one of paragraphs 1 to 15, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0407] 20. The immune cell according to any one of paragraphs 1 to 15, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% identity thereto.

[0408] 21. The immune cell according to any one of paragraphs 1 to 15, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 19 or a variant having at least 80% identity thereto.

[0409] 22. The immune cell according to any preceding paragraph, wherein the antigen binding molecule is capable of binding to an Fc receptor.

[0410] 23. The immune cell according to any preceding paragraph, wherein the antigen binding molecule comprises an Fc region or a modified Fc region.

[0411] 24. The immune cell according to paragraph 23, wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 94.

[0412] 25. The immune cell according to any one of paragraphs 1 to 21 , wherein the antigen binding molecule is not capable of binding to an Fc receptor, optionally wherein the antigen binding molecule comprises a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 93. 26. The immune cell according to any one of paragraphs 1 to 23, wherein the antigen binding molecule is an scFv-Fc fusion protein, optionally wherein the scFv-Fc fusion protein comprises an amino acid sequence according to SEQ ID NO: 91 or SEQ ID NO: 92.

[0413] 27. The immune cell according to paragraph 26, wherein the antigen binding molecule is an scFv-Fc fusion protein comprising an amino acid sequence according to SEQ ID NO: 92.

[0414] 28. A pharmaceutical composition comprising an immune cell according to any one of the preceding paragraphs, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0415] 29. The pharmaceutical composition according to paragraph 28, for use as a medicament.

[0416] 30. The pharmaceutical composition according to paragraph 28, for use in the treatment of cancer.

[0417] 31. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to paragraph 28.

[0418] 32. The pharmaceutical composition for use according to paragraph 30, or the method according to paragraph 31 , wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and B-cell lymphoma.

[0419] 33. An antigen binding molecule comprising heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID

[0420] NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

[0421] 34. The antigen binding molecule according to paragraph 33, wherein the antigen binding molecule comprises an scFv, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, an Fv, a dAb, an Fd, a dsFv, a ds-scFv, an scFv2, a bi-specific T-cell engager, a nanobody, a DARPin, an antibody mimetic, a diabody, a triabody, or a tetrabody.

[0422] 35. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

[0423] 36. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

[0424] 37. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

[0425] 38. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

[0426] 39. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 14, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% identity thereto.

[0427] 40. The antigen binding molecule according to paragraph 33 or paragraph 34, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 19 or a variant having at least 80% identity thereto.

[0428] 41. The antigen binding molecule according to any one of paragraphs 33 to 40, wherein the antigen binding molecule is capable of binding to an Fc receptor

[0429] 42. The antigen binding molecule according to any one of paragraphs 33 to 41 , wherein the antigen binding molecule comprises an Fc region or a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 94.

[0430] 43. The antigen binding molecule according to any one of paragraphs 33 to 40, wherein the antigen binding molecule is not capable of binding to an Fc receptor, optionally wherein the antigen binding molecule comprises a modified Fc region, optionally wherein the modified Fc region comprises an amino acid sequence according to SEQ ID NO: 93.

[0431] 44. The antigen binding molecule according to any one of paragraphs 33 to 41 , wherein the antigen binding molecule is an scFv-Fc fusion protein, optionally wherein the scFv-Fc fusion protein comprises an amino acid sequence according to SEQ ID NO: 91 or SEQ ID NO: 92.

[0432] 45. A pharmaceutical composition comprising an antigen binding molecule according to any one of paragraphs 33 to 44, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0433] 46. One or more nucleic acid sequence(s) encoding an antigen binding molecule according to any one of paragraphs 33 to 44.

[0434] 47. A vector comprising the one or more nucleic acid sequence(s) according to paragraph 46. The vector according to paragraph 47, further comprising a nucleic acid sequence encoding a cytokine, optionally wherein the cytokine is an I L-15Ra-l L-15 fusion protein. The vector according to paragraph 47 or paragraph 48, further comprising a nucleic acid sequence encoding an expression marker, optionally wherein the expression marker is a truncated CD19. A cell comprising an antigen binding molecule according to any one of paragraphs 33 to 44, or one or more nucleic acid sequence(s) according to paragraph 46, or a vector according to any one of paragraphs 47 to 49. A method of producing an immune cell according to any one of paragraphs 1 to 27, comprising introducing one or more nucleic acid sequence(s) according to paragraph 46, or a vector according to any one of paragraphs 47 to 49 into an immune cell. A method for manufacturing a population of immune cells, comprising: i. providing a population of immune cells; ii. incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL-15; and iii. introducing into the immune cells a nucleic acid encoding an I L-15Ra-IL-15 fusion protein.

Claims

CLAIMS1. An immune cell which comprises one or more nucleic acid sequence(s) encoding an antigen binding molecule; wherein the antigen binding molecule comprises heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

2. The immune cell according to claim 1 , wherein the immune cell is capable of antibody dependent cellular cytotoxicity (ADCC).

