Advanced treatment modalities for Anti-prame TCR-engineered immune cells

Abstract

The disclosure provides novel treatment regimen for PRAME-specific TCR-T cells for the treatment of cancer. A kit is also provided.

Description

[0001] Advanced Treatment Modalities for Anti-PRAME TCR-Engineered Immune Cells

[0002] FIELD OF THE DISCLOSURE

[0003] The present invention relates to a method of treating cancer, to immune cells for cancer treatment, and to cancer treatment regimen.

[0004] BACKGROUND

[0005] Cancer immunotherapy has emerged as a transformative approach in oncology, leveraging the body’s immune system to recognize and eliminate malignant cells. Among various immunotherapeutic strategies, T cell-based therapies have gained particular attention due to their specificity and potency. One such approach is T cell receptor (TCR) engineered T cell therapy (TCR-T), which involves modifying a patient’s T cells to express a tumor-specific TCR, thereby enhancing their ability to target and destroy cancer cells.

[0006] TCR-T therapy is distinct from chimeric antigen receptor (CAR) T cell therapy in that it targets intracellular tumor antigens presented on major histocompatibility complex (MHC) molecules, rather than cell surface antigens. This allows TCR-T to address a broader range of tumor-associated antigens, making it a promising strategy for treating solid tumors, which have been less responsive to CAR-T therapies.

[0007] T-cell based immunotherapy targets represent peptide epitopes derived from tumor- associated or tumor-specific proteins, which are presented by molecules of the major histocompatibility complex (MHC). These tumor associated antigens (TAAs) can be peptides derived from all protein classes, such as enzymes, receptors, transcription factors, etc. which are expressed and, as compared to unaltered cells of the same origin, usually up-regulated in cells of the respective tumor.

[0008] A TAA of particular relevance is the Preferentially Expressed Antigen of Melanoma (PRAME). PRAME is encoded by the PRAME gene, which is expressed at a high level in a large proportion of tumors. PRAME is the best characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family whereas in other vertebrate genomes only one PRAME-like LRR protein was identified. PRAME is a cancer / testis antigen that is expressed at very low levels in normal adult tissues except testis but at high levels in a variety of cancer cells.

[0009] Specific elements of the cellular immune response are capable of specifically recognizing and destroying tumor cells. The isolation of T-cells from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in natural immune defense against cancer. CD8-positive T-cells in particular, which recognize class I molecules of the major histocompatibility complex (MHC)- bearing peptides of usually 8 to 10 amino acid residues derived from proteins or defective ribosomal products (DRiPs) located in the cytosol, play an important role in this response. The MHC-molecules of the human are also designated as human leukocyte- antigens (HLA).

[0010] There are two classes of MHC-molecules, MHC class I and MHC class II. Complexes of peptide and MHC class I are recognized by CD8-positive T-cells bearing the appropriate T-cell receptor (TCR), whereas complexes of peptide and MHC class II molecules are recognized by CD4-positive-helper-T-cells bearing the appropriate TCR. Since both types of response, CD8 and CD4 dependent, contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor- associated antigens and corresponding T cell receptors is important in the development of cancer immunotherapies such as vaccines and cell therapies.

[0011] In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T-cells bearing specific T-cell receptors (TCR). Therefore, TAAs are a starting point for the development of T-cell based immunotherapies.

[0012] Approximately 90 percent of peripheral blood T cells express a TCR consisting of an a polypeptide and a [3 polypeptide. A small percentage of T cells (about 5% of total T cells) have been shown to express a TCR consisting of a y polypeptide and a 5 polypeptide. A TCR is a heterodimeric cell surface protein of the immunoglobulin super-family, which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in a|3 and y5 forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The extracellular portion of native heterodimeric alpha beta TCR and gamma delta TCRs each contain two polypeptides, each of which has a membrane-proximal constant domain, and a membrane-distal variable domain. Each of the constant and variable domains include an intra-chain disulfide bond. The chains of the T cell antigen receptor are each composed of a unique combination of domains designated variable (V), [diversity (D),] joining (J), and constant (C). In each T cell clone, the combination of V, D and J domains of both the alpha and the beta chains or of both the delta and gamma chains participates in antigen recognition in a manner which is uniquely characteristic of that T cell clone and defines a unique binding site which is formed by the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies in each variable domain. In contrast, the C domain does not participate in antigen binding. The use of TCR gene therapy overcomes a number of current hurdles. It allows equipping patients' own T cells with desired specificities and generation of sufficient numbers of T cells in a short period of time, avoiding their exhaustion. The TCR will be transduced into T cells, which are subsequently transfused into patients. Despite its promise, TCR-T therapy faces several challenges. Enhancements are still required to improve T cell persistence and functionality, and developing treatment regimen and combination approaches to enhance therapeutic efficacy. Accordingly, there remains a need for improved TCR-T therapies that address these challenges and provide enhanced safety and efficacy for cancer patients. The present invention relates to novel advancements in TCR-T cell therapy that seek to overcome existing limitations and expand the applicability of this promising treatment modality.

[0013] SUMMARY OF THE INVENTION

[0014] The present disclosure relates to novel cancer treatment strategies for TCR- engineered immune cells expressing a PRAME-004 TCR (“PRAME immune cells”), as well as TCR-engineered immune cells expressing a PRAME-004 TCR and CD8 (“PRAME CD8 immune cells”).

[0015] In one aspect, the present disclosure therefore relates to “PRAME CD8 immune cells” and provides an immune cell or population of immune cells for use in a method of treatment of cancer in a patient, wherein said immune cell expresses 1 ) a TCR or a derivative or fragment thereof that is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24), and 2) at least one heterologous CD8 chain. Additionally or alternatively, the immune cell(s) may comprise at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above, and at least one heterologous CD8 chain. The treatment comprises administering at least a single dose of about 1 -10 x 109cells to said patient.

[0016] Advantageously, patients may not require PRAME-004 testing prior to the treatment. However, patients may require testing for HLA-A*02:01 expression prior to treatment.

[0017] The cancer to be treated may be characterized by a medium prevalence of PRAME-004 expression of less than about 80%, 70%, 60%, 50%, 40%, 30% or 20%. Such cancers may be treated by administering at least a single dose of about 1 -10 x 109cells, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 x 109cells. Patients suffering from such cancers may require testing for PRAME-004 expression prior to treatment.

[0018] Alternatively, the cancer may be characterized by a high prevalence of PRAME- 004 expression of at least about 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99%. Such cancers may be treated by administering at least a single dose of about 1 - 10 x 109cells, preferably such as about 1 , 2, 3, 4, or 5 x 109cells. Advantageously patients suffering from such cancers may not require testing for PRAME-004 expression prior to treatment.

[0019] Patients may further have received at least one line of treatment prior to PRAME CD8 immune cell therapy. Specifically, patients may have previously received checkpoint inhibitor treatment, and have optionally relapsed under checkpoint inhibitor treatment.

[0020] The treatment may further comprise administering at least one single dose of about 1 million III interleukin (IL)-2. Typically, IL-2 is administered after the administration of the at least one single dose of immune cells. Preferably, IL-2 is administered once daily or twice daily. For instance, IL-2 may be administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Preferably, a dose of 1 million IU IL-2 is administered once daily on days 1 to 5 after administering said immune cell(s), and / or twice daily on days 6 to 10 after administering said immune cell(s).

[0021] Alternatively, the treatment regimen may not require the administration of IL-2 during and / or after PRAME CD8 immune cell treatment.

[0022] The PRAME-004 peptide recognized by the PRAME-004 TCR (or its fragment or derivative) is preferably presented on MHC-I molecules, typically on the surface of cancer cells.

[0023] The immune cells are preferably lymphocytes, more preferably T lymphocytes or T lymphocyte progenitors, and most preferably CD4 or CD8 positive T-cells.

[0024] It is further preferred that the PRAME-004 TCR (or its fragment or derivative), and / or said nucleic acid(s) encoding said TCR (or its fragment or derivative) are heterologous to said immune cell(s). Preferably, the PRAME-004 TCR and at least one heterologous CD8 chain are stably expressed by said immune cell(s).

[0025] The PRAME-004 TCR, or its fragment or derivative, preferably comprises six complementarity determining regions (CDRs): a CDR1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

[0026] The PRAME-004 TCR (or its fragment or derivative) preferably further comprises a TCR alpha variable domain, preferably comprising CDR1 a, CDR2a and CDR3a, and a TCR beta variable domain, preferably comprising CDR1 b, CDR2b and CDR3b. The TCR alpha variable domain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. The TCR beta variable domain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 . The PRAME-004 TCR (or its fragment or derivative) preferably comprises a TCR alpha chain and a TCR beta chain. The TCR alpha chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. The TCR beta chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0027] The at least one heterologous CD8 chain expressed by the immune cell(s) preferably comprises or consists of a heterologous CD8 alpha chain and / or a heterologous CD8 beta chain. The CD8 alpha chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to an amino acid sequence comprised in the sequence according to SEQ ID NO: 19, and the CD8 beta chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to an amino acid according to and SEQ ID NO: 20.

[0028] The TCR alpha variable domain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and / or the TCR alpha chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

[0029] The TCR beta variable domain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 16; and / or the TCR beta chain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 17;

[0030] The CD8 alpha chain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 21 ; and / or the CD8 beta chain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 22.

[0031] Specifically, the nucleic acid(s) encoding the PRAME-004 TCR (as comprised by the immune cell(s) may preferably comprise or consist of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 18. Preferably, the immune cell(s) may comprise one heterologous nucleic acid encoding the PRAME-004 TCR alpha chain and beta chain, and a heterologous CD8 alpha and beta chain. In such embodiments, the nucleic acids sequence may comprise or consist of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 23.

[0032] The nucleic acid(s) encoding said PRAME-004 TCR (or its fragment or derivative) and / or said heterologous CD8 chain(s) is / are preferably introduced into said immune cell(s) the form of a vector, preferably a viral vector, more preferably a lentiviral vector. The heterologous nucleic acid(s) may therefore be integrated into the genome of the immune cell(s). Alternatively, the heterologous nucleic acids may be present episomally.

[0033] The immune cells are typically administered as a single dose to the patient. However, it is also envisaged that the immune cells are administered in multiple single doses, such as two or more administrations, to the patient. In any event, the immune cells are typically administered parenterally, preferably intravenously, to the patient.

[0034] IL-2 is preferably administered parenterally, preferably subcutaneously or intravenously, to the patient.

[0035] Treatment may further comprise administering a checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. However, treatment may also not require administering a checkpoint inhibitor.

[0036] Treatment may further lymphodepletion, preferably prior to PRAME CD8 immune cell treatment.

[0037] The immune cell(s) is / are typically provided in the form of a pharmaceutical composition, optionally in soluble or cryopreserved form.

[0038] In view of the above, the present disclosure thus provides, in specific embodiments, an immune cell or population of immune cells for use in a method of treating cancer, wherein said immune cell(s) a) express at least one heterologous CD8 chain and a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or c) said immune cell(s) comprise at least one nucleic acid encoding at least one heterologous CD8 chain and a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises

[0039] 1 ) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and

[0040] 2) PRAME CD8 immune cell treatment at a single dose of 1 -10 x 109cells,

[0041] 3) optional IL-2 treatment at one or several doses of 1 million III per day.

[0042] The cancer may be selected from ovarian cancer, including ovarian carcinoma, ovarian serous cystadenocarcinoma, ovarian tube cancer (fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC), and primary peritoneal cancer (PPC); uterine cancer, including uterine carcinosarcoma (UCS), uterine endometrial carcinoma, uterine corpus cancer (UCC), uterine sarcoma, endometrial carcinoma, endometrial clear cell carcinoma, and endometrial serous carcinoma; triple-negative breast cancer; lung cancer, including squamous and non-squamous non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); melanomas, including cutaneous melanoma, uveal melanoma, and mucosal melanoma; and synovial sarcoma.

[0043] For many cancers, such as cutaneous melanoma, uveal melanoma, mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma, the patient may not require PRAME-004 testing prior to treatment.

[0044] In further aspects, the present disclosure relates to a method of treating cancer in a patient, comprising administering an immune cell or population of immune cells to said patient, wherein said immune cell(s) express at least one heterologous CD8 chain; and a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or comprise at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above and at least one heterologous CD8 chain; wherein said treatment comprises 1 ) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and

[0045] 2) PRAME CD8 immune cell treatment at a single dose of 1 -10 x 109cells,

[0046] 3) optional IL-2 treatment at one or several doses of 1 million III per day, preferably once per day on day 1 to day 5 after PRAME immune cell treatment, and twice per day on day 6 to day 10 after PRAME immune cell treatment.

[0047] In another aspect, the present disclosure relates to “PRAME immune cells” and provides an immune cell or population of immune cells for use in a method of treatment of cancer in a patient who has not been previously tested for PRAME-004 expression, wherein said immune cell expresses 1 ) a TCR or a derivative or fragment thereof that is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24). Alternatively or additionally, the immune cell(s) may comprise at least one nucleic acid that encodes a TCR or a derivative or fragment thereof as defined above. Treatment comprises administering at least a single dose of about 1 -10 x 109cells to said patient.

[0048] The cancer to be treated is preferably characterized by a high prevalence PRAME- 004 expression of at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99%. The cancer may be selected from cutaneous melanoma, uveal melanoma, mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

[0049] Advantageously, patients may not require PRAME-004 testing prior to the treatment. However, patients may require testing for HLA-A*02:01 expression prior to treatment.

[0050] Patients may further have received at least one line of treatment prior to PRAME immune cell therapy. Specifically, patients may have previously received checkpoint inhibitor treatment, and have optionally relapsed under checkpoint inhibitor treatment.

[0051] The treatment may further comprise administering at least one single dose of about 1 million III interleukin (IL)-2. Typically, IL-2 is administered after the administration of the at least one single dose of immune cells. Preferably, IL-2 is administered once daily or twice daily. For instance, IL-2 may be administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Preferably, a dose of 1 million IU IL-2 is administered once daily on days 1 to 5 after administering said immune cell(s), and / or twice daily on days 6 to 10 after administering said immune cell(s).

[0052] Alternatively, the treatment regimen may not require the administration of IL-2 during and / or after PRAME immune cell treatment. The PRAME-004 peptide recognized by the PRAME-004 TCR (or its fragment or derivative) is preferably presented on MHC-I molecules, typically on the surface of cancer cells.

[0053] The immune cells are preferably lymphocytes, more preferably T lymphocytes or T lymphocyte progenitors, and most preferably CD4 or CD8 positive T-cells.

[0054] It is further preferred that the PRAME-004 TCR (or its fragment or derivative), and / or said nucleic acid(s) encoding said TCR (or its fragment or derivative) is heterologous to said immune cell(s). Preferably, the PRAME-004 TCR is stably expressed by said immune cell(s).

[0055] The PRAME-004 TCR, or its fragment or derivative, preferably comprises six complementarity determining regions (CDRs): a CDR1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

[0056] The PRAME-004 TCR (or its fragment or derivative) preferably further comprises a TCR alpha variable domain, preferably comprising CDR1 a, CDR2a and CDR3a, and a TCR beta variable domain, preferably comprising CDR1 b, CDR2b and CDR3b. The TCR alpha variable domain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8. The TCR beta variable domain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 .

[0057] The PRAME-004 TCR (or its fragment or derivative) preferably comprises a TCR alpha chain and a TCR beta chain. The TCR alpha chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. The TCR beta chain preferably comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0058] The TCR alpha variable domain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and / or the TCR alpha chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. The TCR beta variable domain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 16; and / or the TCR beta chain is preferably encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 17;

[0059] Specifically, the nucleic acid(s) encoding the PRAME-004 TCR (as comprised by the immune cell(s) may preferably comprise or consist of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

[0060] The nucleic acid(s) encoding said PRAME-004 TCR (or its fragment or derivative) is / are preferably introduced into said immune cell(s) the form of a vector, preferably a viral vector, more preferably a lentiviral vector. The heterologous nucleic acid(s) may therefore be integrated into the genome of the immune cell(s). Alternatively, the heterologous nucleic acids may be present episomally.

[0061] The immune cells are typically administered as a single dose to the patient. However, it is also envisaged that the immune cells are administered in multiple single doses, such as two or more administrations, to the patient. In any event, the immune cells are typically administered parenterally, preferably intravenously, to the patient.

[0062] IL-2 is preferably administered parenterally, preferably subcutaneously or intravenously, to the patient.

[0063] Treatment may further comprise administering a checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. However, treatment may also not require administering a checkpoint inhibitor.

[0064] Treatment may further lymphodepletion, preferably prior to PRAME immune cell treatment.

[0065] The immune cell(s) is / are typically provided in the form of a pharmaceutical composition, optionally in soluble or cryopreserved form.

[0066] In view of the above, the present disclosure thus provides, in specific embodiments, an immune cell or population of immune cells for use in a method of treating cancer in a patient who has not been tested for PRAME-004 expression, wherein said immune cell(s) a) express a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or c) comprise at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises

[0067] 1 ) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and

[0068] 2) PRAME immune cell treatment at a single dose of 1 -10 x 109cells,

[0069] 3) optional IL-2 treatment at one or several doses of 1 million III per day, preferably once per day on day 1 to day 5 after PRAME immune cell treatment, and twice per day on day 6 to day 10 after PRAME immune cell treatment.

[0070] The cancer is preferably a cancer that is known or determined to have a high prevalence of PRAME-004 expression of at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least 98%, or at least about 99%.

[0071] The cancer may be selected from cutaneous melanoma, uveal melanoma, mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

[0072] In further aspects, the present disclosure relates to a method of treating cancer in a patient who has not been tested for PRAME-004 expression, comprising administering an immune cell or population of immune cells to said patient, wherein said immune cell(s) a) express a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or c) comprise at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises

[0073] 1) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and

[0074] 2) PRAME immune cell treatment at a single dose of 1-10 x 109cells, 3) optional IL-2 treatment at one or several doses of 1 million III per day, preferably once per day on day 1 to day 5 after PRAME immune cell treatment, and twice per day on day 6 to day 10 after PRAME immune cell treatment.

[0075] BRIEF DESCRIPTION OF THE FIGURES

[0076] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0077] For a list of abbreviations used throughout the specification, refer to Table 1 below.

[0078] FIGURE 1 : PRAME mRNA expression. FIG. 1 a depicts the PRAME target expression distribution (dark gray histograms) based on TCGA RNAseq data where available, patient data (dots, colors indicate BOR according to RECIST1.1 ) based on in-house qPCR testing of screening biopsies. Numbers in parentheses indicate the number of TCGA patient samples used for TCGA prevalence calculation. PRAME target prevalence is based on TCGA RNAseq data combined with a MS-guided RNA expression threshold. PRAME target prevalence in uveal melanoma based on inhouse qPCR testing of screening biopsies from clinical trial patients (n=33) demonstrates substantial higher prevalence of 91 % compared to prevalence based on TCGA data of 50%, TCGA: early & late-stage primary tumor samples; Immatics clinical trials: late-stage / metastatic tumor samples, indicated elevated expression of PRAME in late-stage uveal mpelanoma patients. FIG. 1 b shows homogenous PRAME expression across many tumor tissues. Spatial mRNA PRAME expression was analyzed using in situ hybridization RNAscope 2.5 LS Reagent Kit-RED assays (322150, Advanced Cell Diagnostics) on the Leica BOND RX platform (6.0.1.14) (Leica Biosystems). For tumor tissues, the tumor and stroma regions within the tissue were determined by analyzing hematoxylin and eosin staining. Corresponding serial sections were stained for PRAME expression (lower panel). PRAME expression is seen as red punctate dots homogenously distributed throughout the tumor regions. The staining was quantified using immunoreactivity score depicted in the adjacent table.

[0079] FIGURE 2: PRAME-004 TCR demonstrates deep objective responses across many tumors indications and deep and durable responses in melanoma. FIG. 2a: Best percent change in sum of diameter of target lesions from baseline and BOR by RECIST 1.1 in PRAME-004 monotherapy population. Each bar represents an individual patient. Left: BOR of patients treated in the dose escalation (DL1 to DL4; n=27), continued in FIG. 3. Right: BOR of patients treated in the dose extension (DL4 and DL5; n=13). Ovarian cancer patient 35 (DL5) received one dose of nivolumab. ‘Maximum change of target lesions and RECIST 1.1 response at different timepoints. FIG. 2b Percent change in sum of diameter of target lesions from baseline over time. Response over time in PRAME-004 TCR monotherapy dose escalation population (n=27). #Synovial sarcoma patient 19 (DL3) PD at week 6 not shown as target lesions were not evaluable. FIG. 2c: Response over time in PRAME-004 TCR monotherapy dose extension population (n=13), continued in FIG. 3. Ovarian cancer patient 35 (DL5) received one dose of nivolumab. ‘Response of patient 30 (DL4) until 5.7 months post-infusion, target lesion response assessment not available (external assessment). Each line represents one patient with the dots representing tumor assessments. The arrow indicates ongoing response at data cut-off. Colors indicate BOR according to RECIST1 .1 . The data show deeper and more durable responses in the dose extension cohort compared to dose escalation. DL1 =40 x106to 60 x106cells / m2BSA, DL2 = 120 x106to 180 x106cells / m2BSA, DL3 = 200 x106to 480 x106cells / m2BSA, DL4 =200 x106to 1200 x106cells / m2BSA, DL5 =1201 x106to 4700 x106cells / m2BSA.

[0080] FIGURE 3: PRAME-004 TCR demonstrates durable responses 2+ Years after treatment in heavily pretreated patients during dose extension (DL4, DL5). FIG 3a: BOR by RECIST 1.1 in PRAME-004 monotherapy population in continued dose extension (n = 28). FIG. 3b: Response over time in PRAME-004 TCR monotherapy continued dose extension (n=28). The median total infused dose was 5.09 x 109cells / patient (1 to 10.2 x 109cells / patient). FIG. 3c: BOR by RECIST 1.1 as in FIG. 3a, grouped by cancer.

[0081] FIGURE 4: Significant shift in mPFS and mOS between melanoma patients treated during the dose escalation and dose expansion phase. mPFS in dose escalation is comparable to reported data in 2L+ cut. melanoma population. mOS in dose escalation is shorter than reported mOS for 2L+ cut. melanoma population. At data cut-off, all patients in the dose escalation group died, and 20 / 28 patients are alive in dose expansion

[0082] FIGURE 5: Best overall response and depth of response were significantly associated with high degree of tumor infiltration. T-cell infiltration correlates with clinical efficacy. FIG. 5a: Bar graph (mean ± SD) showing PRAME-004 TCR T-cell infiltration into tumors in patients at day 42 post-infusion biopsies (n=22). Genomic DNA isolated from pre- and post-infusion biopsies was analyzed by qPCR using lentiviral Psi sequence-specific primers and results are expressed as vector copies detected per pg of gDNA. Mean ± SD. Two-tailed Mann Whitney statistical test was used. The p-values are depicted in the respective graphs. FIG. 5b: PRAME-004 T-cell infiltration [vector copies / pg gDNA] values for each patient with post-infusion biopsies plotted against depth of response max % change in sum of the longest diameter of target tumor lesions from baseline (n=22). FIG. 5c: Best % change in sum of diameter of target lesions compared to baseline according to RECIST 1.1 response plotted against PFS. The p-value was determined by two-sided Spearman correlation. The p- values and correlation coefficients are depicted in the respective graphs. Each point represents one patient and the respective BOR (color-coded) according to RECIST1.1. Triangles indicate censored patients (PFS; n=39).

[0083] FIGURE 6: Shrinkage of metastatic lesions throughout the body after PRAME- 004 TCR application. FIG. 6a: Lesion shrinkage under PRAME-004 TCR treatment in different locations throughout the body. Best percent change in diameter of target lesions from baseline of all measured target lesions (one to five per patient) in PRAME- 004 TCR monotherapy population (n=21 in lung, n=31 in liver, n=10 in pleura, n=6 in abdomen / peritoneum, n=5 in skin, n=21 in lymph node and n=31 in other); “Other” includes amongst others adrenal gland, bladder, kidney, spleen, pelvis, bone, brain and muscle. Box plots represent box bounds as the first and third quartiles, with horizontal lines inside the boxes depicting the median. Whiskers reach the minimum and maximum value of the data and each point represents the best change of one individual target lesion. Colors indicate the BOR according to RECIST 1.1 of the patient. FIG. 6b: Case study: CT scan and target lesion measurement (baseline and post-treatment) of patient 38 (DL5; cutaneous melanoma, post-baseline scan ~9 months post-T-cell infusion), patient 34 (DL5; uveal melanoma, post baseline scan ~12 months post-T-cell infusion), and patient 14 (DL2; synovial sarcoma, postbaseline scan ~24 months post-T-cell infusion). Circles mark target lesion and nontarget lesion, respectively.

[0084] FIGURE 7: Higher PRAME-004 TCR T dose is associated with durable responses. Dose, i.e., number of infused TCR T-cells (transduced viable CD8+ T- cells) is depicted for non-responders / short term responders (PD / SD / PR, n=27) and confirmed durable responders (cPR, n=11 ). Group comparisons were performed using two-sided Mann-Whitney U statistical test. Box plots depict median as horizontal lines within boxes, with box bounds as the first and third quartiles. Whiskers range from minimum to maximum values.

[0085] FIGURE 8: Rapid T-cell engraftment and long-term persistence observed in pharmacokinetic studies. PRAME-004 TCR persisted up to 743 days post-infusion which was determined using qPCR-based assay. Genomic DNA was isolated from post-infusion PBMC and PRAME-004 TCR T-cells were detected by using absolute qPCR assay with lentiviral Psi sequence of vector. Number of T-cells is reported per pg of gDNA used in the assay.

[0086] FIGURE 9: PRAME-004 CD8 TCR-T demonstrates objective responses across many tumors indications. FIG. 9a: Best percent change in sum of diameter of target lesions from baseline and BOR by RECIST 1 .1 in PRAME-004 CD8 TCR monotherapy population in patients treated at dose level DL3 (200 x106to 480 x106cells / m2BSA), DL4a (480 x106to 800 x106cells / m2BSA), and DL4b (801 x106to 1200 x106cells / m2BSA). Each bar represents an individual patient. FIG. 9b: Percent change in sum of diameter of target lesions from baseline over time. Each line represents one patient with the dots representing tumor assessments. The arrow indicates ongoing response at data cut-off.

[0087] FIG. 10: Opportunity of PRAME-004 CD8 TCR-T in medium-level PRAME- expressing cancers. FIG. 10a: Deep responses with PRAME-004 CD8 TCR-T at low doses, comparing PRAME-004 TCR T cells at a total infused dose of 5x109cells / patient to treatment with T cells expressing the same PRAME-004 TCR and additionally also CD8 at a total infused dose of 1.5 x 109cells / patient. FIG 10b: Potential for targeting medium-level PRAME expressing tumors with PRAME-004 CD8 TCR-T. FIG. 11 : Exemplary treatment regimen according aspects of the disclosure.

[0088] DEFINITIONS, ABBREVIATIONS AND ACRONYMS

[0089] As used herein in the specification, claims and Figures, terms, abbreviations and acronyms will have the following meanings.

[0090] Abbreviations Table 1: Abbreviations

[0091] Units

[0092] The unit “(immune) cells / m2BSA (body surface area) can be converted to total immune cell dose per patient using the Mosteller formula: Height (cm) x Weight (kg) 3600

[0093] Approximate total immune cell doses per patient can be calculated using the average BSA of 1 .73 m2.

[0094] Definitions

[0095] Where an indefinite or definite article is used when referring to a singular noun, e.g., “a”, “an” or “the”, does not exclude a plurality, unless something else is specifically stated. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Accordingly, the articles “a”, “an” or “the” preceding an element or component are intended to be non-restrictive regarding the number of instances (i.e. , occurrences) of the element. Therefore, “a”, “an” or “the” is to be read to include one or at least one, and the singular word form of the element also includes the plural unless the number is obviously meant to be singular.

[0096] The terms “about” or “approximately” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, ±15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, for instance within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. The terms “about” and “approximately” also include the concrete value, e.g., "about 50" includes the value "50".

[0097] The term “adoptive cell therapy” (“ACT”) is a type of immunotherapy in which T cells are administered to a patient to treat a disease, such as cancer. In autologous ACT, T cells that have been extracted from a patient are cultured in vitro and are typically genetically modified and are then returned to the same patient for therapy. Comparatively, allogeneic ACT involves T cells isolated and expanded from a donor or donors different from the patient receiving the T cells for therapy.

