WO2025202653A1

WO2025202653A1 - T-cell therapy targetting mutant calreticulin

Info

Publication number: WO2025202653A1
Application number: PCT/GB2025/050661
Authority: WO
Inventors: Alexandros RAMPOTAS; Martin Pule; Claire RODDIE; Isaac GANNON; Jonathan Richard LAMBERT; Bethan PSAILA; Zoe C. WONG
Original assignee: University of Oxford; UCL Business Ltd
Current assignee: University of Oxford; UCL Business Ltd
Priority date: 2024-03-27
Filing date: 2025-03-27
Publication date: 2025-10-02
Anticipated expiration: 2026-09-27

Abstract

The invention relates to an isolated chimeric antigen receptor (CAR) comprising an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin. Disclosed are CAR-T cells for use in the treatment of a myeloid malignancy, methods for their production, and their use.

Description

T-CELL THERAPY TARGETTING MUTANT CALRETICULIN

Field of invention

The present invention relates to an isolated chimeric antigen receptor (CAR). More particularly, the invention relates to an isolated CAR for use in the treatment of a myeloid malignancy.

Introduction

Myelofibrosis (MF) is a chronic disorder categorized as one of the Philadelphia-negative Myeloproliferative Neoplasms (MPNs). It arises from a malignant clone of disordered stem cells and megakaryocyte progenitor cells. In the United Kingdom alone, approximately 750 new cases of MF are diagnosed each year¹. Many patients with MF have previously been diagnosed with a milder form of MPN known as Polycythaemia vera or Essential Thrombocythemia, while others present with de novo primary MF.

With a median prognosis of around five years², MF carries a dire outlook for patients. It is characterized by ineffective haematopoiesis and has the potential to transform into Acute Myeloid Leukaemia (AML). The majority (over 95%) of MF cases are initiated by the acquisition of specific disease-driving mutations, including JAK2 V617, Calreticulin, and MPL. These mutations result in the continuous activation of the TpoR receptor through the JAK-STAT pathway, leading to increased proliferation of megakaryocytes³. Chronic activation of this pathway instigates inflammation, marked by elevated levels of TGF-b, ultimately resulting in fibrosis. The persistent inflammation and weakened immune surveillance in MF have been proposed as factors contributing to the acquisition of secondary pro-leukemic mutations, such as TP53 and ASXL1 , that drive disease progression⁴.

The natural history of MF can be metaphorically represented as a train, set in motion by the disease-driving mutation which occurs in a malignant stem cell⁵. The train gains momentum with the acquisition of secondary mutations as the disease progresses. Although several JAK inhibitors have been developed in recent years, with three approved for MF treatment in the UK, these targeted therapies have not succeeded in altering the course of the disease. They have shown efficacy in improving disease-associated symptoms and potentially prolonging overall survival in specific subgroups of patients⁶⁷. However, the only curative therapeutic option available remains allogeneic bone marrow transplantation (alloHSCT), which is suitable only for younger, medically fit patients. Even in this context, the outcome is generally poor⁸.

Consequently, there is an urgent necessity to develop more effective treatment strategies to address the challenges posed by MF. Further research is needed to identify improved therapies that can target the underlying molecular mechanisms driving the disease. These treatments should not only alleviate symptoms but also halt or reverse disease progression. Similar to the approach used in Chronic Myeloid Leukaemia, where targeting the BCR-ABL mutation has led to effective disease ‘cure’ and regression, our approach aims to emulate this success by specifically targeting the stem mutation of Calreticulin in Myelofibrosis.

Evidence in the literature support the notion that individuals without myelofibrosis (MF) harbour T-cells that specifically recognize mutated Calreticulin⁹. Furthermore, patients with MF associated with mutated Calreticulin exhibit detectable anti-Calreticulin antibodies in their serum which aligns with the reported immunogenic properties of Calreticulin¹⁰. Studies have also suggested that the acquisition of the Calreticulin mutation can occur at an early age, with the disease phenotype remaining latent for many years. Notably, the occurrence of MF in twin siblings who acquired the mutation at birth and developed the disease later in life, at the ages of 38 and 39 respectively, underscores the potential involvement of native immunity in recognizing mutated Calreticulin, but also environmental factors that may dictate the natural history of the disease¹¹. Other factors, such as suppressed T-cell response, may allow the disease clone to evade immune surveillance and progress¹². Mutated Calreticulin has also been found to hinderthe function of Dendritic cells, acting as a "don't eat me" signal, which can impede MHC-dependent immune responses¹³. Given the chronic nature of the disease and the awareness of the immune system to the presence of mutated Calreticulin on the cell surface, approaches relying solely on native immunity may not be effective due to its immunosuppressive effects¹⁴. In this context, a chimeric antigen receptor (CAR) T-cell therapy approach may be more appropriate.

Chimeric antigen receptor (CAR) therapy involves grafting the specificity of a monoclonal antibody onto immune effector cells, such as T cells. CAR T cells possess the ability to expand and persist post-administration, thereby enabling sustained elimination of target cells. This is exemplified by the long-lasting B-cell aplasia observed following CD19 CAR therapy as well as their detection for many months post infusion¹⁵.

In the case of MF, the frameshifted peptide of mutated Calreticulin, bound to TpoR on the aberrant stem cell/ megakaryocyte progenitor surface, could be targeted using CAR therapy. CAR T cells offer several advantages over other approaches targeting mutated Calreticulin: they possess greater potency compared to therapeutic monoclonal antibodies or antibody-drug conjugates, and unlike transgenic T-cell receptor approaches, CARs are not limited by human leukocyte antigen (HLA) types. The CAR receptor circumvents the requirement for MHC-related antigen presentation and exerts a more direct killing effect¹⁶.

Moreover, the most effective curative approach for Myelofibrosis is an allogeneic transplant, which harnesses the graft-versus-tumour effect to eliminate malignant clones of the disease. Remarkably, in up to 60% of cases, this effect leads to long-term remission and reversal of marrow fibrosis¹⁷. Additionally, Donor Lymphocyte Infusions can salvage patients with relapse post-transplant or enhance chimerism levels, further deepening the remission¹⁸. These successful strategies highlight that the fibrotic and inflammatory environment of the Myelofibrosis bone marrow should not hinder the homing of CAR-T cells or impede their ability to eliminate malignant target cells expressing mutated Calreticulin.

CARs are synthetic receptors typically consisting of a targeting/binding moiety that is associated with one or more signaling domains in a single fusion molecule. The binding moiety of a CAR typically consists of an antigen-binding domain of a single-chain antibody (scFv) comprising paired antibody light chain and heavy chain variable domains (VL and VH) that are fused into a single polypeptide chain via a short flexible linker. The scFv retains the same specificity and a similar affinity as the full antibody from which it was derived and is capable of binding to the specific target of interest. In addition to an extracellular antigen-binding domain CARs also comprise a transmembrane domain and signaling molecules such as costimulatory endodomains and CD3 chain.

CARs combine antigen-specificity and T cell activating properties in a single fusion molecule. First generation CARs typically included the cytoplasmic region of the CD3zeta or Fc receptor y chain as their signalling domain. First generation CARs have been tested in phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, where they have induced modest responses (Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009). Second generation CARs, which contain the signalling domains of both CD28 and CD3zeta, provide dual signalling to direct combined activating and co-stimulatory signals. Third generation CARs are more complex with three or more signalling domains.

In summary, the integration of CAR T-cell therapy represents a promising and viable treatment strategy for Myelofibrosis, particularly when targeting mutated Calreticulin. Exploiting the potent cytotoxicity and prolonged persistence of CAR T cells, as well as their capacity to bypass MHC- dependent antigen presentation, this therapeutic approach offers a novel and effective avenue to address the challenges associated with Myelofibrosis. Furthermore, the demonstrated success of allogeneic transplants and Donor Lymphocyte Infusions emphasizes the potential of CAR T-cell therapy to navigate the fibrotic and inflammatory milieu of the disease and achieve favourable clinical outcomes.

Summary of the Invention

According to a first aspect of the invention, there is provided an isolated chimeric antigen receptor (CAR) comprising an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin, wherein said antibody or antigen binding portion thereof comprises a heavy chain variable (VH) region comprising a CDR1 comprising SEQ ID NO. 1 or SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or SEQ ID NO. 11 or a sequence with at least 40% homology thereto and a light chain variable (VL) region comprising a CDR1 comprising SEQ ID NO. 5 or SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or SEQ ID NO. 15 or a sequence with at least 40% homology thereto.

In one embodiment the heavy chain variable (VH) region may comprise a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 5 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or a sequence with at least 40% homology thereto.

In one embodiment the heavy chain variable (VH) region may comprise a CDR1 comprising SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 11 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 15 or a sequence with at least 40% homology.

In one embodiment the heavy chain variable (VH) region may comprise a CDR1 comprising a sequence with at least 100% homology of SEQ ID NO 1 and a CDR2 comprising a sequence with at least 82% homology of SEQ ID NO. 2.

In one embodiment the heavy chain variable (VH) region may comprise a CDR2 comprising a sequence with at least 43% homology of SEQ ID NO. 10 and wherein the light chain variable (VL) region comprises a CDR3 comprising a sequence with at least 67% homology of SEQ ID NO. 15.

In one embodiment the VH region may comprise SEQ ID NO. 4 or 12 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto and the VL region may comprise SEQ ID NO. 8 or 16 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto. In one embodiment the CAR may comprise a hinge region. In one embodiment the hinge region comprise may comprise a CD8alpha domain.

In one embodiment the CAR may comprise a transmembrane domain. In one embodiment the transmembrane domain may be selected from the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, PD-1 , 4-1 BB, 0X40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD3zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

In one embodiment the CAR may comprise an intracellular signalling domain. In one embodiment said intracellular signalling domain may comprise one or more of the following domains: CD28, 0X40, LCK, LAT, FYN, SLP76, ZAP70 and/or CD3zeta endodomain.

In one embodiment the CAR may comprise one or more co-stimulatory domains. In one embodiment the costimulatory domain may be a signaling region of CD28, CD8, 0X40, 4-1 BB, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1 (CD1 la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM11, B7-H3, CDS, ICAM- I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof.

In one embodiment the CAR may comprise a protein encoded by suicide gene. In one embodiment the protein encoded by a suicide gene may comprise iCasp9, CD20, RapaCasp9 or RQR8.

According to a second aspect of the invention, there is provided an isolated nucleic acid encoding a CAR according to the invention. In one embodiment the VH region may comprise SEQ ID NO. 32 or 34 and the VL region may comprise SEQ ID NO. 33 or 35 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. According to a third aspect of the invention, there is provided a nucleic acid according to the invention. In one embodiment the vector may be a retroviral vector, a DNA vector, a plasmid, a RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.

According to a fourth aspect of the invention, there is provided a host cell which may comprise a nucleic acid according to the invention or a vector according to the invention. In one embodiment the host cell may be a bacterial, yeast, viral or mammalian cell.

According to a fifth aspect of the invention, there is provided an isolated cell or cell population which may comprise one or more CAR according to the invention, a nucleic acid according to the invention or a vector according to the invention. In one embodiment the cell may be an immune cell. In one embodiment the immune cell may be selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR-expressing cell, dendritic cell, or NK-T cell and a regulatory T cell.

In one embodiment the cell may be an autologous T cell. In one embodiment the autologous T cell is transduced either in vivo or ex vivo.

In one embodiment the cell may be an allogeneic T cell.

According to a sixth aspect of the invention, there is provided a pharmaceutical composition comprising a cell or cell population as defined according to the invention and a pharmaceutical acceptable carrier, excipient or diluent.

According to a seventh aspect of the invention, there is provided a method for treating a malignancy comprising administering a cell or cell population according to the invention or a pharmaceutical composition according to the invention.

According to an eighth aspect of the invention, there is provided a use of a cell or cell population according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament in the treatment of a malignancy.

According to a ninth aspect of the invention, there is provided a cell or cell population according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a malignancy. In one embodiment the malignancy may be a myeloid malignancy and wherein said myeloid malignancy may be a Philadelphia-negative Myeloproliferative Neoplasm (MPN), hairy cell leukemia, Pro lymphocytic leukemia, Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, a histiocytic disorder and a mast cell disorder. In one embodiment the MPN may be selected from the list comprising acute myeloid leukemia, chronic myeloid leukemia, primary myelofibrosis, secondary myelofibrosis, pre-fibrotic fibrosis, polycythemia vera, essential thrombocythemia, chronic neutrophilic leukemia and chronic eosinophilic leukemia.

In one embodiment the method, use or the cell or cell population according to the invention, may further comprise the step of measuring mutant calreticulin expression in a sample from said subject. In one embodiment the method, use or the cell or cell population according to the invention, may further comprise at least one further therapy. In one embodiment the method, use or the cell or cell population according to the invention, wherein the further therapy may be selected from the list comprising administering an immunotherapy, chemotherapy, cellular therapy, biological agents, cytokine therapy, gene therapy and/or a stem cell transplant. In one embodiment the further therapy may be administered before, after or at the same time as the cell or cell population.

According to a tenth aspect of the invention, there is provided a method for stimulating a T cell- mediated immune response to a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of a cell or cell population according to the invention or a pharmaceutical composition according to the invention.

According to an eleventh aspect of the invention, there is provided a method of providing an anti- myeloid malignancy immunity in a subject, the method comprising administering to the subject an effective amount of a cell or cell population according to the invention or a pharmaceutical composition according to the invention.

According to a twelfth aspect of the invention, there is provided an ex vivo method for generating a population of cells for use in adaptive immunotherapy comprising transforming said cell with a nucleic acid encoding a CAR as defined according to the invention, a nucleic acid according to the invention or a vector according to the invention.

According to a thirteenth aspect of the invention, there is provided a kit comprising a CAR as defined according to the invention, a nucleic acid according to the invention or a vector according to the invention or a cell or cell population according to the invention. According to a fourteenth aspect of the invention, there is provided a combination therapy comprising an effective amount of a cell or cell population according to the invention or a pharmaceutical composition according to the invention and an effective amount of a further therapy selected from the list comprising an immunotherapy, a chemotherapy a cytokine therapy, gene therapy and/or a stem cell transplant.