3. The immune cell according to claim 1 or claim 2, wherein the immune cell comprises an exogenous nucleic acid sequence encoding a cytokine, preferably wherein the cytokine is Interleukin-15 (IL-15) or a modified IL-15.

4. The immune cell according to claim 3, wherein the modified IL-15 is an IL-15Ra-IL-15 fusion protein, preferably wherein the modified IL-15 comprises an amino acid sequence according to SEQ ID NO: 99.

5. The immune cell according to any preceding claim, wherein the immune cell comprises an exogenous nucleic acid sequence encoding an expression marker, preferably wherein the expression marker is a truncated CD19.

6. The immune cell according to any preceding claim, wherein the immune cell is an NK cell.

7. The immune cell according to any of claims 1-5, wherein the immune cell is a gamma delta T cell.

8. The immune cell according to claim 7, wherein the gamma delta T cell is a V51+ gamma delta T cell, a V52+ gamma delta T cell, or a V51- / V52- gamma delta T cell, preferably a Vy9V52 T cell.

9. The immune cell according to any preceding claim, wherein the immune cell does not express a chimeric antigen receptor (CAR).

10. The immune cell according to any preceding claim, wherein the antigen binding molecule comprises an scFv, a Fab, a modified Fab, a Fab', a modified Fab', a F(ab')2, an Fv, a dAb, an Fd, a dsFv, a ds-scFv, an scFv2, a bi-specific T-cell engager, a nanobody, a DARPin, an antibody mimetic, a diabody, a triabody, or a tetrabody.

11. The immune cell according to any preceding claim, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 16, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 17, or a variant having at least 80% identity thereto.

12. The immune cell according to any one of claims 1 to 10, wherein the antigen binding molecule comprises a heavy chain variable region (VH) comprising an amino acid sequence according to SEQ ID NO: 8, or a variant having at least 80% identity thereto, and a light chain variable region (VL) comprising an amino acid sequence according to SEQ ID NO: 9, or a variant having at least 80% identity thereto.

13. The immune cell according to any one of claims 1 to 10, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 10, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 11 , or a variant having at least 80% identity thereto.

14. The immune cell according to any one of claims 1 to 10, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 12, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 13, or a variant having at least 80% identity thereto.

15. The immune cell according to any one of claims 1 to 10, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO:14, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% identity thereto.

16. The immune cell according to any one of claims 1 to 10, wherein the antigen binding molecule comprises a VH comprising an amino acid sequence according to SEQ ID NO: 18, or a variant having at least 80% identity thereto, and a VL comprising an amino acid sequence according to SEQ ID NO: 19 or a variant having at least 80% identity thereto.

17. The immune cell according to any one of claims 1 to 16, wherein the antigen binding molecule is an scFv-Fc fusion protein, optionally wherein the scFv-Fc fusion protein comprises an amino acid sequence according to SEQ ID NO: 91 or SEQ ID NO: 92.

18. A pharmaceutical composition comprising an immune cell according to any one of the preceding claims, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

19. The pharmaceutical composition according to claim 18, for use in the treatment of cancer.

20. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 18.

21. The pharmaceutical composition for use according to claim 19, or the method according to claim 20, wherein the cancer is selected from melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, gastric cancer, kidney cancer, liver cancer, biliary cancer, thyroid cancer, mesothelioma, prostate cancer, breast cancer, endometrial cancer, oesophageal cancer, cervical cancer, ovarian cancer, colorectal cancer, pancreatic cancer, head and neck squamous cell carcinomas (HNSCC), neuroblastoma, Ewing sarcoma, osteosarcoma, soft tissue sarcoma, rhabdomyosarcoma, medulloblastoma, glioma, glioblastoma, multiple myeloma, acute myeloid leukaemia, acute lymphoblastic leukaemia, T-cell lymphoma, and B-cell lymphoma.

22. An antigen binding molecule comprising heavy chain complementarity determining regions (HCDRs) 1-3 and light chain complementarity determining regions (LCDRs) 1-3, wherein: i. HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 , ii. HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2, iii. HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 or SEQ IDNO: 4,iv. LCDR1 comprises an amino acid sequence according to SEQ ID NO: 5, v. LCDR2 comprises an amino acid sequence according to SEQ ID NO: 6, and vi. LCDR3 comprises an amino acid sequence according to SEQ ID NO: 7; optionally, wherein one or more of the HCDRs and / or LCDRs comprise one, two or three amino acid mutations relative to the recited sequences.

23. A pharmaceutical composition comprising an antigen binding molecule according to claim 22, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

24. One or more nucleic acid sequence(s) encoding an antigen binding molecule according to claim 22.

25. A method of producing an immune cell according to any one of claims 1 to 17, comprising introducing one or more nucleic acid sequence(s) according to claim 24 into an immune cell.

26. A method for manufacturing a population of immune cells, comprising: i. providing a population of immune cells; ii. incubating the immune cells in the presence of zoledronic acid and / or IL-2 and / or IL-15; and iii. introducing into the immune cells a nucleic acid encoding an I L-15Ra-IL-15 fusion protein.

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