[0098] The term “administration” of an agent (e.g., host cells or polynucleotides of the disclosure or compositions or formulations comprising such host cells or polynucleotides) to a subject (e.g., a subject in need thereof) includes any suitable route of introducing or delivering the agent to a subject to perform its intended function. Administration of a therapeutic agent and compositions containing same can be performed in one dose or in several doses, continuously or intermittently throughout the course of treatment. Methods of determining the most effective routes and means of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, and the disease and subject being treated.

[0099] The term “administered in combination” or “combined administration” or “combination therapy” means that two or more therapeutic agents are administered to a patient at the same time or within an interval such that there can be an overlap of an effect of each agent on the patient.

[0100] “Affinity” is defined, in the context of the present disclosure by the equilibrium binding between the antigen binding protein and its antigen, e.g. the PRAME-004 TCR and the PRAME-004 peptide in a complex with a MHC protein. Affinity is usually expressed as equilibrium dissociation constant (KD).

[0101] The term “amino acid” refers to one of the 20 naturally occurring amino acids or any non-natural analogues. Preferably, the term “amino acid” refers to one of the 20 naturally occurring amino acids. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0102] “Amino acid mutations” may be deletions, insertions or substitutions.

[0103] The term “amino acid substitution” refers to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. An amino acid can be substituted in a parent or reference sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. In the context of the present disclosure, substitutions (even when they referred to as amino acid substitution) are typically conducted at the nucleic acid level, i.e., substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.

[0104] Accordingly, a reference to a “substitution at position X” refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position X, and Y and Z are alternative substituting amino acid residue. Amino acid substitutions may be conservative or non-conservative. For instance, and specifically in the context of antigen binding proteins, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. An amino acid substitution may also be a post-translational modification of the antigen binding protein and is herein also encompassed.

[0105] A conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class, for example, (non-polar amino acids substituted by other non-polar amino acids. A conservative amino acid substitution may be made in accordance with Table

[0106] 1. Methods for predicting tolerance to protein modification may be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA, 101 (25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0107] Conservative Amino Acid substitutions

[0108]

[0109] An antigen binding protein of the present disclosure can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and may include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S- acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, [3-phenylserine [3-hydroxyphenylalanine, phenylglycine, a- naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a,y- diaminobutyric acid, a,[3-diaminopropionic acid, homophenylalanine, and a-tert- butylglycine.

[0110] An antigen binding protein or nucleic acid(s) encoding the antigen binding protein of the present disclosure can be recombinant, isolated, engineered and / or purified.

[0111] The term “analogue” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0112] The term “and / or,” as used herein should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. In other words, the term “and / or” is to be interpreted as encompassing that one or more of the cases it connects may occur. Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0113] The term “antibody” as used herein is meant to include native and engineered antibodies. The term “engineered antibody includes functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

[0114] A “native antibody” comprises two heavy and two light chains, wherein the heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct domains (also referred to as regions). The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four or five domains depending on the antibody isotype; a variable domain (VH) and three or four constant domains (CHI , C 2 and CH3, and optionally CH4, collectively referred to as C ). The variable domains of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant domains of the light (C ) and heavy (C ) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0115] The specificity of the antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are made up of residues that are primarily from the “antibody complementarity determining regions” (CDRs) or hypervariable regions. Occasionally, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and hence the binding site. CDRs refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native antibody binding site. The light and heavy chains of an antibody each have three CDRs, designated CDR1 -L, CDR2-L, CDR3-L and CDR1 -H, CDR2-H, CDR3-H, respectively. An antibody antigen binding site, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. “Antibody framework regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e. to those portions of antibody light and heavy chain variable regions that are relatively conserved among different antibodies in a single species. The light and heavy chains of an antibody each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1 -H, FR2-H, FR3-H, FR4-H, respectively. Accordingly, the light chain variable domain may be described as (FR1 -L)-(CDR1 -L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L) and the heavy chain variable domain may be described as (FR1 -H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3- H)-(CDR3-H)-(FR4-H). As used herein, a "human framework region" is a framework region that is substantially identical (about 85%, or more, in particular 90%, 95%, 97%, 99% or 100%) to the framework region of a naturally occurring human antibody. In the context of the disclosure, CDR / FR in a TCR is determined based on IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1 ):55-77; www.imgt.org). Accordingly, amino acid sequences of the CDR1 , CDR2 and CDR3 of a given variable chain and the amino acid sequences of the framework regions (e.g. FR1 , FR2, FR3, and FR4) are indicated according to said IMGT definition in the herein provided disclosure.

[0116] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule or complex that is capable of being bound by an antigen binding site, wherein said antigen binding site is present in an antigen binding protein, preferably an antigen binding protein of the present disclosure. A target antigen may generally be a protein or antigenic peptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound.

[0117] The term “antigen binding protein” (occasionally abbreviated to “ABP”) herein refers to a polypeptide or a complex of two or more polypeptides comprising an antigen binding site that specifically binds to an antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein, and that polypeptide or the two or more polypeptides comprise(s) the CDRs as herein provided, such as CDRal , CDRa3, and optionally CDRa2, and CDRbl , CDRb3, and optionally CDRb2. The two or more polypeptides of the antigen binding protein may be covalently or non- covalently linked together. As used in the context of the present specification, the term antigen binding protein includes antigen binding proteins that comprise fragment(s) of the herein provided TCRs. The herein provided antigen binding proteins may be used in different formats as also described below, such as membrane bound antigen binding proteins, fusion proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins.

[0118] The term includes antigen binding proteins having the overall structure of a TCR, an antibody and / or a chimeric antigen receptor (CAR). The antigen binding protein can comprise TCR-derived CDRs, in particular a variable domain VAcomprising TCR-derived CDRal , CDRa3, and optionally CDRa2, and a variable domain VBcomprising TCR-derived CDRbl , CDRb3, and optionally CDRb2. Antigen binding proteins can comprise a variable domain VAcomprising complementarity determining regions (CDRs) CDRal , CDRa2, and CDRa3, e.g. on a first polypeptide, and a variable domain VBcomprising CDRbl , CDRb2, and CDRb3, e.g. on a second polypeptide, wherein CDRal , CDRa2, CDRa3, CDRbl , CDRb2 and CDRb3 form an “antigen binding domain A”. “Antigen binding domain A” denotes a binding domain that binds to the antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein. For instance, the entire VAdomain and / or the entire VBdomain may be TCR-derived and thus be TCR alpha, beta, gamma or delta variable domains (Va, V|3, Vy or Vb). Preferably, the antigen binding protein may be a TCR or functional fragment(s) thereof, e.g. the variable domains VAand VBof the TCR. In some instances, the antigen binding protein can comprise CDRs and optionally the VAand VBas herein provided, and can comprise further (an) additional domain(s) fused directly or indirectly to V or VB. The further domains may form (an) additional binding domain(s) or (a) binding site(s). For example and in particular instances, the additional binding domains may form antigen binding domain B. Further binding domains may be comprised that, e.g., form further antigen binding domains, e.g. antigen binding domain C, etc. The additional / further domains comprised in the antigen binding protein may also be a further protein.

[0119] The antigen binding protein thus also includes fusion proteins wherein fragment(s) of the herein provided TCRs further comprise other binding domains. Examples of additional domains comprised in an antigen binding protein of the disclosure that is a fusion protein are listed below.

[0120] The term “antigenic peptide in a complex with an MHC protein”, herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. In particular, the antigenic peptide is located to a “peptide-binding groove” formed by the MHC molecule. A complex of an MHC molecule and an antigenic peptide is herein also referred to as “peptide-MHC complex” or “pMHC complex”. For instance, in the case of the PRAME antigenic peptide, the complex is also referred to as “PRAME antigenic peptide-MHC complex” or “PRAME:MHC complex”.

[0121] An “antigen-expressing” cancer” (also referred to as an antigen “positive” cancer), is characterized by the over-presentation of the antigenic peptide (e.g., PRAME-004) in cancer cells. Some cancer indications are known to express a certain antigenic peptide. Others reguire testing of the patient. In such cases, a cancer biopsy can be used and the antigenic peptide can be identified using the XPresident® and related methods (according to WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, US 7,811 ,828, US 9,791 ,444, and US 2016 / 0187351 , the contents of each are hereby incorporated by reference in their entirety.

[0122] An “antigenic peptide presenting cell” or “antigenic peptide:MHC complex presenting cell” refers to a cell that presents on its surface the a certain antigenic peptide (e.g., PRAME-004) in a complex with an MHC molecule. Specifically, the antigenic peptide:MHC complex presenting cell may be a tumor cell, wherein the tumor is preferably a cancer as defined herein. In the context of the present disclosure, an antigenic peptide:MHC complex is “over-presented” on the cell surface of a an antigenic peptide:MHC complex presenting cell, compared to levels of said complex on the surface of cells in normal (healthy) tissue (also referred to as “healthy cells”) or on the surface of control cells loaded with a different antigen presenting peptide or no peptide. By "over-presented" is meant that the antigenic peptide:MHC complex is present at a level at least 2-fold, preferably between 5-fold to 10-fold of the level present in healthy tissue or control cells.

[0123] The term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain instances, the term “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0124] As used herein with respect to a disease, the term “associated with” means that the symptom, measurement, characteristic, or status in question is linked to the diagnosis, development, presence, or progression of that disease. As association can, but need not, be causatively linked to the disease. For example, symptoms, sequelae, or any effects causing a decrease in the quality of life of a patient having cancer are considered associated with the cancer and in some instances of the present disclosure can be treated, ameliorated, or prevented by administering the polynucleotides of the present disclosure to a subject in need thereof.

[0125] The term “at least one” herein refers to one or more of the specified elements such as 1 , 2, 3, 4, 5 or 6 or more of the specified elements. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to 1 , 2, 3, 4, 5 or 6 or more A, with no B present (and optionally including elements other than B); in another embodiment, to 1 , 2, 3, 4, 5 or 6 or more B, with no A present (and optionally including elements other than A); in yet another embodiment, to 1 , 2, 3, 4, 5 or 6 or more A, and 1 , 2, 3, 4, 5 or 6 or more B (and optionally including other elements); etc.

[0126] The term “binding affinity” refers to the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Without wishing to be bound by theory, affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term “avidity,” which refers to the strength of the antigen-antibody bond after formation of reversible complexes (e.g., either monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, comprising use of binding experiments to calculate affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). A low-affinity complex contains an antibody that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an antibody that generally tends to remain bound to the antigen for a longer duration. Antibody activity in functional assays (e.g., flow cytometry assay) is also reflective of antibody affinity. Antibodies and affinities can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assay).

[0127] The term “biocompatible” means compatible with living cells, tissues, organs or systems posing little to no risk of injury, toxicity or rejection by the immune system. The term “biodegradable” means capable of being broken down into innocuous products by the action of living things.

[0128] The phrase “biologically active” refers to a characteristic of any substance that has activity in a biological system and / or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. For instance, a polynucleotide (e.g. mRNA) or antigen binding protein can be considered biologically active if even a only a portion of the polynucleotide or antigen binding protein retains or mimics the desired biological effect.

[0129] The term “(pharmaceutically acceptable) carrier” refers to a type of excipient used to deliver a therapeutic agent to a specific target site in the patient’s body. It acts as a vehicle or medium to transport the therapeutic agent, and it can help to enhance the solubility, stability, and bioavailability of the therapeutic agent. Carriers are usually inert substances that do not interfere with the therapeutic action of the therapeutic agent.

[0130] The carrier typically serves as a vehicle or medium to transport the therapeutic agent to the target site and ensure its proper distribution and absorption in the body. Examples of pharmaceutically acceptable carriers include solvents, diluents, binders, and lubricants, among others. These carriers are typically selected based on their compatibility with the therapeutic agent and other components of the composition or formulation, as well as their safety and effectiveness in delivering the therapeutic agent to the patient. The use of a pharmaceutically acceptable carrier can help to ensure the stability, efficacy, and safety of the final product. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They can be selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. In the context of polynucleotides, e.g. mRNAs, pharmaceutically acceptable carriers include nanoparticles, such as an polymeric nanoparticle carrier or an lipid nanoparticle (LNP). The terms “coding region” and “region encoding” and grammatical variants thereof, refer to an Open Reading Frame (ORF) in a polynucleotide that upon expression yields a polypeptide or protein.

[0131] The terms “codon substitution” or “codon replacement” in the context of seguence optimization refer to replacing a codon present in a reference nucleic acid seguence with another codon. A codon can be substituted in a reference nucleic acid seguence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a "substitution" or "replacement" at a certain location in a nucleic acid seguence (e.g., an mRNA) or within a certain region or subseguence of a nucleic acid seguence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon.

[0132] The term “complementarity” with respect to seguences refers to two nucleotide seguences which, when aligned anti-parallel to each other, contain multiple individual nucleotide bases which pair with each other. Pairing of nucleotide bases forms hydrogen bonds and thus stabilizes the double strand structure formed by the complementarity seguences. It is not necessary for every nucleotide base in two seguences to pair with each other for seguences to be considered “complementarity”. Seguences may be considered complementarity, for example, if at least 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the nucleotide bases in two seguences pair with each other. In some instances, the term complementarity refers to 100% of the nucleotide bases in two seguences pair with each other. In addition, seguences may still be considered “complementarity” when the total lengths of the two seguences are significantly different from each other. For example, a primer of 15 nucleotides may be considered “complementarity” to a longer polynucleotide containing hundreds of nucleotides if multiple individual nucleotide bases of the primer pair with nucleotide bases in the longer polynucleotide when the primer is aligned anti-parallel to a particular region of the longer polynucleotide. Nucleotide bases paring is known in the field, such as in DNA, the purine adenine (A) pairs with the pyrimidine thymine (T) and the pyrimidine cytosine (C) always pairs with the purine guanine (G); while in RNA, adenine (A) pairs with uracil (U) and guanine (G) pairs with cytosine (C). Further, the nucleotide bases aligned anti-parallel to each other in two complementarity seguences, but not a pair, are referred to herein as a mismatch.

[0133] The term “comprising” is to be interpreted as encompassing all specifically mentioned elements as well optional, additional, unspecified elements. In other words, the term “comprising” does not exclude other elements. For the purposes of the present disclosure, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments. The term “consisting of” refers to the inclusion of exactly one element of a number or list of elements, and preferably excluding more than trace elements of other ingredients other than the element or list of elements.

[0134] The term “consisting essentially of” when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like.

[0135] The term “contacting” means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a compound means that the mammalian cell and compound are made to share a physical connection. Methods of contacting cells with entities both in vivo and ex vivo are known in the biological arts. For example, contacting a compound and a mammalian cell disposed within a mammal can be performed by varied routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve varied amounts of compound. Moreover, more than one mammalian cell can be contacted by a compound.

[0136] The term “copy number” refers to the number of antigenic peptide / MHC complexes as defined in the context of the present disclosure that are present on the cell surface of a cell, such as a antigenic peptide / MHC presenting cell, for example a cancer cell, or a healthy cell. Copy numbers of a protein can be determined by a variety of art known methods including FACS analysis of diseased cells with fluorescently labelled antigen binding proteins.

[0137] The term "cytotoxic agent" refers to a compound, molecule, or biological entity that induces cell death or inhibits cell proliferation by disrupting essential cellular processes. Cytotoxic agents may function through various mechanisms, including but not limited to, DNA damage, inhibition of mitosis, induction of apoptosis, disruption of protein synthesis, or generation of reactive oxygen species. Examples include chemotherapeutic drugs (e.g., doxorubicin, paclitaxel), immunotoxins, oncolytic viruses, radionuclides, and targeted payloads in antibody-drug conjugates (ADCs). Cytotoxic agents may be used in therapeutic, diagnostic, or research applications, particularly in the treatment of cancer, autoimmune diseases, or other pathological conditions involving aberrant cell growth.

[0138] The term “delivering” means providing an entity to a destination. For example, delivering a polynucleotide or host cell to a subject can involve administering a composition including the polynucleotide or host cell to the subject (e.g., by an intravenous, intramuscular, intradermal, or subcutaneous route). The term "disease" or "disorder" refers to any condition that would benefit from treatment with the therapeutic agent(s) of the disclosure. The term may include chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question.

[0139] The term “diagnostic agent” as used herein refers to a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any other labels known in the art that provide (either directly or indirectly) a signal.

[0140] A “domain” may be any region of a protein or nucleic acid sequence, generally defined on the basis of sequence homologies and often having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions). The terms “domain” and “region” may be used interchangeably herein.

[0141] The term "dosage" or "unit dose" or refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses in association with its administration, i.e. , the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the result and / or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition. Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described herein.

[0142] The term “(therapeutically / pharmaceutically) effective amount” refers to a quantity of a therapeutic agent, either alone or on combination with one or more other therapeutic agents, sufficient to treat a disease as defined elsewhere herein. In the context cancer treatment, an effective amount of the therapeutic agent preferably produces at least one desired response, which includes inhibiting the progression of the cancer, either by slowing its progression temporarily, or by stopping its progression permanently. The effective amount of a therapeutic agent administered to the subject can depend on the type and severity of the disease or condition and on the characteristics of the individual, such as general health, age, sex, body weight, effective concentration of the polynucleotide(s) administered, and tolerance to drugs. It can also depend on the degree, severity, and type of disease, the time of administration, route of administration, and rate of excretion, exhaustion or degradation of the therapeutic agent; the duration of the treatment; drugs used in combination or coincidental with the specific therapeutic agent employed; and other factors. The skilled artisan will be able to determine the effective amount depending on these and other factors. An effective amount can be administered to a subject in one or more doses. The terms “(therapeutically / pharmaceutically) effective amount” can be used interchangeably with “(therapeutically / pharmaceutically) effective dose”, respectively.

[0143] The term “(therapeutic) efficacy” refers to as a parameter that describes the capability of a therapeutic agent to exert its desired therapeutic effect, e.g. for a host cell expressing an antigen binding protein to kill cancer cells. The efficacy can be determined in a functional assay, for example a live-cell monitoring cytotoxicity assay as described below.

[0144] The term “E:T ratio” refers to the ratio of effector cells (i.e. immune cells, in particular T cells, expressing the antigen binding protein, in particular the TCR) to target cells. In some instances, the E:T ratio corresponds to the seeding ratio, i.e. the ratio of the total number of immune cells, in particular T cells, to target cells. In some instances, the E:T ratio is lower than the seeding ratio. This applies to cases where not all immune cells express the antigen binding protein, i.e. not all immune cells are effector cells, for example due to a low electroporation efficiency. In some instances, the seeding ratio is used as approximation of the E:T ratio. In some instances, the E:T ratio is determined by adjusting the seeding ratio taking into account the electroporation efficiency.

[0145] In an example of a lactate dehydrogenase (LDH)-release assay, the effector cells are immune cells. These effector cells are co-cultured with tumor cells endogenously expressing and presenting the antigenic peptide and optionally additionally loaded with the antigenic peptide. In some instances, the tumor cells are SKMEL-5 cells, RPMI7951 cells or SCC25 cells. In some instances of the LDH-release assay, the seeding ratio of total immune cells and target cells is 10:1 . The efficacy of an effector cell or its antigen binding protein is considered high if in a LDH-release assay as defined above, killing of tumor cells (as determined LDH release) is observed at an E:T ratio of 10: 1 . Alternatively, the efficacy of an effector cell or its antigen binding protein is considered high if in a cytotoxicity assay, preferably a LDH-release assay as defined above, the cytotoxic activity of the effector cells against the target cells at the highest concentration of the antigen binding protein tested is at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of the cytotoxic activity of a control toxic reagent. The skilled in the art is aware that the cytotoxic activity can be higher than 100%. This is due to the fact that 100% cytotoxic activity is defined by a “maximum lysis control”, which refers to incubation of the target cells with the toxic reagent. In some instances, the toxic reagent is a detergent, e.g. Triton-Xi 00, Tween-20, Tween-80 or NP-40, that effects lysis of the target cells. In some specific examples, the maximum lysis control comprises adding a 0.9% Triton-X100 solution to the target cell culture. The cytotoxic activity of the toxic reagent, i.e. the number of target cells killed by the toxic reagent is defined as 100%. Since the target cells can still proliferate during the co-culture, the effector cells may eventually kill an even higher number of target cells during the cytotoxicity assay than the toxic reagent killed during the maximum lysis control. In such instances, the calculated cytotoxic activity will be higher than 100%.

[0146] In an example of a cytokine production assay and cytokine release assay, the effector cells are immune cells expressing the antigen binding protein as host cells. These effector cells are co-cultured with cells (either target cells, e.g. tumor / cancer cells, or peptide loaded cells, e.g. T2 cells) with different expression of the antigenic peptide in complex with MHC, such as an HLA protein, for instance HLA-A*02. Preferably, the effector cells and the target cells are seeded, e.g. at a ratio between 10:1 and 1 :1. For the cytokine release assay, after a defined time of co-culture, e.g. 24-48 hours, preferably about 48 hours, the supernatants of the co-culture (effector cells + target cells) are collected and subjected to a cytokine release ELISA assay, for example IFN-gamma, TNF alpha, IL-2 and Granzyme B, to determine the amount of cytokine released by the effector cells. In order to determine the efficacy, a cytokine production assay can be applied using target cells, e.g. tumor or cancer cells. Alternatively, the killing of a target cell / cancer cell / tumor cell can be determined in e.g. an LDH assay or a live cell imaging assay. The efficacy of an antigen binding protein may be considered high if the antigen is capable of activating effector cells in a cytokine production assay, in particular if the amount of produced cytokines upon coculture with target cells is at least about 100 pg / ml, at least about 300 pg / ml, preferably at least about 500 pg / ml, more preferably at least about 1000 pg / ml.

[0147] The term “engineered” or “genetically engineered” in the context of a nucleic acid, protein, cell or organism, means that the nucleic acid, protein, cell or organism has been modified using biotechnological methods. Such genetically modified nucleic acids, proteins, cells or organisms typically do not occur in nature. For instance, genetically engineered nucleic acids, proteins, cells or organisms can be obtained by introducing a DNA vector or an RNA encoding a heterologous gene.

[0148] The term “epitope”, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system. As used herein, the term epitope comprises the terms “structural epitope” and “functional epitope”. The term “structural epitope” refers those amino acids of the antigen, e.g. peptide-MHC complex, that are covered by the antigen binding protein when bound to the antigen. Typically, all amino acids of the antigen are considered covered that are within 5 A of any atom of an amino acid of the antigen binding protein. The structural epitope of an antigen may be determined by art known methods including X-ray crystallography or NMR analysis. The structural epitope of an antibody typically comprises 20 to 30 amino acids. The structural epitope of a TCR typically comprises 20 to 30 amino acids. A “functional epitope” as herein defined is a subset of those amino acids forming the structural epitope and comprises the amino acids of the antigen that are critical for formation of the interface with the antigen binding protein, either by directly forming non-covalent interactions such as H-bonds, salt bridges, aromatic stacking or hydrophobic interactions or by indirectly stabilizing the binding conformation of the antigen and is, for instance, determined by mutational scanning. In the context of the present disclosure, the functional epitope is also referred to as “binding motif”. Typically, the functional epitope of an antigen bound by an antibody comprises between 4 and 6 amino acids. Typically, the functional epitope of a peptide-MHC complex comprises between 2 to 6 or 7 amino acids of the peptide and 2 to 7 amino acids of the MHC molecule. Since MHC I presented peptides typically have a length between 8 to 10 amino acids only a subset of amino acids of each given peptide is part of the functional epitope of a peptide-MHC complex. The epitope, in particular the functional epitope bound by the antigen binding protein comprises or consists of the amino acids of the antigen that are required for formation of the binding interface.

[0149] Term “excipient” refers to a substance that is added to a therapeutic agent to facilitate its manufacturing, administration, stability or therapeutic efficacy. Excipients are typically inactive substances that are used, inter alia, as carriers, solvents, fillers, binders, disintegrants, lubricants, or flavoring agents.

[0150] The term "expression system" means a host cell and compatible expression vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

[0151] An “expression vector” refers to vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0152] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.

[0153] “Fluorescent molecules” are known in the art include fluorescein isothiocyanate (FITC), phycoerythrin (PE), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or uv laser (e.g. Pacific blue, pacific orange). A “format” in relation to an antigen binding protein relates to a defined spatial arrangement of domains, in particular of variable and optionally constant domains. Characteristics of such antigen binding protein formats are the number of polypeptide chains (single chain, double chain or multiple chains), the type and length of linkers connecting different domains, the number of variable domains (and thus the number of valences), the number of different variable domains (and thus the number of specificities for different antigens, e.g. bispecific, or multispecific), and the order and orientation of variable domains (e.g. cross-over, parallel).

[0154] The term "formulation" refers to a specific therapeutic product that has been developed with a particular set of ingredients, dosage form, and delivery method to meet specific therapeutic needs. It is a final product that has typically undergone several steps of formulation development, such as selecting the appropriate ingredients, optimizing the drug delivery system, and ensuring its safety and efficacy. Different formulations of the same therapeutic agents may have different pharmacokinetic and pharmacodynamic properties, and may be used to treat different conditions or patient populations.

[0155] A "fragment," as used herein, refers to a portion of a nucleic acid or protein. For example, fragments of proteins can comprise polypeptides obtained by digesting full- length protein isolated from cultured cells. A fragment may be a subsequence of a full- length protein wherein N-terminal, and / or C-terminal, and / or internal subsequences have been deleted. Preferably, fragments of a protein are functional fragments (i.e., exhibit or mimic the protein’s desired biological activity), or exhibit a novel desired biological activity.

[0156] The term “free of”, as used herein, means not comprising the referenced component. For example, when a composition, solution, or formulation is described as being “free of X”, the composition, solution, or formulation does not comprise X.

[0157] In a “functional assay”, an antigen binding protein is, for example, expressed in an “effector cell (E)”, and the effector cell is co-cultured with “target cells (T)”, i.e. with antigen presenting cells presenting a peptide:MHC complex. Functional assays can thus also be described as “co-culture assays”. For all cell culture assays described herein, the cell culture temperature preferably is at about 37°C. Preferably, the effector cell is a T cell. The target cells may be cells that are artificially loaded with the antigenic peptide (e.g. T2 cells) or may be cells that endogenously present the target antigenic peptide on their surface (e.g. cancer cells expressing PRAME). Binding of the antigen binding protein to the peptide:MHC complex leads to activation of the effector cell. Depending of the type of functional assay, there are different readouts for measuring the degree of activation. In a cytokine production assay or cytokine release assay, such as an ELISA, the production of cytokines (e.g. TNF-a, IFN-y, CD107a+, IL-2 and / or Granzyme B) by the effector cells is determined. In a cytotoxicity assay, the killing of target cells by the effector cells is determined, e.g. by measuring a decline in proliferation of target cells, in particular cancer cells or by measuring the release of intracellular proteins from the target cells. Suitable intracellular proteins to be measured in a cytotoxicity assay can be endogenous proteins, e.g. LDH release assay.

[0158] “Functional avidity” is defined, in the context of the present disclosure, as a parameter that describes the capability of an antigen binding protein to activate an effector cell, preferably a T cell, upon binding to its target antigenic peptide in a complex with MHC. The activation of the effector cell, preferably T cell, can be measured in a functional assay, e.g., a cytokine production assay or a cytotoxicity assay as described below. In some embodiments, the functional avidity of an antigen binding protein is considered high if the EC50determined in a functional assay is low, such as less than about 50 nM, less than about 20 nM, or less than about 5 nM in a cytotoxicity assay as described below, and / or the activity determined in a functional assay is high, such as at least 50%, at least 60%, at least 70%, at least 75%, preferably at least 80%, at least 85%, at least 90%, or at least 95% of a maximal activity defined in the respective functional assay. Depending on the functional assay, the maximal activity may be the activity of a reference protein with known high functional avidity or the activity of a “maximum lysis control” as described below.

[0159] The term "gene" means a DNA sequence that codes for, or corresponds to, a particular sequence of amino acids which comprises all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. In particular, the term gene may be intended for the genomic sequence encoding a protein, i.e. a sequence comprising regulator, promoter, intron and exon sequences.

[0160] “Half maximal effective concentration” also called “ECso”, typically refers to the concentration of a molecule, which induces a response halfway between the baseline and maximum after a specified exposure time. EC50and affinity are inversely related, the lower the EC50value the higher the affinity of the molecule.