According to a fifteenth aspect of the invention, there is provided a method of making a population of cells according to the invention, the method comprising:

(i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells;

(ii) contacting the population of cells (for example, T cells) with the nucleic acid molecule according to the invention, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and

(iii) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration.

According to a sixteenth aspect of the invention, there is provided a combination of an effective amount of a cell or cell population genetically modified to express a CAR and at least one cytokine selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics

According to a seventeenth aspect of the invention, there is provided a method for treating a malignancy comprising administering a combination according to the invention.

According to an eighteenth aspect of the invention, there is provided a use of the combination according to the invention for the manufacture of a medicament in the treatment of a malignancy.

According to a nineteenth aspect of the invention, there is provided a combination according to the invention for use in the treatment of a malignancy.

Figures

Figure 1 Pathogenesis of CALR mutated MF and CAR-T targeting of mutated novel sequence on C-terminus. (A) WT CALR remains in the endoplasmic reticulum (ER) as it carries a KDEL motif. (B) Mutations in CALR result in KDEL loss and mutcalr is able to escape ER. It activates the MPL receptor on the cell surface which leads to aberrant megakaryocyte production via JAK2/STAT5 activation and fibrosis. (C) The mutated C-terminus prevents the P- Domain from inhibiting the N-Domain from binding to the Thrombopoietin receptor and hence binding is achieved. All described mutations are frameshift mutations which result to a common terminal sequence on the mutated C-domain SPARPRTSCREACLQGWTEA.

Figure 2. Co-culture killing assay of RPG-4, RPG-11 , FMC63 CAR-T cells and Nontransduced CAR-T cells against Ba/F3 cell line targets. Targets included non-transduced cell line, transduced with Thrombopoietin receptor (TpoR), Wild-type Calreticulin (CALR WT), mutated Calreticulin (CALR mt), Thrombopoietin receptor and wild type Calreticulin and Thrombopoietin receptor and mutated Calreticulin (TpoR+mtCALR). First graph is readout at 72h post co-culture of 1 :1 effector cells to target cells ratio, while the second graph is at 72h post co-culture of 1 :4 effector cells to target cells ratio.

Figure 3. ELISA for IL-2 and IFNy of supes coming from co-cultures of RPG-4, RPG-11 , FMC63 CAR-T cells and Non-transduced CAR-T cells against Ba/F3 cell line targets. Targets included non-transduced cell line, transduced with Thrombopoietin receptor (TpoR), Wild-type Calreticulin (CALR WT), mutated Calreticulin (CALR mt), Thrombopoietin receptor and wild type Calreticulin and Thrombopoietin receptor and mutated Calreticulin (TpoR+mtCALR). Readout is from supes obtained at 24h post co-culture of 1 :4 effector cells to target cells ratio.

Figure 4. Co-culture killing assay of RPG-4, RPG-11 , FMC63 CAR-T cells and Nontransduced CAR-T cells against human cell line targets. Targets included non-transduced Marimo (Marimo-NT), Marimo transduced with Thrombopoietin receptor (Marimo-TpoR), UT7- TPO transduced with mutated Calreticulin (UT7-CALRmt), transduced with both Thrombopoietin receptor and mutated Calreticulin (UT7-TpoR+CALRmt) and non-transduced SupTls. First graph is readout at 24h post co-culture of 1 :1 effector cells to target cells ratio, while the second graph is at 72h post co-culture of 1 :4 effector cells to target cells ratio.

Figure 5. ELISA for IL-2 and IFNy of supes coming from co-cultures of RPG-4, RPG-11 , FMC63 CAR-T cells and Non-transduced CAR-T cells against human cell line targets. Targets included non-transduced Marimo (Marimo NT), Marimo transduced with Thrombopoietin receptor (Marimo-TpoR), Non-transduced UT7-TPO (UT7 NT), UT7-TPO transduced with mutated Calreticulin (UT7 CALRmt), UT7-TPO transduced with Thrombopoietin receptor and mutated Calreticulin (UT7 TpoR+mtCALR) and non-transduced SupTls (SupT1 NT). Readout is from supes obtained at 24h post co-culture of 1 :1 effector cells to target cells ratio.

Figure 6. 24h CAR-T activation measured by relative expression of PD-1 and CD69 in CD4 T-cells. % expression of PD1 + on gated CD4 T-cells during co-culture with human cell lines at 1 :1 ratio. Readout at 24h. Target cell lines included non-transduced Marimo (Marimo NT), Marimo transduced with Thrombopoietin receptor (Marimo-TpoR), Non-transduced UT7-TPO (UT7 NT), UT7-TPO transduced with mutated Calreticulin (UT7 CALRmt), UT7-TPO transduced with Thrombopoietin receptor and mutated Calreticulin (UT7 TpoR+mtCALR) and nontransduced SupTls (SupT1 NT).

Figure 7. CAR-T proliferation on day 6 measured by Mean fluresence intensity of CAR-T marker gene RQR8 during co-culture with human cell lines at 1 :1 ratio. Target cell lines included non-transduced Marimo (Marimo NT), Marimo transduced with Thrombopoietin receptor (Marimo-TpoR), Non-transduced UT7-TPO (UT7 NT), UT7-TPO transduced with mutated Calreticulin (UT7 CALRmt), UT7-TPO transduced with Thrombopoietin receptor and mutated Calreticulin (UT7 TpoR+mtCALR) and non-transduced SupTls (SupT1 NT).

Figure 8. CD4 phenotype on T-cells obtained from co-culture with Marimo TpoR target cells. Phenotype was determined via flow cytometry using the markers CCR7 and CD45RA. Naive: CCR7+/CD45RA+; Central Memory (CM): CCR7+/CD45RA-; Effector Memory (EFM): CCDR7-/CD45-; Effector (EFF): CCDR7-/CD45+.

Figure 9. NSGMarimoTpoR - Exp1. Average radiance measured for cohorts A1 and A2. A1 cohort received 5x10^A6 FMC63 CAR-T cells on D1 post Marimo TpoR-Fluc (200,000 cells) inoculation. Cohort A2 received 5x10^A6 RPG-11 CAR-T cells on D1 post Marimo TpoR-Fluc (200,000 cells) inoculation. Radiance was measured via bioluminescence imaging.

Figure 10. NSGMarimoTpoR - Exp1. Average radiance measured for cohorts B1 and B2. B1 cohort received 5x10^A6 RPG-4 CAR-T cells on D1 post Marimo TpoR-Fluc (200,000 cells) inoculation. Cohort B2 received 5x10^A6 FMC63 CAR-T cells on D1 post Marimo TpoR-Fluc (200,000 cells) inoculation. Radiance was measured via bioluminescence imaging.

Figure 11. NSGMarimoTpoR - Exp1. Survival analysis using Kaplan-Meier method for all cohorts (A1 , A2, B1 and B2).

Figure 12. NSGMarimoTpoR - Exp2. Average radiance of BLI imaging for all cohorts. Cohort A, B, C and received 5x10^A6 RPG-11 CAR-T , RPG-4 CAR-T, FMC63 CAR-T and Non transduced T-cells respectively, 3 days post cell line inoculation. Average radiance was measured via bioluminescence imaging.

Figure 13. NSGMarimoTpoR - Exp2. Mean percentage and standard deviation of human CD3 positive cells in the bone marrow of the 6 mice of each cohort upon sacrifice. Mean percentage and standard deviation of target cell line in the bone marrow of the 6 mice of each cohort, measured by BFP2 market gene. Figure 14. NSGMarimoTpoR - Exp2. Mean percentage and standard deviation of human CD3 positive cells in the spleen of the 6 mice of each cohort upon sacrifice. Mean percentage and standard deviation of target cell line in the spleen of the 6 mice of each cohort, measured by BFP2 market gene.

Figure 15. 24h readout of FACS based killing assay against the non-transduced Marimo and Marimo cell line transduced with thrombopoietin receptor. Bars correspond to mean with Standard deviation. Co-culture with Marimo TpoR and Marimo non transduced cell line was performed at 1 :1 ratio.

Figure 16. 96h readout of FACS based killing assay against the non-transduced Marimo and Marimo cell line transduced with thrombopoietin receptor. Bars correspond to mean with Standard deviation. Co-culture with Marimo TpoR and Marimo non transduced cell line was performed at 1 :1 ratio.

Figure 17. Binding of our two binders RPG-4 and RPG-against Ba/F3 cell line expressing TpoR and mutated Calreticulin. Binding was only observed when Ba/F3s were transduced with both TpoR and mutCALR to facilitated surface expression of mutCALR. Staining of Ba/F3 was done with commercial TPOR-APC antibody for TpoR expression (A) and with our two purified binders against mutated Calreticulin (RPG-11 and RPG-4) (B). Cell lines tested are Ba/F3 with TpoR and mutCALR expression (TpoR_CALRmut), Ba/F3 with TpoR and WT CALR expression (TpoR_CALRWT), Ba/F3 with mutCALR expression (CALRmut), Ba/F3 with WT CALR expression (CALRWT), Ba/F3 with TpoR expression (TpoR), Non transduced Ba/F3 (NT). Ba/F3s expressing both TpoR and mutCALR were also stained with an isotype control antibody (Isotype) on each experiment.

Figure 18. Binding of our two binders RPG-4 and RPG-against human cell lines expressing TpoR and mutated Calreticulin. Binding was only observed against Marimo cell line (naturally expressing mutated Calreticulin) transduced with TpoR and against UT7-TPO (naturally expressing TpoR) transduced with mutated Calreticulin or transduced with both mutated Calreticulin and TpoR.

Figure 19. RPG-11 binding to purified mutated C-terminus kinetics.

Figure 20. RPG-4 binding to purified mutated C-terminus kinetics.

Figure 21. ELISA readings for RPG-11 and RPG-4. Anti-WT Calreticulin antibody, RPG-4 and RPG-11 were tested at various concentrations (x-axis) tested against 1 ug/ml of purified WT Calreticulin, 0.1 ug/ml and 1 ug/ml of purified mutated Calreticulin C-terminus. Figure 22. Co-culture of CAR-T cells with CD34 cells from myelofibrosis patients. FBKA analysis of co-culture of 50,000 CAR-T cells and primary CD34+ cells from myelofibrosis patients. E:T ratio utilised was 1 :1 and readouts were obtained after 48h. mtCALR: chronic phase myelofibrosis or pre-fibrotic myelofibrosis, A/BP mtCALR: Accelerated phase or blast phase myelofibrosis, JAK2: JAK2 V617F myelofibrosis. Comparisons were made using 2-way ANOVA. P-value: * (<0.05), ** (<0.01), *** (<0.001), **** (<0.0001).

Figure 23. Co-culture of CAR-T cells with CD34+ cells from myelofibrosis in 3d organoids FBKA analysis of co-culture of 25,000 CAR-T cells and primary CD34+ cells from myelofibrosis patients in 3d organoid environment. E:T ratio utilised was 1 :1 and readouts were obtained after 48h. mtCALR: chronic phase myelofibrosis or pre-fibrotic myelofibrosis, JAK2: JAK2 V617F myelofibrosis. Comparisons were made using 2-way ANOVA. P-value: * (<0.05), ** (<0.01), *** (<0.001), **** (<0.0001).

Figure 24. Example of assessing memory and exhaustion phenotype.

Figure 25. Both RPG-4 and RPG-11 are able to kill well after 3 rounds of stimulation against Marimo TpoR. RPG-11 was able to kill sufficiently up to round 6 of stimulation. Against Marimo NT, both binders there were able to control the cell line growth up to round 5 of stimulation. RQR8 counts were maintained up to the third round of stimulation. There was no killing of the negative control SupT1 cell line.

Figure 26. Expression of exhaustion markers on CD4 CAR T-cells when co-cultured with Marimo TpoR on Day 0 and after Stimulation round 1, 3 and 5. There are similar exhaustion profiles between RPG-4 and RPG-11 .

Figure 27. Expression of exhaustion markers on CD8 CAR-T T-cells when co-cultured with Marimo TpoR on Day 0 and after Stimulation round 1, 3 and 5. There are similar exhaustion profiles between RPG-4 and RPG-11 .

Figure 28. Expression of exhaustion markers on CD4 CAR-T T-cells when co-cultured with Marimo NT on Day 0 and after Stimulation round 1, 3 and 5. There are similar exhaustion profiles between RPG-4 and RPG-11 .

Figure 29. Expression of exhaustion markers on CD8 CAR-T T-cells when co-cultured with Marimo NT on Day 0 and after Stimulation round 1, 3 and 5. There are similar exhaustion profiles between RPG-4 and RPG-11 . Figure 30. T-cell phenotypes after each round of stimulation with negative control cell line SupT1.

Figure 31. T-cell phenotypes after each round of stimulation with Marimo NT.

Figure 32. T-cell phenotypes after each round of stimulation with Marimo TpoR.

Figure 33. Summary results of T-cell phenotypes against negative control cell line (SupT1), low antigen target (Marimo NT) and high antigen target (Marimo TpoR)

Figure 34. Platebound assay results. Platebound assay for RPG-4, RPG-11 and FMC63 CAR- T Cells using variable amount of purified mutated C terminus. Readouts included RQR8+ count, percentage and MFI of CD25.

Figure 35. Staining of CD34+ stem cells isolated from two patients with Myelofibrosis with anti-TpoR antibody. In our co-culture with our RPG4 CAR-T, there was minimal killing against the cells from 001/351 , while there was significant killing against the cells from 001/236. The reduced killing corresponds to lower TpoR expression as show on this histogram.

Figure 36. TpoR expression of the MarimoTpoR cell line after addition of increasing concentration of Thrombopoietin (TPO), Romiplostim and Eltrombopag. At 100 and 500nM of Eltrombopag, there is significant increase in surface TpoR expression, while expression is reduced at higher concentrations of TPO and Romiplostim.

Figure 37. Co-culture of RPG4 CAR-T and negative control FMC63 CAR-T against the Marimo TpoR cell line in the presence of no drug or 500nM of TPO, Eltrombopag or Romiplostim.