[0161] The term “heterologous” refers to an element, such as a nucleic acid sequence, a gene or a protein that is foreign to its genomic location and / or host in that it originates from a different cell, genomic location, organism or species. The term is, inter alia, used in the context of “heterologous” gene expression, which is typically achieved through recombinant DNA technology. Essentially, a gene from one cell or organism is optionally engineered and inserted into a different cell or organism, which his then also referred to as a “recombinant” cell or organism. A “heterologous” proteins is produced in a system (e.g., a cell) different from their native host. The term “HLA-A*02” signifies a specific HLA allele, wherein the letter A signifies the allele and “*02” indicates the A2 serotype.

[0162] The term "homology" refers to the overall relatedness between polymeric molecules, e.g., between polynucleotides (e.g., DNA molecules and / or RNA molecules) and / or between (poly-)peptides. Generally, the term "homology" implies an evolutionary relationship between two molecules. Thus, two molecules that are homologous will have a common evolutionary ancestor. In the context of the present disclosure, the term homology encompasses both to identity and similarity. As used herein, polymeric molecules may be considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers in the molecule are identical (exactly the same monomer) or are similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0163] A “host cell” is a cell that comprises a heterologous nucleic acid, gene or protein. Specifically, a host cell can express a heterologous nucleic acid. The host cell may be transfected, infected or transduced or transformed, in particular with nucleic acid(s) and / or a vector(s) as described herein. Host cells include immune cells and other types of cells.

[0164] The term "human," when referring to an antigen binding protein, such as a TOR, or any component of a TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, alpha chain, and / or beta chain), means a TCR (or component thereof), which is derived from a human unrearranged corresponding gene locus, such as a TCR locus, respectively. Similarly, the term “murine” refers to an antigen binding protein (or a component thereof) which is derived from a corresponding murine unrearranged gene locus, respectively.

[0165] The term “humanized” in the context of an antigen binding protein, such as a TCR or antibody refers to an antigen binding protein which is completely or partially of non-human origin and which has been modified by replacing certain amino acids, in particular in the framework regions of the heavy and light chains, in order to avoid or minimize an immune response in humans. The constant domains of a humanized antigen binding protein are mainly human C and C domains. Numerous methods for humanization of an antibody sequence are known in the art. For example, a “humanized” antigen binding protein can be made by the introduction of conservative substitutions, consensus sequence substitutions, germ line substitutions and / or back mutations, see, e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and the review by Almagro & Fransson (2008) Front Biosci. 13: 1619- 1633. One commonly used method is CDR grafting, which, for example, involves grafting of the CDR sequences of a donor antibody, generally a mouse antibody, into the framework scaffold of a human antibody of different specificity. Since CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody, back mutations may be introduced at selected positions of the CDR grafted antibody in order to retain the binding specificity and affinity of the parent antibody. Identification of positions for possible back mutations can be performed using information available in the literature and in antibody databases. An alternative humanization technique to CDR grafting and back mutation is resurfacing, in which non-surface exposed residues of non-human origin are retained, while surface residues are altered to human residues. Another alternative technique is known as “guided selection” (Jespers et al. (1994) Biotechnology 12, 899) and can be used to derive from for example a murine or rat antibody a fully human antibody conserving the epitope and binding characteristics of the parental antibody. A further method of humanization is the so-called 4D humanization. The 4D humanization protocol is described in the patent application US20110027266 A1 (the content of which is incorporated by reference in its entirety) (W02009032661A1 ) and is exemplified in the following applying the 4D humanization to humanize the rat antibody variable light (VL) and heavy (VH) domains. Such techniques are equally applicable to other antigen binding proteins, such as TCRs, mutatis mutandis.

[0166] For chimeric antigen binding proteins, such as antibodies or TCRs, humanization typically involves modification of the framework regions of the variable region sequences. Knowing the amino acid sequence of the CDRs an antigen binding protein of the disclosure, e.g., a TCR, one skilled in the art can easily determine the framework regions, such as the TCR framework regions. In cases where the CDRs are not indicated, the skilled in the art can first determine the CDR amino acid sequences based on the IMGT definition for TCRs and then determine the amino acid sequences of the framework regions.

[0167] The term “immunoglobulin (Iq) domain” refers to a protein domain that consists of a 2-layer sandwich of 7-9 antiparallel [3-strands arranged in two [3-sheets with a Greek key topology. Proteins containing Ig domains are subsumed into the immunoglobulin superfamily, including e.g. antibodies, T cell receptors (TCRs) and cell adhesion molecules. Examples of Ig domains are the variable and constant domains of antibodies and TCRs.

[0168] The term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0169] The term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0170] The term “isolated” refers to the separation of a specific molecule or entity from its natural environment or context. For nucleic acids, isolation can refer to the separation of a specific DNA or RNA sequence from the rest of the naturally occurring genomic material. This can be done through techniques such as PCR, gel electrophoresis, or hybridization. An “isolated” nucleic acid can be present in solution, or in a host cell. For polypeptides, isolation can refer to the purification of a specific protein from a complex mixture of proteins or cell culture components. This can be done using techniques such as chromatography, electrophoresis, or immunoaffinity purification. For host cells, isolation can refer to the removal of a specific cell type from a mixed population of cells. This can be done using techniques such as fluorescence- activated cell sorting (FACS), magnetic-activated cell sorting (MACS), or microfluidic cell sorting.

[0171] “KD” is the equilibrium dissociation constant, a ratio of kOff / kOn, between an antigen binding protein and its antigen. KDand affinity are inversely related. The KDvalue relates to the concentration of an antigen binding protein and the lower the KDvalue, the higher the affinity of an antigen binding protein. The KDvalue can be experimentally assessed by a variety of known methods, such as measuring association and dissociation rates with surface plasmon resonance (SPR) or bio-layer interferometry (BLI). For example, an antigen binding protein can be produced as soluble molecule, for instance, by removing the transmembrane domain and introducing an artificial disulfide bridge (Boulter et al. 2003; Stable, soluble T-cell receptor molecules for crystallization and therapeutics; Protein Engineering vol. 16 no. 9 pp. 707-711 ) or dimerization via a leucin zipper domain. (Willcox et al. 1999; TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface; Immunity, Vol. 10, 357-365). Binding interactions can be measured at a broad range of settings, including, but not limited to, a temperature range of 25°C to 37°C and a shake speed range of 500 rpm to 1500 rpm using a suitable buffer that minimizes nonspecific binding and maintains protein stability. Examples of such buffers are phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS), or other physiological buffers, with or without additives such as Tween, BSA, DMSO, or EDTA. The analyte can be immobilized on various sensors at a concentration range, including, but not limited to, 1 pg / ml to 100 pg / ml for a duration range of 30 s to 300 s. KDdetermination can be measured at various molarities of the analyte sample for detecting potential off-target reactivities with high sensitivity. Exemplary determination of the KD is herein provided in the Examples. For example the KD may be determined by the following bio-layer interferometry (BLI) method: using a 384-well tilted bottom microplate, black and loading with 50 pg / ml pMHC (100 pl / well). The antigen binding protein can then be added, e.g. as soluble TCR in 7 concentrations as well as a reference well (only HEPES / Tween): 50 pM; 25 pM; 12,5 pM; 6,25 pM; 3,1 pM; 1 ,6 pM; 0,8 pM with 60 pl / well. The association of off-target TCRs can be against NYESO1 -001 at 25pM TCR concentration. Then, the dissociation of soluble TCR can be 100 pl / well HEPES / Tween. The measurements can be conducted by 16 streptavidin sensors: duplicates measured with different sensors (sensor offset: 3).

[0172] A “linker”, as long as it is not specified otherwise in the respective context, can be from at least 1 to 30 amino acids in length. For instance, a linker can be 2-25, 2- 20, or 3-18 amino acids long. In some instances, a linker can be a peptide of a length of no more than 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, or 3 amino acids. In other instances, a linker can be 5-25, 5-15, 4-11 , 10-20, or 20-30 amino acids long. In other instances, a linker can be about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20,

[0173] 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In a particular instance, a linker can be less than 24, less than 20, less than 16, is less than 12, less than 10, for example from 5 to 24, 10 to 24 or 5-10 amino acid residues in length. In some instances, said linker is equal to 1 or more amino acid residues in length, such as more than 1 , more than 2, more than 5, more than 10, more than 20 amino acid residues in length, more than 22 amino acid residues in length. In preferred instances, the linker is an alanine linker, i.e. a linker consisting of or essentially consisting of one or more alanine residues. In some instances, the linker is a single alanine linker. In some instances, a linker comprises or consists of 2 alanine residues. In some instances, a linker comprises or consists of 3 alanine residues.

[0174] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of nonlimiting example, B cells, T cells, NKT cells, and NK cells.

[0175] The “Major Histocompatibility Complex” (MHC) is a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or just HLA). Thus, the terms “MHC” and “HLA” can be used interchangeably. The MHC gene family is divided into three subgroups: class I, class II, and class III. Complexes of peptide and MHC class I molecules (MHC I) are usually recognized by CD8-positive T cells (CD8+ T cells) bearing the appropriate T cell receptor (TCR), whereas complexes of peptide and MHC class II molecules (MHC II) are usually recognized by CD4-positive helper-T cells (CD4+ T cells) bearing the appropriate TCR. CD4 and CD8 usually function as coreceptors of a TCR in binding to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptide and MHC I are recognized by CD8-negative (in particular CD8-negative, CD4-positive) T cells (Soto et al., 2013, Cancer Immunol Immunother. 2013 Feb; 62(2): 359-369). Since the responses of CD8-positive and CD4-positive T cells contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens and corresponding T cell receptors is important in the development of cancer immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger, a-chain, constituent of HLA-A. Variation of HLA-A a-chain is key to HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of certain structures, greater variety of HLAs means greater variety of antigens to be 'presented' on the cell surface. The MHC class I HLA protein in the context of the present disclosure may be an HLA-A, HLA-B or HLA-C protein, suitably HLA-A protein, for example HLA-A*02. In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T cells bearing specific T cell receptors (TCR).

[0176] The term “messenger RNA” or “mRNA” refers to any polynucleotide (a ribonucleic acid) which encodes a (poly-)peptide of interest and which is capable of being translated to produce the encoded (poly-)peptide of interest in vitro, in vivo, in situ, or ex vivo. Typically, the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail.

[0177] The term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Nucleotides are referred to by their commonly accepted singleletter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

[0178] The term “open reading frame”, abbreviated as “ORF”, refers to a segment or region of an mRNA molecule that encodes a (poly-)peptide. The ORF comprises a continuous stretch of non-overlapping, in-frame codons, beginning with the initiation codon and ending with a stop codon, and is translated by the ribosome.

[0179] As used herein, “operably linked” with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a (poly-)peptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0180] Herein a phrase of the form "optionally substituted X" is intended to be equivalent to "X, wherein X is optionally substituted". It is not intended to mean that the feature "X" per se is optional.

[0181] As used herein, a "part" or "region" of a polynucleotide or polypeptide is defined as any portion of the polynucleotide or polypeptide that is less than the entire length of the polynucleotide or polypeptide, respectively.

[0182] As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component.

[0183] The terms "pharmaceutical composition" as used herein refers to a mixture or formulation of one or more therapeutic agent(s), typically along with one or more excipients, which are added to the formulation to aid in its manufacture, stability, or administration. The pharmaceutical composition is typically designed to deliver the therapeutic agent(s) in a safe, effective, and convenient manner for the treatment or prevention of a particular disease or medical condition. Pharmaceutical compositions can take various forms, such as tablets, capsules, injections, creams, ointments, or inhalers, and can be administered by various routes, including oral, topical, intravenous, intramuscular, or inhalation. The composition may need to meet regulatory requirements for safety, efficacy, and quality before it can be approved for marketing and distribution to patients.

[0184] "Pharmaceutically acceptable" refers to the suitability of a therapeutic agent for use in a pharmaceutical formulation, which typically means that the substance is safe, effective, and compatible with the other ingredients in the formulation. A therapeutic agent that is considered "pharmaceutically acceptable" may need to meet certain criteria, including purity, stability, and absence of harmful impurities or contaminants, and it must not cause any adverse effects on the patient's health. In addition, the substance may need to be able to perform its intended function within the formulation and be compatible with the manufacturing process, packaging, and storage conditions.

[0185] The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double and single stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double stranded form and each of two complementarity single stranded forms known or predicted to make up the double stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides encoding antigen binding proteins are typically provided as part of vectors. In some instances, nucleic acids may be isolated nucleic acids. In some instances, nucleic acids may be a recombinant or heterologous nucleic acids. In some instances, nucleic acids may be present in whole cells, in a cell lysate, or may be nucleic acids in a partially purified or substantially pure form.

[0186] As used herein, a “polymeric lipid” refers to a lipid comprising repeating subunits in its chemical structure. In some instances, the polymeric lipid is a lipid comprising a polymer component. In some instances, the polymeric lipid is a PEG lipid. In some instances, the polymeric lipid is not a PEG lipid. In some instances, the polymeric lipid is Brij or OH-PEG-stearate.

[0187] The term "prevent" refers to partially or completely delaying onset of an infection, disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying progression from an infection, a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and / or condition.

[0188] The terms "prophylactic" or “prophylaxis” refer to a therapeutic agent or measure used to prevent the spread of disease.

[0189] The term “proliferative diseases refers to a type of disease characterized by an uncontrolled or abnormal growth of cells, which can lead to the formation of tumors or other forms of abnormal tissue growth. This growth can occur in various parts of the body. Examples of proliferative diseases include cancer, benign tumors, and conditions such as hyperplasia and dysplasia. The causes of proliferative diseases can vary, but they may be related to genetic mutations, exposure to toxins or radiation, or other environmental factors.

[0190] The terms "protein" and “polypeptide” are used interchangeably herein to refer to polymers of amino acids of any length. A protein can be a native protein, that is, a protein produced by a naturally-occurring and non-recombinant cell; or it can be produced by a genetically-engineered or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and / or substitutions of one or more amino acids of the native sequence. The protein can be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, proteins containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a monomer or can be a multi-molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0191] The term "purified" in the context of nucleic acids, polypeptides, and host cells, refers to the separation of a specific molecule or entity from its (natural) environment or context. Purification is a process of removing impurities, contaminants, or unwanted molecules to obtain a more homogeneous or highly concentrated form of the molecule or entity of interest. “Purified” thus denotes a degree of separation that is higher than isolation. Isolation can be the first step in the purification process, but it does not necessarily imply that the molecule or entity has been completely purified. A “purified” or “biologically pure” nucleic acid, polypeptide, or host cell is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the nucleic acid, polypeptide, or host cell. That is, a nucleic acid, polypeptide or host cell is purified if it is substantially free of cellular material, viral material, culture medium or other supplements and additives.

[0192] For nucleic acids, purification can involve removing proteins, lipids, and other cellular debris to obtain a highly concentrated and pure DNA or RNA sample. This can be done using techniques such as column chromatography, gel electrophoresis, or spin column purification. For polypeptides, purification can involve removing other proteins, nucleic acids, or contaminants to obtain a highly purified form of the polypeptide of interest. This can be done using techniques such as affinity chromatography, size exclusion chromatography, or ion exchange chromatography.

[0193] For host cells, purification can involve removing dead cells, debris, and other unwanted materials to obtain a highly purified population of cells. This can be done using techniques such as density gradient centrifugation, magnetic separation, or fluorescence-activated cell sorting (FACS).

[0194] Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0195] The term “radioactive isotope” or “radioactive molecule” is intended to include radioactive isotopes suitable for treating cancer, such as At211, Bi212, Er169, I131, I125, Y90, In111, P32, Re186, Re188, Sm153, Sr89, and radioactive isotopes of Lu. Such radioisotopes generally emit mainly beta-radiation. For instance, the radioactive isotope can be an alpha-emitter isotope, more precisely Thorium 227 which emits alpha-radiation. The term also includes spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine- 123, indium-111 , fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0196] The term "recombinant" refers to any nucleic acid, protein, cell or organism that has been genetically engineered using molecular biology techniques, such as through the artificial modification, insertion, or alteration of genetic material. A “recombinant” nucleic acid, protein, cell or organism is not naturally occurring. A “recombinant” cell can be modified to express a heterologous protein.

[0197] The term "reference sequence" refers to a starting nucleic acid or amino acid sequence that can be sequence optimized. A reference nucleic acid sequence may be a wild-type nucleic acid sequence, a fragment or a variant thereof, or a previously sequence optimized nucleic acid sequence.

[0198] As used herein, “regulatory sequence” of a nucleic acid molecule means a cisacting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression (i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0199] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EF1 -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. For instance, the promoter can be a constitutive promoter. The term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus 40 (SV40) promoter, a polyubiquitin C (UBC) promoter, an EF1 -alpha promoter, a PGK promoter and a CAG promoter. Alternatively, the promoter can be a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. The conditional promoter may be an immune cell specific promoter, which allows for continual transcription of the coding sequence or gene in an immune cell. Non-limiting examples of the immune cell specific promoters include a promoter of a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-|3 gene promoter, a WASP gene promoter, a T-cell receptor [3-chain gene promoter, a V9 y (TRGV9) gene promoter, a V2 5 (TRDV2) gene promoter, and the like.

[0200] Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0201] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0202] The term “safety profile” herein refers to the capacity to distinguish tumor cells from healthy tissue cells and this is often determined by determining the safety window.

[0203] The term “safety window” or “therapeutic window” herein refers to a factor that compares the half maximal concentration of a compound that is required for inducing 100% cytotoxicity in a tumor cell line in comparison to the half maximal concentration of a compound that is required for inducing 100% cytotoxicity healthy tissue cells. If for an antigen binding protein of interest the EC50determined for a tumor cell line is 1 pM and the EC50value determined for, for instance, primary cells is 1000 pM then the safety window is 1000 since the EC50 for the tumor cell line is 1000 times smaller than the EC50for the primary cells.

[0204] The term "sample" or "biological sample" refers to a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further can include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. A sample further refers to a medium, such as a nutrient broth or gel, which can contain cellular components, such as proteins or nucleic acid molecule.

[0205] The term "stable" when used in the contex of compounds or compositions refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and in some cases capable of formulation into an efficient therapeutic agent.

[0206] The term "stabilize," "stabilized," "stabilized region" means to make or become stable.

[0207] The term “sequence optimization” refers to a process or series of processes by which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties, e.g., improved protein expression or decreased immunogenicity of the nucleic acid itself.

[0208] In general, the goal in sequence optimization is to produce a synonymous nucleotide sequence than encodes the same polypeptide sequence encoded by the reference nucleotide sequence. Thus, there are no amino acid substitutions (as a result of codon optimization) in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the reference nucleotide sequence. In some instances, codon optimization is achieved by modification of a coding sequence according to at least one of the following; (i) replacing the naturally occurring codon sequence with an alternative codon that retains the amino acid sequence encoding the protein but alters the composition and / or structure of the encoding RNA; (ii) adjusting the guanosine cytosine content of the coding sequence relative to the naturally occurring guanosine cytosine content of the coding sequence; (iii) adjusting the number of CpG sites of the coding sequence relative to the number of CpG sites in the naturally occurring coding sequence; (iv) substituting the naturally occurring codon sequence with an alternative codon relative to (ii) guanosine cytosine content and / or (iii) number of CpG sites. Codon optimization may include adjusting codons in the context of tRNA expression in a particular tissue and / or may include methods for evading the effects of natural, tissue-specific shRNAs or miRNAs.

[0209] The term “sequential” therapeutic use refers to administration of at least two therapeutic agents at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the therapeutic agents before administration of the other or others commences. It is thus possible to administer one of the therapeutic agents over several minutes, hours, or days before administering the other therapeutic agent or ingredients. There is no simultaneous treatment in this case.

[0210] As used herein, the phrases "signal sequence," "signal peptide," and "transit peptide" are used interchangeably and refer to a sequence that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal sequence polypeptide and the nucleic acid sequence encoding the signal sequence. Thus, references to a signal sequence in the context of a nucleic acid refer in fact to the nucleic acid sequence encoding the signal sequence polypeptide.

[0211] The term “sequence identity” refers to a measure of the similarity between two biological sequences, such as polynucleotide or polypeptide sequences. It is expressed as a percentage of the number of identical positions in the two sequences divided by the total number of positions. In other words, sequence identity indicates how much two sequences match each other in terms of the order and composition of their building blocks, such as nucleotides or amino acids. If the two sequences to be compared are not of equal length, they can be aligned to give the best possible fit, allowing the insertion of gaps or alternatively, truncation at the ends of the nucleic acid sequences or amino acid sequences. The skilled person will acknowledge that various means for comparing sequence identity are available (see below). A higher sequence identity may indicate a closer evolutionary relationship between the two sequences, or a higher degree of functional similarity or homology. Sequence identity is commonly used in bioinformatics and molecular biology to compare and analyze the structure and function of biological molecules, such as proteins, and to infer their evolutionary history and relationships.

[0212] For example, in the context of the present disclosure, a sequence that is “at least 85% identical to a reference sequence” may be a sequence having, over its entire length, 85%, or more, in particular 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of a reference sequence (e.g., a variable domain disclosed herein). Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% Identical” to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence.

[0213] In the context of the present disclosure, the sequence identity can be calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle” program, which uses the Needleman- Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides or polynucleotides, in accordance with the disclosure, can be calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

[0214] In the context of the present disclosure “similar peptides” herein refers to potential off-target peptides, i.e. peptides that may potentially be bound by the antigen binding proteins of the disclosure based on their biochemical / biophysical characteristics, including but not limited to a homologous sequence or a similar motif. Similar peptides comprise typically 8 to 12 amino acids in length. The similar peptides in the context of the present disclosure are typically MHC, in particular MHC I, presented. Furthermore, similar peptides in the context of the present disclosure include peptides that comprise or consist of an amino acid sequence that is similar to the amino acid sequence of the PRAME antigenic peptide, more particular, peptides that, in comparison to the epitope of the PRAME antigenic peptide, comprise an epitope wherein some or all amino acids have identical and / or similar biochemical / biophysical characteristics as the amino acids that constitute the epitope of the PRAME antigenic peptide. In some examples, similar peptides investigated in the context of the present disclosure were selected from a database of tumor and normal tissue-presented HLA-A*02 bound peptides (XPRESIDENT® database) using a similarity scoring within the binding-relevant positions of PRAME and the requirement of at least one detection on normal tissues. Binding of an antigen binding protein to a similar peptide presented by an MHC protein may lead to adverse reactions. Such adverse reactions may be “off-tumor” side effects, such as crossreactivity of a specific TCR with a similar peptide in healthy tissues as reported in Lowdell et al., Cytotherapy, published on December 4, 2018).

[0215] In particular, the “similar” peptides disclosed in WO2018172533 A1 are similar peptides in the context of the present disclosure.

[0216] The skilled person is aware that among the similar peptides, there are some that are not bound by the antigen binding proteins of the disclosure to a detectable degree, e.g. peptides for which no binding signal during affinity determination or no response in a functional assay beyond the background level is detectable. “Background level” in this context refers to a response in a functional assay observed for the co-culture of target cells and effector cells at the respective E:T ratio without the addition of bispecific TCR-antibody fusion protein.

[0217] For other similar peptides, a low, but non-significant binding may be detectable. These latter similar peptides may also be described as "potentially relevant" similar peptides. An antigen binding protein is considered to not significantly bind to a similar peptide and to be specific for its target antigenic peptide if at least one of the following applies when binding to the similar peptide and the target antigenic peptide is compared under similar, preferably identical experimental conditions:

[0218] The functional avidity in response to the similar peptide, determined in a functional assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the functional avidity in response to the target antigenic peptide.

[0219] The cytotoxic activity in response to the similar peptide, determined in a cytotoxicity assay as described above, is 25% or less, 20% or less, 15% or less, 10% or less of the cytotoxic activity in response to the target antigenic peptide.

[0220] The EC50of the similar peptide, determined in a functional assay, preferably a cytotoxicity assay, as described above, is increased by a factor of at least 50, at least 100, at least 200 or at least 500, compared to the EC50of the target antigenic peptide.

[0221] The KDfor the similar peptide is increased by a factor at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100, compared to the KDfor the target antigenic peptide.

[0222] The relative response signal for the similar peptide is not higher than 30%, not higher than 25%, not higher than 20%, or not higher than 15%, compared to the response signal to the target antigenic peptide.

[0223] The term “simultaneous” therapeutic use refers to the administration of at least two therapeutic agents by the same or different routes and at the same time or at substantially the same time. A "single unit dose" is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event.

[0224] The term “specificity” or “specific binding” or “specifically binds” or “specifically targets” refers to the ability of an antigen binding molecule such as a TCR to recognize and bind to a single target (antigen) while avoiding interactions with unrelated targets. A specific TCR binds only to its intended target and does not or not substantially crossreact with other proteins or similar epitopes. For instance, the term "specificity" or "antigen specificity" or "specific for" a PRAME-004 peptide in the context of TCRs means that the TCR can specifically bind to said PRAME-004 peptide, more preferably with high avidity, when said PRAME peptide is presented by HLA, preferably by HLA A2. Typically, an antigen binding protein is considered “specific” for a target peptide, if binding to the target peptide presented in an MHC molecule occurs with a significantly higher affinity and / or higher functional avidity than the binding to similar peptides. The specificity of the antigen binding protein is determined by the amino acid seguences CDRal , CDRa3, CDRbl and CDRb3. The amino acid seguences of CDRa2 and CDRb2 contact the MHC molecule and may in some instances not be reguired for antigen specificity. Therefore, the term “specifically binds” or “specifically binds to” or “specifically target” can be exhibited, for example, by a molecule having a Kd for the molecule to which it binds to of about 10-4M, 10-5M, 10-6M, 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10’12M.

[0225] The term “specific delivery,” “specifically deliver,” or “specifically delivering”, in particular in the context of polynucleotides (e.g., mRNAs) means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a polynucleotide by a nanoparticle to a target tissue of interest (e.g., mammalian liver) compared to an off-target tissue (e.g., mammalian spleen). The level of delivery of a nanoparticle to a particular tissue can be measured by comparing the amount of protein produced in a tissue to the weight of said tissue, comparing the amount of polynucleotide in a tissue to the weight of said tissue, comparing the amount of protein produced in a tissue to the amount of total protein in said tissue, or comparing the amount of polynucleotide in a tissue to the amount of total polynucleotide in said tissue. For example, for renovascular targeting, a polynucleotide is specifically provided to a mammalian kidney as compared to the liver and spleen if 1 .5, 2-fold, 3-fold, 5-fold, 10-fold, 15 fold, or 20 fold more polynucleotide per 1 g of tissue is delivered to a kidney compared to that delivered to the liver or spleen following systemic administration of the polynucleotide. It will be understood that the ability of a nanoparticle to specifically deliver to a target tissue need not be determined in a subject being treated, it can be determined in a surrogate such as an animal model (e.g., a rat model).

[0226] The term “stable expression” or “stably expressed refers to the sustained and reproducible production of a nucleic acid, RNA, or protein in a host cell, organism, or system over multiple generations, passages, or extended periods without the need for continuous transduction. Stable expression may result from genomic integration of the nucleic acid sequence, the use of self-replicating vectors, or other mechanisms that allow for long-term maintenance and inheritance of the expressed sequence. The term includes expression in prokaryotic or eukaryotic systems, whether natural, engineered, or recombinant.

[0227] The term “start codon”, used interchangeably with the term “initiation codon”, refers to the first codon of an open reading frame that is translated by the ribosome and is comprised of a triplet of linked adenine-uracil-guanine nucleobases. The initiation codon is depicted by the first letter codes of adenine (A), uracil (U), and guanine (G) and is often written simply as “AUG”. Although natural mRNAs may use codons other than AUG as the initiation codon, which are referred to herein as “alternative initiation codons”, the initiation codons of polynucleotides described herein use the AUG codon. During the process of translation initiation, the sequence comprising the initiation codon is recognized via complementarity base-pairing to the anticodon of an initiator tRNA (Met-tRNAiMet) bound by the ribosome. Open reading frames may contain more than one AUG initiation codon, which are referred to herein as “alternate initiation codons”.

[0228] As used herein, by "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, prophylaxis or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferably, the subject is a human subject.

[0229] The term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness in many biological and chemical characteristics.

[0230] The term “substantially equal” as it relates to time differences between doses, the term means plus / minus 2%.