Figure 38. Staining of CD34+ stem cells isolated from two patients with Myelofibrosis with anti-TpoR antibody. In our co-culture with our RPG4 CAR-T, there was minimal killing against the cells from 001/351 , while there was significant killing against the cells from 001/236. The reduced killing corresponds to lower TpoR expression as show on this histogram.

Figure 39. TpoR expression of the MarimoTpoR cell line after addition of increasing concentration of Thrombopoietin (TPO), Romiplostim and Eltrombopag. At 100 and 500nM of Eltrombopag, there is significant increase in surface TpoR expression, while expression is reduced at higher concentrations of TPO and Romiplostim. Figure 40. Impact of Eltrombopag on CAR-T cell killing against the Marimo-TpoR cell line at a ratio of 1 :8. Ratio 1 :8 is unfavourable and usually leads to less than complete killing of RPG4 CAR-T against the MarimoTpoR cell line. The use of Eltrombopag has boosted the killing of the RPG4 CAR-T against the cell line in a dose response manner.

Figure 41. Eltrombopag and Romiplostin led to increased killing of our RPG4 CAR-T against CD34+ cells isolated from a patient with Type 1 CALR mutation. rhTPO led to reduced killing, while as expected, no killing was observed against the control JAK2 V617F CD34+ cells or with our control anti-CD19 CAR-T.

Figure 42. Difference in BLI average measurements between the RPG4 (n=6) and FMC63 (n=6) treated mice.

Figure 43. Survival curves for RPG4 and CD19 treated cohorts.

Figure 44. Reduction in mutCALR variant allele frequency (VAF) (up to 78.9% change) was demonstrated in sorted mutCALR myelofibrosis (MF) cells after co-culture with mutCALR CAR-Ts. VAF was determined by digital droplet PCR (ddPCR) and normalized to the VAF observed after control treatment (either non-transduced T cells or CD19 CAR-T). Four patients were tested and baseline clinical mutCALR VAF and co-mutations are indicated on the right of each datapoint. No change in JAK2 V617F VAF was detected (n=1).

Figure 45. Reduction in mutant clone burden of the human mutCALR+ MARIMO cell line was demonstrated in cell line mixtures with high (90%, 50%) or low (10%) starting ratio of mutCALR : wild-type CALR cells (n = 3). VAF was determined by fragment analysis and normalized to the VAF observed after control treatment (non-transduced T cells). CAR-Ts were generated from n = 3 healthy donors. (Left panel). Reduction in mutant clone burden of primary HSPCs was demonstrated with high (50%) or low (10%) starting ratios of cells from patients with mutCALR+ myelofibrosis (n=2) mixed with cells from a healthy donor (repeated in 2 independent experiments). VAF was determined by ddPCR and normalized to the control condition where non-transduced T cells were added. CAR-Ts were generated from n = 2 healthy donors. (Right panel).

Figure 46. To stratify level of response, the starting mutCALR variant allele frequency (VAF) as determined by the clinical diagnostic assay was correlated with percent killing after CAR-T treatment in vitro. The majority of patients have heterozygous CALR mutations. The expected cytotoxicity correlation for a heterozygous mutation is y=x for 50% killing (light blue) and y=2x for 100% killing (dark blue). CAR-T cytotoxicity of all patients with baseline VAF information correlated to > 50% killing (assuming a heterozygous mutation, 100% killing if homozygous).

Figure 47. Human bone marrow organoids were generated from induced pluripotent stem cells (iPSCs) and engrafted with mutCALR+ HSPCs from a myelofibrosis patient or mutCALR+ HSPCs and anti-mutCALR CAR-Ts. Genetic SNP-based demultiplexing was performed bioinformatically (using the souporcell pipeline) and this plot shows cells from the myelofibrosis patient analyzed separately (13,331 cells). Cell types were annotated using reference datasets and visualized using uniform manifold approximation and projection (UMAP).

Figure 48. CALR mutational status was determined bioinformatically using VarTrix. Co- engraftment with anti-mutCALR CAR-T resulted in an almost complete ablation of mutCALR+ primary cells.

Figure 49. The differential abundance of cell types when co-engrafted with or without mutCALR CAR-Ts was calculated. Addition of mutCALR CAR-Ts resulted in a decrease of Eosinophil/Basophil/Mast cells (EBMs), Myeloid/Erythroid Progenitors (MEPs), and Hematopoietic Stem and Progenitor Cells (HSPCs) and a relative increase in monocytes, late erythroid cells, and erythroid progenitors.

Figure 50. Separate UMAPs were generated for primary patient cells that were coengrafted into bone marrow organoids with (right) or without (left) mutCALR CAR-Ts. The mutCALR type 2 mutation was detected bioinformatically (VarTrix) and mutCALR (red) and WT cells (grey) were plotted, showing how the mutant cells were almost completely removed by the mutCALR targeting CAR-T cells.

Detailed Description

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2013)). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, immunology, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The invention relates to CAR constructs that include an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin and has the sequences described herein as well as to related products, methods and uses.

According to one aspect of the invention, there is provided an isolated chimeric antigen receptor (CAR) comprising an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin, wherein said antibody or antigen binding portion thereof comprises a heavy chain variable (VH) region comprising a CDR1 comprising SEQ ID NO. 1 or SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or SEQ ID NO. 11 or a sequence with at least 40% homology thereto and a light chain variable (VL) region comprising a CDR1 comprising SEQ ID NO. 5 or SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or SEQ ID NO. 15 or a sequence with at least 40% homology thereto.

According to one aspect of the invention, there is provided an isolated chimeric antigen receptor (CAR) comprising an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin, wherein said antibody or antigen binding portion thereof comprises a) a VH region comprising a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 5 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or a sequence with at least 40% homology thereto; or b) a VH region comprising a CDR1 comprising SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 1 1 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 15 or a sequence with at least 40% homology.

In one embodiment the VH region comprises a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto and wherein the VL region comprises a CDR1 comprising SEQ ID NO. 5 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or a sequence with at least 40% homology thereto. In one embodiment the VH region comprises a CDR1 comprising a sequence with at least 100% homology of SEQ ID NO 1 and a CDR2 comprising a sequence with at least 82% homology of SEQ ID NO. 2.

In one embodiment the VH region comprises a CDR1 comprising SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 11 or a sequence with at least 40% homology thereto and wherein the VL region comprises a CDR1 comprising SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 15 or a sequence with at least 40% homology. In one embodiment the VH region comprises a CDR2 comprising a sequence with at least 43% homology of SEQ ID NO. 10 and wherein the VL region comprises a CDR3 comprising a sequence with at least 67% homology of SEQ ID NO. 15.

In one embodiment the CDR comprises a sequence with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity thereto.

In some embodiments, CDR1 comprises any of the CDR1 sequences describe herein or with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity thereto.

In some embodiments, CDR2 comprises any of the CDR2 sequences describe herein or with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity thereto.

In some embodiments, CDR3 comprises any of the CDR3 sequences describe herein or with at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity thereto.

The properties of the CARs comprising an antibody or antigen binding portions thereof of the invention can be exploited in therapeutic methods and uses as well as in pharmaceutical formulations as described herein.

Calreticulin (CALR) is an endoplasmic reticulin chaperone protein that is also involved in intracellular calcium regulation. Its protein structure has three domains: an amino-terminal domain, which contains a signal peptide and is essential for chaperone function; a central proline-rich P domain; and a carboxy-terminal domain, which contains an endoplasmic reticulum (ER) retention signal (KDEL motif).

Mutations in CALR have been identified as having oncogenic properties. While most cases of MF are driven by mutations in JAK2 and MPL, in about one third of MF cases, mutated CALR is thought to lead to dysregulated megakaryopoiesis through constitutive JAK2/STAT5 signalling via activation of the Thrombopoietin receptor (MPL).

While multiple CALR mutations are described, conveniently, the mutations all cause a +1 bp frame shift. This results to an alternative reading frame translated into neoantigen C-terminus which includes the sequence ‘SPARPRTSCREACLQGWTEA.’; this frame-shift “scrambles” the C-terminal KDEL motif so the mutated CALR can now escape ER retention and is translocated to the cell surface. Mutated Calreticulin binds to immature TpoR in the cytoplasm and they are both expressed together on the cell surface (mutCALR/TpoR) (Figure 1). Secreted mutated Calreticulin from non-TpoR expressing cells acts like a rogue cytokine and induces the proliferation of other Megakaryocytes that also carry the mutation. Interestingly, there is no bystander expression, as it doesn’t bind to non-mutated Megakaryocytes. This is likely due to its ability to bind to the immature TpoR receptor within the ER as they are expressed together on the surface¹². Introduction of the mutated Calreticulin either in murine haematopoiesis leads to features of Myelofibrosis or essential thrombocythemia (ET) like phenotype and a few models have been developed¹³. For patients who bear the mutation, mutCALR is an attractive target for immunotherapy since it is a driving mutation, is absent on normal tissues and is expressed on the malignant cells surface. The term mutant calreticulin means a calreticulin protein comprising at least one mutation relative to an unmutated calreticulin protein. As used herein, “mutant calreticulin” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type mutant calreticulin.

In one embodiment the mutation results in a +1 bp frame shift. In one embodiment the mutation results in alternative reading frame. In one embodiment the mutant calreticulin protein comprises the sequence SEQ ID NO: 17. In one embodiment the mutant calreticulin protein comprises the sequence SEQ ID NO: 70.

SEQ ID NO: 17 SPARPRTSCREACLQGWTEA

SEQ ID NO: 70 RRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA

In one embodiment the CAR is specific for mutant calreticulin. In one embodiment the CAR of the invention is specific for mutant calreticulin. The term "specific binding" or "specifically binds to" or is "specific for" a mutant calreticulin polypeptide or an epitope on a mutant calreticulin as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10-6 M, alternatively at least about 10-7 M, alternatively at least about 10-8 M, alternatively at least about 10-9 M, alternatively at least about 10-10 M, alternatively at least about 10-11 M, alternatively at least about 10-12 M, or lower. In one embodiment, the KD is at least about 10-8 M to about 10-9 M, e.g. In one embodiment, the KD is in the nanomolar range. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a mutant calreticulin polypeptide or epitope on a mutant calreticulin without substantially binding to any other polypeptide or polypeptide epitope. For example, the CAR does not substantially bind to unmutated calreticulin. The terms KD and KD are used interchangeably herein. Further binding affinities are set out elsewhere herein.

The terms "mutant calreticulin binding molecule/protein/polypeptide/agent/moiety”, "mutant calreticulin antigen binding molecule molecule/protein/polypeptide/agent/moiety”, “anti- mutant calreticulin single chain antibody”, “anti- mutant calreticulin single immunoglobulin variable domain”, or “anti- mutant calreticulin antibody” all refer to a molecule capable of specifically binding to the human mutant calreticulin antigen. The binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity. Binding is to human mutant calreticulin unless otherwise defined.

An antibody or antigen binding portion thereof as described herein, "which binds" or is “capable of binding” an antigen of interest, e.g. human mutant calreticulin, is one that binds the antigen with sufficient affinity such that the CAR with the antibody or antigen binding portion thereof is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen mutant calreticulin as described herein. Binding is to the extracellular domain of mutant calreticulin.

“Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g, antibody or antigen -binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD).

Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of koff/kon, whereas KA is calculated from the quotient of kon/koff. Kon refers to the association rate constant of, e.g, an antibody or antigen-binding fragment thereof to an antigen, and koff refers to the dissociation of, e.g, an antibody or antigenbinding fragment thereof from an antigen. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The kon and koff can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.

The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody.

The degree of competition can be expressed as a percentage of the reduction in binding. Such competition can be measured using a real time, label-free bio-layer interferometry assay, e.g., on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assays) or SPR (surface plasmon resonance), HTRF; flow cytometry; fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.

The term "antibody" as used herein broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

The term "CDR" refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art. The numbering system described by Kabat is used herein unless otherwise stated. Also, as used herein, the term VH or "variable domain" refers to immunoglobulin variable domains defined by Kabat et al. The terms "Kabat numbering", "Kabat definitions" and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (/.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

The invention extends to CARs comprising antigen binding portions or antigen binding fragments of an antibody. The terms “binding portion” and “fragment” are used interchangeably herein. An antibody fragment/portion is a portion of an antibody, for example as F(ab')2, Fab (Fragment, antibody), scFv (single chain variable chain fragments), single domain antibodies (dAbs), Fv, sFv, and the like. Functional fragments of a full-length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments have been used to develop therapeutics as an alternative to therapeutics based on mAbs. scFv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domains are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible linker and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL -linker- VH or may comprise VH -linker-VL.

In one embodiment the linker is a peptide linker. In one embodiment the peptide linker is capable of being expressed as a single chain polypeptide.

The linker is, for example, a peptide linker with GS residues such as (Gly4Ser)n, where n=from 1 to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 as further described below.

The term "peptide linker" as used in the various embodiments described herein refers to a peptide comprising one or more amino acids. A peptide linker comprises 1 to 44 amino acids, more particularly 2 to 20 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, linkers that include G and/or S residues, (G4S)n, (SG4)n or G4(SG4)n peptide linkers, wherein "n" is generally a number between 1 and 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment, the peptide is for example selected from the group consisting of GGGGS (SEQ ID NO: 18), GGGGSGGGGS (SEQ ID NO: 19), GGGGSGGGGSGGGGS (SEQ ID NO: 20), SGGGGSGGGG (SEQ ID NO: 21), GGGGSGGGGSGGGG (SEQ ID NO: 22), GSGSGSGS (SEQ ID NO: 23), GGSGSGSG (SEQ ID NO: 24), GGSGSG (SEQ ID NO: 25), GGSG (SEQ ID NO: 26). In one embodiment the linker is (G4S)3.

The portion of the CAR composition comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding portion is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.). In one aspect, the antigen binding portion of a CAR comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv. In one embodiment, the CAR comprises a humanised VH-VL in scFv format.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigenbinding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.

These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

The invention also extends to CARs comprising antibody mimetics that comprise a sequence of the invention.

The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to a CAR or cell or population that is substantially free of other CARs, cells or cell populations. Moreover, an isolated single domain antibody may be substantially free of other cellular material and/or chemicals.