[0231] The term “sub-therapeutic dose” refers to a dose of an agent that does not achieve a particular therapeutic effect (e.g., wherein the particular therapeutic effect is achieved using a therapeutically effective amount). Typically, a sub-therapeutic dose of an agent is an amount of a therapeutic dose that is less than a therapeutically effective amount of the agent.

[0232] The term “suffering from” refers to a subject who is “suffering from” a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0233] A subject who is "susceptible to" a disease, disorder, and / or condition has not been diagnosed with and / or cannot exhibit symptoms of the disease, disorder, and / or condition but harbors a propensity to develop a disease or its symptoms. In some instances, a subject who is susceptible to a disease, disorder, and / or condition (for example, cancer) can be characterized by one or more of the following: (1 ) a genetic mutation associated with development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microbe associated with development of the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some instances, a subject who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0234] In the context of the present disclosure, the term “T2 cell” refers to a cell that expresses an MHCI molecule (HLA-A2) that lacks TAP function. T2 cells can be easily artificially loaded with different concentrations of exogenous antigenic peptides. T2 cell are described e.g. in (Hosken and Bevan, Science 1990 Apr 20;248(4953):367- 70). T2 cells are commercially available, e.g. from ATCC (American Type Culture Collection). Loading of T2 cells can be achieved under standard cell culture conditions known to the skilled in the art by incubating the T2 cells for about 2 hours with a desired concentration of antigenic peptide. In the context of the present disclosure, T2 cells that are incubated with a certain concentration of antigenic peptide, such as 1 pM, 100 nM, 10 nM, 1 nM, 100 pM, 10 pM, 1 pM, are referred to as T2 cells loaded with said concentration of antigenic peptide, e.g. T2 cells incubated with 10 pM of antigenic peptide are referred to as T2 cells loaded with 10 pM of antigenic peptide.

[0235] The term “TCR” as used herein includes both native and engineered TCRs.

[0236] A “native” TCR refers to a wildtype TCR that can be isolated from nature, whereas an “engineered” TCR may be a protein resembling a native TCR, but comprising further modifications e.g. in the variable and / or constant domains compared to the naturally occurring seguence, e.g. a humanized TCR or a TCR with altered characteristics (e.g. altered binding, heterodimerization or expression level). Native TCRs are heterodimeric cell surface proteins of the immunoglobulin super-family, which are associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Native heterodimeric TCRs exist in a|3 and y5 forms, which are structurally similar but have distinct locations and probably functions. The terms “a / p TCR” or a ”y / b TCR” thus refer to a TCR comprising an a-chain and a p- chain as described above, or a y-chain and a b-chain, respectively. Such TCRs may also be described as “full length TCRs” or “conventional TCRs”. An a / p TCR or a y / 5 TCR may be a native TCR or may be an engineered TCR that retains the structure of a native TCR, i.e. an engineered TCR comprising minor modifications in the variable and / or constant domains as described above, such as a humanized TCR.

[0237] Native, full-length ap heterodimeric TCRs consist of an a-chain and a p-chain. The a-chain comprises a variable region (V region) encoded by a TRAV gene, a joining region (J region) encoded by a TRAJ gene, and a constant region (C region) encoded by a TRAC gene. The p-chain comprises a variable region (V region) encoded by a TRBV gene, a joining region (J region) encoded by a TRBJ gene and a constant region (C region) encoded by a TRBC gene, and usually a short diversity region (D region) encoded by a TRBD gene between the V and J regions, although this D region is often considered as part of the J region (Lefranc, (2001 ), Curr Protoc Immunol Appendix 1 : Appendix 10). The genes encoding different a-chain and p-chain variable, joining and constant regions are referred to in IMGT nomenclature by unique numbers (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1 ): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83-96; LeFranc and LeFranc, (2001 ), "T cell Receptor Factsbook", Academic Press). Further information on TCR genes can be found in the international ImMunoGeneTics information system®, Lefranc M-P et al., (Nucleic Acids Res. 2015 Jan;43(Database issue): D413-22; and http: / / www.imgt.org / ).

[0238] The alpha chain TRAC constant domain sequence and the beta chain TRBC1 or TRBC2 constant domain are in the following, also referred to as TCR constant domain sequences. The TCR constant domain sequences may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human. The TCR constant domain sequences may be modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. Also, further stabilizing mutations as known from the state of the art (e.g. WO 2018 / 104407, PCT / EP2018 / 069151 , WO 2011 / 044186, WO 2014 / 018863) may be introduced, such as replacement of unfavorable amino acids in the variable regions and / or the introduction of a disulfide bridge between the TCR C domains and the removal of unpaired cysteine. Thus, the disclosure also relates to proteins that may comprise the antigen binding domains (e.g. the CDRs as provided herein) and further comprise domains / amino acid sequences that are not found in the naturally occurring TCR.

[0239] On the protein level, TCR a-, p-, y- and 5-chains comprise two immunoglobulin domains, the variable domain and the constant domain. The variable domain corresponds to the V(D)J region. The constant domain corresponds to the C region. The constant domain is the membrane-proximal domain and in the context of the present disclosure also includes the transmembrane (TM) domain and a short cytoplasmic tail. Each of the constant and variable domains include an intra-chain disulfide bond. The variable domains (Vaand Vpin a|3 TCRs and VYand V5in y5 TCRs) contain highly polymorphic loops comprising the complementarity determining regions (CDRs).

[0240] Each TCR variable domain comprises three “TCR complementarity determining regions (CDRs)” embedded in a framework seguence, one being the hypervariable region named CDR3. In the context of the present disclosure, CDRal , CDRa2 and CDRa3 denote a-chain CDRs, and CDRbl , CDRb2 and CDRb3 denote [3-chain CDRs. The seguences encoding CDRal and CDRa2 are comprised in TRAV, the seguences encoding CDRa3 are comprised in TRAV and TRAJ, the seguences encoding CDRbl and CDRb2 are comprised in TRBV, and the seguences encoding CDRb3 are comprised in TRBV, TRBD and TRBJ. In TCRs, the CDR1 and CDR3 amino acid residues make contact with the antigenic peptide, while the CDR2 amino acid residues mainly contact the HLA molecule (Stadinski et al., J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et al., J Biol Chem. 2014 Jan 10;289(2):628-38). The antigen specificity of a TCR is thus defined by the CDR3 and CDR1 seguences. The CDR2 seguences are not reguired for the determination of antigen specificity, but may play a role in the overall affinity of a TCR towards a peptide:MHC complex.

[0241] “TCR framework regions” (FRs) refer to amino acid seguences interposed between the CDRs, i.e. to those portions of the variable domains that are to some extent conserved among different TCRs. The a-, (3-, y- and 5-chain variable domains each have four FRs, herein designated FR1 -a, FR2-a, FR3-a, FR4-a (for an a- or y- chain), and FR1 -b, FR2-b, FR3-b, FR4-b (for a [3- or 5-chain), respectively. Accordingly, an a-chain or y-chain variable domain may be described as (FR1-a)- (CDRa1 )-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a) and a [3- or 5-chain variable domain may be described as (FR1 -b)-(CDRb1 )-(FR2-b)-(CDRb2)-(FR3-b)-(CDRb3)- (FR4-b). In the context of the present disclosure, the CDR / FR seguences in an a-, (3, y- or 5-chain variable domain is determined based on IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1 ):55-77; www.imgt.org). Accordingly, CDR / FR amino acid positions when related to TCR or TCR-derived domains are indicated according to said IMGT definition. Preferably, the IMGT position of the CDR / FR amino acid positions of the variable domain Va is given in analogy to the IMGT numbering of TRAV24*01 and / or the IMGT position of the CDR / FR amino acid positions of the variable domain V(3 is given in analogy to the IMGT numbering of TRBV12-3*01 .

[0242] A “fragment” of a TCR” refers to a fragment of a TCR that retains or substantially retains the affinity, functional avidity and / or specificity of the parental TCR from which it is derived for a target antigen. In other words, a “fragment” of a TCR is preferably a “functional” fragment of the TCR. The term “parental TCR” in this context refers to a full length TCR from which a functional fragment may be derived.

[0243] As binding to the target antigenic peptide is defined by the CDR1 and CDR3 sequences, and binding to the target antigenic peptide MHC complex is defined by CDR1 , CDR2 and CDR3, antigen binding proteins comprising the CDR1 and CDR3 and optionally CDR2 sequences of a TCR retain the affinity, functional avidity and / or specificity of the parental TCR for a target antigen. The person skilled in the art is aware that the CDRs have to be interspersed with framework regions (FRs), however the specific amino acid sequences of the framework regions are not directly involved in target antigen specificity. Examples of functional TCR fragments include single variable domains, such as TCR alpha, beta, gamma or delta variable domains, or fragments of the a, (3, 5 or y chain, such as an a, (3, 5 or y chain without transmembrane domain and short cytoplasmic tail. The term “fragment” as used herein refers to naturally occurring fragments (e.g. splice variants or peptide fragments) as well as artificially constructed fragments, in particular to those obtained by gene-technological means.

[0244] A functional fragment of a TCR is considered to have retained or substantially retained the affinity for a target antigen, if, for example, the KDfor binding to the target antigen measured as outlined below is identical to the KDof the TCR or is increased or reduced, preferably reduced, no more than 10x, 5x, 3x, or 2x.

[0245] A functional fragment of a TCR may have retained or substantially retained the functional avidity for a target antigen, if, for example, the functional avidity for the target antigen is identical to that of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1 %. In particular, a functional fragment of a TCR is considered to have retained or substantially retained the functional avidity for a target antigen, if, for example, its cytotoxic activity in response to the target of the parent protein measured in a cytotoxicity assay is identical to the cytotoxic activity of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1 %, preferably 10%, 8%, 5%, 3%, 2% or 1 %.

[0246] A functional fragment of a TCR is considered to have retained or substantially retained the specificity for a target antigen (i.e. the ability to specifically bind to a target antigen), if it does not significantly bind to peptides other than the target antigenic peptide of the TCR.

[0247] “Does not significantly bind” in the context of antigenic peptide variants and in the context of antigen binding proteins of the disclosure, denotes, typically, a functional avidity was determined for the antigen binding protein binding to an antigenic peptide variant that is not higher than 30%, not higher than 25%, not higher than 20%, not higher than 15%, preferably not higher than 20% of the functional avidity obtained for binding to the target antigenic peptide consisting of the amino acid sequence of SEQ ID NO: 24, preferably in the same experimental conditions. For instance, the functional avidity obtained for the antigen binding protein binding to a similar peptide is not higher than 30% of the signal obtained in the same experimental conditions for the antigen binding protein binding to the target antigenic peptide consisting of the amino acid sequence of SEQ ID NO: 24 (see WO2018172533 A1 ). The skilled person knows how to determine whether an antigen binding protein does not significantly bind to an antigenic peptide. An exemplary method is herein disclosed below and exemplified in the appended examples.

[0248] “Single chain TOR (scTCR)” as used herein denotes a TOR in which the variable domains of the TOR are located on a single polypeptide. Typically, the variable domains in scTCRs are separated by a linker, wherein said linker typically comprises 10 to 30 amino acids, such as 25 amino acids.

[0249] The term "therapeutic agent" or “active agent” refers to an agent that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect and / or elicits a desired biological and / or pharmacological effect. For example, a host cell expressing an antigen binding protein, or an mRNA encoding an antigenic peptide can be a therapeutic agent.

[0250] The term "therapeutically effective outcome" means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0251] The term "transcription" refers to methods to produce mRNA (e.g., an mRNA sequence or template) from DNA (e.g., a DNA template or sequence).

[0252] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or heterologous) nucleic acid such as a gene, DNA or RNA to a host cell, so that the host cell will express the introduced nucleic acid to produce a desired substance, e.g. the PRAME antigenic peptide or the antigen-binding protein described herein. A host cell that receives and expresses introduced DNA or RNA bas been "transformed". The nucleic acid can be introduced into the host cell by means of non-viral methods (e.g., via plasmids, electroporation, chemical transfection, heat shock, calcium phosphate, microinjection) or viral methods (i.e., infection with genetically engineered viruses). Non-virally introduced DNA can remain episomal (plasmid-based), typically leading to transient (short-term) expression, or be integrated into the genome for stable expression. Similarly, virally introduced DNA can integrate into the genome (retroviruses) or remain episomal (adenoviruses), but is typically stably expressed in either case. Transformation can combine viral and non-viral methods (e.g., electroporation for viral infection). The introduction of foreign nucleic acids into host cells via viral methods is also referred to as “transduction” and is often preferred for mammalian cells. Throughout the specification, the terms “transformation” and “transformed” include the terms “transduction” and “transduced”, respectively. The term "treating" or "treatment" or "therapy" refers to the application or administration of a composition including one or more therapeutic agents to a subject with the aim of curing, healing, preventing, reducing, delaying, slowing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting a condition, disorder or disease such as cancer and / or its symptoms. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. For instance, cancer treatment may include, without limitation, inhibiting the recurrence of cancer, alleviating its symptoms, diminishing any direct or indirect pathological consequences, preventing or reducing metastases, decreasing the rate of cancer progression, ameliorating or palliation of the disease state, and remission or improved prognosis.

[0253] The term “treatment regimen” refers to a protocol or plan for administering a therapeutic agent to a patient. A treatment regimen may encompass parameters such as the dosage amount, frequency of administration, duration of treatment, and the method or route by which the therapeutic agent is delivered.

[0254] As used herein, “unmodified” refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the “unmodified” starting molecule for a subsequent modification.

[0255] The term “variant” in relation to a nucleic acid or amino acid sequence refers to both natural variants (e.g., polymorphisms, isoforms, etc.) and artificial variants in which at least one nucleic or amino acid residue in a native or starting sequence (e.g., a wild type sequence) has been removed and a different nucleic or amino acid inserted in its place at the same position, respectively. These variants can be described as “substitutional variants.” The substitutions can be single, where only one nucleic or amino acid in the molecule has been substituted, or they can be multiple, where two or more nucleic or amino acids have been substituted in the same molecule. If nucleic or amino acids are inserted or deleted, the resulting variant would be an “insertional variant” or a “deletional variant” respectively.

[0256] “Va” in the context of the present disclosure refers to a variable domain of a TCR a-chain.

[0257] “VA” or Vain the context of the present disclosure refers to a TCR variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (Va), [3-chain (Vp), y-chain (VY) or b-chain (V5), preferably from a Va. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, [3-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR a-chain.

[0258] The CDR and framework sequences of the VAdomain may not necessarily be derived from the same TCR chain. For example, the CDRs derived from one TCR variable domain (of the donor TCR) could also be grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VAencoded by TRAV5 and TRAJ17, and the acceptor TCR may comprise a VAencoded by TRAV14 and TRAJ33.

[0259] ”VP” in the context of the present disclosure refers to a variable domain of a TCR [3-chain.

[0260] “VB” or Vbin the context of the present disclosure refers to a variable domain comprising TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (Va), [3-chain (Vp), y-chain (VY) or b-chain (V5), preferably from a Vp. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, [3-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR [3-chain. In the examples, various framework and CDR mutations / substitutions are shown.

[0261] The CDR and framework sequences of the VBdomain in context of the present disclosure may not necessarily be derived from the same TCR. For example, the CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VBencoded by TRBV2 and TRBJ2-1 , and the acceptor TCR may comprise a VBencoded by TRBV27 and TRBJ1 -5.

[0262] CDRs may not only be exchanged / grafted between different alpha variable domains or different beta variable domains, but also may be grafted from a TCR alpha to a TCR beta, gamma or delta variable domain, or from a TCR beta to a TCR alpha, gamma or delta variable domain.

[0263] ”VY” in the context of the present disclosure refers to a variable domain of a TCR y-chain.

[0264] ”Vs” in the context of the present disclosure refers to a variable domain of a TCR b-chain.

[0265] ”VL” in the context of the present disclosure refers to a variable domain of an antibody light chain.

[0266] “VH” in the context of the present disclosure refers to a variable domain of an antibody heavy chain. The terms "vector" includes "cloning vectors" and "expression vectors" and refers to a vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Preferably, and unless specified otherwise, the term “vector” refers to expression vectors.

[0267] The term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle and encodes at least an exogenous nucleic acid. The vector and / or particle can be utilized for the purpose of transferring a nucleic acid of interest into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art. Useful viral vectors include vectors based on retroviruses, lentiviruses, adenoviruses, adeno- associated viruses, herpes viruses, vectors based on SV40, papilloma virus, Epstein Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV). Recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861 ,719, US 5,278,056 and WO 94 / 19478.

[0268] The terms “of the disclosure” as used herein are intended to refer to all aspects and embodiments disclosed and / or claimed herein. Any aspects, items or embodiments referred to herein as being “disclosed herein” or “described herein” are to be understood as being aspects, items or embodiments “of the disclosure” or “according to the disclosure”.

[0269] The term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either”, “one of”, “only one of”, or “exactly one of”.

[0270] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0271] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within this disclosure. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0272] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0273] The invention will now be described in more details with reference to the following figures and examples. All literature and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the foregoing description, the examples are to be considered illustrative or exemplary and not restrictive.

[0274] DESCRIPTION

[0275] The present disclosure relates to treatment regimen, medical uses, methods and patient groups in the context of cell therapy for treating cancer with immune cells expressing a PRAME-004 TCR (“PRAME immune cells”, specifically PRAME-004 TCR-T cells) or a PRAME-004 TCR and heterologous CD8 (PRAME CD8 immune cells”, specifically PRAME-004 TCR T cells).

[0276] “PRAME” immune cells and “PRAME CD8” immune cells both express the same PRAME TCR, which specifically binds to the PRAME peptide SLLQHLIGL (SEQ ID NO: 4). Such TCRs are referred to as “PRAME-004 TCRs” or “PRAME TCRs” for short herein. PRAME CD8 immune cells additionally express heterologous CD8. Both PRAME and PRAME CD8 immune cells are particularly useful for treating PRAME- 004 positive cancers.

[0277] The present inventors have developed advantageous treatment regimen for both “PRAME” immune cells and “PRAME CD8” immune cells.

[0278] A first aspect of the invention relates to treatment regimen, indications and patient groups for “PRAME” immune cell treatment.

[0279] A second aspect of the invention relates to treatment regimen, indications and patient groups for “PRAME CD8” immune cell treatment.

[0280] Both aspects have proven safe and effective in a clinical setting, leading to durable patient responses.

[0281] The first and second aspect of the invention relate to cancer therapy using immune cells that express or comprise a PRAME-004 TCR.

[0282] For PRAME immune cells, the inventors have determined a treatment regimen with a suitable cell dose and an advantageous low-dose lnterleukin-2 treatment schedule to support immune cell function while minimizing side effects, specifically in patients who have not undergone previous testing for PRAME-004 expression, and typically suffer from high PRAME-004 expressing tumors.

[0283] For PRAME CD8 immune cells, the present inventors have determined a treatment regimen which does not necessarily require IL-2 treatment and preferably allows either administering lower cell doses, or treatment of low to medium PRAME- 004 expressing tumors.

[0284] PRAME “PRAME” refers to Preferentially Expressed Antigen of Melanoma, preferably human PRAME (UniProtKB Accession No. P78395, UniProt Consortium, www.uniprot.org / uniprot / P78395 and its variants). The PRAME-004 antigenic peptide is derived from the amino acid sequence of said PRAME protein and comprises or consists of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24). Typically, PRAME-004 is presented in a complex with a MHC protein, such as an HLA protein, for instance HLA-A*02. Unless denoted otherwise, “PRAME” will be used short for “PRAME-004” throughout the present disclosure to preferably refer to the PRAME-004 peptide having the amino acid sequence of SLLQHLIGL (SEQ ID NO: 24). In instances where the PRAME antigenic peptide comprises further amino acids in addition to the amino acid sequence SLLQHLIGL (SEQ ID NO: 25), it is preferred that the overall length of the PRAME antigenic peptide does not exceed 30 or 20 amino acids, more preferably does not exceed 15 amino acids, even more preferably does not exceed 12 amino acids. In instances where the PRAME antigenic peptide comprises further amino acids in addition to SEQ ID NO: 24, the amino acids of SEQ ID NO: 25 are preferably located within the peptide binding groove of the MHC protein when the PRAME-004 antigenic peptide is in a complex with an MHC protein. The person skilled in the art is aware that antigenic peptides presented on MHC I are usually no longer than 12 amino acids.

[0285] The PRAME-004 antigenic peptide SLLQHLIGL (SEQ ID NO: 24) was found to be over-presented in cancers, as disclosed in US20160250307, US20170165337, and US20170253633, respectively; the contents of which are hereby incorporated by reference in their entireties.

[0286] Immune cells

[0287] The terms “immune cell” and “host cell” are used interchangeably herein to include any cell that is involved in the immune response, including but not limited to leukocytes such as lymphocytes (e.g., T cells, B cells, and natural killer cells), macrophages, dendritic cells, neutrophils, monocytes, and other antigen-presenting cells, and any precursor cells of the foregoing. These cells may be naturally occurring, genetically modified, or artificially engineered for therapeutic, diagnostic, or research purposes. Preferably, the term “immune cell” refers to a lymphocyte, such as a T cell or T cell progenitor. Preferably, the T cell is a human T cell. Preferably, the T cell is a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD3- positive, CD4-positive and / or CD8- positive, CD4-positive helper T cells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g., cytotoxic T cells), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like. Preferably, the T cell is a CD3-positive T cell, a CD8-positive T cell and / or a CD4-positive T cell. Preferably, the T cell is an alpha beta T cell. Typically, the immune cells are autologous to the patient. However, in some instances, the immune cells may be allogeneic to the patient. The immune cell or population of immune cells comprises or expresses a TCR or derivative or fragment thereof and is obtainable by isolating immune cells from a patient; genetically engineering the immune cells by introducing one or more nucleic acid molecules, preferably in the form of expression vectors, encoding a PRAME-004 specific TCR; expanding the genetically engineered immune cell under suitable conditions to produce a plurality of genetically engineered PRAME-004 TCR expressing immune cells. Throughout the present disclosure, the term “comprising” a TCR and “expressing” a TCR are used interchangeably to refer to the production, synthesis, and / or presence of the TCR in the immune cell or on its surface, typically resulting from transcription of the corresponding nucleic acid sequence into mRNA and subsequent translation into the protein. The TCR or its fragment or derivative may be expressed with an N-terminal leader or signal peptide sequence that directs the nascent TCR polypeptide to the endoplasmic reticulum, facilitating proper folding and assembly of the TCR complex. The TCR alpha variable domain or TCR alpha chain may be expressed with a signal peptide according to SEQ ID NO: 25, or a sequence having at least 80%, 90%, 95% or 99% sequence identity thereto. The TCR beta variable domain or TCR beta chain may be expressed with a signal peptide according to SEQ ID NO: 26, or a sequence having at least 80%, 90%, 95% or 99% sequence identity thereto. Other signal peptide sequences can also be used as long as they facilitate TCR targeting and translocation to the cell membrane. Signal peptides are typically cleaved off by signal peptidases in the endoplasmic reticulum and do not form part of the mature TCR or derivative or fragment thereof.

[0288] TCR expression may be natural, induced, constitutive, transient, or stable. Preferably, the immune cell or population of immune cells stably express said TCR or derivative or fragment thereof. The term “stable” expression has the meaning as defined elsewhere herein.

[0289] The immune cell(s) express a TCR or a derivative or fragment thereof.

[0290] TCR

[0291] A TCR “derivative” refers to a T cell receptor (TCR) that has been modified or engineered to alter its properties. These modifications can include changes to the TCR's amino acid sequence, structure, or binding characteristics, aiming to enhance its affinity, specificity, stability, or expression. Such derivatives may involve alterations in the variable regions, constant regions, or other domains of the TCR to improve its therapeutic efficacy or applicability. Furthermore, encompassed are TCRs having alternative domains, such as an alternative membrane anchor domain instead of the endogenous transmembrane region. Also encompassed are TCRs having point mutations in the TCR variable domain or constant domain in order to improve TCR expression or stability and / or chain pairing. Also encompassed are TCRs having an additional functional domains, such as a label or a therapeutically active substance. These modifications can be achieved through various techniques, including site- directed mutagenesis, domain swapping, or the incorporation of synthetic sequences.

[0292] A TCR “fragment” refers to a portion of a TCR, including, without limitation, isolated variable regions. Such fragments may be utilized in various therapeutic or diagnostic applications, especially when the full-length TCR is not required or when a smaller fragment offers advantages in stability, specificity, or manufacturability. For instance, a TCR fragment might comprise only the variable domains of the alpha and beta chains, which are primarily responsible for antigen specificity, without the constant regions or transmembrane domains. Alternatively, it could include engineered segments designed to enhance binding affinity or modify specificity.

[0293] A TCR derivative or fragment retains the ability to specifically bind to the target antigenic peptide PRAME-004 and optionally performs a desired function associated with the full-length or parent TCR. For instance, derivatives may have in the TCR alpha or beta variable domain at least one mutation relative to a TCR alpha or beta variable domain shown in Table 1 , respectively.

[0294] Stable TCR expression is typically achieved through the use of a suitable vector, preferably a viral vector, more preferably a lentiviral vector. Other suitable vectors may include y-retroviral vectors, Sleeping Beauty (SB) transposons, PiggyBac transposons, Adeno-Associated Virus (AAV) vectors, preferably in combination with targeted genome integration. CRISPR / Cas-assisted integration can be used to insert the TCR gene (e.g. carried by an AAV) into a suitable genomic target site.

[0295] As described above, the immune cell or population of immune cells is preferably obtained using genetic engineering. Therefore, the immune cell or population of immune cells are preferably recombinant with respect to at least the TCR or derivative or fragment thereof as described herein. In other words, the immune cell or population of immune cells preferably comprise recombinant DNA encoding at least the TCR or derivative or fragment thereof as described herein. The TCR or derivative or fragment thereof is therefore preferably heterologous to said immune cell(s).

[0296] Additionally or alternatively to expressing the TCR or derivative or fragment thereof, the immune cell comprises at least one nucleic acid(s) encoding said TCR or fragment or derivative thereof. The at least one nucleic acid may be a vector, in particular a viral vector as described elsewhere herein. The immune cell may comprise one or several nucleic acids, specifically (viral) vectors, where for instance different TCR domains or chains are expressed by the same or different nucleic acid(s) or (viral) vector(s). For instance, the TCR alpha variable domain or TCR alpha chain may be expressed by a first nucleic acid or vector, and the TCR beta variable domain or TCR beta chain may be expressed by a second nucleic acid or vector.

[0297] The TCR or derivative or fragment thereof is preferably a TCR as described in WO 2018 / 172533 A1 , which is incorporated herein in its entirety. The terms “derivative” and “fragment” of a TCR shall have the meaning as defined elsewhere herein.

[0298] The PRAME-004 specific TCR or derivative or fragment thereof is also referred to as “PRAME-004 TCR” or “PRAME TCR” herein. The term “specifically binding” has the meaning as defined elsewhere herein. Preferably, the PRAME-004 antigenic peptide is presented on an major histocompatibility complex (MHC) molecule, meaning that said antigenic peptide is bound to and displayed by a MHC molecule, preferably on the surface of a cell, such as a cancer cell. This presentation facilitates recognition by immune cells, particularly T cells through their TCR. Preferably, the PRAME-004 peptide recognized and bound by the TCR or derivative or fragment thereof is preferably presented on an MHC-I molecule. The PRAME-004 peptide may be presented via recombinant or synthetic MHC complexes, whether in vivo or in vitro. In the patient, the PRAME-004 peptide is preferably presented on cancer cells through endogenous pathways involving intracellular processing and loading onto MHC class I molecules, such as HLA- A*02:01 molecules. Preferably, the PRAME-004 peptide may be presented on a HLA- A*02:01 molecule. In other words, The immune cell’s TCR is able to recognize the PRAME-004 antigenic peptide in a major histocompatibility complex (MHC) class l-dependent manner. "MHC class l-dependent manner," as used herein, means that the TCR elicits an immune response upon binding to the PRAME peptide within the context of an MHC class I molecule. The MHC class I molecule can be any MHC class I molecule known in the art, e.g., HLA- A molecules. Preferably, the MHC class I molecule is an HLA-A*02 molecule.