As used herein, the terms sequence "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, for example with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST, FASTA, the Smith-Waterman algorithm, or the TBLASTN program. In particular, the psi-Blast algorithm may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments can be performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms.

By "amino acid" herein is meant one of the 20 naturally occurring amino acids or any non- natural analogues that may be present at a specific, defined position. Amino acid encompasses both naturally occurring and synthetic amino acids. Although in most cases, when the protein is to be produced recombinantly, only naturally occurring amino acids are used. In one embodiment the VH region may comprise SEQ ID NO. 4, 12, 72-75 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto and the VL region may comprise SEQ ID NO. 8 or 16, 77-80 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. In one embodiment the VH region or VL region may comprise a sequence as described herein or with at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

Table 1 Mutant calreticulin binders

Elements of the CAR

The terms "Chimeric antigen receptor" or "CAR" or "CARs" as used herein refer to engineered receptors, which graft an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combination thereof) thus combining the antigen binding properties of the antigen binding domain with the lytic capacity and self renewal ofT cells. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. The term “antigen binding domain or “antigen-specific targeting domain" as used herein refers to the region of the CAR which targets and binds to specific antigens as explained above. When a CAR is expressed in a host cell, this domain forms the extracellular domain (ectodomain).

A skilled person would know that a CAR comprises additional elements.

A skilled person would also know that such elements of a CAR (other than antigen-specific targeting domain described herein) are well known in the art. Thus, the invention is not limited to specific domains of the CAR in addition to the antigen-specific targeting domain described herein.

As mentioned above, the first generation CARs have been tested in various phase I clinical studies in patients with cancer. Second generation CARs and third generation CARs have also been described are more complex with three or more signalling domains (reviewed references 4 and 5 and in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009, Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11 , 69, 2021). CARs are also described in US2004043401 , WO2019200007 and WO2021108613, all incorporated herein by reference.

For example, the CAR of the invention may comprise a molecule of the general formula: Mutant calreticulin binding scFv - transmembrane domain- Intracellular signaling domain. Exemplary domains are listed below. As will also be apparent, the CAR may comprise additional domains as explained below.

In one embodiment, the CAR may comprise a mutant calreticulin binding scFv as described herein, an extracellular domain (which may comprise a “hinge” domain), a transmembrane domain, and an intracellular signaling domain.

The Intracellular (Cytoplasmic) Domain

The intracellular (cytoplasmic) domain of the CAR can provide activation of at least one of the normal effector functions of the immune cell. The CAR of the invention may thus further comprise an intracellular signaling domain. An "intracellular signaling domain", "cytoplasmic domain" or “endodomain” is the domain that transmits activation signals to T cells and directs the cell to perform its specialized function.

An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CART cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

The intracellular domain may comprise at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof. The antigen binding molecule, i.e. the mutant calreticulin binding antibody or antigen binding portion thereof may be engineered such that it is located in the extracellular portion of the molecule/construct, such that it is capable of recognizing and binding to its target or targets.

Examples of domains that transduce the effector function signal and can be used according to the invention include but are not limited to the chain of the T-cell receptor complex or any of its homologs (e.g., q chain, FcsRIy and chains, MB1 (Igalpha) chain, B29 (Igbeta) chain, human CD3zeta chain, CD3 gamma or other CD3 polypeptides (A, 6 and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5, 0X40 and CD28.

It will be appreciated that suitable intracellular molecules may also include but are not limited to, 4-1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-I, CDI-la/CDI8), CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

In some embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.

The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.

The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBan Acc. No. BAG36664.1 , or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1.

In one embodiment said intracellular signalling domain may comprise one or more of the following domains: CD28, 0X40 and/or CD3zeta endodomain. In one embodiment said intracellular signalling domain comprises a CD3zeta endodomain. In one embodiment the CD3zeta endodomain comprises SEQ ID NO: 27 as shown below:

SEQ ID NO: 27

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYN ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

The extracellular signaling domain

The CAR may also comprise an extracellular signaling domain. The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4-1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 , CDI-la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, IT GAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRAN CE/R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.

Hinge region

In one embodiment, the CAR of the invention further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. In particular, extracellular domains often comprise a hinge portion. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of the hinge or spacer region that can be used according to the invention include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer region will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In one embodiment, the hinge is an lgG4 hinge or a CD8A hinge, an immunoglobulin (Ig) sequence or other suitable molecule to achieve the desired special distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T, or the EC domain of CD28.

In one embodiment the hinge region comprise may comprise a CD8a domain. In one embodiment the CD8a hinge comprises SEQ ID NO: 28 as shown below:

SEQ ID NO: 28: PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

The transmembrane domain

The CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.

A "transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane and is connected to the endoplasmic signaling domain and the antigen binding domain, in case of the latter optionally via a hinge. In one embodiment, the transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In one embodiment, the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain.

In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4- 1 BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-I, CDI-la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHO class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM- 1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

In one embodiment the CAR may comprise a transmembrane domain. In one embodiment the transmembrane domain may be selected from the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, PD-1 , 4-1 BB, 0X40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD3zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. In one embodiment the transmembrane domain is a CD8 transmembrane domain. In one embodiment the CD8 transmembrane domain comprises SEQ ID NO: 29 as shown below: SEQ ID NO: 29: IYIWAPLAGTCGVLLLSLVIT

Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR. In one embodiment, the CAR of the invention further comprises one or more co- stimulatory domains to enhance CAR-T cell activity after antigen specific engagement. Inclusion of this domain in the CAR of the invention enhances the proliferation, survival and/or development of memory cells. The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. The co-stimulatory domain is located intracellularly.

In one embodiment the CAR may comprise a co-stimulatory domain. In one embodiment the costimulatory domain may be a signaling region of CD28, CD8, 0X40, 4-1 BB, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1 (CD1 la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM1 I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Multiple co- stimulatory domains can be included in a single CAR to recruit multiple signaling pathways. In one embodiment, the co-stimulatory domain is obtained from 4- 1 BB. The term "4-1 BB" refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2. In one embodiment, the term "4-1 BB costimulatory domain" refers to amino acid residues 214-255 of GenBank Acc. No. AAA62478.2.

In one embodiment, the CAR of the invention further comprises a "linker domain" or "linker region" that connects different domains of the CAR. This domain includes an oligo- or polypeptide region from about 1 to 100 amino acids in length. Suitable linkers will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N- terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain during cellular processing and localization of the CAR to the cellular membrane. In one embodiment, the leader sequence is a T2A. In one embodiment the leader sequence comprises the sequence SEQ ID NO: 30.

SEQ ID NO: 30: EGRGSLLTCGDVEENPGP

Suicide gene

In one embodiment the CAR comprises a protein encoded by a suicide gene. A suicide-gene is a genetically encoded mechanism which allows selective destruction of adoptively transferred cells, such as T-cells, in the face of unacceptable toxicity. Non-limiting examples of suicide genes include RQR8, Herpes Simplex Virus thymidine kinase (HSVtk), inducible Caspase 9 (iCas9), and rapaCasp9. The suicide gene preferably allows cells expressing the suicide gene to be selectively deleted in response to administration of a substance. For example, RQR8 facilitates selective deletion of cells expressing this gene upon exposure to rituximab. Similarly, iCasp9 facilitates selective deletion of cells expressing this gene upon exposure to APi 903. Thymidine kinase allows cells expressing this gene to be killed using ganciclovir. In one embodiment the suicide gene comprises iCasp9, CD20, RapaCasp9, HSVtk or RQR8.

The suicide gene may be expressed as a single polypeptide with the CAR, for example by using a self-cleaving peptide between the two sequences.

In one embodiment the RQR8 comprises the sequence SEQ ID NO: 31 .

SEQ ID NO: 31

MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTAC PYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPL AGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVV

In one embodiment the CAR does not comprise a suicide gene.

The CAR may further include a label, for example a label that facilitates imaging, such as a fluorescent label or other tag. This can, for example, be used in methods for imaging tumor binding. The label may be conjugated to the antigen binding domain.

Suitable detectable labels which may be conjugated to antibody molecules are known in the art and include radioisotopes such as iodine-125, iodine-131 , yttrium-90, indium-11 1 and technetium-99; fluorochromes, such as fluorescein, rhodamine, phycoerythrin, Texas Red and cyanine dye derivatives for example, Cy7 and Alexa750; chromogenic dyes, such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; phosphor or laser dyes with spectrally isolated absorption or emission characteristics; and chemical moieties, such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

The CARs described herein may be synthesized as single polypeptide chains. In this embodiment, the antigen-specific targeting regions are at the N- terminus, arranged in tandem and are separated by a linker peptide.

In one embodiment, the CAR comprises the CD8a hinge and CD8 transmembrane domain, the 41 -BB costimulatory domain and CD3 intracellular signaling domain.

Polynucleotides, cells and methods for generating cells

In another aspect, the invention relates to an isolated nucleic acid molecule or construct encoding a CAR as defined above. Such construct comprises the nucleic acid encoding an antibody or antigen binding portion thereof that targets mutant calreticulin as described herein and additional nucleic acids which encode elements of the CAR.

In one embodiment the VH region may comprise SEQ ID NO. 32 or 34 and the VL region may comprise SEQ ID NO. 33 or 35 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto. In one embodiment the VH region may comprise SEQ ID NO. 32 and the VL region may comprise SEQ ID NO. 33 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto. In one embodiment the VH region may comprise SEQ ID NO. 34 and the VL region may comprise SEQ ID NO. 35 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto.

In one embodiment the VH region or VL region may comprise a sequence as described herein or with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% for example at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

The term "nucleic acid," "polynucleotide," or "nucleic acid molecule" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA. RNA includes in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein.

In another aspect, the invention relates to an isolated nucleic acid construct comprising a nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce nonproliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases.

In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2013). A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses such as adenovirus vectors can be used. In one embodiment, a lentivirus vector is used. The RD114 retroviral vector is demonstrated in the examples.

In a further aspect, the invention also relates to an isolated cell or cell population comprising one or more nucleic acid construct or vector as described above. In one embodiment, the cell is an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, plant, mammalian or other suitable host cell.

Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.

The invention also provides an isolated and genetically engineered cell or cell population which comprises and stably express a CAR nucleic acid construct or vector of the invention. Thus, the cell or cell population comprises the nucleic acid molecule encoding a CAR molecule having an antigen binding domain as described herein and further optional domains as described herein, e.g. an intracellular domain, a transmembrane domain and an extracellular domain.

In one embodiment, the cell is an immune cell. In one embodiment, the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR-expressing cell, dendritic cell, or NK-T cell and a regulatory T cell, hematopoietic stem cells and/or pluripotent embryonic/induced stem cells. T cells may be isolated from a patient for transfection with a CAR nucleic acid construct of the invention.

In one embodiment, the cell is an autologous T cell or allogeneic T cell. In one embodiment the autologous T cell is transduced either in vivo or ex vivo

In some embodiments, a source of cells, e.g., T cells or natural killer (NK) cells, can be obtained from a subject, e.g. a human patient. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present invention disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing. The cells may be washed with PBS. As will be appreciated, a washing step may be used, such as by using a semiautomated flowthrough centrifuge, for example, the Cobe™ 2991 cell processor, the Baxter CytoMate™, or the like. After washing, the cells may be resuspended in a variety of biocompatible buffers, or other saline solution with or without buffer. In certain embodiments, the undesired components of the apheresis sample may be removed.

In certain embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28⁺ CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺T cells can be further isolated by positive or negative selection techniques known in the art. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4<+>cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDI lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present invention.

PBMCs may be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.

In some embodiments, CD8⁺ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8⁺ cells. In some embodiments, the expression of phenotypic markers of central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, central memory T cells are CD45RO⁺, CD62L⁺, CD8⁺T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin. In certain embodiments, CD4⁺ T cells are further sorted into subpopulations. For example, CD4⁺T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

The immune cells, such as T cells, can be genetically modified following isolation using known methods, or the immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modified. In another embodiment, the immune cells, such as T cells, are genetically modified with the chimeric antigen receptors described herein (e.g., transduced with a viral vector comprising one or more nucleotide sequences encoding a CAR) and then are activated and/or expanded in vitro.

For example, cells can be transfected with the nucleic acid of the invention ex vivo. Various methods produce stable transfectants which express CARs of the invention. In one embodiment, a method of stably transfecting and re-directing cells is by electroporation using naked DNA. Additional methods to genetically engineer cells using naked DNA encoding a CAR of the invention include but are not limited to chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and/or hydrodynamic delivery) and/or particle-based methods (e.g., impalefection, using a gene gun and/or magnetofection). The transfected cells demonstrating presence of an integrated un-rearranged vector and expression of the CAR may be expanded ex vivo. Viral transduction methods may also be used to generate redirected cells which express the CAR of the invention.

In another aspect, the invention relates to a method, e.g. an ex vivo or in vitro for producing a genetically modified cell or cell population comprising expressing in said cell or cell population a CAR nucleic acid construct of the invention. The method may include introducing into the cell a nucleic acid as described herein (e.g., an in vitro transcribed RNA or synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein). In embodiments, the RNA expresses the CAR polypeptide transiently. In one embodiment, the cell is a cell as described herein, e.g., an immune effector cell (e.g., T cells or NK cells, or cell population). Cells produced by such methods are also within the scope of the invention.

The present invention also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by poly A addition, to produce a construct containing 3' and 5' untranslated sequence (“UTR”), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR.

In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.

In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.

In some embodiments, the T cells may be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines.

Cells of the invention may be cryopreserved such that the cells remain viable upon thawing. A fraction of the cells expressing the CARs can be cryopreserved by methods known in the art to provide a permanent source of such cells for the future treatment of patients afflicted with a malignancy. When needed, the cryopreserved transformed immune cells can be thawed, grown and expanded for more such cells.

Cells described above can be used in adaptive immunotherapy for treatment of disease as further explained below.