[0299] In the aspects of the disclosure, the TCR or derivative or fragment thereof expressed by the immune cell(s) and / or encoded by the nucleic acid(s) comprised by the immune cell(s) preferably comprises a complementarity determining region (CDR) 1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

[0300] Alternatively, the TCR or derivative or fragment thereof expressed by the immune cell(s) and / or encoded by the nucleic acid comprised by the immune cell(s) may comprise a complementarity determining region (CDR) 1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTL (SEQ ID NO. 7), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

[0301] The TCR or derivative or fragment thereof preferably at least one alpha variable (V) domain harbouring three complementarity-determining regions (CDR1 a, CDR2a and CDR3a), and at least one beta variable (V) domain harbouring three complementarity-determining regions (CDR1 b, CDR2b and CDR3b) as defined elsewhere herein.

[0302] Accordingly, the immune cell(s) may express a TCR alpha variable domain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and / or a TCR beta variable domain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 . Preferably, the TCR alpha variable domain comprises the CDR1 a, CDR2a and CDR3a as disclosed herein, and the TCR beta variable domain comprises the CDR1 b, CDR2b and CDR3b as disclosed herein.

[0303] The TCR or derivative or fragment thereof may further comprise at least one alpha constant (C) domain, alpha transmembrane (TM) domain and / or alpha cytoplasmic tail. The TCR or derivative or fragment thereof may further comprise at least one beta constant (C) domain, beta transmembrane (TM) domain and / or beta cytoplasmic tail.

[0304] The TCR constant region(s), transmembrane region(s) and cytoplasmic tail(s) may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse. Preferably, the PRAME-004 TCR comprises a human constant region, a human transmembrane region and a human cytoplasmic tail.

[0305] The constant region of the TCR may be slightly modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. The variable domains of the TCR may be slightly modified, for example, by the introduction of single point mutations to optimize the TCR stability and / or to enhance TCR chain pairing. A modified amino acid residue may be selected from an amino acid insertion, deletion or substitution. If the modification is a substitution, the substitution is preferably a conservative amino acid substitution.

[0306] The TCR may be chimeric, and comprise sequences from multiple species. Preferably, a TCR of the disclosure may comprise an a chain comprising a human variable region of an alpha chain and, for example, a murine constant region of a murine TCR alpha chain. Preferably, however, the TCR is a human TCR comprising human variable domains as defined herein and human constant and transmembrane domain.

[0307] The immune cell(s) may therefore express a TCR alpha chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, and / or a TCR beta chain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0308] The immune cell(s) may comprise a TCR-encoding nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

[0309] The TCR may comprise CDR(s), alpha / beta variable domains and / or alpha / beta chains having an amino acid sequence as set out in Table 2, or an amino acid sequence having at least 80 %, 85%, 90%, 95%, 99% sequence identity thereto.

[0310] The TCR may be encoded by at least one nucleic acid having a nucleic acid sequence as set out in Table 2, or a nucleic acid sequence having at least 80 %, 85%, 90%, 95%, 99% sequence identity thereto.

[0311] The TCR or derivative or fragment thereof is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24).

[0312] CD8

[0313] PRAME CD8 immune cells according to the second aspect of the present disclosure additionally express CD8 and / or comprise a nucleic acid encoding CD8. Preferably, said CD8 is heterologous to said immune cell(s). Specifically, PRAME CD8 immune cells according to the second aspect of the present disclosure additionally express at least one heterologous CD8 chain and / or comprise a nucleic acid encoding CD8. Without wishing to be bound by theory, it is thought that a heterologous CD8 together with the PRAME-004 TCR adds functional CD4+ T cells and boosts cytotoxicity. Therefore, it is speculated that PRAME-004 CD8 TCR expressing immune cell(s) could be useful for targeting both high- and medium-level PRAME expressing cancers while reducing dose-limiting toxicity.

[0314] “CD8” refers to a transmembrane glycoprotein composed of either a homodimer (CD8aa) or a heterodimer (CD8a[3). Each monomer typically consists of an extracellular immunoglobulin-like domain, a stalk region, a transmembrane domain, and a cytoplasmic tail. The extracellular domain facilitates binding to major histocompatibility complex class I (MHC-I) molecules, while the cytoplasmic domain interacts with intracellular signaling pathways to modulate T cell activation. The CD8a and CD8[3 subunits are encoded by the CD8A and CD8B genes, respectively." In the context of the present disclosure, CD8 preferably refers to human CD8, preferably the CD8 heterodimer composed of a CD8 alpha chain as referenced in UniProtKB under Accession No. P01732 and its variants, and a CD8 beta chain as referenced in UniProtKB under Accession No. P10966 and its variants. Different CD8alpha and CD8beta chain sequences have been described. The skilled person will select a CD8alpha and / or CD8beta chain that is capable of enhancing MHC-I binding and T cell activation through the PRAME-004 TCR.

[0315] CD8 functions as a co-receptor that enhances the sensitivity and specificity of T cell receptor (TCR) signaling in cytotoxic T lymphocytes. It facilitates the interaction between the TCR complex and major histocompatibility complex class I (MHC I) molecules presented on target cells, thereby promoting antigen recognition and subsequent T cell activation. CD8 typically exists as a heterodimer composed of CD8a and CD8[3 chains, although homodimeric forms may also be functional.

[0316] Accordingly, in instances of the present disclosure, the CD8 alpha chain expressed by the immune cell(s) comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19, and / or the CD8 beta chain expressed by the immune cell(s) comprises or consists of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. However, other CD8alpha and CD8beta chains may also be used.

[0317] The immune cell or population of immune cells described herein comprises or expresses CD8 and is obtainable by introducing, in addition to one or more nucleic acid molecules encoding a T cell receptor (TCR), one or more nucleic acid molecules encoding CD8 into the patient’s immune cells, preferably via expression vectors. As used throughout the present disclosure, the terms “comprising CD8” and “expressing CD8” are intended to encompass the production, synthesis, and / or presence of functional CD8 protein within or on the surface of the immune cell, typically resulting from transcription of the CD8-encoding nucleic acid into mRNA and subsequent translation into protein. The term “functional CD8” refers to a CD8 molecule that may consist of a CD8 alpha and CD8 beta chain, two CD8 alpha chains, or one or more variants of CD8 alpha and / or CD8 beta chains, and preferably retains the functional characteristics of naturally occurring CD8a / p in facilitating TCR-MHC interaction, antigen recognition and / or T cell activation.

[0318] The CD8 chain(s) may be expressed with N-terminal leader or signal peptide sequences that direct the nascent CD8 polypeptide(s) to the endoplasmic reticulum, facilitating proper folding and assembly of the CD8 dimer.

[0319] The CD8 alpha chain may be expressed with a signal peptide according to SEQ ID NO: 27, or a sequence having at least 80%, 90%, 95% or 99% sequence identity thereto. The CD8 beta chain may be expressed with a signal peptide according to SEQ ID NO: 28, or a sequence having at least 80%, 90%, 95% or 99% sequence identity thereto. Other signal peptide sequences can also be used as long as they facilitate CD8 targeting and translocation to the cell membrane. Signal peptides are typically cleaved off by signal peptidases in the endoplasmic reticulum and do not form part of the mature CD8 chain(s).

[0320] CD8 expression may be induced, constitutive, transient, or stable. Preferably, the immune cell or population of immune cells stably express said CD8. The term “stable” expression has the meaning as defined elsewhere herein and can be achieved as described in the context of the first aspect of the disclosure.

[0321] As described above, the immune cell(s) described throughout the specification are preferably obtained using genetic engineering. Therefore, the PRAME CD8 immune cell(s) preferably recombinant with respect to the TCR or derivative or fragment thereof and the CD8 as described herein. In other words, the PRAME CD8 immune cell(s) preferably comprise recombinant DNA encoding at least the TCR or derivative or fragment thereof and the CD8 as described herein. The TCR or derivative or fragment thereof and the CD8 are therefore preferably heterologous to said PRAME CD8 immune cell(s).

[0322] Additionally or alternatively to expressing the CD8, the PRAME CD8 immune cell(s) may comprise at least one nucleic acid(s) encoding said CD8. The at least one nucleic acid may be a vector, in particular a viral vector as described elsewhere herein. The PRAME CD8 immune cell may comprise one or several nucleic acids, specifically (viral) vectors, where for instance different CD8 chains are expressed by the same or different nucleic acid(s) or (viral) vector(s). For instance, the CD8 alpha chain may be expressed by a first nucleic acid or vector, and the CD8 beta chain may be expressed by a second nucleic acid or vector. Alternatively, the TCR or derivative or fragment thereof and the CD8 may all be encoded by the same nucleic acid or vector.

[0323] Preferably, a nucleic acid encoding said TCR and said CD8 comprises a nucleotide sequence S1 encoding a CD8 alpha chain, a nucleotide sequence S2 encoding a CD8 beta chain, a nucleotide sequence S3 encoding a TCR alpha chain, and a nucleotide sequence S4 encoding a TCR beta chain, wherein the nucleotide sequences are arranged in a 5’ to 3’ orientation selected from S1 -S2-S3-S4, S1-S2- S4-S3, S2-S1 -S3-S4, S2-S1 -S4-S3, S3-S4-S1-S2, S3-S4-S2-S1 , S4-S3-S1 -S2, and S4-S3-S2-S1 , preferably in S2-S1 -S4-S3 orientation. Said nucleotide sequences are typically arranged on a suitable expression vector, such as a lentiviral vector.

[0324] The PRAME CD8 immune cell(s) may comprise a nucleic acid as described in WO 2020 / 243134 A1 , which is incorporated by reference in its entirety.

[0325] In view of the above, the PRAME CD8 immune cell(s) for use in the method of the second aspect of the present disclosure may comprise a) a TCR alpha variable domain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and / or a TCR alpha chain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15; b) a TCR beta variable domain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 16; and / or a TCR beta chain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 17; c) a CD8 alpha chain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 21 ; and / or a CD8 beta chain encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 22.

[0326] The PRAME CD8 immune cell(s) may comprise a TCR- and CD8-encoding nucleic acid comprising or consisting of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 23.

[0327] The CD8 may comprise alpha / beta chains having an amino acid sequence as set out in Table 2, or an amino acid sequence having at least 80 %, 85%, 90%, 95%, 99% sequence identity thereto.

[0328] The CD8 may be encoded by at least one nucleic acid having a nucleic acid sequence as set out in Table 2, or a nucleic acid sequence having at least 80 %, 85%, 90%, 95%, 99% sequence identity thereto.

[0329] Any features and embodiments relating to the TCR, PRAME immune cells, and treatment regimen described in the context of the first aspect of the disclosure applicable, mutatis mutandis, equally applicable to the second aspect of the disclosure unless stated otherwise.

[0330] Cell dose

[0331] The treatment regimen of the present disclosure preferably comprises administering an effective amount of immune cells to the patient to be treated. The term “effective amount” has the meaning as defined elsewhere herein. The inventors of the present disclosure have found that a flat, high dose of TCR-engineered immune cells can be administered to cancer patients for an enhanced therapeutic effect.

[0332] Cell doses may be the same or different for PRAME immune cells and PRAME CD8 immune cells.

[0333] PRAME immune cells are administered at a single dose of about 1 -10x109cells per patient, preferably at a single dose of about 2, 3, 4, more preferably about 5, 6, 7, 8, 9 or 10 x109cells. Alternatively, PRAME immune cells may be administered in several doses. Other suitable doses include about 12 to 18, preferably about 40 to 60 x 106immune cells / m2BSA, more preferably about 120 to 180, about 200 to 480 or preferably up to about 1200 x 106immune cells / m2BSA, even more preferably about 1201 to 4700 x 106immune cells / m2BSA. The present inventors have observed that a single, flat dose of about 1 -10 x 109PRAME immune cells per patient and treatment can be successfully applied for cancer treatment. Advantageously, high cell doses of PRAME immune cells of about 5 x 109or higher have proven safe and effective in a clinical setting.

[0334] PRAME CD8 immune cells are administered at a single dose of about 1 -10x109cells, preferably at a single dose of about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 x109cells per patient. Alternatively, PRAME CD8 immune cells may be administered in several doses. Other suitable doses include about 12 to 18, preferably about 40 to 60 x 106immune cells / m2BSA, more preferably about 120 to 180, about 200 to 480, about 481 to 800, about 801 to 1200, or more preferably about 1200 to 2000, about 1200 to 4700 or even more preferably about 2001 to 4000 or about 4001 to 5500 x 106immune cells / m2BSA. The present inventors have observed that a single, flat dose of about 1- 10 x 109PRAME CD8 immune cells per patient and treatment can be successfully applied for cancer treatment. Advantageously, PRAME CD8 immune cells can be used for treating cancer with a medium prevalence for PRAME-004 expression, preferably at a dose of above 5 x 109cells / patient. Advantageously, PRAME CD8 immune cells can also be used for treating cancer with a high prevalence for PRAME-004 expression, preferably at a dose of up to 5 x 109cells / patient, if desired. Suitable PRAME CD8 immune cell flat doses for treating cancers having any prevalence of PRAME-004 expression may include about 0.4 to 2.5 x 109cells / patient, preferably about 0.4 to 3 x 109cells / patient or 0.4 to 4 x 109cells / patient, more preferably about 0.4 to 5 x 109cells / patient, more preferably about 0.4 to 6 x 109cells / patient. In some instances, up to about 10 x 109cells / patient, such as about 9 x 109cells / patient, 8 x 109cells / patient, 7 x 109cells / patient or 6 x 109cells / patient, 0.4 to 10 x 109cells / patient, preferably 0.4 to 6 x 109cells / patient, including 0.4 to 5 x 109cells / patient, 0.4 to 4 x 109cells / patient, 0.4 to 3 x 109cells / patient, 0.4 to 2.5 x 109cells / patient immune cells / patient.

[0335] The PRAME or PRAME CD8 immune cells or a pharmaceutical composition comprising the PRAME or PRAME CD8 immune cells may be provided in any suitable dosage form, such as a cryopreserved cell suspension in a suitable container, such as a cryobag. A dose of about 1 -10 x 109PRAME or PRAME CD8 cells may be supplied in one or more containers. Preferably, a single dose of PRAME or PRAME CD8 immune cells is administered to the patient, typically on day 0 of the cancer treatment. Alternatively, two or more doses of PRAME or PRAME CD8 immune cells are administered to the patient The dose(s) can be supplied in one or more containers. Preferably, the PRAME or PRAME CD8 immune cells or pharmaceutical composition comprising the PRAME or PRAME CD8 immune cells is administered parenterally, in particular intravenously, to the patient.

[0336] Interleukin-2

[0337] The term “interleukin-2” or “IL-2” preferably refers to human interleukin-2, UniProtKB Accession No. P60568 (UniProt Consortium, www.uniprot.org / uniprot / P60568) and its variants, in particular recombinant lnterleukin-2 variants, and engineered IL-2 molecules, such as PEGylated IL-2 and IL- 2 fusion proteins. Human interleukin-2 (IL-2) for therapeutic use is typically provided as a recombinant protein in the form of a pharmaceutical composition, particularly a lyophilized powder or solution for injection. A lyophilized powder requires reconstitution before administration. An FDA-approved IL-2 therapy is aldesleukin (Proleukin®), a recombinant IL-2 used for cancer and immune disorders. The target dose may be provided in one or more containers.

[0338] In TCR-T cell therapy, interleukin-2 (IL-2) is commonly administered to facilitate the in vivo expansion, activation, and persistence of the engineered T cells following infusion. IL-2 can promote proliferation and enhance the cytotoxic activity of the transferred T cells against target cells, thereby contributing to the therapeutic efficacy of the treatment. Accordingly, any IL-2 variant employed in the treatment regimen described herein preferably exhibits one or more of these functional properties, including but not limited to supporting T cell expansion, enhancing effector function, and sustaining cellular persistence.

[0339] PRAME immune cell treatment may further comprise administering an effective amount of interleukin (IL)-2 to the patient.

[0340] The inventors of the present disclosure have found that a low dose of interleukin-2 cells is effective to support the therapeutic effect of PRAME immune cells. Specifically, the treatment regimen includes administering at least one single dose of 1 million IU interleukin (IL)-2 per patient. Human interleukin-2 (IL-2) for therapeutic use is typically provided as a recombinant protein in the form of a pharmaceutical composition, particularly a lyophilized powder or solution for injection. A lyophilized powder requires reconstitution before administration. One dose of 1 million IU per patient may be provided in one or more containers. Preferably, several IL-2 doses of 1 million IU IL-2 per patient are administered over the course of several days, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Preferably, several IL-2 doses of about 1 million IU IL-2 per patient are administered over the course of several consecutive days, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive days. Preferably, a dose of about 1 million III IL-2 is administered once daily or twice daily over the course of several days, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive days. Preferably, a dose of about 1 million IU IL-2 is administered once daily for days 1 -5 after administering the PRAME immune cell(s) on day 0, and / or twice daily at days 6-10 after administering said immune cell(s) on day 0. Preferably, IL-2 treatment starts within about 24 hours after immune cell treatment.

[0341] Accordingly, PRAME immune cell treatment may comprise administering IL-2 according to one of the following schedules: i) 1 million IU IL-2 once daily for 5 days, beginning about 24 hours after immune cell administration, followed by 1 million IU IL-2 twice daily for 5 days; ii) 1 million IU IL-2 once daily for 10 days, beginning about 24 hours after immune cell administration; iii) 1 million IU IL-2 once daily for 3 days, beginning about 72 hours after immune cell administration, followed by twice daily for 5 days; iv) 1 million IU IL-2 once daily for 8 days, beginning about 72 hours after immune cell administration; or v) No IL-2 administration.

[0342] Typically, IL-2 or the pharmaceutical composition comprising IL-2 is administered parenterally, preferably intravenously or more preferably subcutaneously, to the patient.

[0343] Specifically, the cancer treatment regimen according to the present disclosure may comprise: administering 1-10 x 109PRAME immune cells at day 0 of the treatment regimen, administering 1 million IU IL-2 once daily on days 1-5 after administering said PRAME immune cell(s) on day 0, and twice daily on days 6-10 after administering said PRAME immune cell(s) on day 0. Alternatively, the cancer treatment regimen for PRAME immune cells may not involve administering IL-2 at all.

[0344] PRAME CD8 immune cell treatment may or may not further comprise administering an effective amount of interleukin (IL)-2 to the patient.

[0345] The inventors of the present disclosure have found that a low dose of interleukin-2 cells may be effective to support the therapeutic effect of PRAME CD8 immune cells. However, the inventors have observed that, due to their high potency, PRAME CD8 T cells may not require additional IL-2 treatment. If the treatment regimen relies on IL-2, it will include administering at least one single dose of 1 million IU interleukin (IL)-2 per patient. Preferably, several IL-2 doses of 1 million III IL-2 per patient are administered over the course of several days, such as 1 , 2, 3, 4, 5, 6, 7, 8,

[0346] 9 or 10 days. Preferably, several IL-2 doses of about 1 million IU IL-2 per patient are administered over the course of several consecutive days, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive days. Preferably, a dose of about 1 million IU IL-2 is administered once daily or twice daily over the course of several days, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, preferably 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive days. Preferably, a dose of about 1 million IU IL-2 is administered once daily for days 1 -5 after administering the PRAME immune cell(s) on day 0, and / or twice daily at days 6-

[0347] 10 after administering said immune cell(s) on day 0. Preferably, IL-2 treatment starts within about 24 hours after immune cell treatment.

[0348] Accordingly, PRAME CD8 immune cell treatment may comprise administering IL-2 according to one of the following schedules: vi) 1 million IU IL-2 once daily for 5 days, beginning about 24 hours after immune cell administration, followed by 1 million IU IL-2 twice daily for 5 days; vii) 1 million IU IL-2 once daily for 10 days, beginning about 24 hours after immune cell administration; viii) 1 million IU IL-2 once daily for 3 days, beginning about 72 hours after immune cell administration, followed by twice daily for 5 days; ix) 1 million IU IL-2 once daily for 8 days, beginning about 72 hours after immune cell administration; or x) No IL-2 administration.

[0349] Typically, IL-2 or the pharmaceutical composition comprising IL-2 is administered parenterally, preferably intravenously or more preferably subcutaneously, to the patient.

[0350] Specifically, the cancer treatment regimen according to the present disclosure may comprise: administering 1-10 x 109PRAME immune cells at day 0 of the treatment regimen, administering 1 million IU IL-2 once daily on days 1-5 after administering said PRAME immune cell(s) on day 0, and twice daily on days 6-10 after administering said PRAME immune cell(s) on day 0. Alternatively, the cancer treatment regimen for PRAME CD8 immune cells may not involve administering IL-2 at all.

[0351] Checkpoint inhibitor PRAME immune cell treatment may or may not further comprise administering an effective amount of at least one checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to the patient.

[0352] PRAME CD8 immune cell treatment may or may not further comprise administering an effective amount of at least one checkpoint inhibitor, preferably a PD- 1 or PD-L1 inhibitor, to the patient.

[0353] A ’’checkpoint inhibitor” is a molecule, including but not limited to antibodies, small molecules, peptides, or nucleic acids, that interferes with an immune checkpoint pathway to modulate immune responses. Checkpoint inhibitors typically block inhibitory signaling interactions between immune checkpoint proteins, such as PD- 1 / PD-L1 , CTLA-4, LAG-3, TIM-3, and TIGIT, thereby enhancing immune cell activation, proliferation, or function. The term includes naturally occurring, recombinant, engineered, or synthetic molecules that inhibit checkpoint signaling. Many checkpoint inhibitors are available and well established for cancer treatment.

[0354] Preferably, the checkpoint inhibitor is a PD-1 / PD-L1 checkpoint inhibitor. A PD- 1 / PD-L1 checkpoint inhibitor is a molecule that blocks the interaction between programmed cell death protein 1 (PD-1 ) and its ligand PD-L1 (or PD-L2) to enhance immune responses, particularly by restoring T cell activity against cancer or other diseases. Exemplary PD-1 / PD-L1 checkpoint inhibitors include, without limitation, the anti-PD-1 monoclonal antibodies pembrolizumab (Keytruda®), nivolumab (Opdivo®), and cemiplimab (Libtayo®), as well as the anti-PD-L1 monoclonal antibodies atezolizumab (Tecentriq®), durvalumab (Imfinzi®), and avelumab (Bavencio®).

[0355] Preferably, the checkpoint inhibitor is nivolumab (Opdivo®). Preferably, nivolumab is administered at an effective dose, preferably at a maximum of about 480 mg nivolumab per month, or about 6000 mg nivolumab per year, to said patient. Preferably, nivolumab is administered at a dose of a about 240 mg nivolumab every 2 weeks or about 480 mg nivolumab every 4 weeks.

[0356] Typically, nivolumab is administered parenterally, preferably intravenously, to the patient. Typically, nivolumab is provided as a pharmaceutical composition in the form of a solution for infusion in single-dose vials (40 mg / 4 mL, 100 mg / 10 mL, or 240 mg / 24 mL) and requires dilution with 0.9% NaCI or 5% dextrose before administra tion.

[0357] In view of the above, the present disclosure thus relates to a use or method for the treatment of cancer, wherein said treatment comprises administering 1 -10 x 109PRAME immune cells, at least one single dose of 1 million IU interleukin (IL)-2 and at least one checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. Additionally, the present disclosure relates to a use or method for the treatment of cancer, wherein said treatment comprises administering 1 -10 x 109PRAME immune cells and at least one checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. It may be preferred that PRAME immune cell treatment does not comprise administering a checkpoint inhibitor to the patient.

[0358] Further, the disclosure thus relates to a use or method for the treatment of cancer, wherein said treatment comprises administering an effective amount of PRAME CD8 immune cells, and at least one checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. Additionally, the disclosure relates to a use or method for the treatment of cancer, wherein said treatment comprises administering an effective amount of PRAME CD8 immune cells, at least one single dose of 1 million III interleukin (IL)-2 and at least one checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient. It may be preferred that PRAME CD8 immune cell treatment does not comprise administering a checkpoint inhibitor to the patient.

[0359] Lymphodepletion

[0360] The PRAME immune cell treatment regimen of the present disclosure may further comprise administering at least one lymphodepletion agent to said patient.

[0361] The PRAME CD8 immune cell treatment regimen of the present disclosure may further comprise administering at least one lymphodepletion agent to said patient.

[0362] A “lymphodepletion agent” or “lymphodepleting agent” refers to any compound, biologic, or treatment regimen that reduces or depletes lymphocytes, including but not limited to T cells, B cells, and natural killer (NK) cells, in a subject. Such agents may include chemotherapeutic drugs (e.g., cyclophosphamide, fludarabine), monoclonal antibodies (e.g., anti-CD52, anti-CD3), radiation therapy, or any other modality that results in a reduction of lymphocyte populations. Lymphodepletion agents may be used to enhance the efficacy of adoptive cell therapies, reduce immune responses, or facilitate engraftment of transplanted cells or tissues.

[0363] Preferably, the at least one lymphodepletion agent is a chemotherapeutic drug. Preferably, the at least one lymphodepletion agent is administered intravenously.

[0364] Preferably, the lymphodepletion agent is fludarabine and / or cyclophosphamide. More preferably, the use or method of the present disclosure involves administering both fludarabine and cyclophosphamide at an effective dose to said patient, wherein fludarabine is preferably administered at a dose of about 30 mg / m2or less, such as 25 mg / m2or 20 mg / m2and cyclophosphamide is preferably administered at a dose of about 500 mg / m2or less, such as about 250 mg / m2to the patient.

[0365] Preferably, the at least one lymphodepletion agent, preferably fludarabine and cyclophosphamide, are administered at an effective dose for 1 , 2, 3 or 4 consecutive days before administering said immune cell(s) to said patient. Preferably, patients with solid tumors except HCC, adequate renal function and adequate bone marrow reserve receive daily doses of about 30 mg / m2fludarabine, and about 500 mg / m2 cyclophosphamide for 4 consecutive days before administering the immune cells. Preferably, HCC patients with adequate renal function and adequate bone marrow reserve receive daily doses of about 25 mg / m2fludarabine, and about 400 mg / m2cyclophosphamide for 4 consecutive days before administering the immune cells. Preferably, patients with solid tumors except HCC, moderate renal impairment and adequate bone marrow reserve receive daily doses of about 25 mg / m2fludarabine, and about 500 mg / m2cyclophosphamide for 4 consecutive days before administering the immune cells. Preferably, HCC patients with moderate renal impairment and adequate bone marrow reserve receive daily doses of about 20 mg / m2fludarabine, and about 400 mg / m2cyclophosphamide for 4 consecutive days before administering the immune cells. Preferably, patients with solid tumors except HCC, adequate renal function and reduced bone marrow reserve receive daily doses of about 25 mg / m2fludarabine, and about 400 mg / m2cyclophosphamide for 4 consecutive days before administering the immune cells. Preferably, HCC patients with adequate renal function and reduced bone marrow reserve receive daily doses of about 20 mg / m2fludarabine, and about 300 mg / m2cyclophosphamide for 4 consecutive days before administering the immune cells. “Adequate renal function” is characterized by a creatinine clearance of >70 ml / min / 1.73 m2. “Moderate renal impairment” is characterized by a creatinine clearance of <70 ml / min / 1 .73 m2. Patients >70 years and / or with heavy pre-treatments or other conditions impacting bone marrow reserve are classified as having “reduced bone marrow reserve”. If clinically required, minor reductions in fludarabine and / or cyclophosphamide dose(s) may be applied.

[0366] Preferably, said lymphodepleting agent is administered to said patient before administration of the PRAME or PRAME CD8 immune cell(s).

[0367] Specifically, the present disclosure thus includes a use or method of treating cancer in a patient, where in a first step at least one lymphodepletion agent is administered, followed by administration of about 1 -10 x 109PRAME immune cells, and at least one single dose of about 1 million III interleukin ( I L)-2.

[0368] Specifically, the present disclosure thus includes a use or method of treating cancer in a patient, where in a first step at least one lymphodepletion agent is administered, followed by administration of about 1 -10 x 109PRAME CD8 immune cells, and at least one single dose of about 1 million IU interleukin ( I L)-2. Additionally, the present disclosure thus includes a use or method of treating cancer in a patient, where in a first step at least one lymphodepletion agent is administered, followed by administration of about 1 -10 x 109PRAME CD8 immune cells, and no additional administration of IL-2.

[0369] Cancer indications

[0370] Treatment with PRAME immune cells or PRAME CD8 immune cells as disclosed herein is suitable for treating a variety of cancers. The cancer may be selected from Acute lymphoblastic leukemia; Acute lymphocytic cancer; Acute myeloid leukemia; Adenoid cystic carcinoma; Adrenocortical carcinoma; AIDS-related cancers; AIDS-related lymphoma; Alveolar rhabdomyosarcoma; Anal cancer; Angiosarcoma; Appendix cancer; Astrocytomas; Ardenocarcinoma; Atypical meningioma; Basal cell carcinoma; Bladder cancer, including Bladder urothelial carcinoma; Bone cancer; Brain cancer; Brain tumors; Breast cancer, including Breast carcinoma; Bronchial adenomas; Burkitt lymphoma; Cancer of the anus; anal canal; or anorectum; Cancer of the eye; Cancer of the intrahepatic bile duct; Cancer of the joints; Cancer of the neck; gallbladder; or pleura; Cancer of the nose; nasal cavity; or middle ear; Cancer of the oral cavity; Cancer of the oropharynx; Cancer of the penis; Cancer of the uterus; Cancer of the vagina; Cancer of the vulva; Cancer of unknown primary origin; Carcinoma of the neck; gallbladder; or pleura; Central nervous system lymphoma; Cervical cancer, including Cervical carcinoma, Cervical squamous cell carcinoma and endocervical adenocarcinoma; Childhood cancers; Cholangiocarcinoma (Bile duct cancer); Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloid cancer; Chronic myeloproliferative disorders; Colon cancer; Colorectal cancer; Cutaneous T- cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Epithelial cancer of the larynx; Esophageal cancer, including Esophageal carcinoma; Ewings sarcoma; Extranodal T / NK-cell lymphomas; Fibrosarcoma; Gallbladder cancer; Gastric cancer; Gastrointestinal cancer; Gastrointestinal carcinoid tumor; Gastrointestinal stromal tumor; Germ cell tumor; Glioblastoma; Glioma; H. pylon- induced MALT Non-Hodgkin’s lymphoma; Hairy cell leukemia; Head and neck cancer, including Head and neck adenocarcinoma and Head and neck squamous cell carcinoma (HNSCC); Heart cancer; Hepatocellular cancer, including Hepatocellular carcinoma (HCC); Hodgkin lymphoma; Hypopharyngeal cancer; Intraocular melanoma; Islet cell carcinoma; Kaposi sarcoma; Kidney cancer, including Kidney carcinoma, Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma; Laryngeal cancer; Larynx cancer; Leukemia; Lip and oral cavity cancer; Liposarcoma; Liver cancer; Liver hepatocellular carcinoma; Lung cancer, including Non-small cell lung cancer (NSCLC), including squamous cell Non-small cell lung cell carcinoma, non-squamous Non-small cell lung cell carcinoma Non-small cell lung cell carcinoma and Non-small cell lung adenocarcinoma; Lymphoma; Macroglobulinemia; Malignant fibrous histiocytoma of the bone (MFH); Malignant mesothelioma; Malignant peripheral nerve sheath tumor (MPNST); Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin; Merkel cell carcinoma; Mesothelioma; Metastatic squamous neck cancer with occult primary; Mouth cancer; Multiple endocrine neoplasia syndrome; Multiple myeloma; Myelodysplastic syndromes; Myeloid leukemia; Myxoid liposarcoma; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Nasopharynx cancer; Neuroblastoma; Neuroendocrine tumors (NET), including Large cell neuroendocrine lung cancer (LCNEC); NonHodgkin lymphoma; Oral cancer; Oral carcinoma; Oral cavity carcinomas; Oral squamous carcinoma; Oropharyngeal cancer; Osteosarcoma;; Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); Pancreatic cancer; Pancreatic cancer (islet cell); Papillary thyroid carcinoma; Parathyroid cancer; Penile cancer; Peritoneum, omentum and mesentery cancer; Pharyngeal cancer; Pharynx cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pituitary adenoma; Plasma cell neoplasia; Pleuropulmonary blastoma; Primary brain cancer; Primary central nervous system lymphoma; Prostate cancer; Pulmonary sarcomatoid carcinoma; Rectal cancer; Renal cell carcinoma; Renal pelvis and ureter transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma; Salivary duct carcinoma; Salivary gland cancer; Sarcoma, including Synovial sarcoma and Soft tissue sarcoma; Skin cancer; Small cell lung cancer (SLC); Small intestine cancer; Small cell neuroendocrine cancer (SNC); Soft tissue cancer; Stomach adenocarcinoma; Stomach cancer; T-cell lymphoma; Testicular cancer; Testicular germ cell tumors; Throat cancer; Thymic carcinoma; Thymoma; Thyroid cancer; Triple-negative breast cancer (TNBC); Trophoblastic tumor (gestational); Ureter cancer; Urethral cancer; Urinary bladder cancer; Uterine cancer, including Uterine Carcinoma, Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma; Vaginal cancer; Vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.

[0371] Preferably, the cancer is selected from Adrenocortical carcinoma; Ardenocarcinoma; Bladder cancer, including Bladder urothelial carcinoma; Breast cancer, including Breast carcinoma and Triple-negative breast cancer (TNBC); Cervical cancer, including Cervical carcinoma, Cervical squamous cell carcinoma and endocervical adenocarcinoma; Cholangiocarcinoma; Esophageal cancer, including Esophageal carcinoma; Head and neck cancer, including Head and neck adenocarcinoma and Head and neck squamous cell carcinoma (HNSCC); Hepatocellular cancer, including Hepatocellular carcinoma (HCC); Kidney cancer, including Kidney carcinoma, Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma; Lung cancer, including Non-small cell lung cancer (NSCLC), including squamous cell Non-small cell lung cell carcinoma, non-squamous Non-small cell lung cell carcinoma and Non-small cell lung adenocarcinoma; Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin; Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); Sarcoma, including Synovial sarcoma; Small cell lung cancer (SLC); and Uterine cancer, including Uterine Carcinoma, Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma.

[0372] Preferably, the cancer is selected from a PRAME-004 positive Adrenocortical carcinoma; Ardenocarcinoma; Bladder cancer, including Bladder urothelial carcinoma; Breast cancer, including Breast carcinoma and Triple-negative breast cancer (TNBC); Cervical cancer, including Cervical carcinoma, Cervical squamous cell carcinoma and endocervical adenocarcinoma; Cholangiocarcinoma; Esophageal cancer, including Esophageal carcinoma; Head and neck cancer, including Head and neck adenocarcinoma and Head and neck squamous cell carcinoma (HNSCC); Hepatocellular cancer, including Hepatocellular carcinoma (HCC); Kidney cancer, including Kidney carcinoma, Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma; Lung cancer, including Non-small cell lung cancer (NSCLC), including squamous cell Non-small cell lung cell carcinoma, non-squamous Non-small cell lung cell carcinoma Non-small cell lung cell carcinoma and Non-small cell lung adenocarcinoma; Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin; Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); Sarcoma, including Synovial sarcoma; Small cell lung cancer (SLC); and Uterine cancer, including Uterine Carcinoma, Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma.

[0373] Preferably, the cancer is selected from Cholangiocarcinoma; Breast cancer, including triple negative breast cancer (TNBC); Hepatocellular cancer, including Hepatocellular carcinoma (HCC); Head and neck squamous cell carcinoma (HNSCC) Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin; Synovial Sarcoma; Lung cancer, including Squamous and Non-squamous non-small cell lung cancer (NSCLC) and Small cell lung cancer (SCLC); Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); and Uterine cancer, including Uterine Carcinoma, Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma.

[0374] The cancer may preferably be selected from Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin. Preferably, the cancer may be selected from Cutaneous Melanoma (CM), and Acral Melanoma.

[0375] In some instances of the first aspect, the cancer is selected from Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin, preferably, the cancer is selected from Cutaneous Melanoma (CM), and Acral Melanoma. In particular preferred embodiments of the first aspect, the cancer is selected from Cutaneous Melanoma (CM), Uveal Melanoma (UM), and Mucosal Melanoma. The cancer may preferably be selected from Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC.

[0376] The cancer may preferably be selected from Uterine cancer, including Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma.

[0377] The cancer may preferably be selected from Breast cancer, including triple negative breast cancer (TNBC).

[0378] The cancer may preferably be selected from Lung cancer, including Squamous and Non-squamous non-small cell lung cancer (NSCLC) and Small cell lung cancer (SCLC).

[0379] In some instances of the second aspect, the cancer is selected from Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin, preferably, the cancer is selected from Cutaneous Melanoma (CM), and Acral Melanoma; Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); Uterine cancer, including Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC) and Uterine sarcoma; Triple-negative breast cancer; and Lung cancer, including Squamous and Non-squamous non-small cell lung cancer (NSCLC) and Small cell lung cancer (SCLC). In particular preferred embodiments of the second aspect, the cancer is selected from Cutaneous Melanoma (CM), Uveal Melanoma (UM), and Mucosal Melanoma.

[0380] In alternative or even more preferred embodiments of the first and / or second aspect of the present disclosure, the cancer to be treated is selected from adrenocortical carcinoma, adenoid cystic carcinoma, non-squamous anal carcinoma, squamous anal carcinoma, basal cell carcinoma, bladder urothelial carcinoma, Her2- enriched breast carcinoma, luminal a breast carcinoma, luminal b breast carcinoma, triple negative breast carcinoma (TNBC), cervical adenocarcinoma, cervical adenosquamous cell carcinoma, cervical squamous cell carcinoma, cholangiocarcinoma, diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, esophageal adenosquamous carcinoma, esophageal small cell carcinoma, esophageal squamous carcinoma, head and neck squamous cell carcinoma (HNSCC), kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, malignant rhabdoid tumor, Wilms tumor (nephroblastoma), acute myeloid leukemia (AML), hepatocellular carcinoma, large cell lung carcinoma (LCLC), large cell neuroendocrine lung carcinoma (LCNEC), adenocarcinoma non-small cell lung carcinoma (adenocarcinoma NSCLC), squamous cell NSCLC, adenosquamous NSCLC, sarcomatoid carcinoma of the lung, small cell lung cancer (SCLC), acral melanoma, mucosal melanoma, cutaneous melanoma, spindle cell melanoma, uveal melanoma, Merkel cell carcinoma (MCC), mucoepidermoid carcinoma, neuroblastoma, ovarian clear cell carcinoma, ovarian endometrioid carcinoma, ovarian leiomyosarcoma, ovarian mucinous carcinoma, ovarian serous cystadenocarcinoma, pancreatic neuroendocrine adenocarcinoma, prostate neuroendocrine adenocarcinoma, head and neck salivary duct carcinoma, head and neck salivary gland carcinoma, malignant peripheral nerve sheath tumor (MPNST), alveolar rhabdomyosarcoma, angiosarcoma, Ewing sarcoma, fibrosarcoma, liposarcoma, myxoid liposarcoma, osteosarcoma, embryonal rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, cutaneous squamous cell carcinoma, stomach adenocarcinoma, testicular germ cell tumor (seminoma and nonseminoma), anaplastic thyroid carcinoma, thymoma, endometrial clear cell carcinoma, endometrial sarcoma, endometrioid endometrial carcinoma, endometrial serous carcinoma, and uterine carcinosarcoma.

[0381] In preferred embodiments, the cancer to be treated is a “PRAME-004 positive cancer”. Preferably, the PRAME-004 positive cancer is a solid tumor. Preferably, the PRAME-004 positive cancer is a recurrent and / or refractory cancer. The PRAME-004 positive cancer may be a metastatic cancer.

[0382] The term “PRAME-positive cancer”, as used herein, refers to a cancer that exhibits expression of the Preferentially Expressed Antigen of Melanoma (PRAME) at a level equal to, or above, a clinically relevant threshold.

[0383] The term “clinically relevant threshold”, as used herein in connection with the expression of PRAME (or in connection with the presentation of the PRAME-004 peptide on the cancer cell surface, as further discussed below), refers in its broadest sense to a level at or above which there is a meaningful likelihood of a therapeutic benefit or response to the herein disclosed PRAME-targeting therapeutic means and methods. This threshold is typically established through rigorous clinical validation and is used to guide personalized treatment decisions, ensuring that patients most likely to benefit from a targeted therapy are accurately identified. More specific forms of, and embodiments relating to, the term “clinically relevant threshold” are provided below.

[0384] A person skilled in the art is aware of, and capable of routinely applying, various methods for assessing the expression of PRAME in cancer cells, including, without limitation, determining the level of PRAME mRNA using techniques such as quantitative reverse transcription PCR (qRT-PCR), RNA sequencing (RNA-seq), quantitative Polymerase Chain Reaction (qPCR), in-situ hybridization (ISH), microarrays and / or Northern Blotting; or evaluating PRAME protein expression levels using immunodetection methods, such as immunohistochemistry (IHC), Western Blotting, enzyme-linked immunosorbent assay (ELISA), and / or mass spectrometry (e.g., LC-MS / MS). In view of PRAME being expressed intracellularly, the assessment of PRAME expression will typically be conducted in vitro through analysis of sample(s) isolated from a subject’s (prospective patient’s) body, such a biopsy sample from the cancer tissue. However, in view of PRAME peptide fragments thereof being presented on the cancer cell surface, it is also conceivable, and expressly contemplated herein, to assess PRAME expression in vivo, for example, by using molecular imaging techniques which allow non-invasive visualization of gene or protein expression in a subject’s body, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT) using radiolabelled antibodies or similar probes.

[0385] In a similar vein to the term “PRAME-004 positive cancer”, as used herein, is to be understood as referring to a PRAME-positive cancer which cells (or at least a significant fraction thereof) present the herein referred PRAME-004 peptide (7.e. , a fragment of the PRAME protein comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24)) in complex with an MHC protein (such as an HLA protein, preferably an HLA class I protein, more preferably an HLA-A*02, more preferably an HLA-A*02:01 ) on their cell surface and at a level (in this case, the number of copies of PRAME-004 peptide presented per cell) equal to, or above, a clinically relevant threshold. Said level may be assessed, for example, based on the number of copies of the PRAME-004 peptide that is presented per cell, as determined, for example, by quantitative peptide presentation analysis mass spectrometry (e.g., by AbsQuant®, as described, for example, in US10545154B2, the contents of which are hereby incorporated by reference).

[0386] It will be appreciated by the skilled artisan that in the instance of a cancer being positive for PRAME-004 expression (i.e. , a “PRAME-004 positive cancer”), said cancer can, with all likelihood, be expected to also be positive with respect to the presentation of the PRAME-004 peptide in complex with MHC on the cancer cells’ surface (i.e., a “PRAME-004-positve cancer”) and vice versa. In other words, it can be expected that the intracellular PRAME mRNA / protein expression levels correlate with the number of PRAME-004 peptides presented on the surface of the same cell. It will thus also be appreciated that once the number of PRAME-004 peptides presented on a given cell and the amount / level of (intracellular) PRAME-encoding mRNA / protein of said cell has been quantified, it should in general be possible to infer the number of copies of PRAME-004 peptide presented on the surface of other cells from which merely the PRAME mRNA and / or protein level is known or has been quantified.

[0387] In connection with the present disclosure, it is preferably to be understood that in order for a threshold level, i.e., the level of PRAME mRNA / protein expression or of PRAME-004 peptide(s) presented on the cell surface, to classify as “clinically relevant”, said level is at least, with increasing preference, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (2-fold), 150% (2.5-fold), 200% (3-fold), 300% (4- fold), 400% (5-fold), 500% (6-fold), 600% (7-fold), 700% (8-fold), 800% (9-fold), 900% (10-fold), 1000% (11 -fold), 1100% (12-fold), 1200% (13-fold), 1300% (14-fold), 1400% (15-fold), or more increased as compared to the level (i.e. , a reference level) which is or has been determined from a cell or cells of corresponding tissue from (i) one or more healthy individuals; (ii) one or more individuals not afflicted by a cancer; or (iii) one or more individuals at least not afflicted by a cancer of the same organ, tissue or cell type.

[0388] In particularly preferred embodiments, a cancer is a “PRAME-004-positive cancer”, if:

[0389] - said cancer comprises or consists of cancer cells that present at least 20, preferably at least 30, more preferably at least 40, and even more preferably at least 50 copies of the PRAME-004 peptide per cell on their cell surface, as preferably determined by quantitative peptide presentation analysis mass spectrometry (e.g., by AbsQuant®, as described in US10545154B2, the contents of which are hereby incorporated by reference; see also Wermke M, et al., Nat Med. 2025 Apr 9. doi: 10.1038 / s41591 -025-03650-6, online ahead of print)', and / or

[0390] - if said cancer comprises or consists of cells which possess a PRAME mRNA and / or protein expression level equal to or above a PRAME mRNA and / or protein expression level of a cell which has been determined to present a respective number copies of the PRAME-004 peptide per cell on their cell surface, as preferably determined by quantitative peptide presentation analysis mass spectrometry (see also Example 1).

[0391] In alternative or more preferred embodiments, a cancer is a “PRAME-004- positive cancer”, if said cancer is known or identified as to comprise or consist of cells having a PRAME mRNA and / or protein expression level(s) that exceed(s) the, with increasing preference, 90th-percentile, 91st-percentile, 92nd-percentile, 93rd-percentile, 94thpercentile, 95thpercentile, 96thpercentile, 97th-percentile, 98th-percentile, 99th- percentile, 99.5th-percentile, 99.6th-percentile, 99.7thpercentile, 99.8thpercentile, or most preferably the 99.9th-percentile of the PRAME mRNA and / or protein expression level within a healthy reference population. For example, the PRAME-004 mRNA level of a cancer may be compared to the PRAME-004 mRNA levels in one or more normal (healthy) tissues (e.g., from at least 3, preferably at least 4, more preferably from at least 5, even more preferably from at least 6, and even more preferably from 7 different healthy tissues, selected from, or including, skin, lung, breast, salivary gland, esophagus, liver, and bladder) as, e.g., available from the GTEx (Genotype-Tissue Expression) database as a reference (http: / / www.gtexportal.org; preferably from the GTEx portal release V6, 2015, which was used herein), and the aforementioned percentile may be used as “threshold” (herein alternatively referred to as “healthy threshold”), which, when exceeded by the determined PRAME mRNA level, classifies the cancer as “PRAME-positive cancer” and / or “PRAME-004-positive cancer”.

[0392] The above threshold can be used to determine the prevalence for a cancer of a specific type to be “PRAME-004-positive”. In this connection, the term “prevalence”, as used herein, refers to the proportion or percentage of cancers of a specified cancer type that comprise or consist of cells that are “PRAME-004-positive”; as preferably assessed by comparing the PRAME expression level (i.e., the PRAME-004 mRNA and / or protein level, preferably the PRAME-004 mRNA level) throughout a population of reference samples from cancers of the same type. In this connection, the “prevalence” can also be interpreted as corresponding to, or at least as providing a strong indication or predictive measure of, the percentage of patients within a defined patient population diagnosed with a specific type of cancer who can be expected to respond successfully to a treatment with the herein disclosed PRAME-targeting therapeutic means and methods.

[0393] For example, in connection with the present disclosure, the “prevalence” of a specific cancer type for being “PRAME-004-positive” may be assessed through comparison of PRAME mRNA levels from cancer samples of a selected type - e.g., available from databases, for example, from The Cancer Genome Atlas Program (TCGA) database (https: / / cancergenome.nih.gov / , version 24-SEP-2019), or the TEMPUS database (commercial product, version AUG-2023), or determined from cancer samples of a selected cancer type from a group patients participating in a clinical trial - vs. the above referred "healthy threshold", whereby a cancer sample is classified as being “PRAME-004-positive”, if the PRAME-004 mRNA level is found to exceed(s) the 99th-percentile (or any lower or higher percentile value from those listed above) of the PRAME mRNA expression levels of a healthy reference population, preferably the mRNA levels from one or more healthy tissues (more preferably the mRNA levels from selected healthy tissues, preferably selected from or including healthy skin, lung, breast, salivary gland, esophagus, liver, and bladder tissues), as available, for example, from publicly accessible databases, such as the GTEx (Genotype-Tissue Expression) database (http: / / www.gtexportal.org), preferably from the GTEx portal release V6, 2015, from which herein used data were retrieved.

[0394] It will be understood by those skilled in the art that in a practical medical setting, a cancer patient and prospective future recipient of the herein disclosed therapeutic means may first be tested on whether in that specific instance the cancer is indeed PRAME-004 positive. This serves to identify who will likely benefit from the treatment, ensuring that only patients who test positive would then be considered eligible for a respective therapeutic intervention. However, besides the additional cost and personal resources required for such testing, in cases where the disease progresses rapidly, the time required to complete such testing may disadvantage the patient by delaying the initiation of therapy, potentially compromising the effectiveness of the treatment and adversely affecting clinical outcomes. Moreover, in certain cases, obtaining a tumor sample (biopsy) may not be feasible due to the inaccessibility of the tumor’s location within the patient’s body. In such and other cases, it may be advantageous if the initial step of testing can be omitted. This may be particularly feasible in instances wherein a specific cancer type due to its high prevalence can be expected, without prior testing, to be PRAME-positive and / or PRAME-004 positive. Thus, it is particularly contemplated, in preferred embodiments, that a patient or subject having any of the cancers (i.e., any of the different types of cancers) mentioned in the present disclosure may be treated by the herein disclosed means without prior testing or confirmation of the cancer for being PRAME-004 positive.

[0395] Respective embodiments are particularly contemplated in such instances where a cancer is of a specific cancer type that is known to have a high prevalence (i.e., for being PRAME-004 positive), wherein a high prevalence preferably means a prevalence of at least, with increasing preference, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%. Preferably, said prevalence is determined by classifying a cancer as PRAME-004 positive if said cancer comprises or consists of cell(s) which PRAME-004 mRNA expression level exceed(s) the 99th-percentile (or any lower or higher percentile value from those listed above) of the PRAME mRNA expression level of a healthy reference population, preferably the mRNA levels from selected healthy tissues (e.g., from at least 3, preferably from at least 4, more preferably from at least 5, even more preferably from at least 6, and even more preferably from 7 different healthy tissues, preferably selected from or including skin, lung, breast, salivary gland, esophagus, liver, and bladder), as, e.g., available from publicly accessible databases, for example, the GTEx (Genotype-Tissue Expression) database (http: / / www.gtexportal.org), preferably from the GTEx portal release V6, 2015.

[0396] Cancers known or determined to have a medium prevalence (i.e., for being PRAME-004 positive) of 10 %, 20%, 30 %, 40 %, 50 % or 60 % may require prior testing or confirmation of the cancer for being PRAME-004 positive.

[0397] In connection with the present invention, the inventors found that there are certain types of cancers which, among a statistically significant population of cancer patients, consistently exhibit a higher prevalence than other cancer types. For example, the following cancer types were thereby identified as possessing extraordinarily high prevalence (>85%) for being “PRAME-positive” and / or “PRAME- 004-positive”: Thus, in preferred embodiments, the cancer to be treated with PRAME or PRAME CD8 immune cells is one having a prevalence (i.e., for being “PRAME-004- positive”) of, with increasing preference, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, wherein preferably said prevalence is or has been determined if the PRAME mRNA level of the cancer exceeds the 99th-percentile (or any lower or higher percentile value from those listed above) of the PRAME-004 mRNA expression level of a healthy reference population, preferably the mRNA level from selected healthy tissues (e.g., from at least 3, preferably from at least 4, more preferably from at least 5, even more preferably from at least 6, and even more preferably from 7 different healthy tissues, preferably selected from or including skin, lung, breast, salivary gland, esophagus, liver, and bladder), as, e.g., available from publicly accessible databases, preferably, the GTEx (Genotype-Tissue Expression) database (http: / / www.gtexportal.org), more preferably from the GTEx portal release V6, 2015.

[0398] In such embodiments, the patient to be treated may not require testing for PRAME-004 expression prior to treatment.

[0399] In such embodiments, patients may be treated with PRAME immune cell doses within the full dose range, i.e. at least about 1 , at least about 2, at least about 3, at least about 4 at least about 5, at least about 6, at least about 7, at least about 8, at least about 9 or up to about 10 x 109cells / patient.

[0400] Because of their potency, PRAME CD8 immune cells may be used at lower doses when treating cancers with a high prevalence of PRAME-004 expression. In such embodiments, patients may be treated with PRAME CD8 immune cell doses in the lower range of the treatment dose, i.e. about 5, about 4, about 3, about 2 or about 1 x 109cells / patient.

[0401] In particular preferred embodiments of the present invention, the cancer to be treated is selected from the group consisting of cutaneous melanoma, uveal melanoma (more preferably, metastatic uveal melanoma), mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

[0402] In preferred embodiments of the latter embodiments, the treatment of the subject or patient is performed without prior testing of the cancer for being “PRAME- 004-positive”.

[0403] In particularly preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is cutaneous melanoma (CM). In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is uveal melanoma (UM), more preferably metastatic uveal melanoma.

[0404] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is mucosal melanoma.

[0405] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is endometrial carcinoma.

[0406] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is uterine carcinosarcoma.

[0407] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is endometrial clear cell carcinoma.

[0408] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is endometrial serous carcinoma.

[0409] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is ovarian cancer.

[0410] In other particular preferred embodiments, the cancer to be treated without prior testing for being “PRAME-004-positive” is synovial sarcoma.

[0411] In further preferred embodiments, the cancer to be treated with PRAME or PRAME CD8 immune cells is one having a prevalence ( / .e., for being “PRAME-004- positive”) of, with increasing preference, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, wherein preferably said prevalence is or has been determined if the PRAME mRNA level of the cancer exceeds the 99th-percentile (or any lower or higher percentile value from those listed above) of the PRAME-004 mRNA expression level of a healthy reference population, preferably the mRNA level from selected healthy tissues (e.g., from at least 3, preferably from at least 4, more preferably from at least 5, even more preferably from at least 6, and even more preferably from 7 different healthy tissues, preferably selected from or including skin, lung, breast, salivary gland, esophagus, liver, and bladder), as, e.g., available from publicly accessible databases, preferably, the GTEx (Genotype-Tissue Expression) database (http: / / www.gtexportal.org), more preferably from the GTEx portal release V6, 2015.

[0412] In such embodiments, the patient to be treated may require testing for PRAME- 004 expression prior to treatment.

[0413] In such embodiments, patients may be treated with PRAME immune cell doses in the upper range of the dose range, i.e. at least about 6, at least about 7, at least about 8, at least about 9 or about 10 x 109cells / patient. Because of their potency, PRAME CD8 immune cells may be particularly suitable for treating cancers with a medium prevalence of PRAME-004 expression. In such embodiments, patients may be treated with PRAME CD8 immune cell doses within the full dose range, i.e. at least about 1 , at least about 2, at least about 3, at least about 4 at least about 5, at least about 6, at least about 7, at least about 8, at least about 9 or up to about 10 x 109cells / patient.

[0414] Patient groups

[0415] Patients treated with PRAME or PRAME CD8 immune cells are preferably HLA- A*02:01 positive. The HLA-A*02:01 status of a patient can be determined by commonly available means and methods. The HLA-A*02:01 status is typically determined from the patient’s blood prior to treatment.

[0416] Patients treated with PRAME or PRAME CD8 immune cells are preferably PRAME-004 positive. When referring to “PRAME-004 positive” patients, it is meant that their cancer(s) is / are PRAME-004 positive and thus addressable by immune cell treatment, as defined above. Some cancer indications, in particular cancer indications having a high prevalence (i.e., for being “PRAME-004-positive”) of, with increasing preference, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, may not require testing of the patient for PRAME-004 expression in their cancer prior to immune cell treatment. Other cancer indications, in particular cancer indications having a medium prevalence (i.e., for being “PRAME- 004-positive”) or 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, may require testing of the patient for PRAME-004 expression in their cancer prior to immune cell treatment.

[0417] As described elsewhere herein, the cell dose used for treating cancers with medium or high prevalence for being PRAME-004 positive may differ for PRAME and PRAME CD8 immune cells.

[0418] In some embodiments, the patients have previously received checkpoint inhibitor treatment, and / or have optionally relapsed under checkpoint inhibitor treatment.

[0419] Treatment regimen

[0420] The present disclosure provides treatment regimen for PRAME and PRAME CD8 immune cells for treating cancer.

[0421] In some aspects, the present disclosure provides PRAME immune cell cancer treatment. Specifically, the treatment regimen may comprise administering a single dose of about 1 -10 x 109PRAME immune cells, followed by administration of at least a single dose of about 1 million III IL-2 to said patient. Preferably, the PRAME immune cell cancer treatment may comprise: administering to a patient about 20-30 mg / m2fludarabine and about 300-500 mg / m2of cyclophosphamide for 4 consecutive days, about 1 -10 x 109immune cells on day 0 of the treatment regimen, about 1 million III IL-2 once daily on days 1 -5 after administering said immune cell(s) on day 0, and twice daily on days 6-10 after administering said immune cell(s) on day 0. Preferably, said cancer treatment regimen is the treatment regimen depicted in FIG. 1 e.