Nucleic acid constructs of the invention are as follows:

SEQ ID NO: 32 RPG-4 VH

GACGTACAGCTTCAGGAAAGTGGCCCCGGTCTCGTCAAGCCAAGCCAGAGCCTTTCACT TACCTGCACAGTTACGGGCTATTCAATCACGTCCGACTACGCTTGGAATTGGATAAGACA ATTTCCAGGTAATAAACTGGAGTGGATGGGCTACATCGACTACTCTGGAAACACTAATTAC AATCCTAGCCTCAAAAGCAGAATATCTATTACTAGGGATACGTCCAAAAACCAGTTTTTTC TTCAACTCAGTTCAGTGACGACGGAAGATACCGGGACGTATTACTGCTCTCGGGGAATTA CCGGATATTGGGGACGAGGCACTACCCTGACCGTGTCAAGC

SEQ ID NO: 33 RPG-4 VL

CAAGCGGTAGTTACGCAAGAATCCGCGTTGACCACGAGCCCTGGCGAAACCGTGACGCT CACCTGCCGATCTTCTACGGGCGCAATTACCACCAGTAATTACGCAAATTGGGTACAAGA GAAATCTGATCATTTGTTCACGGGATTGATCGGAGGAGCGAAGAACCGGGCCCCTGGCG TACCAGCCCGCTTTTCTGGCAGTCTTATAGGAGACAAGGCTGCTCTCACTATTACGGGTG CCCAGACGGAGGACGAAGCAATCTATTTTTGCGCACTCTGGTATTCAAATCATTGGGTCT TTGGAGGTGGGACCAAACTGACCGTCCTT

SEQ ID NO: 34 RPG-11 VH

CAGGTACAGCTGCAGCAATCTGGGGCTGAACTGGTGAAGCCTGGGTCCTCAGTGAAAAT TTCCTGCAAGGCTTCTGGCTACACCTTCACCCGTAACTTTATACACTGGATAAAACAGCAG CCTGGAAATGGCCTTGAGTGGATTGGGTGGATTTTTCCTGGAGATGGTGATACAGAGTAC AATCAAAAGTTCAATGGGAAGGCAACACTCACTGCAGACAAATCGTCCAGCACAGCCTAT ATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTGCAAGAGGAAAT TACAACTACGAGTACTTTGATTACTGGGGCCAAGGAgtcatggtcacagtctccagc SEQ ID NO: 35 RPG-11 VL gacatccagatgacacagtctccgGCTTCCCTGTCTGCATCTCTGGGAGAAACTGTCTCCATCGAGT GTCTAGCAAGTGAGGACATTTACAGTTATTTAGCATGGTATCAACAGAAGCCAGGGAAAT CTCCTCAGCTCCTGATCTTTGCTGCAAATAGGTTGCAAGATGGGGTCCCATCACGGTTCA GTGGCAGTGGATCTGGCACACAGTTTTCTCTCAAGATCAGCGGCATGCAACCTGAAGATG AAGGGGATTATTTCTGTCTACAGGGTTCCAAGTTTCCGTACACCTTTGGACctgggaccaagct ggaactgaac

Pharmaceutical compositions

In another aspect of the present invention, there is provided a pharmaceutical composition comprising a nucleic acid encoding a CAR as described herein, a vector comprising a nucleic acid encoding a CAR as described herein, a CAR as described herein or an isolated cell or cell population comprising a CAR according to the present invention and optionally a pharmaceutically acceptable carrier.

The genetically modified cells or pharmaceutical composition of the present invention can be administered by any convenient route, including parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Compositions can take the form of one or more dosage units.

The composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. The liquid compositions of the invention, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

The amount of the pharmaceutical composition of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. The compositions of the invention comprise an effective amount of a binding molecule of the present invention such that a suitable dosage will be obtained. The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and its particular site, host and the disease being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose.

Typically, this amount is at least about 0.01 % of a binding molecule of the present invention by weight of the composition.

Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding molecule of the present invention.

For intravenous administration, the composition can comprise from typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight.

Suitable treatment with cells is also described below.

The present compositions can take the form of suitable carriers, such aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

The pharmaceutical compositions can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a binding molecule of the present invention with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

The pharmaceutical composition of the invention can be co-administered with other therapeutics, for example anti-cancer agents.

Methods of treatment

As described above, mutant calreticulin is an attractive target for immunotherapy since it is a driving mutation, is absent on normal tissues and is expressed on the malignant cells surface. The molecules and cells described herein are therefore expected to find application in the treatment of disease, in particular a myeloid malignancy.

In one embodiment, the disease is a disease associated with expression of mutant calreticulin. The molecules of the invention may preferentially bind to mutant calreticulin present on the surface of a malignant cell. The malignancy to be treated using an CAR of the invention therefore preferably expresses, or has been determined to express, mutant calreticulin. More preferably, cells of the malignancy to be treated comprise, or have been determined to comprise, mutant calreticulin at their cell surface, i.e. to comprise cell-surface bound mutant calreticulin. Methods for determining the presence of an antigen on a cell surface are known in the art and include, for example, flow cytometry.

In one embodiment, the disease is a malignancy and the invention thus relates to methods for the prevention and/or treatment of a malignancy, comprising administering to a subject a cell or cell population comprising a CAR or a pharmaceutical formulation as described herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a cell and/or of a pharmaceutical composition of the invention. In one embodiment there is provided a method for treating a proliferative disease such as a cancer or malignancy, a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or a non-cancer related indication associated with a cell which expresses mutant calreticulin.

The invention also relates to the use of a cell or cell population according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament in the treatment of a malignancy. In one embodiment there is provided a use of a cell or cell population according to the invention or a pharmaceutical composition according to the invention for the manufacture of a medicament in the treatment of a proliferative disease such as a cancer or malignancy, a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or a non-cancer related indication associated with a cell which expresses mutant calreticulin.

The invention also relates to a CAR, a cell or cell population comprising a CAR or a pharmaceutical formulation as described herein for use in therapy. The invention also relates to a CAR or a cell or cell population comprising a CAR or a pharmaceutical formulation as described herein for use in the treatment of a malignancy. In one embodiment there is provided a cell or cell population according to the invention or a pharmaceutical composition according to the invention for use in the treatment of a proliferative disease such as a cancer or malignancy, a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or a non-cancer related indication associated with a cell which expresses mutant calreticulin.

Myeloid malignant diseases are clonal diseases arising in hematopoietic stem or progenitor cells. In one embodiment the malignancy is a myeloid malignancy and wherein said myeloid malignancy is a Philadelphia-negative Myeloproliferative Neoplasm (MPN), hairy cell leukemia, Prolymphocytic leukemia, Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B-cell leukemia (B-cell acute lymphoid leukemia, BALL), acute lymphoblastic T- cell leukemia (T-cell acute lymphoid leukemia (TALL), a histiocytic disorder (e.g., a mast cell disorder or a blastic plasmacytoid dendritic cell neoplasm) or a mast cell disorder (e.g., systemic mastocytosis or mast cell leukemia). In one embodiment the MPN is selected from the list comprising acute myeloid leukemia, chronic myeloid leukemia, primary myelofibrosis, secondary myelofibrosis, pre-fibrotic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic neutrophilic leukemia and chronic eosinophilic leukemia. In one embodiment the malignancy is a cancer or a solid tumor. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. The cancer may be haematological or non haematological.

In one embodiment the cancer is cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In one embodiment the method, use or the cell or cell population according to the invention, may further comprise the step of measuring mutant calreticulin expression in a sample from said subject. As referred to above, there are many methods of measuring mutant calreticulin expression in a sample from said subject which are not limited to the invention, for example flow cytometry. Other examples include Western Blot, ELISA or cytometry by time of flight (CyTOF).

In one embodiment the method, use or the cell or cell population according to the invention, may further comprise at least one further therapy. Typically, the further therapy is an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of a CAR-T or pharmaceutical composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include checkpoint inhibitors, antineoplastic agents, interferons and in particular interferon-alpha, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor- specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy or interferons. In one embodiment, the CAR-T or pharmaceutical composition of the invention is used in combination with surgery.

In one embodiment, an immune checkpoint inhibitor is also administered with the cell or cell population or pharmaceutical composition. The immune checkpoint inhibitor may be an anti- PD1 , anti PDL-1 , anti PDL-2, anti CTL-4, anti-TIM-3 or anti LAG-3 antibody. In another embodiment, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, cemiplimab, avelumab, durvalumab, or atezolizumab, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Pidilizumab, Toripalimab, Ipilimumab or Tremelimumab. In another embodiment, the immune checkpoint inhibitor is an interfering nucleic acid molecule, a small molecule or a PROteolysis TArgeting Chimera (PROTAC).

In one embodiment the method, use or the cell or cell population according to the invention, wherein the further therapy may be selected from the list comprising administering an immunotherapy, chemotherapy, cellular therapy, biological agents, cytokine therapy, gene therapy and/or a stem cell transplant. Suitable stem cells include haematological stem cells or mesenchymal stem cells. A biologic is a drug or vaccine made from a living organism. Any biologic is considered for example a microorganism. In one embodiment, the further therapy is a cytokine therapy and is selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics. In one embodiment, the cytokine therapy is Eltrombopag. In one embodiment the further therapy may be administered before, after or at the same time as the cell or cell population.

In another aspect, the invention relates to a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to a subject an effective amount of a cell or cell population that expresses a CAR of the invention, wherein the antigen binding domain is selected to specifically recognize the target cell population or tissue. The term "stimulation," refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-0, and/or reorganization of cytoskeletal structures, and the like.

In another aspect, the invention relates to a method of providing an anti- myeloid malignancy immunity in a subject, the method comprising administering to the subject an effective amount of a cell or cell population according to the invention or a pharmaceutical composition according to the invention. In one embodiment the method comprises administering to the subject an effective amount of a cell or cell population genetically modified to express a CAR of the invention or a pharmaceutical formulation described herein, thereby providing an anti- myeloid malignancy immunity in the subject. The term “anti- myeloid malignancy immunity” as used herein means the ability to provide an immunological response to a myeloid malignancy.

All methods described above may be carried out in vivo, ex vivo or in vitro.

Suitable treatment amounts of cells in the composition is generally at least 2 cells (for example, at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset) or is more typically greater than 10² cells, and up to 10⁶, up to and including 10⁹or 10⁹ cells and can be more than 10¹⁰ cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 10⁶ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹² cells. In some aspects of the present invention, particularly since all the infused cells will be redirected to the desired target antigen (mutant calreticulin), lower numbers of cells, in the range of I0⁶/kilogram (10⁶- 10¹¹ per patient) may be administered. CAR treatments may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy. The CAR expressing cell populations of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Combination

In one aspect, the invention relates to the combination of an effective amount of a cell or cell population genetically modified to express a CAR and at least one cytokine selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics. In one embodiment, the addition of at least one cytokine, for example thrombopoietin or thrombopoietin mimetics, such as Romiplostim and eltrombopag, is used as an agonist to selectively boost protein expression to provide a more efficacious CAR-T cell therapy.

In one embodiment, the at least one cytokine comprises Eltrombopag. In one embodiment, the thrombopoietin is recombinant thrombopoietin.

In one embodiment, the at least one cytokine is administered before, after or at the same time as the cell or cell population. In one embodiment, the at least one cytokine is administered before, the cell or cell population.

In one embodiment, the cell or cell population is a cell or cell population as described above, for example a T cell. In one embodiment, the cell or cell population is an immune cell. In one embodiment, the immune cell is selected from from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR- expressing cell, dendritic cell, or NK-T cell and a regulatory T cell. In one embodiment, the immune cell is a T cell and the T cell is an autologous T cell or allogeneic T cell.

In one aspect, the invention relates to a method for treating a malignancy comprising administering a combination according to the above.

In one aspect, the invention relates to a use of a combination according to the above for the manufacture of a medicament in the treatment of a malignancy.

In one aspect, the invention relates to a combination according to the above for use in the treatment of a malignancy.

In one embodiment the malignancy is a myeloid malignancy and wherein said myeloid malignancy is a Philadelphia-negative Myeloproliferative Neoplasm (MPN), hairy cell leukemia, Prolymphocytic leukemia, Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, a histiocytic disorder and a mast cell disorder. In one emobdiment the MPN is selected from the list comprising acute myeloid leukemia, chronic myeloid leukemia, primary myelofibrosis, secondary myelofibrosis, pre-fibrotic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic neutrophilic leukemia and chronic eosinophilic leukemia.

Kits and methods

According to a further aspect of the invention, there is provided a kit comprising a CAR as defined according to the invention, a nucleic acid according to the invention or a vector according to the invention or a cell or cell population according to the invention. In one embodiment the kit is for detecting a myeloid malignancy, for example an MPN for diagnosis, treatment, prognosis or monitoring comprising a genetically modified cell or pharmaceutical composition of the invention. The kit may also comprise instructions for use. In one embodiment, the CAR-T or pharmaceutical composition comprises a label and one or more compounds for detecting the label. The invention in another aspect provides a binding molecule of the invention packaged in lyophilized form, or packaged in an aqueous medium.

In another aspect, the invention relates to a method of making a population of cells as described herein, the method comprising:

(ii) contacting the population of cells (for example, T cells) with the nucleic acid molecule of described herein, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and

In one aspect, the disclosure provides a method of manufacturing an effective dose of engineered T cells for CAR T-cell therapy comprising: (a) preparing a population of engineered T cells comprising CAR described herein; (b) measuring the T cell expansion capability of the population; and (c) preparing an effective dose of engineered T cells for CAR T-cell therapy for treating a malignancy in a patient in need thereof based on the T cell expansion capability of the population. In some embodiments, the T cell expansion capability relates to in vivo expansion. In some embodiments, the T cell expansion capability relates to in vitro expansion. In some embodiments, the T cell expansion capability is measured during the manufacturing process.

In another aspect, the disclosure provides a method of determining whether a patient will respond to the CAR T cell therapy comprising: (a) measuring in vivo CAR T-cell expansion after administration of CAR T-cells relative to pretreatment tumor burden to obtain a value and (b) determining if the patient will achieve durable response based on the value. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents and sequence database identifiers mentioned in this specification are incorporated herein by reference in their entirety.

"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. For example, "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The invention is further described in the non-limiting examples.

Examples

Example 1 : Killing co-culture experiment

Introduction

We have demonstrated the efficacy of binders RPG-4 and RPG-11 in targeting Ba/F3 cells engineered to express both the thrombopoietin receptor (TpoR) and mutant calreticulin, leading to selective cell death induction (Figure 2). However, their specificity towards TpoR/CALRmt coexpressing targets necessitates further investigation. To address this, we conducted a comprehensive evaluation of the killing specificity of these binders across various target cell combinations, including wild-type calreticulin (CALR WT) and mutant calreticulin (CALR mutant), alongside TpoR, utilizing a FACS-based killing assay followed by ELISA analysis of 24-hour supernatants. Methods

Peripheral blood samples from three healthy donors were collected, and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation of the buffy coat. T cells were isolated using a negative selection kit and resuspended at a concentration of 1x10^A6 cells/mL in an activation cocktail containing anti-CD3 and anti-CD28 antibodies, supplemented with IL7 and IL15 cytokines. Subsequently, T cells were transduced with FMC63, RPG-11 , RPG-4, or RPMI (mock transduction) at a multiplicity of infection (MOI) of 1. Transduced cells were then harvested, replated with fresh media supplemented with IL7 and IL15, and evaluated for transduction efficiency using flow cytometry. Target cell lines were prepared and added to wells according to the experimental plan. After 24 hours of co-culture, supernatants were collected and stored for subsequent ELISA testing, while cell pellets were stained and analyzed by flow cytometry. This process was repeated after an additional 72 hours of incubation. Supernatant samples were later thawed and tested for interleukin-2 (IL-2) and interferon-gamma (IFN-y) secretion. The positive target cell line was Ba/F3s transduced with mutated Calreticulin and Thrombopoietin receptor, while the negative targets were non-transduced Ba/F3s and Ba/F3s transduced with Wild Type Calreticulin, Thrombopoietin receptor, mutated Calreticulin and Wild Type Calreticulin and Thrombopoietin receptor.

Results

RPG-4 and RPG-11 both achieved a mean killing of 99.4% when co-cultured with BaF3 TpoR/mtCALR) positive target cells in 1 :1 ratio at 24h and a 93.1 % and 89.5% respectively in 1 :4 ratio at 72h readout (Figure 2). There was no significant off-target killing of the other targets when compared with the negative control FMC63 CAR-T cells. Both RPG-4 and RPG-11 achieved good secretion of IL-2 and IFNy measured with ELISA at 24h and at 1 :4 co-culture ratio. RPG-4 had 887.8 and 2249pg/ul IL-2 and IFNy secretion respectively, while RPG-11 had 751 .8 and 2007.3pg/ul (Figure 3). There was negligible IL-2 and IFNy secretion when co-cultured with negative targets, while there was no secretion in any condition when the negative control FMC63 CAR-T was tested.

Conclusion:

Our methodology facilitated a comprehensive assessment of killing specificity towards TpoR/CALRmt co-expressing targets by the mutant calreticulin binders RPG-4 and RPG-11 , highlighting their potential therapeutic utility in relevant pathological contexts. Both RPG-4 and RPG-11 binder-based chimeric antigen receptor (CAR) constructs demonstrated specific and comparable killing efficacy against TpoR_CALRmt co-expressing Ba/F3 targets, with minimal background killing observed against other targets. Furthermore, ELISA results confirmed similar specificity in terms of IL-2 and IFN-y secretion towards these targets, underscoring the promise of RPG-4 and RPG-11 in targeted cancer therapy. Example 2: Killing co-culture experiment

Introduction

Previous demonstrations have exhibited the efficient and specific killing ability of anti-Calreticulin mutant CARs against Ba/F3 cell lines co-expressing TpoR and mutant Calreticulin. However, these target lines, originating from murine cancer cells, were engineered to express the target antigen. The current study aims to investigate the potential translatability of this efficacy to human cell lines with minimal or no engineering required for mutant calreticulin expression. To assess this, Fluorescence-based killing (FBK) assays were conducted at various ratios and time points, complemented by T cell phenotypic analysis and cytokine secretion measurements.

Methods

Blood samples from three healthy donors were processed to isolate peripheral blood mononuclear cells (PBMCs) and subsequently, T cells were further purified using a negative selection kit. These T cells were activated with an anti-CD3/CD28 cocktail and IL7/IL15 cytokines. On Day 3, the T cells were transduced with FMC63, RPG-11 , RPG-4, or RPMI (mock transduction), followed by cell replating and incubation. Transduction efficiency was assessed on Day 7, followed by cell trace violet labelling to monitor proliferation. T cells and target cell lines were co-cultured, and supernatant was collected at 24 hours for cytokine analysis. On subsequent days, cell pellets were stained for flow cytometry analysis to evaluate activation markers PD1 and CD69 (Day 8), PD1 and CD25 (Day 10), and T cell phenotype (Day 13). T- cell phenotype was assessed by expression of CCR7 and CD45RA; Naive phenotype: double expression, Central memory: CCR7 expression; Effector memory: CD45RA expression and Effector cells: no expression of either CCR7 or CD45RA. Flow cytometry data was analyzed to elucidate the killing specificity and functional characteristics of the anti-Calreticulin mutant CARs against human cell lines expressing mutant Calreticulin. Positive target cell lines included Marimo transduced with Thrombopoietin receptor, UT7-TPO transduced with mutated Calreticulin or transduced with both thrombopoietin receptor and mutated Calreticulin and non transduced Marimos which is a low antigen target as there is very little TpoR expression and hence very limited surface mutated Calreticulin expression. Negative target cell lines included UT7-TPO non transduced and SupTls non-transduced.

Results

RPG-11 demonstrated robust killing efficacy, achieving mean killing rates of 86%, 61.6%, and 55.6% against Marimo-TpoR, UT7-CALRmt, and UT7-TpoR+CALRmt, respectively, at a 1 :1 ratio with a 24-hour readout (Figure 4). Notably, at 72 hours and a 1 :4 ratio, killing rates were significantly augmented, reaching 97.2% against Marimo-TpoR, while remaining at 41.8% and 73% against UT7-CALRmt and UT7-TpoR+CALRmt, respectively (Figure 4). RPG-4 exhibited a killing efficacy of 59.8% against Marimo-TpoR at 24 hours and a 1 :1 ratio, which escalated to 88.7% at 72 hours and a 1 :4 ratio. Against UT7-TpoR and UT7-TpoR+CALRmt, RPG-4 achieved killing rates of 39.9% and 58.5%, respectively. Additionally, RPG-4 displayed significant killing activity (77.6%) against the low antigen target Marimo NT at 72 hours and a 1 :4 ratio. Negative controls demonstrated negligible killing, and there was no notable non-specific killing of the negative cell lines (Figure 4). ELISA analysis for IFN-gamma and IL-2 confirmed specific activation against positive targets exclusively (Figure 5). Proliferation and activation assays, as evidenced by CD69, PD1 , and RQR8 expression, exhibited activation and proliferation exclusively in the presence of positive targets, without any indication of non-specific activation. Lastly, RPG-4 CAR-T cells displayed a more naive phenotype, whereas RPG-11 retained a central memory phenotype during co-culture with the antigen-high target Marimo TpoR (Figures 6-8).

Conclusion

CAR-T cells bearing RPG-4 and RPG-11 showed excellent killing efficacy against human cell line targets with little to no off-target killing. The RPG-4 bearing CAR-T was also superior when the low antigen target (Marimo NT) was targeted as it was able to achieve killing of >80% after 72h even when co-cultured at 1 :4 ratio. ELISA for IFNy and IL-2 confirmed activation when cocultured with positive targets. CAR-T cell activation and proliferation also measured by CD69, PD-1 and marker gene RQR8 was optimal and specific to co-cultures where there was a positive target.

Example 3

NSG MarimoTpoR exp1

Eighteen NSG (NOD.Cg-PrkdcSCID H2rgtm1 Wjl/SzJ) mice obtained from Charles River UK were divided into 4 cohorts, each comprising 3 mice. All mice were administered 200,000 cells of the Marimo cell line transduced with Thrombopoietin receptor and Flue. Cohorts A1 and A2 received 5x10^A6 RPG-11 CAR-T cells and FMC63 CAR-T cells, respectively, on Day 1 post cell line injection. Conversely, cohorts B1 and B2 were administered 5x10^A6 RPG-11 and FMC63 CAR-T cells on Day 6 post cell line injection. Throughout the experiment, mice were closely monitored using clinical scoring as per the animal license protocol, regular weight measurements, and BLI imaging. Humane care was provided to all animals, and all experimental procedures adhered to local guidelines. The mice were housed in a controlled environment with 12-hour light-dark cycles, humidity maintained between 30-70%, and an ambient temperature ranging from 20-26 degrees Celsius. Both study and control animals were housed in the same room, within a specific pathogen-free facility. The health status of the mice was assessed through clinical scoring and observations outlined in the project license protocol. Weight measurements were recorded every 2-3 days, with a clinical scoring above 1 triggering euthanasia using CO2 overdose followed by cervical dislocation. For bioluminescence imaging, D-luciferin firefly potassium salt was dissolved in sterile PBS at a concentration of 10mg/ml and injected intraperitoneally (200 pl per mouse) into anesthetized mice. The average radiance was measured using a Xenogen VivoVision IVIS Lumina camera (see figures 9 and 10). Survival analysis was conducted using Kaplan-Meier curves (refer to figure 11).

NSG MarimoTpoR exp2

Twenty-four NSG (NOD.Cg-PrkdcSCID H2rgtm1 Wjl/SzJ) mice sourced from Charles River UK were divided into 4 cohorts, with each cohort comprising 6 mice. All mice received an injection of 200,000 cells of the Marimo cell line, which had been transduced with the Thrombopoietin receptor and Flue. Cohorts A and B were administered 5x10^A6 RPG-11 and RPG-4 CAR-T cells, respectively, while the negative control cohorts C and D received 5x10^A6 FMC63 CAR-T cells and non-transduced cells, respectively. CAR-T cells were introduced on day 3 post cell line injection. Throughout the experiment, mice were meticulously monitored using clinical scoring protocols outlined in the animal license, along with regular weight measurements and BLI imaging. Humane care was provided to all animals, and experimental procedures strictly adhered to local guidelines. Mice were housed in an environment with 12-hour light-dark cycles, humidity maintained between 30-70%, and an ambient temperature ranging from 20-26 degrees Celsius. Both study and control animals shared the same room within a specific pathogen-free facility. Health status was continually assessed through clinical scoring and observations in accordance with the project license protocol. Weight measurements were conducted every 2-3 days, with a clinical score above 1 triggering euthanasia using CO2 overdose followed by cervical dislocation. For bioluminescence imaging, D-luciferin firefly potassium salt, dissolved in sterile PBS at a concentration of 10mg/ml, was injected intraperitoneally (200 pl per mouse) into anesthetized mice. Average radiance was measured using a high-sensitivity camera: Xenogen, VivoVision IVIS Lumina (figure 12). When a discernible difference in average radiance between the target and control cohorts was observed, mice were sacrificed for tissue harvesting. Spleens and bone marrows were collected, and the number of cells from the cell line as well as CAR-T cells was quantified and compared between each group (Figure 13 and 14). The cell line was identified via the marker gene BFP2 in the transgene construct, while CAR-T cells were identified using CD3 markers. Conclusion

RPG-4 and RPG-11 were able to control the growth of the cell line and achieve reduction of tumour volume as measured by BLI. On experiment 1 , mice were able to live longer when treated with RPG-11 , especially when it was given on day 1 post cell line injection, but also when it was given at day 6 post cell line injection. On experiment 2, it was demonstrated that there was better control of the cell line growth with RPG-4 and this was confirmed as upon sacrifice there were more human T-cells in the spleen and marrow indicating expansion and persistence of the CAR- T cells, while there were less target cell line cells in the marrow and spleen. This experiments shows that RPG-4 and RPG-11 are able to achieve killing efficacy against mutated Calreticulin bearing targets in an NSG in vivo model when compared with non-transduced T-cells or negative control CAR-T cells (FMC63).

Example 4 Comparing RPG-4 and RPG-11 to ELSTAR binders H1 and M2

Introduction

Previous demonstrations have exhibited the efficient and specific killing ability of anti-Calreticulin mutant CARs against Ba/F3 cell lines co-expressing TpoR and mutant Calreticulin and human cell lines engineered to express mutated Calreticulin. This experiment aims to assess if the two binders RPG-4 and RPG-11 are demonstrate better killing efficacy than two of the ESLTAR binders named for the purposes of the experiment H1 (Elstar Therapeutics Inc., patent application US20210137982A1 - heavy chain: seq1 and light chain: seq3) and M2 (Elstar Therapeutics Inc., patent application US20210137982A1 - heavy chain: seq 5 and light chain: seq 6). The ELSTAR binders have been recreated n single chain variable fragments and inserted into a 2^nd generation 4-1 BB CAR-T construct which is the same as the construct that bears the RPG-4 and RPG-11 binders. We decided to perform a co-culturing killing experiment as the most relevant for the efficacy of the CAR-T as other surrogates (activation, proliferation, binding) may be informative but don’t always translate to killing efficacy.

Methods

Blood samples from three healthy donors were processed to isolate peripheral blood mononuclear cells (PBMCs) and subsequently, T cells were further purified using a negative selection kit. These T cells were activated with an anti-CD3/CD28 cocktail and IL7/IL15 cytokines. On Day 3, the T cells were transduced with FMC63, RPG-11 , RPG-4, H1 , M2 or RPMI (mock transduction), followed by cell replating and incubation. Transduction efficiency was assessed on Day 7, followed by cell trace violet labelling to monitor proliferation. T cells and target cell lines were co-cultured. The two target cell lines selected were the Non Transduced Marimo and the Marimo transduced with thrombopoietin receptor. The former cell line represents a low antigen target cell line, while the latter a high antigen target cell line. Read outs were done at 24 and 96h post co-culture.