[0422] In view of the above, the present disclosure relates to uses and methods for PRAME immune cell cancer treatment, wherein said immune cells a) express a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO.

[0423] 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6), and / or b) said immune cell comprising at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises administering to said patient:

[0424] 1 ) Lymphodepletion, optionally by administering about 30 mg / m2fludarabine and 500 mg / m2cyclophosphamide daily for 4 consecutive days;

[0425] 2) PRAME immune cell treatment with a single dose of about 1-10 x 109PRAME immune cells (dO);

[0426] 3) IL-2 treatment with a single dose of about 1 million IU interleukin-2 (IL- 2) once daily on days 1 , 2, 3, 4 and 5 (d1 -5) after immune cell administration and a single dose of about 1 million IU interleukin-2 (IL-2) twice daily on days 6, 7, 8, 9 and 10 (d6-10) after immune cell administration;

[0427] 4) Optional checkpoint inhibitor treatment with a single dose of nivolumab at a dose of about 240 mg nivolumab on day 14 after immune cell administration (d14) or at a dose of about 480 mg nivolumab on day 30 after immune cell administration.

[0428] In some embodiments of the first aspect of the present disclosure, treatment does not comprise administering a checkpoint inhibitor.

[0429] It is envisaged that the PRAME immune cell treatment regimen described above and elsewhere herein are particularly applicable to patients that have not been previously tested for PRAME-004 expression. Preferably, the treated cancer indications in such patients are cancers known or determined to have a high prevalence for PRAME-004 expression. Preferably, said cancers are selected from cutaneous melanoma, uveal melanoma (more preferably, metastatic uveal melanoma), mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

[0430] In some aspects, the present disclosure provides PRAME CD8 immune cell cancer treatment. Specifically, the treatment regimen may comprise administering a single dose of about 1 -10 x 109PRAME CD8 immune cells, optionally followed by administration of at least a single dose of about 1 million III IL-2 to said patient. Alternatively, the treatment regimen may comprise administering a single dose of about 1 -10 x 109PRAME CD8 immune cells, but does not include administration of at least a single dose of about 1 million III IL-2 to said patient.

[0431] Specifically, the cancer treatment regimen according to second aspect of the present disclosure may comprise: administering to a patient i) about 20-30 mg / m2fludarabine and about 300-500 mg / m2of cyclophosphamide for 4 consecutive days, ii) an effective amount of PRAME CD8 immune cells on day 0 of the treatment regimen, iii) optionally at least one dose of about 1 million IU IL-2.

[0432] In view of the above, the second aspect of the present disclosure includes a method of treating cancer, or to an immune cell or population of immune cells for use in a method of treatment of cancer in a patient, said immune cell a) expressing at least one heterologous CD8 chain and a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6), and / or b) said immune cell comprising at least one nucleic acid encoding CD8 and a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises administering to said patient:

[0433] 1 ) Lymphodepletion, optionally by administering about 30 mg / m2fludarabine and 500 mg / m2cyclophosphamide daily for 4 consecutive days;

[0434] 2) PRAME CD8 immune cell treatment with a single dose of about 1 -10 x 109PRAME CD8 immune cells (dO); 3) Optional IL-2 treatment with a single dose of about 1 million III interleukin-2 (IL-2) once daily on days 1 , 2, 3, 4 and 5 (d1-5) after immune cell administration and a single dose of about 1 million IU interleukin-2 (IL-2) twice daily on days 6, 7, 8, 9 and 10 (d6-10) after immune cell administration;

[0435] 4) Optional checkpoint inhibitor treatment with a single dose of nivolumab at a dose of about 240 mg nivolumab on day 14 after immune cell administration (d14) or at a dose of about 480 mg nivolumab on day 30 after immune cell administration.

[0436] Advantageously, due to their potency, PRAME CD8 immune cells may not require additional IL-2 treatment. Further preferred embodiments do not require checkpoint inhibitor treatment.

[0437] It is envisaged that the PRAME CD8 immune cell treatment regimen described above and elsewhere herein are particularly applicable to patients that have not been previously tested for PRAME-004 expression. Preferably, the treated cancer indications in such patients are cancers known or determined to have a high prevalence for PRAME-004 expression. Preferably, said cancers are selected from cutaneous melanoma, uveal melanoma (more preferably, metastatic uveal melanoma), mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma. Advantageously, PRAME CD8 immune cells may be used at the lower end of the dose range of up to about 5 x 109immune cells for treating such cancers, if desired.

[0438] It is further envisaged that the PRAME CD8 immune cell treatment regimen described above and elsewhere herein are particularly applicable to patients that have been previously tested for PRAME-004 expression. Due to their potency, PRAME CD8 immune cells are particularly suitable for treating cancers known or determined to have a medium prevalence for PRAME-004 expression. Advantageously, PRAME CD8 immune cells may be used at the full dose range of up to about 10 x 109immune cells for treating such cancers.

[0439] In the context of the present disclosure, cancer treatment with PRAME or PRAME CD8 immune cells is preferably achieved by T cell receptor T cell therapy (TCR-T), which is a type of adoptive T-cell therapy. Typically, the adoptive T cell therapy is autologous T cell therapy. In some aspects of the disclosure, the TCR-T can be in vivo therapy. In such aspects, one or several suitable vectors encoding the TCR or TCR and CD8 is / are administered to the patient.

[0440] PRAME or PRAME CD8 immune cell(s) of the first and / or second aspect of the present disclosure are preferably provided in the form of a pharmaceutical composition. The pharmaceutical composition may be provided in soluble or cryopreserved form and may be thawed before use. The pharmaceutical composition may comprise suitable pharmaceutically acceptable carrier(s), excipient(s) and / or stabilizer(s). Exemplary suitable pharmaceutically acceptable carriers include any isotonic carrier such as, for example, saline (about 0.90% w / v of NaCI in water, about 300 mOsm / L NaCI in water, or about 9.0 g NaCI per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. The pharmaceutical composition may further include excipients such as dimethyl sulfoxide (DMSO) and dextran 40. In some instances, the pharmaceutically acceptable composition may be supplemented with human serum albumen.

[0441] In a further aspect, the present disclosure relates to a kit comprising the immune cell or population of immune cell(s) described herein, and optionally a) at least one lymphodepletion agent, preferably fludarabine and cyclophosphamide; b) interleukin- 2; and / or c) a checkpoint inhibitor, preferably a PD-1 or PD-1 L inhibitor, more preferably nivolumab.

[0442] SEQUENCES

[0443] Table 2: Amino acid and Nucleic acid sequences

[0444]

[0445]

[0446]

[0447] EXAMPLES

[0448] EXAMPLE 1: PRAME-004 TCR-T DOSE ESCALATION AND EXPANSION

[0449] Target identification and expression in solid tumors

[0450] The PRAME-derived HLA-A*02: 01 -presented target peptide PRAME-004 with the peptide sequence SLLQHLIGL was identified using a mass spectrometry (MS)- based antigen discovery platform. Abundant PRAME-004 presentation could be demonstrated on patient-derived native (i.e., unmodified, shock-frozen) primary tumor tissue and metastases while not quantifiable on 41 different normal tissue types (data not shown). The mRNA threshold equals to PRAME target peptide copy numbers of about 40 to 50 copies per cell according to quantitative peptide presentation analysis. Applying this cutoff to The Cancer Genome Atlas (TCGA) RNA sequencing (RNAseq) and own quantitative real-time (RT-q) PCR expression data, a high prevalence of PRAME expression across various tumor types could be confirmed. In indications such as cutaneous melanoma, synovial sarcoma, uterine carcinoma and ovarian carcinoma, expression of the target antigen was particularly strong and could be demonstrated in the majority of patients (Fig. 1a). PRAME expression was found to be homogenous applying mRNA in situ hybridization (Fig. 1 b).

[0451] Derivation and optimization of the PRAME-004 T-cell receptor

[0452] The PRAME-004 TCR was derived from a natural TCR targeting PRAME-004, identified by screening of more than 20 healthy donors using in vitro priming of T-cells followed by single cell sorting and rapid amplification of complementarity DNA (cDNA) ends (RACE) for individual TCR a- and [3-chains. 52 TCRs were selected for characterization after re-expression in healthy donors. Among those, the parental TCR for PRAME-004 recognized PRAME-004 peptide-loaded T2 cells with relatively high avidity (EC50 of 0.8 nM, data not shown). The engineered TCR used for PRAME-004 is a pairing-optimized and affinity-enhanced variant of the parental TCR, with ~4-fold enhanced avidity towards peptide-loaded T2 cells (EC50 = 0.2 nM, data not shown). The engineering comprised only two charge-complementarity single amino acid substitutions at the a- (W44K) and [3-chain (Q44E) interface for promoting pairing of both chains through electrostatic interactions. Rational design was used to identify these interface substitutions and, despite their locations being distant from the TCRs complementarity determining regions (CDRs), they enhanced the binding properties of the TCR, potentially via improving overall TCR stability. As a result, the engineered PRAME-004 TCR exhibited an improved binding affinity for PRAME-004 (5 pM by biolayer interferometry analysis compared to 18 pM for the parental TCR, data not shown).

[0453] The PRAME-004 TCR has a broad binding motif, recognizing six out of the nine target peptide residues, as determined by positional scanning. The TCR did not react against a set of ten naturally expressed, MS-detected normal tissue peptides with high sequence similarity to the target peptide. The engineered PRAME-004 TCR showed improved recognition of tumor cell lines with low endogenous PRAME-004 target expression (data not shown) but no reactivity towards nonmalignant human primary cells (data not shown), except for a very low signal on human cortical renal epithelial cells with one of two donors. Noteworthy, no specific renal toxicity was observed in patients up to date.

[0454] Patient characteristics and PRAME expression

[0455] Patient tumors had to express PRAME, as assessed by an assay based on a reverse transcriptase qPCR analysis of a fresh tumor biopsy specimen stored in RNAIater™ stabilization solution (ThermoFisher Scientific). For PRAME, a correlation between mRNA and immunopeptidome levels were established as demonstrated before (Fritsche, J., et al. Translating Immunopeptidomics to Immunotherapy- Decision-Making for Patient and Personalized Target Selection. Proteomics 18, e1700284 (2018)). From this correlation, a reads per kilobase per million mapped reads threshold was generated and translated into a RT-qPCR assay threshold in which PRAME was considered positive if expression levels were above a targetspecific DCt threshold. A threshold (DCt of 4.75 for PRAME) was chosen to maximize the sensitivity and specificity of prediction of peptide presentation as described previously (Fritsche et al., 2018).

[0456] A total of 85 HLA-A*02:01 + patients with advanced PRAME+ solid tumors underwent leukapheresis. Thereof, 41 patients started treatment, i.e. lymphodepletion and 40 patients received PRAME-004 TCR-T. A total of 27 patients were treated dose escalation across four dose levels (DLs) ranging from 0.04 to 1.2 x109 / m2PRAME- 004 TCR T-cells. Since observed manageable toxicity was observed until DL4 after clearance of this level, a protocol amendment was introduced to expand to higher doses. An additional 13 patients were treated in dose extension aimed to further evaluate DL4 and a higher DL5 (doses of 1.2 x109to 4.7 x109 / m2PRAME-004 TCR T-cells).

[0457] Patients with melanoma (n=16, including cutaneous melanoma, n=11 ; uveal melanoma, n=3; unknown primary, n=1 ; and mucosal melanoma, n=1 ) were most frequent, followed by synovial sarcoma (n=8), head and neck squamous cell carcinoma (HNSCC, n=5) and ovarian carcinoma (n=4). The median age was 53.5 years (range 18 to 79 years). Patients were heavily pretreated with a median of four prior lines of systemic therapies. Median tumor burden (sum of diameters of target lesions) was assessed at 108.9 mm and 60% of patients had serum lactate dehydrogenase (LDH) levels above the upper limit of normal at baseline. Of note, 42.5% of patients had liver lesions and 20% had preexisting brain lesions, which were required to be asymptomatic and stable prior to PRAME-004 TCR-T treatment.

[0458] Patients treated in dose escalation received a median total dose of 0.409 x109PRAME-004 TCR T cells (range 0.078 x109- 2.090 x109TCR T-cells) across DL1 to DL4, whereas the median dose applied in dose extension was considerably higher (median: 4.16 x109PRAME-004 TCR T-cells, range 1 .3 x109- 8.84 x109PRAME-004 TCR T-cells).

[0459] Manufacturing and Characterization of PRAME-004 TCR T cell drug product

[0460] Patients underwent leukapheresis and PRAME-004 TCR-T drug product was manufactured under current Good Manufacturing Practice-compliant conditions. Briefly, Overall, manufacturing starts with cryopreservation of the starting material i.e. , PBMC or leukapheresis. Briefly, whole or monocyte-depleted PBMC or T-cells selected from thawed leukapheresis were activated with anti-CD3 and anti-CD28 antibodies. Activated T-cells were genetically modified by transducing with a lentiviral vector encoding the PRAME-004 specific TCR (SEQ ID NO: 18) as described in WO 2018 / 172533 A1. Transduced cells were expanded until day 7 before being harvested, washed, concentrated, and formulated into the drug product. All products underwent in-process and product release testing prior to infusion and are stable for >12 months. Prior to infusion, release testing was performed to ensure sterility, safety and quality of the final product.

[0461] Phenotype analysis

[0462] For flow cytometry-based ex vivo immunomonitoring and phenotype analysis, isolated and cryopreserved cells collected at different time points before and after infusion were subjected to pHLA multimer and cell surface staining. Memory T-cell subsets were classified using the markers CD197 (CCR7) and CD45RA, with naive being CCR7+CD45RA+, central memory being CCR7+CD45RA-, effector memory being CCR7-CD45RA- and terminally differentiated effector memory cells being CCR7-CD45RA+. PRAME-004 T cells post-infusion were detected as CD3+CD8+ tetramer-positive cells or CD3+CD8+ tetramer-positive V[38-positive cells (data not shown).

[0463] The final product was highly enriched in T-cells (median 97.4% viable CD3+ cells) and nearly depleted of B cells, NK cells, NK T-cells and y5 T-cells as well as monocytes (grouped median 2.2%). Final drug products had median 47.2% dextramer-positive out of CD3+ CD8+ T-cells, median 5 vector copies per transduced cell and median 89.9% post-thaw viability (data not shown). The drug product was enriched in memory phenotype with a favorable costimulatory / activation profile (high CD62L, CD28 and less CD57) while resulting in less terminally differentiated T cells.

[0464] Cytotoxicity

[0465] Manufactured patient drug products were thawed, washed, and resuspended in culture medium and then treated with benzonase nuclease for 15 minutes. After a wash, cells were rested overnight in culture medium at 37°C, 5% CO2 and added to red fluorescent protein (RFP)-labeled tumor cell lines, UACC257, hs695T, and A375 at specific effector-to-target ratios; 2:1 for UACC257, 4:1 for hs695T, and 8:1 for A375. The co-culture plates were then placed at 37°C, 5% C02 and imaged every 4 h for the duration of the assay. Drug products were functionally active and demonstrated in vitro cytotoxicity against high, medium or low PRAME-expressing tumor cell lines (data not shown). Overall, the PRAME-004 TCR T cell manufacturing process delivered functionally active T cells.

[0466] Treatment

[0467] A total of five DLs was tested starting with 40 x106to 60 x106transduced T- cells (CD3+CD8+ dextramer-positive T-cells) per m2BSA (DL1 ) and then escalating to 120 x106to 180 x106cells / m2BSA (DL2), 200 x106to 480 x106cells / m2BSA (DL3), 200 x106to 1200 x106cells / m2BSA (DL4), and 1201 x106to 4700 x106cells / m2BSA (DL5). Additionally, patients were allowed to be enrolled at DLs already cleared for safety or at any intermediate DLs to better understand the safety and tolerability of PRAME-004 TCR T cells and to provide a T-cell product to patients in need.

[0468] A standard 3+3 trial design with a maximum of six investigated DLs (including DL-1 ) and with at least 36 subjects was utilized.

[0469] Lymphodepletion with fludarabine and cyclophosphamide was applied from day -6 to day -3 before PRAME-004 TCR T cell infusion at day 0. The IL-2 dosing regimen of 1 x106IU (approximately 5.5 x105IU / m2) IL-2 being administered subcutaneously (s.c.) approximately 6 h after PRAME-004 TCR T cell infusion followed by s.c. injection approximately every 12 h for 14 days was adapted in the course of the trial to s.c. administration starting approximately 24 h after PRAME-004 TCR T cell infusion, followed by s.c. injection every 24 h for 5 days and then approximately every 12 h for another 5 days. The planned number of IL-2 applications was modified from originally 28 doses to 15 doses. IL-2 administration was interrupted at the discretion of the investigator in case of toxicities. IL-2 treatment could be resumed until day 10 at the longest, if IL-2 administration had to be paused. Injection of IL-2 was paused if >Grade 2 CRS was suspected or it was decided to apply tocilizumab. Before continuing IL-2 administration, patient had to recover to at least Grade 1 CRS. When resumed, IL-2 dose may have been adapted to any lower dose for safety reasons.

[0470] Prophylaxis for infections started on day -6 continuing as clinically indicated and the prophylaxis for allergic reactions (acetaminophen [paracetamol] 500 to 650 mg and diphenhydramine hydrochloride 25 to 50 mg orally or intravenously; according to institutional guidelines) started on day 0 continuing as clinically indicated.

[0471] After PRAME-004 TCR T cell infusion, patients were closely observed during the treatment and observation phase until month 12, progressive disease, death or early discontinuation. Thereafter, the follow up started which ranged up to 25.6 months (median 1.9 months). No patients were lost to follow up, but three patients withdrew consent after progression. During follow up, patients were evaluated for changes in health status, vital signs, and physical examination, tumor assessment and OS. In addition, blood samples were collected to be tested for replication competent lentivirus and monitoring of T-cell persistence was continued.

[0472] Safety was assessed by physical examinations (e.g., assessment of cardiovascular, respiratory, gastrointestinal, and neurological systems), measurement of vital signs (i.e., temperature, systolic and diastolic blood pressure, pulse rate, respiratory rate, and oxygen saturation), electrocardiograms and transthoracic echocardiogram, pulmonary function tests, ECOG-PS and clinical safety laboratory assessments (among others, hematology, clinical chemistry, urinalysis, thyroid function, and coagulation). Early assessment, grading and treatment of CRS followed the recommendations by Neelapu, S.S., et al. (Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol 15, 47-62 (2018)) and Lee, D.W., et al. (Current concepts in the diagnosis and management of cytokine release syndrome. Blood 124, 188-195 (2014)). Early assessment and management of neurotoxicity followed published guidance from Neelapu et al. with more aggressive treatment being possible for patients with high fever (temperature >39.5°C). Interruption of IL-2 application was recommended in case of CRS >Grade 2 or tocilizumab was applied.

[0473] Tumor response was assessed according to RECIST 1.1 (Eisenhauer, E.A., et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1 ). Eur J Cancer 45, 228-247 (2009)).

[0474] ORR (for patients with at least one scan post-infusion or clinical progression) and cORR (patients with at least two tumor assessments post-infusion or with clinical / radiological progressions at any timepoint) were determined according to RECIST 1.1 and evaluated using a Clopper-Pearson 95% confidence interval. DOR was defined as the time from first documented partial response (PR) until first documented date of disease progression or death due to any cause, whichever occurred first in patients with confirmed response. PFS was defined as the time between T-cell infusion and event (progression or death, whatever occurs first). A patient who experiences any form of progression (radiological and clinical) as determined by the local investigator was evaluated as a patient with an event. Patients who did not record disease progression or death as of the cut-off date for the analysis were censored at the cut-off date. OS was defined as the time between T-cell infusion and death of the patients. Patients who did not record death as of the cut-off date for the analysis were censored at the last date they were known to be alive. Median followup of confirmed responses was defined as the time from first documented PR that patients with confirmed responses were followed up on and was calculated using the reverse Kaplan-Meier method. Patients with disease progression were censored at time-point of progressive disease and patients without documented progressive disease at the cut-off date for the analysis were counted as events at data cut-off. DOR, PFS, and OS were summarized using the Kaplan-Meier method to estimate the median survival time including minimum and maximum values and 95% confidence intervals based on Brookmeyer and Crowley methodology. Formal statistical testing was not prespecified. In the post-hoc analysis, difference between two independent proportions were analyzed using Fisher’s exact test, medians of two groups were compared using Mann-Whitney U test, multiple comparisons were performed using Kruskal-Wallis-test, correlations were calculated using the Spearman’s rank correlation. The p-values were considered statistically significant for p<0.05. 95% exact confidence intervals were provided where appropriate.

[0475] Statistical analyses were conducted using GraphPad Prism v9 and R v4.3.0. No data were excluded from the analyses, but datapoints may be missing in case patient samples were not available. Sample numbers are mentioned in the respective Figure legends.

[0476] An independent Data and Safety Monitoring Board closely monitored the safety of this trial.

[0477] Primary end point: Safety

[0478] Safety analysis comprised 41 + patients who received at least one dose of lymphodepletion, including one patient not receiving PRAME-004 due to an intercurrent infection. Treatment-Emergent Adverse Event (TEAE) were predominantly of mild to moderate intensity (not shown). A formal MTD was not reached for PRAME-004 and the highest dose levels (DL4 / DL5), and an equivalent range of 1 x109to 10 x109total transduced TCR T-cells was selected for further clinical development.

[0479] Secondary endpoint: Clinical efficacy

[0480] Until the data cut-off, all 40+ patients treated with PRAME-004 TCR T-cells had at least one tumor response assessment post-T-cell infusion. Target lesions were evaluable for 39 patients (FIG. 2a). For patients treated during the dose escalation part (n=27), the overall response rate (ORR) consisting of patients with unconfirmed and confirmed response (u / cORR) according to RECIST 1.1 was 48.1 % (13 / 27; 95% Cl: 28.7%-68.1 %) and the confirmed ORR (cORR) equalled 18.5% (5 / 27; 95% Cl: 6.3%-38.1 %). The median duration of response (mDOR) in this cohort was 4.4 months (range: 2.4 - 23.0; 95% Cl:: 2.4 - not reached), median progression-free survival (PFS) 2.8 months (range: 1.3 - 24.4; 95% Cl: 2.4 - 3.1 ) and median overall survival (OS) 7.5 months (range: 1 .9 - 34.2+; 95% Cl: 4.0 - 10.9). For patients treated in dose extension (n=13), the u / cORR increased to 61.5% (8 / 13; 95% Cl: 31.6% - 86.1 %) with a cORR of 54.5% (6 / 11 ; 95% Cl: 23.4% - 83.3%). At the time of data cut-off, four of eight responses remained ongoing with two patients being in partial response (PR) more than a year after T-cell infusion (FIG. 2b, c). With a median follow-up of 11 .5 months, the mDOR was 8.1 months (range 2.6 - 11.8+; 95% Cl: 2.6 - not reached) and the median PFS was 5.7 months (range 1.2 - 13.1 +; 95% Cl: 2.4 - not reached). The median OS was not reached at data cut-off for FIG. 2.

[0481] A post-hoc analysis of melanoma patients (n=10) treated at DL4 / DL5 across dose escalation and dose expansion cohorts revealed a u / cORR of 70% (7 / 10; 95% Cl: 34.8% - 93.3%) with a cORR of 50% (5 / 10; 95% Cl 18.7-81.3). The mDOR for this cohort was not reached at data cut-off for FIG. 2 with three of seven responses ongoing at 9+ months (one patient) and 12+ months (two patients) post-T-cell infusion as of data cut-off.

[0482] The mOS and mDOR for the dose expansion cohort are discussed in the context of FIG. 3.

[0483] Taken together, these data suggest that PRAME-004 administered at DL4 / DL5 has significant clinical activity in heavily pretreated PRAME-004 positive solid tumors including checkpoint-inhibitor relapsed / refractory melanoma.

[0484] Clinical predictors of response to PRAME-004 TCR-T

[0485] Response to PRAME-004 TCR-T was not limited to certain body organs suggesting successful trafficking of TCR T-cells to lung, liver, pleura, peritoneum, skin, lymph node, adrenal gland, bladder, kidney, spleen, and muscle (FIG. 6).

[0486] No significant association between number of prior lines of treatment or baseline LDH with response was observed (data not shown). However, confirmed responses were significantly more frequent in patients with a higher TCR T dose to tumor burden ratio (data not shown).

[0487] Secondary endpoint: PRAME-004 TCR-T cell pharmacokinetics

[0488] Pharmacokinetics (PK) characterization of PRAME-004 TCR T cells revealed rapid PRAME-004 T-cell engraftment in all patients (median peak day 3.5; range day 1 to day 15) irrespective of tumor indication (Fig. 8). Higher T-cell dose led to higher maximal PRAME-004 TCR transgene levels in blood (Cmax) and higher Cmax was significantly associated with a higher percentage of TCR-positive T-cells in the drug product (data not shown). PRAME-004 TCR T cells in peripheral blood declined over time, but no patient showed complete loss during the period of assessment. The persistence of PRAME-004 T-cells was observed for more than 2 years in two patients who underwent long-term follow up. There was no correlation observed of response with Cmax and the integrated transgene levels over the first 4 weeks (AUC0-28d) at data cut-off. However, Cmax and AUCO-28 normalized to tumor burden were higher in responders compared to non-responders (data not shown).

[0489] Dynamics of PRAME-004 immunophenotype In the PRAME-004 T-cell product, with the exception of TEMRA, the relative abundance of TN, CM and TEM within the transduced CD8+ compartment was not associated with response, neither was the expression of activation / differentiation (CD62L, CD27, CD28, CD45RO, CD57) or activation / exhaustion markers (PD-1 , TIM- 3, LAG-3, TIGIT; data not shown).

[0490] In patients’ blood, a gradual increase in TEMRA associated with a decrease in TN, CM, and TEM subsets after infusion was observed, suggesting in vivo activation and differentiation of PRAME-004 T cells (data not shown). Two weeks post-infusion, responders trended to retain higher degree of CM phenotype as compared to nonresponders (data not shown). After infusion, in vivo activation of transduced CD8+ T- cells could be followed by loss of CD62L, CD27, and CD28 expression over time while temporal upregulation of CD45RO and gradual increase of CD57 was seen (Supplementary Fig. 16). Two weeks post-infusion, responders trended to have more CD27 and CD45RO expressing transduced TCR T-cells and depth of response trended to correlate with higher CD27 and CD45RO expression at week 2 (data not shown).

[0491] A transient upregulation of PD-1 and TIGIT and gradual downregulation of TIM- 3 and LAG-3 expression in peripheral blood T-cells after PRAME-004 infusion was observed (data not shown). Two weeks post-infusion, upregulation of PD-1 was more prominent in responders together with upregulation of TIGIT by trend compared to non-responders, and depth of response correlated with ratio of upregulation of PD-1 and TIGIT expression at week 2 compared to final product (data not shown). None of these activation / exhaustion related markers (PD-1 , TIM-3, LAG-3, TIGIT) were upregulated 8 weeks after infusion and thus, no evidence of PRAME-004 exhaustion was observed in the periphery.

[0492] PRAME-004 in tumor microenvironment

[0493] T-cell infiltration into tumor tissues was tested through preplanned post-infusion biopsies available from 21 treated patients. TCR-transgenic T-cells could be detected in all evaluable post-infusion biopsies except one independent of the cancer type. Responders were shown to have a significantly higher degree of PRAME-004 TCR T cell infiltration than non-responders, and a higher degree of tumor infiltration correlated with deeper response and longer PFS (Fig. 5).

[0494] Taken together, the PRAME-004 TCR was found to be safe, well tolerable and no treatment-related fatalities occurred in 40 patients receiving up to 8.8 x109TCR T- cells. The mDOR was 4.4 months during dose escalation and 8.1 months in the extension cohort with responses exceeding 12 months suggesting a clear doseresponse relationship. Clinical responses were observed in heavily pre-treated cutaneous, mucosal and uveal melanoma, sarcoma, HNSCC, and ovarian cancer suggesting that PRAME-004 has therapeutic potential across immunologically hot (melanoma, head and neck) and cold tumors (ovarian, synovial sarcoma). There was no obvious organ restriction to PRAME-004 and infiltration of transduced cells into tumor tissue was observed in all post-treatment biopsies assessed. This suggests that PRAME-004 cells were activated and expressed the chemokine receptor signature required to overcome stromal or vasculature barriers and effectively engage with PRAME target. A significant correlation between tumor T- cell infiltration and response as well as depth of response was observed. Deeper responses to PRAME-004 predicted for longer duration of response.