Results

RPG-4 had a mean 40.6% and 29% killing efficacy against Marimo TpoR and Marimo NT at 24h respectively (Figure 15) which was increased to 82.1 % and 95.5% at 96h (Figure 16). RPG-11 had a mean killing efficacy of 32.6% and 34.9% against Marimo TpoR and Marimo NT at 24h respectively (Figure 15), which was increased to 83.2% and 96.8% at 96h (Figure 16). M2 CAR- Ts had mean 16.7% and 25.1 % killing at 24h (Figure 15) and 68.4% and 84.6% at 96h against Marimo TpoR and Marimo NT respectively (Figure 16). While, H1 CAR-Ts achieved 13.3% and 18.8% at 24h (Figure 15) which changed to 19.9% and 8.7% at 96h against Marimo TpoR and Marimo NT respectively (Figure 16).

Conclusion

RPG-4 and RPG-11 are superior binders for CAR-T cell killing against a low antigen and high antigen mutated Calreticulin cell line such as Marimo and Marimo transduced with TpoR. H1 is an incapable binder for CAR-T cell killing, while the M2 binder demonstrated killing but less superior to the RPG-4 and -11 binders.

Example 5

Binding against cell lines

RPG-4 and RPG-11 in single-chain variable fragments (scFvs), were cloned into SFG expression vectors in a secreted scFv-Fc format. The Fc region used was generally derived from murine sources, and the vectors included marker genes such as eGFP or eBFP2. HEK293T cells were transiently transfected using these plasmids, as previously detailed, and the supernatant was collected after 48 hours, then filtered to remove any remaining 293T cells and purified using GraviTrap protein G columns. To assess binding, the target cell line that either expressed or lacked the antigen of interest (mutated C terminus) was used. Between 50,000 and 100,000 target cells were harvested into FACS tubes and incubated with 1 ml of the supernatant, both with and without the test protein, for 30 minutes with shaking. After incubation, the cells were washed and stained with a viability dye and a secondary anti-murine Fc antibody (unless specified otherwise) for 30 minutes in the dark. Binding studies were initially done using purified RPG-4 and RPG-11 binders against the Ba/F3 cell line which showed specific binding to the co-transduced construct without non-specific binding (Figure 17).

The binding of both RPG-4 and RPG-11 was also further confirmed on Marimos and UT7-TPO expressing mutated Calreticulin (Figure 18).

Biacore

The binding affinities of these single-chain variable fragments (scFvs) (RPG-11 and RPG-4) were characterized using surface plasmon resonance technology on a Biacore T200 machine. The binding kinetics analyses involved the recombinant mutant C terminus peptide of the mutated Calreticulin. For these experiments, HEK293T cells were transiently transfected to generate supernatants containing scFvs linked to a murine lgG2a and then they were purified using antibody columns (GraviTrap protein G columns, Cytiva 28-9852-55). Both binders demonstrated binding affinities in the nanomolar range with RPG-4 showing a higher sensitivity, but RPG-11 demonstrating the highest affinity (Figure 19 and Figure 20, Table 3).

ELISA

Elisa tests were conducted using the mutated form of the C-terminal segment of calreticulin and wild type Calreticulin (without the KDEL motif) as a negative control. Supernatant obtained from a simple transfection of 293T HEK cells. Additionally, an isotype control antibody and a purified antibody against the wild-type calreticulin were employed as secondary controls. There was significant binding observed at even low concentrations of the peptide (0.1 ug/ml) with the RPG- 4 binder. Both binders demonstrated strong interactions at a concentration of 1 pg/ml of the mutated C-terminus. There was no interaction detected with the wild-type calreticulin. The anti- wild-type CALR antibody specifically bound only to the wild-type CALR as expected, whereas the isotype control did not exhibit any binding to the proteins tested (Figure 21).

Conclusion

All of these studies, show that there is specific binding of both RPG-4 and RPG-1 1 against the mutated C terminus of mutated calreticulin protein, while on cell lines it only binds to that when expressed on the surface in conjunction with thrombopoietin receptor. Biacore and ELISA tests show that RPG-4 appears to be able to bind to the mutated protein even at very low levels of the protein, but at higher levels binding is similar. Both of these binders are suitable for CAR-T cell development against mutated Calreticulin. Example 6

CAR-T vs primary cells

We assessed our CAR-T killing against stem cells from MF patients. CAR-T cells were cocultured with CD34+ cells from patients with myelofibrosis either on the chronic phase, pre- fibrotic myelofibrosis, accelerated or blast phase, while CD34+ cells from patients with JAK2 mutated myelofibrosis were used as a control. Details of the patients tested are presented on table 1 with their variable allele frequency and relevant anti-mf therapies. Effector and target cells were co-cultured on 1 :1 (25,000:25,000) ratio and facs readings were obtained after 48h. Car-T cells were identified as CD3+ and CD34+ (when RQR8 was expressed as part of the CAR transgene) while primary CD34+ cells were identified as CD3- CD34+. Countbright beads were added for accurate cell measurement. CAR-T cells were obtained from 5 healthy donors and killing percentage was normalised against co-culture with non-transduced T-cells. We tested our Car-T cells from 5 healthy donors against 6 patients with chronic phase or pre-fibrotic MF, 2 patients with accelerated or blast phase MF and 4 negative controls with JAK2 mutated MF. A CD19 CAR-T cell was used as a control (Figure 22 and Table 4).

Testing in an organoid environment

To challenge the CAR-T killing we conducted a pilot co-culture experiment using the CAR-T against CD34+ isolated cells in an organoid environment. The T-cells were sourced from healthy donors. Organoids were seeded with primary cells and CAR-T cells were added 72h later at E:T ratio of 1 :1 (25,000:25,000) to the original amount of cells added. Readouts were obtained 48h afterwards. CAR-T cells were generated via four healthy donors T-cells and they were tested against 3 mutated Calreticulin chronic phase myelofibrosis patients and 2 JAK2 mutated myelofibrosis patients (Figure 23 and Table 5).

Conclusion

Both RPG-4 and RPG-11 CAR-T cells have demonstrated specific targeting and killing of CD34+ cells in patients with Myelofibrosis. Considering that the variant allele frequency (VAF) is less than 50% due to heterozygosity, the observed killing ratio likely reflects targeting solely of the malignant cell population. There was no off-target killing observed against JAK2 mutated CD34+ cells, and the negative control CAR-T cell line, FMC63, showed no activity against these cells. Interestingly, minimal to no killing was observed in two patients in accelerated and blast phases of Myelofibrosis, respectively. This finding suggests that in advanced stages of the disease, the malignancy may no longer rely on the Calreticulin mutation or may downregulate the expression of mutated Calreticulin which can be one of the tools employed for immune escape and disease progression.

Example 7

Re-stimulation, memory phenotype and exhaustion.

Our current mutant calreticulin targeting agents, RPG4 and RPG11 , have shown effectiveness and selectivity when tested against various cell lines. RPG4 is particularly responsive to cells with low levels of the target antigen (Marimo NT), maintaining consistent CD45RA expression and elevated CCR7 levels, which may suggest a higher presence of naive and mixed effector memory expressing CD45RA (TEMRA) T-cell populations. In contrast, RPG11 exhibits less sensitivity to cells with low antigen levels, with its capacity to eliminate such cells varying between donors. We performed a restimulation assay to check the resilience of both RPG-4 and RPG-11 against low antigen (Marimo NT) and high antigen (Marimo TpoR) targets.

During the re-stimulation experiment against the MarimoTpoR and Marimo NT cell line, we performed phenotypic characterisation of CD4+ and CD8+ T-cells at baseline and after each round of stimulation. We investigated also for exhaustion by measuring the exhaustion markers TIM3, LAG3 and PD1.

Methods

T-cell were isolated and transduced with the CAR constructs as described above. Transduction efficiency was assessed on Day 7 using FACS, staining with aCD34-APC, aCD3-PE/Cy7, and live/dead dye ef780. Post-staining, cells were resuspended at 210^A6/mL in PBS with celltrace violet, incubated at 37°C in the dark for 20 minutes, then diluted in R10 and incubated again for 15 minutes. Samples were normalized to the lowest transduction efficiency and adjusted to specific cell concentrations for subsequent assays. T cells were combined with target cells at designated ratios and volumes according to the experimental plan, and duplicate plates were prepared for each effector-to-target (E:T) ratio. On Days 4, 7, 1 1 , 14, and 18, 21 and 25 postsetup, one plate for each E:T ratio was processed: cells were centrifuged, supernatants removed, and cell pellets were stained with specific antibodies and counting beads, then analysed by FACS. These corresponded to stimulation rounds 1 , 2, 3, 4, 5, 6 and 7 respectively. Remaining plates were treated similarly, with new targets added for continued co-culture, repeating the process until the final stimulation. This meticulous procedure allowed for detailed monitoring and analysis of T cell functionality and response over the course of the experiment. Memory phenotype was assessed on the basis of expression of CCR7 and CD45RA as depicted in Figure 24. Exhaustion phenotype was assessed as either single positive, double positive or triple positive for expression of either of LAG3, TIM3 and PD1 while non-exhausted was triple negative.

Results (Figures 25-33)

Based on the results of the experiment, it appears that there are similar killing profiles observed in the higher Ag Marimo-TpoR. RPG11 managed to control the lower Ag Marimo-NT at a rate comparable to that of RPG4. Although RPG4 showed a generally higher CD45% and MFI, as well as increased levels of CD95 and CD62L, indicating a higher presence of naive and stem cell memory (SCM) populations, this did not translate into functional differences in this assay. Notably, RPG4 did not exhibit reduced exhaustion markers compared to RPG11. Both binders were able to kill both low and high antigen cell lines for at least 3 rounds of stimulations with RPG-11 maintaining killing up to round 5, while their exhaustion profile was similar.

Example 8

Plate bound assay

Using plate-bound antigens facilitates the assessment of CAR-T cell activation sensitivity across various antigen densities, eliminating the variability introduced by target cells. This approach also allows for the selection of specific antigen densities that trigger sub-optimal activation under different immune conditions.

In this context, the mutant C-terminal peptide of calreticulin was evaluated to determine the range of activation for RPG-11 and RPG-4 41 BBz CAR-T cells. An activation dynamic range between 19-312ng/mL was identified, with median activation points approximately at 40ng/mL for RPG-4 and 80ng/mL for RPG-11 , respectively. To achieve a median level of activation, a concentration of 60ng/mL can be used as a compromise when administering both CAR-T variants at the same time.

Methods

Recombinant mutant calreticulin C-terminus was diluted to 10ug/mL in PBS from a 1 mg/mL stock. Ten serial dilutions ranging from 10ug to 0.0195ug/mL were made in a 96-well plate, leaving two columns with only PBS as controls. After a 2-hour incubation at 37°C, the wells were washed and CAR-T cells at 25,000 cells/well were added. One PBS-only column served as a non-activated control, while the other was treated with transact as a positive control. Post a 48- hour incubation, cells were centrifuged, supernatants removed, and cell pellets stained with aCD34-APC, aCD3-PE/Cy, aCD25-APC/Cy7, and 7AAD, with counting beads included in the staining mix. Stained samples were analysed by FACS to assess CAR-T cell activation and proliferation.

Results

Results are shown in Figure 34.

Conclusion

PRG-4 CAR-T is more sensitive and is activated event at low levels of mutated Calreticulin protein. At higher levels of protein the activation of both RPG-4 and RPG-11 is comparable.

Example 9

Combination of thrombopoietin mimetics with our mutated Calreticulin targeting CAR-T therapy

Given that our CAR-T therapy targets mutated Calreticulin on the surface of malignant cells expressed with thrombopoietin receptor (tpoR), we hypothesised that it may not be effective if TpoR expression is not strong enough. We hypothesized that treatment with TpoR agonists might increase TpoR expression on AP/BP-MPN HSPCs, by promoting megakaryocytic differentiation. When we tested our CAR-T against CD34 cells isolated from myelofibrosis patients, we noticed that it is less effective against patients in the accelerated or blast phase of the disease. We have tested one of our worse responders for TpoR expression and it was significantly lower than one of our best responders. The assessment was done via flow cytometry using anti-TpoR antibody (cat no 130-128-171).

We then explored whether addition of thrombopoietin or thrombopoietin mimetics (Romiplostim and eltrombopag) could boost TpoR expression and hence mutCALR expression leading to better killing of our CAR-T cell therapy. We have co-cultured the Marimo TpoR cell line with thrombopoietin, Romiplostim and Eltrombopag. We have noticed significant increased expression of TpoR when co-cultured with 500nM of Eltrombopag.

We then performed a co-culture of our RPG4 CAR-T cells at 1 :8 ratio against the Marimo T poR in the presence of 500nM of Eltrombopag, Romiplostim and Thrombopoietin. This is a ration which is unfavourable for the CAR-Ts and the killing is significantly reduced or diminished. We have noticed improved killing when the co-cultured was performed with Eltrobopag, while Thrombopoietin or Romiplostim resulted in either no effect or worse killing. Pre-treatment of cells with Eltrombopag, but not with Romiplostim or rhTPO, increased surface TpoR expression by >50% and improved CAR-T killing, confirming that pharmacologically enhancing TpoR expression might boost vulnerability to anti-mutCALR immunotherapies.

Conclusion

By pre-treating patients with Eltrombopag or other similar thrombopoietin mimetics, TpoR expression may be increased on malignant cells which will lead to higher mutated Calreticulin expression. This will enhance the efficacy of CAR-T cells or other immunotherapies against mutated Calreticulin.

Example 10

Use of Thrombopoietin mimetics to boost function of immunotherapies against myeloproliferative neoplasms

Given that mutated Calreticulin expression is dependant on the expression of thrombopoietin receptor (TpoR), reduced TpoR expression can lead to reduce surface mutCLAR expression. Additionally, leukaemic transformation of MPN is typically myelogenous rather than megakaryoblastic in phenotype, and hence there might be reduced killing against accelerated or blast phase (AP/BP) malignant cells due to lower cell surface expression of TPOR, and therefore mutCALR, on leukaemic blasts. In keeping with this, we detected lower TPOR expression on AP/BP-MPN samples who were ‘poor responders’ to the CAR-T therapy in vitro than patients who were ‘responders’. We hypothesized that pharmacological TPOR stimulation may promote mutCALR expression by driving megakaryocytic differentiation of blasts. Several TPOR agonists are currently in clinical use for the treatment of thrombocytopenia, including small molecule agonists (e.g. eltrombopag) and peptibody therapies (e.g. romiplostim). We evaluated the impact of recombinant human TPO (rhTPO), eltrombopag and romiplostim on cell surface TPOR expression of the Marimo cell line transduced with TpoR and on patient samples. Unexpectedly, while rhTPO drove internalisation of the TPOR, eltrombopag markedly increased TPOR expression and this corresponded with increased mutCALR CAR-T cell mediated killing of both cell lines and patient samples.