[0495] In contrast to other trials using TCR therapies in solid tumors, there was no correlation between the number of prior treatments received and response to PRAME- 004. So far, no correlation was observed between elevated baseline LDH leading to poor response as has been reported in other adoptive T-cell and checkpoint inhibitor trials. PRAME-004 responders were more likely to have a lower tumor burden which fits well with other data suggesting similar associations in lymphoma25and sarcoma42treated with CAR and TCR T-cells, respectively. Of note, a higher dose of PRAME- 004 was clearly correlated with higher likelihood of durable response and patients with a low tumor burden receiving a high TCR T-cell dose were the most likely to derive long-term benefit. A similar association has been described for CAR T-cell therapy in lymphoma, where a higher effector to tumor burden ratio was a key driver of durable response46. In contrast to the majority of CAR T-cell trials and a TCR T-trial targeting NY-ESO1 , no differences in Cmax, Tmax, AUC0.28, T1 / 2or other PK parameters between non-responders and responders were observed. The fact that a correlation between TCR T-cells in the tumor and depth of response was observed, but no correlation was found for classical predictive PK parameters measured in the peripheral blood that are well-described for CAR T-cells may be driven by the biological differences of solid versus liquid cancers and highlight the importance of monitoring T-cell infiltration as an additional key parameter in solid cancer trials.

[0496] Several trials have emphasized the role of CM and TEM T-cell phenotype content in the drug product as drivers of response; however, such differences were not observed in PRAME-004 drug products between responders and non-responders.

[0497] Although responses were seen across all PRAME expressions levels above the threshold, PRAME-004 responders had a higher median target antigen expression, paralleling observations with MAGE-A4 targeting TCR T-cells7and PRAME -directed T-cell engaging bispecifics48. We also observed a trend for deeper responses and longer PFS in patients with higher PRAME expression. To our knowledge, antigen density has so far not been described as one of the known factors predicting durable remissions for CAR T47. This may be because data generated so far are dominated by CD19 and BCMA target antigens which show very high and homogenous expression, a rare feature for tumor-associated antigens in solid tumors. Our data indicate that understanding antigen expression levels is key to designing drug products with defined activation thresholds tailored to the targeted disease and patient. This principle is potentially applicable for both liquid and solid cancers whenever targeted antigens do not show the properties of BCMA and CD19.

[0498] Given the limited availability of post-progression biopsies, it is difficult to speculate on potential mechanisms of resistance to PRAME-004. In contrast to observations in CD19-targeting49and BCMA-targeting CAR T-cells50, there were no obvious signs of loss or significant downregulation of the target antigen PRAME nor of HLA-A or [32M in PRAME-004-treated patients. In contrast to observations in solid tumors treated with checkpoint inhibitors51or TCR T-cells8’17, we did not detect deficiencies in antigen processing machinery or HLA expression in the post-treatment biopsies assessed. On the contrary, upregulation of IFN-y-inducible genes in responders indicate active IFN-y signaling in the tumors of these patients52.

[0499] Patients were subjected to lymphodepletion with 30 mg / m2BSA fludarabine and 500 mg / m2BSA prior to treatment. Subsequently, several cohorts were treated with autologous, genetically engineered T cells expressing the PRAME TCR or PRAME TCR+CD8.

[0500] Patient cohorts were treated with escalating dosages of host cells expressing the PRAME TCR of 1 -10x109total TCR-T cells with or without nivolumab. Further patient cohorts were treated with host cells expressing the PRAME TCR+CD8 at dosages of 0.2-0.48 x 109host cells / m2BSA, 0.481 -0.8 x 109host cells / m2BSA, 0.801 - 1 .2 x 109host cells / m2BSA with and without nivolumab.

[0501] Treatment was well or at least manageably tolerated. Confirmed objective response rates (ORRs) of > 50% were achieved. Durable responses and tumor shrinkage was observed in several patients beyond 12 months.

[0502] EXAMPLE 2: PRAME-004 CD8 TCR-T DOSE ESCALATION

[0503] Patients and tumors evaluated and PRAME-004 CD8 TCR T cells were manufactured and characterized as described in Example 1 above. Briefly, patients underwent leukapheresis and PRAME-004 TCR-T drug product was manufactured under current Good Manufacturing Practice-compliant conditions. Briefly, Overall, manufacturing starts with cryopreservation of the starting material i.e., PBMC or leukapheresis. Briefly, whole or monocyte-depleted PBMC or T-cells selected from thawed leukapheresis were activated with anti-CD3 and anti-CD28 antibodies. Activated T-cells were genetically modified by transducing with a lentiviral vector encoding the PRAME-004 specific TCR and CD8 (SEQ ID NO: 23), as described in WO 2020 / 243134 A1. Transduced cells were expanded until day 7 before being harvested, washed, concentrated, and formulated into the drug product. All products underwent in-process and product release testing prior to infusion and are stable for >12 months. Prior to infusion, release testing was performed to ensure sterility, safety and quality of the final product. At data cut-off, a total of 41 heavily pretreated PRAME-004 positive patients were infused with PRAME-004 TCR CD8 across DL3 (0.2-0.48x109TCR-T cells / m2BSA), DL4a (0.481 -0.8x109TCR-T cells / m2BSA) and DL4b (0.801 -1 .2x109TCR-T cells / m2). The median total infused dose was 1 .48 x 109cells / patient (0.443 to 2.05 x 109cells / patient).

[0504] Initial clinical activity was observed with a cORR of 41 % (14 / 34) and initial ORR of 41 % (17 / 41 ) (RECIST 1.1 ). The mDOR as analyzed by using the Kaplan-Meier method was 9.2 months (min 2.0+ months, max 23.5+ months). At data cut-off, 10 / 17 responses were ongoing including 3 confirmed responses at 1 + year. Reduction of tumor size was observed in 32 out of 38 patients (84%), with deep responses with >50% tumor size reduction in 11 / 17 responders including 2 patients with complete response of target lesions (FIG. 9).

[0505] Translational data showed enhanced pharmacology of PRAME-004 CD8 TCR and a trend towards responses at lower T cell dose and higher tumor burden compared to PRAME-004 TCR without CD8. PRAME-004 CD8 TCR offers similar responses at 1.5 x 109total infused dose as PRAME-004 TCR at 3x higher dose (FIG. 10). With higher doses currently being explored, PRAME-004 CD8 TCR may offer an enhanced opportunity to treat cancers with both high and medium-level PRAME expression.

[0506] ASPECTS OF THE DISCLOSURE

[0507] The disclosure may be defined by one or more of the following aspects:

[0508] 1 . A host cell for use in a method of treating cancer in a patient, said host cell comprising a TCR or a derivative or fragment thereof comprising six complementarity determining regions (CDRs) according to SEQ ID NO: 1 , 2, 3, 4, 5 and 6; and / or a nucleic acid encoding said TCR and optionally a CD8 molecule, wherein said method of treatment comprises administering said host cell to said patient, and one or more of the following: a) lymphodepletion treatment with cyclophosphamide and fludarabine; b) combination therapy with IL-2; and / or c) combination therapy with a checkpoint inhibitor, preferably a PD-1 or PD-1 L inhibitor, more preferably nivolumab.

[0509] 2. The host cell for the use of aspect 1 , wherein said IL-2 combination therapy includes administering a low IL-2 dose, preferably at 1 million IU or less per day.

[0510] 3. The host cell for the use of aspect 1 or 2, wherein IL-2 is administered daily or twice daily.

[0511] 4. The host cell for the use of any one of the preceding aspects, wherein IL-2 is administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0512] 5. The host cell for the use of any one of the preceding aspects, wherein IL-2 is administered daily for days 1 -5 after administering said host cell, and / or twice daily at days 6-10, preferably at a dose of 1 million IU per day.

[0513] 6. The host cell for the use of aspect 1 , wherein said treatment does not comprise administering IL-2 and / or a checkpoint inhibitor.

[0514] 7. The host cell for the use of any one of the preceding aspects, wherein said nivolumab combination therapy comprises administering a maximum of 480 mg nivolumab per month, or 6000 mg nivolumab per year, to said patient

[0515] 8. The host cell for the use of any one of the preceding aspects, wherein said nivolumab combination therapy comprises administering a 240 mg nivolumab every 2 weeks or 480 mg nivolumab every 4 weeks.

[0516] 9. The host cell for the use of any one of the preceding aspects, wherein said lymphodepletion treatment comprises administering 30 mg / m2fludarabine and 500 mg / m2cyclophosphamide.

[0517] 10. The host cell for the use of any one of the preceding aspects, wherein said lymphodepletion treatment is administered for at least 1 , 2, 3 or 4 consecutive days before administering said host cell. 11 . The host cell for the use of any one of the preceding aspects, wherein said host cell is comprised by a population of host cells administered to a total dose of 1- 10x109host cells, preferably of 1 -5x109host cells, such as up to 4.7 x 109host cells per patient.

[0518] 12. The host cell for the use of any one of the preceding aspects, wherein said host cell is comprised by a population of host cells administered at dose of 12-18 x 106host cells / m2BSA, 40-60 x 106host cells / m2BSA, 0.12-0.18 x 109host cells / m2BSA, 0.2- 0.48 x 109host cells / m2BSA, 0.481 -0.8 x 109host cells / m2BSA, 0.801 -1.2 x 109host cells / m2, or 1.2-4.7 x 109host cells / m2BSA, preferably 0.64-2.05x109host cells / m2or 1.07-5.12 x 109host cells / m2BSA.

[0519] 13. The host cell for the use of any one of aspects 1 to 10, wherein said host cell is comprised by a population of host cells administered at a flat dose.

[0520] 14. The host cell for the use of any one of the preceding aspects, wherein said host cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

[0521] 15. The host cell for the use of any one of the preceding aspects, wherein said TCR is capable of specifically binding to Preferentially Expressed Antigen of Melanoma (PRAME) antigenic peptide as shown in SEQ ID NO: 11 .

[0522] 16. The host cell for the use of any one of the preceding aspects, wherein said TCR comprises a TCR a or y chain; and a TCR [3 or 5 chain; wherein the TCR a or y chain and the TCR [3 or 5 chain comprise the sequences as defined in aspect 1 .

[0523] 17. The host cell for the use of any one of the preceding aspects, comprising at least one TCR alpha variable chain region having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 7, and a TCR beta variable chain having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 8.

[0524] 18. The host cell for the use of any one of the preceding aspects, wherein said nucleic acid encoding said TCR and said CD8 molecule comprises a nucleotide sequence S1 encoding a CD8 alpha polypeptide, a nucleotide sequence S2 encoding a CD8 beta polypeptide, a nucleotide sequence S3 encoding a TCR alpha polypeptide, and a nucleotide sequence S4 encoding a TCR beta polypeptide, wherein the nucleotide sequences are arranged in tandem in a 5’ to 3’ orientation selected from S1 -S2-S3-S4, S1 -S2-S4-S3, S2-S1 -S3-S4, S2-S1 -S4-S3, S3-S4-S1 -S2, S3-S4-S2- S1 , S4-S3-S1 -S2, and S4-S3-S2-S1 , preferably in S2-S1 -S4-S3 orientation.

[0525] 19. The host cell for the use of aspect 17, wherein said CD8 alpha polypeptides comprises an amino acid sequence having at least 80% sequence identity to an amino acid sequence comprised in the sequence according to SEQ ID NO: 12, and the CD8 beta polypeptide comprises an amino acid sequence having at least 80% sequence identity to an amino acid according to and SEQ ID NO: 13.

[0526] 20. The host cell for the use of aspect 17 or 18, wherein said nucleic acid encoding the TCR and CD8 molecule comprises the sequence PTE CD8 TCR WPRE according to SEQ ID NO: 14, or a sequence having at least 80% sequence identity to SEQ ID NO: 14.

[0527] 21. The host cell for the use of any one of the preceding aspects, wherein said nucleic acid encoding the TCR and optionally the CD8 molecule is a vector, preferably a viral vector, optionally selected from an adeno-associated virus or a lentivirus.

[0528] 22. The host cell for the use of any one of the preceding aspects, wherein said host cell is provided in the form of a pharmaceutical composition, optionally in cryopreserved from.

[0529] 23. The host cell for the use according to any one of the preceding aspects, wherein said cancer is selected from non-small cell lung cancer, specifically non-squamous non-small cell lung cancer (NSCLC); small cell lung cancer (SCLC); melanoma, specifically cutaneous, mucosal or uveal melanoma; liver cancer or hepatocellular carcinoma (HCC); breast cancer, specifically triple negative breast cancer; uterine cancer; Merkel cell carcinoma; pancreatic cancer; gallbladder cancer; bile duct cancer; colorectal cancer; urinary bladder cancer; kidney cancer; leukemia; ovarian cancer; esophageal cancer; brain cancer; gastric cancer; synovial sarcoma; head and neck squamous cell carcinoma; and prostate cancer, more preferably from cutaneous, mucosal or uveal melanoma; synovial sarcoma; NSCLC; SCLC; head and neck squamous cell carcinoma (HNCC); triple negative breast cancer; ovarian cancer; uterine cancer; or hepatocellular carcinoma (HCC).

[0530] The invention may further be characterized by the following aspects:

[0531] 1 . An immune cell or population of immune cells for use in a method of treatment of cancer in a patient, said immune cell a) expressing a TCR or a derivative or fragment thereof wherein said TCR or derivative or fragment is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24), and / or b) said immune cell comprising at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises administering to said patient i) about 1-10 x 109immune cells, and ii) at least one single dose of about 1 million IU interleukin (IL)-2.

[0532] 2. The immune cell or population of immune cells of aspect 1 , wherein said PRAME-004 peptide is presented on an MHC-I molecule. 3. The immune cell or population of immune cells of aspect 1 or 2, wherein said TCR or derivative or fragment thereof comprises: a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

[0533] 4. The immune cell or population of immune cells for the use of any one of the preceding aspects, wherein IL-2 is administered daily or twice daily.

[0534] 5. The immune cell or population of immune cells for the use of any one of the preceding aspects, wherein IL-2 is administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0535] 6. The immune cell or population of immune cells for the use of any one of the preceding aspects, wherein single dose of about 1 million IU IL-2 is administered daily on days 1-5 after administering said immune cell(s), and / or twice daily on days 6-10 after administering said immune cell(s).

[0536] 7. The immune cell or population of immune cells for the use of aspect 1 , wherein said TCR or derivative or fragment thereof, and / or said nucleic acid(s) encoding said TCR or derivative or fra...

Claims

1. CLAIMS1 . An immune cell or population of immune cells for use in a method of treatment of cancer in a patient, said immune cell a) expressing a TCR or a derivative or fragment thereof wherein said TCR or derivative or fragment is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24), and b) expressing at least one heterologous CD8 chain, and / or c) comprising at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above, and at least one heterologous CD8 chain; wherein said treatment comprises administering at least a single dose of about 1 -10 x 109cells to said patient.

2. The immune cell or population of immune cells of claim 1 , wherein the cancer is characterized by a medium prevalence of PRAME-004 expression of less than about 80%, 70%, 60%, 50%, 30%, or 20%.

3. The immune cell or population of immune cells of claim 1 , wherein the cancer is characterized by a high prevalence of PRAME-004 expression of at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least 98%, at least about 99%, and wherein treatment optionally comprises administering at least a single dose of about 1 -10 x 109 cells, preferably of about 1 , 2,3. 4, or 5 x 109 cells.

4. The immune cell or population of immune cells of any one of the preceding claims, wherein said cancer is selected from ovarian cancer, including ovarian carcinoma, ovarian serous cystadenocarcinoma, ovarian tube cancer (fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC), and primary peritoneal cancer (PPC); uterine cancer, including uterine carcinosarcoma (UCS), uterine endometrial carcinoma, uterine corpus cancer (UCC), uterine sarcoma, endometrial carcinoma, endometrial clear cell carcinoma, and endometrial serous carcinoma; triple-negative breast cancer; lung cancer, including squamous and non-squamous non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC); melanomas, including cutaneous melanoma, uveal melanoma, and mucosal melanoma; and synovial sarcoma.

5. The immune cell or population of immune cells of any one of the preceding claims, wherein the patient has not been tested for PRAME-004 expression prior to treatment, and optionally wherein the cancer is selected from cutaneous melanoma, uveal melanoma, mucosal melanoma, endometrial carcinoma, uterinecarcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

6. The immune cell or population of immune cells of any one of the preceding claims, wherein the patient has been tested for HLA-A*02:01 expression prior to treatment.

7. The immune cell or population of immune cells of any one of claims 1 to 6, wherein treatment further comprises administering at least one single dose of about 1 million III interleukin (IL)-2.

8. The immune cell or population of immune cells of claim 7, wherein said IL-2 is administered after administering the at least one single dose of immune cells.

9. The immune cell or population of immune cells of claim 7 or 8, wherein IL-2 is administered once or twice daily.

10. The immune cell or population of immune cells of any one of claims 7 to 9, wherein IL-2 is administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

11. The immune cell or population of immune cells of any one of claims 7 to 10, wherein a single dose of about 1 million IU IL-2 is administered once daily on days 1 to 5 after administering said immune cell(s), and / or twice daily on days 6 to 10 after administering said immune cell(s).

12. The immune cell or population of immune cells of any one of the preceding claims, wherein the patient does not receive IL-2 during or after treatment.

13. The immune cell or population of immune cells of any one of the preceding claims, wherein said PRAME-004 peptide is presented on an MHC-I molecule.

14. The immune cell or population of immune cells for the use of any one of the preceding claims, wherein said immune cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

15. The immune cell or population of immune cells of any one of the preceding claims, wherein said TCR or derivative or fragment thereof, and / or said nucleic acid(s) encoding said TCR or derivative or fragment thereof are heterologous to said immune cell(s).

16. The immune cell or population of immune cells of any one of the preceding claims, wherein said immune cell(s) stably express said TCR or derivative or fragment thereof and / or said at least one heterologous CD8 chain.

17. The immune cell or population of immune cells of any one of the preceding claims, wherein said TCR or derivative or fragment thereof comprises: acomplementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

18. The immune cell or population of immune cells of claim 17, wherein said TCR or derivative or fragment thereof comprises a TCR alpha variable domain comprising or consisting of an amino acid sequence having at least about about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and / or a TCR beta variable domain comprising or consisting of an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 .

19. The immune cell or population of immune cells of claim 18, wherein said TCR or derivative or fragment thereof comprises a TCR alpha chain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, and / or a TCR beta chain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

20. The immune cell or population of immune cells of any one of the preceding claims, wherein said at least one heterologous CD8 chain comprises or consists of a heterologous CD8 alpha chain or a heterologous CD8 beta chain, wherein said immune cell(s) preferably express a heterologous CD8 alpha chain and a heterologous CD8 beta chain.21 . The immune cell or population of immune cells of claim 20, wherein said CD8 alpha chain comprises an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to an amino acid sequence comprised in the sequence according to SEQ ID NO: 19, and the CD8 beta chain comprises an amino acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to an amino acid according to and SEQ ID NO: 20.

22. The immune cell or population of immune cells of any one of claims 18 to 21 , wherein a) said TCR alpha variable domain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and / or said TCR alpha chain is encoded by a nucleic acid comprising or consisting of a nucleicacid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15; b) said TCR beta variable domain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 16; and / or said TCR beta chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 17; c) said CD8 alpha chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 21 ; and / or said CD8 beta chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 22.

23. The immune cell or population of immune cells of any one of claims 18 to 22, wherein said nucleic acid(s) encoding said TCR comprise(s) or consist(s) of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

24. The immune cell or population of immune cells of any one of claims 18 to 23, wherein said nucleic acid(s) encoding said TCR and said CD8 comprise(s) or consist(s) of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 23.

25. The immune cell or population of immune cells for the use any one of the preceding claims, wherein said immune cell(s) is / are administered once as a single dose to said patient.

26. The immune cell or population of immune cells for the use any one of the preceding claims, wherein said immune cell(s) is / are administered several times as a single dose to said patient.

27. The immune cell or population of immune cells for the use any one of the preceding claims, wherein said immune cell(s) is / are administered parenterally, preferably intravenously, to said patient.

28. The immune cell or population of immune cells for the use of any one of the preceding claims, wherein IL-2 is administered parenterally, preferably subcutaneously or intravenously, to said patient.

29. The immune cell or population of immune cells for the use of any one of the preceding claims, wherein said treatment further comprises administering a checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient.

30. The immune cell or population of immune cells for the use any one of the preceding claims, said nucleic acid(s) encoding said TCR or derivative or fragment thereof and / or said heterologous CD8 chain(s) is / are introduced into said immune cell(s) the form of a vector, preferably a viral vector, more preferably a lentivi ral vector.

31. The immune cell or population of immune cells of any one of the preceding claims, wherein said patient has previously received checkpoint inhibitor treatment, and have optionally relapsed under checkpoint inhibitor treatment. .

32. The immune cell or population of immune cells of any one of the preceding claims, wherein said immune cell(s) is / are provided in the form of a pharmaceutical composition, optionally in soluble or cryopreserved form.

33. An immune cell or population of immune cells for use in a method of treating cancer, wherein said immune cell(s) express at least one heterologous CD8 chain and a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or comprise at least one nucleic acid encoding at least one heterologous CD8 chain and a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises1 ) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and2) immune cell treatment at a single dose of about 1 -10 x 109cells / patient;3) optional IL-2 treatment at one or several doses of 1 million III per day, such as preferably once per day on day 1 to day 5 after immune cell treatment, and twice per day on day 6 to day 10 after immune cell treatment.

34. The immune cell or population of immune cells of claim 33, wherein said treatment further comprises administration of a checkpoint inhibitor.

35. An immune cell or population of immune cells for use in a method of treatment of cancer in a patient who has not been previously tested for PRAME-004 expression, wherein said immune cellexpresses a TCR or a derivative or fragment thereof wherein said TCR or derivative or fragment is capable of specifically binding to a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 24), and / or comprises at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above, wherein said treatment comprises administering at least a single dose of about 1 -10 x 109cells to said patient.

36. The immune cell or population of immune cells of claim 35, wherein the cancer is characterized by a high prevalence PRAME-004 expression of at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least 98%, at least about 99%.

37. The immune cell(s) of any one of claims 35 or 36, wherein said cancer is selected from cutaneous melanoma, uveal melanoma, mucosal melanoma, endometrial carcinoma, uterine carcinosarcoma, endometrial clear cell carcinoma, endometrial serous carcinoma, ovarian cancer, and synovial sarcoma.

38. The immune cell or population of immune cells of any one of claims 35 to 37, wherein the patient has been tested for HLA-A*02:01 expression prior to treatment.

39. The immune cell or population of immune cells of any one of claims 35 to 38, wherein the patient has previously received at least one line of treatment.

40. The immune cell or population of immune cells of any one of claims 35 to 39, wherein treatment further comprises administering at least one single dose of about 1 million III interleukin (IL)-2.41 . The immune cell or population of immune cells of claim 40, wherein said IL-2 is administered after administering the at least one single dose of immune cells.

42. The immune cell or population of immune cells of claim 40 or 41 , wherein IL-2 is administered once or twice daily.

43. The immune cell or population of immune cells of any one of claims 40 to 42, wherein IL-2 is administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

44. The immune cell or population of immune cells of any one of claims 40 to 43, wherein single dose of about 1 million IU IL-2 is administered once daily on days 1 to 5 after administering said immune cell(s), and / or twice daily on days 6 to 10 after administering said immune cell(s).

45. The immune cell or population of immune cells of any one of claims 35 to 44, wherein the patient does not receive IL-2 during or after treatment.

46. The immune cell or population of immune cells of any one of the preceding claims, wherein said PRAME-004 peptide is presented on an MHC-I molecule.

47. The immune cell or population of immune cells for the use of any one of claims 35 to 46, wherein said immune cell is a lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor, more preferably a CD4 or CD8 positive T-cell.

48. The immune cell or population of immune cells of any one of claims 35 to 47, wherein said TCR or derivative or fragment thereof, and / or said nucleic acid(s) encoding said TCR or derivative or fragment thereof are heterologous to said immune cell(s).

49. The immune cell or population of immune cells of any one of claims 35 to 48, wherein said immune cell(s) stably express said TCR or derivative or fragment thereof.

50. The immune cell or population of immune cells of any one of claims 35 to 49, wherein said TCR or derivative or fragment thereof comprises: a complementarity determining region (CDR) 1 a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6).

51. The immune cell or population of immune cells of claim 50, wherein said TCR or derivative or fragment thereof comprises a TCR alpha variable domain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and / or a TCR beta variable domain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 .

52. The immune cell or population of immune cells of claim 51 , wherein said TCR or derivative or fragment thereof comprises a TCR alpha chain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, and / or a TCR beta chain comprising or consisting of an amino acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13.

53. The immune cell or population of immune cells of any one of claims 35 to 52, wherein a) said TCR alpha variable domain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, and / or said TCR alpha chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 15; b) said TCR beta variable domain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 16; and / or said TCR beta chain is encoded by a nucleic acid comprising or consisting of a nucleic acid sequence having at least about 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

54. The immune cell or population of immune cells of any one of claims 35 to 53, wherein said nucleic acid(s) encoding said TCR comprises or consists of a nucleic acid sequence having at least 80%, 90%, 95%, 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

55. The immune cell or population of immune cells for the use any one of claims 35 to 54, wherein said immune cell(s) is / are administered once as a single dose to said patient.

56. The immune cell or population of immune cells for the use any one claims 35 to 55, wherein said immune cell(s) is / are administered several times as a single dose to said patient.

57. The immune cell or population of immune cells for the use any one of claims 35 to 56, wherein said immune cell(s) is / are administered parenterally, preferably intravenously, to said patient.

58. The immune cell or population of immune cells for the use of any one of claims 35 to 57, wherein IL-2 is administered parenterally, preferably subcutaneously or intravenously, to said patient.

59. The immune cell or population of immune cells for the use of any one of claims 35 to 58, wherein said treatment further comprises administering a checkpoint inhibitor, preferably a PD-1 or PD-L1 inhibitor, to said patient.

60. The immune cell or population of immune cells for the use any one of claims 35 to 59, said nucleic acid(s) encoding said TCR or derivative or fragment thereof is introduced into said immune cell(s) the form of a vector, preferably a viral vector, more preferably a lentiviral vector.61 . The immune cell or population of immune cells of any one of claims 35 to 60, wherein said patient has previously received checkpoint inhibitor treatment, and have optionally relapsed under checkpoint inhibitor treatment.

62. The immune cell or population of immune cells of any one of claims 35 to 61 , wherein said immune cell(s) is / are provided in the form of a pharmaceutical composition, optionally in soluble or cryopreserved form.

63. An immune cell or population of immune cells for use in a method of treating cancer, wherein said immune cell(s) express a TCR or a derivative or fragment thereof comprising a complementarity determining region (CDR) 1a comprising or consisting of the amino acid sequence SSNFYA (SEQ ID NO. 1 ), a CDR2a comprising or consisting of the amino acid sequence MTLNGDE (SEQ ID NO. 2), a CDR3a comprising or consisting of the amino acid sequence CALYNNNDMRF (SEQ ID NO. 3), a CDR1 b comprising or consisting of the amino acid sequence SGHNS (SEQ ID NO. 4), a CDR2b comprising or consisting of the amino acid sequence FNNNVP (SEQ ID NO. 5), and a CDR3b comprising or consisting of the amino acid sequence CASSPGSTDTQYF (SEQ ID NO. 6); and / or comprise at least one nucleic acid encoding a TCR or a derivative or fragment thereof as defined above; wherein said treatment comprises1) lymphodepletion, optionally by administering about 30 mg / m2 fludarabine and 500 mg / m2 cyclophosphamide daily for 4 consecutive days; and2) immune cell treatment at a single dose of 1-10 x 109cells,3) optional IL-2 treatment at one or several doses of 1 million III per day, preferably once per day on day 1 to day 5 after immune cell treatment, and twice per day on day 6 to day 10 after immune cell treatment.

64. A kit comprising the immune cell or population of immune cell(s) of any one of the preceding claims, and optionally at least one lymphodepletion agent, preferably fludarabine and cyclophosphamide; interleukin-2; and / or a checkpoint inhibitor, preferably a PD-1 or PD-1 L inhibitor, more preferably nivolumab.

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