Results

Results are shown in Figures 38-41 . Conclusion

Thrombopoietin mimetics can boost TpoR expression which can lead to increased surface mutCALR expression on malignant cells. This can be particularly useful to enhance immunotherapeutic approaches such as CAR-T cells, Bispecific T-cell engagers or monoclonal antibodies targeting mutated Caltericulin on malignant cells.

Example 11

Humanized sequences for RPG4

Introduction

We generated humanised sequences using RPG4 as the template for the antibody generation. Humanised scfvs have less immunogenicity and are potentially more effective. Hence we generated 16 in silico humanised sequences.

Methods

Initially, a homology model of the parental VH and VL regions was constructed in a single chain Fv (scFv) format. The modelling process comprised four main stages: (1) gathering homologous sequences, (2) scanning a fold library, (3) loop modeling, and (4) side chain placement. The resulting three-dimensional model provided the structural framework that guided the subsequent selection of “donor” and “acceptor” amino acids during the humanization process.

For humanization, the parental VH and VL sequences were aligned with a panel of human germline sequences. This panel was pre-filtered to exclude sequences harbouring unwanted liabilities, specifically N-linked glycosylation sites and free cysteine residues. From this curated set, the closest matching germline sequences from two distinct VH and VL families were identified.

A humanization algorithm was then employed to graft the complementarity-determining regions (CDRs) and framework amino acids from the parental (donor) sequences onto the selected human (acceptor) germline sequences. This process yielded four VH and four VL sequence variants, resulting in a total of 16 potential antibody combinations. These variants were ranked in priority order, and the percentage identity to the human germline was calculated using the IMGT Domain Gap Align tool.

Results

Results are shown below (SEQ ID NO: 71-112; table 6). Example 12: NSG MarimoTpoR Survival with RPG-4

Eighteen NSG (NOD.Cg-PrkdcSCID H2rgtm1 Wjl/SzJ) mice obtained from Charles River UK were divided into 2 cohorts, each comprising 6 mice. All mice were administered 100,000 cells of the Marimo cell line transduced with Thrombopoietin receptor and Flue. Cohorts A and B received 5x10^A6 RPG-4 CAR-T cells and FMC63 CAR-T cells, respectively, on Day 1 post cell line injection. Throughout the experiment, mice were closely monitored using clinical scoring as per the animal license protocol, regular weight measurements, and BLI imaging. Humane care was provided to all animals, and all experimental procedures adhered to local guidelines. The mice were housed in a controlled environment with 12-hour light-dark cycles, humidity maintained between 30-70%, and an ambient temperature ranging from 20-26 degrees Celsius. Both study and control animals were housed in the same room, within a specific pathogen-free facility. The health status of the mice was assessed through clinical scoring and observations outlined in the project license protocol. Weight measurements were recorded every 2-3 days, and BLI imaging was performed twice weekly with an average radiance of 1*10^A7 triggering euthanasia using CO2 overdose followed by cervical dislocation. For bioluminescence imaging, D-luciferin firefly potassium salt was dissolved in sterile PBS at a concentration of 10mg/ml and injected intraperitoneally (200 pl per mouse) into anesthetized mice. The average radiance was measured using a Xenogen VivoVision I VIS Lumina camera. Survival analysis was conducted using Kaplan-Meier curves.

Results

Survival was significantly improved with RPG-4 treated mice in comparison to mice who were treated with FMC63 control CAR-T. See Figure 42 and 43.

Example 12: RPG4 CAR-T reduces the mutation burden in mutated primary cells and cell lines

Introduction

We have demonstrated that RPG4 CAR T cells could eliminate between 60-75% of HSPCs from mutCALR myelofibrosis patients. No significant difference in killing was observed between type- 1 or type-2 mutCALR subtypes. No cytotoxicity of JAK2V617F+ myelofibrosis cells was seen. Patients with myelofibrosis generally have heterozygous mutCALR clones and some residual wild-type haematopoiesis hence we sought to quantify the reduction of the mutation burden of myelofibrosis primary cells when co-cultured with CAR-T cells and demonstrate that RPG4 CAR- T selectively eliminates only the malignant cells. Methods

Following co-culture of effector and target cells (primary HSPCs or cell lines), live target cells were sorted using a BD FACSAria Fusion Cell Sorter with FACSDIVA software. Gating of live target cells was determined by forward scatter size profile, CD3 negativity, and celltrace positivity. DNA of target cells was extracted using the QIAamp DNA Micro kit (Qiagen, Cat#56304).

Type 1 and 2 CALR mutations were quantified by droplet digital PCR (ddPCR) using the BioRad QX200 AutoDG Droplet Digital PCR System. Both reactions used the following primers at 900 nM (forward primer 5’-GCAGCAGAGAAACAAATGAAG-3’ (SEQ ID NO: 113) and reverse primer 5’-TCCTCATCCTCCTCATCC-3’) (SEQ ID NO: 114) and 5 units of Haelll restriction enzyme (New England Biolabs, Cat# R0108S). Analysis of ddPCR data was done using QX Manager Software, Standard Edition, v1.2 (Bio-Rad Laboratories).

To detect type 1 CALR mutations, the following probes were used at 250 nM final concentration (WT: /5HEX/CTCCTTAAGCCTCTGCTCCTCG/3BHQ_1 (SEQ ID NO: 115) and Mutant: Z56- FAM/CTCCTTGTCCTCTGCTCCTCG/3BHQ_1/) (SEQ ID NO: 116) with the following cycling protocol: initial enzyme activation for 10 min at 95 °C, 43 cycles of denaturation for 30 sec at 94 °C, annealing/extension for 90 sec at 55 °C and enzyme deactivation for 10 min at 98 °C (ramp rate 2 °C/s at all steps).

To detect type 2 CALR mutations, the following probes were used at 250 nM final concentration (WT: /5HEX/TGTCCTCATCATCCTCCTTGTCCTC/3BHQ_1/ (SEQ ID NO: 117) and Mutant: /56-FAM/CTCATCATCCTCCGACAATTGTCCTC/3BHQ_1/) (SEQ ID NO: 118) with the following cycling protocol: initial enzyme activation for 10 min at 95 °C, 43 cycles of denaturation for 30 sec at 94 °C, annealing/extension for 90 sec at 54 °C and enzyme deactivation for 10 min at 98 °C (ramp rate 2 °C/sec at all steps).

Type 1 -like CALR mutations were quantified following amplification of CALR exon 9 using the previously listed primer set. Samples were run on a high sensitivity D1000 ScreenTape using the Agilent 2200 Tapestation automated electrophoresis system and the A.0202 SR1 software version (Agilent Technologies).

Results

Quantifying the mutant clone burden (variant allele frequency, VAF) before and after CAR T cell co-culture showed a reduction in VAF from 78.9% to 20.2%, only in samples from mutCALR+ patients, reflecting 50-100% clearance of the mutant clone. RPG4 CAR T cells could also clear CALR mutated cells when present at both low and high frequencies, RPG4 CAR T cells were tested against mixtures of mutCALR+ and - cells. RPG4 CAR-T cells depleted 96.7% of mutCALR+ cells when present at only 10% frequency among wild-type haematopoietic cells. See Figures 44 to 46.

Conclusion

Our RPG4 CAR-T cell is able to selectively eliminate the malignant clone by targeting only the mutated primary cells or cell lines. A significant percentage reduction of mutation burden of 50- 100% was achieved in all cases when our RPG4 CAR-T was co-cultured with different percentages of mutated cells.

Example 13: Determining clearing of malignant CD34+ cells from mutated Calreticulin myelofibrosis patients using single cell RNA sequence in bone marrow organoids

Introduction

The human organoid model provided a unique opportunity to examine the functional impact and molecular consequences of CAR-T cell-mediated killing on healthy and malignant haematopoietic clones within the bone marrow microenvironment. Single cell RNA sequencing (scRNAseq) was performed on human organoids engrafted with CD34+ HSPCs from a patient with high-risk mutCALR+ myelofibrosis, with and without addition of RPG-4 CAR-T cells generated from a healthy donor and engrafted 24 hours later. In this system, the haematopoietic niche, myelofibrosis cells and CAR-T cells each derived from different donors, therefore we were able to genetically de-multiplex each cellular compartment using single nucleotide polymorphisms unique to each donor. After QC, 56,896 cells were included in the analysis, including 36,038 iPSC-derived organoid cells, 13,769 myelofibrosis cells and 7,089 CAR-T cells. Cell types were annotated using canonical marker genes

Methods

Single-cell RNA sequencing

Organoids were pooled together by condition (n = 12) and dissociated using Collagenase D on day 18 of the experiment. Live cells were FACS-sorted on a BD FACSAria Fusion (100 pm nozzle, < 5 minutes per sample) and 30,000-40,000 cells were processed using the Chromium Single Cell 5’ High Throughput kit v2, Dual Index (PN-1000263) and reagents according to the 10x Genomics protocol (CG000331). Gel bead-in-Emulsions (GEMs) were generated using the Chromium Controller and reverse transcription was performed within GEMs. GEM-RT products were demulsified and purified, followed by cDNA QC and amplification. Gene Expression Libraries (GEX) were prepared using 100 ng input cDNA and paired-end 150 bp sequencing was performed on a NovaSeq X Plus.

Analysis of single cell RNA sequencing

Demultiplexed FASTQ files were aligned to a custom human reference genome (GRCh38/hg38) that included the CAR-T construct (CellRanger pipeline, 10x Genomics). Post-processing steps included exclusion of technical artifacts and ambient RNA (CellBender), SNP-based genetic demultiplexing and doublet exclusion (Souporcell), and CALR mutation detection (VarTrix). Cells meeting the following QC parameters were included in downstream analyses: percentage of mitochondrial gene expression < 15%, gene expression within 3 median absolute deviations, and identified as a genotypic singlet by Souporcell. Cells passing QC filters were subset based on genotype (MF primary patient cells, iPSC-derived organoid cells, healthy donor CAR-T) and separate Seurat objects were normalized (log vst), scaled, and annotated using reference datasets.

Results

In organoids where RPG4 mutCALR CAR-T cells had been added, >95% of patient cells were wild-type, indicating robust clearance of the mutant clone including of mutant stem/progenitor cells as well as myeloid progenitor clusters, with regeneration of wild-type haematopoiesis

The differential abundance and transcriptional phenotypes of the CAR-T cells engrafted into organoids alone vs. engrafted into organoids containing mutCALR+ myelofibrosis cells were examined. This revealed strong enrichment of activation markers and inflammatory signalling pathways including TNF, IFNg and IL2-STAT in CAR-T cells that had been exposed to mutCALR target cells. See Figure 47-50.

Conclusion

Single cell RNA sequencing showed that RPG4 CAR-T cell is able to selectively eliminate the malignant mutCALR stem cells and myeloid erythroid progenitor cells without impacting the normal wild-type stem cells. This was demonstrated in a 3d bone marrow organoid which is able to emulate the human bone marrow microenvironment.

Table 2 Sequences of H1 and M2 binders from Elstar Therapeutics Inc., patent application US20210137982A1

Table 3. Binding kinetic numbers for binders RPG-11 and RPG-4

Quality

Kinetics Binder 1 : 1 binding

Kinetics Chi² kd KD (M) model (scFv) ka

(RU²)

1: 1 binding RPG-11 3.70e-02 4.64e+04 1.75e-03 3.76e-08

1: 1 binding RPG-4 2.08e-02 1.09e+05 1.77e-03 1.63e-08 Table 4 Patient characteristics of primary cells used in 2d co-culture

PATIENT DIAGNOSIS DRIVER OTHER TREATMENT

MUTATION MUTATIONS

1 Myelofibrosis CALR ins5 ASXL1 , EZH2

2 i Myelofibrosis CLAR del52 CHEK2

3 i Myelofibrosis CALR del 46 CBL, DNMT3A,

4 i Myelofibrosis CALR ins5 Nil Peg- Interferon i myelofibrosis a

7 i Accelerated CALR ins5 i phase i myelofibrosis

(9% blasts)

8 Blast phase CALR ins5 ASXL1 myelofibrosis

| (22%)

9 i Myelofibrosis JAK2 V617F ASXL1

10 i Post PV JAK2 V617F i myelofibrosis (VAF 95%)

11 i Pre-fibrotic JAK2 V617F ASXL1 , EZH2, i myelofibrosis ZRSR1

12 Myelofibrosis JAK2 V617F ASXL1 , SRSF2, Ruxolitinib and

(73%) TET2 Navitoclax or

Placebo Table 5 Patient characteristics of primary cells used in 3d organoid co-culture

PATIENT DIAGNOSIS DRIVER OTHER TREATMENT

SEQ ID NO: 52 H1 VH

CAAGTCCAGCTTGTCCAATCCGGGGCGGAGGTGAAGAAGCCGGGTGCGAGTGTCAAGG

TTAGTTGTAAGGCGAGTGGCTACTCATTCACGGGATATTATATTCACTGGGTGAGACAGG

CGCCAGGGCAGGAGCTCGGGTGGATGGGTTATATATCATGCTATAACGGCGCCTCCAGC

TACAATCAAAAGTTCAAAGGAAGGGTTACGATGACGGTAGACACCAGTATCTCCACGGCT

TACACTGAACTTTCAAGTTTGCGGAGTGAGGATACGGCGACCTATTACTGCGCTTCCAGT

ATGGACTATTGGGGTCAAGGCACACTGGTGACAGTTAGCAGT

SEQ ID NO: 53 H1 VL

GACGTAGTTATGACACAATCCCCACTCAGCCTCCCGGTAACCCTGGGTCAACCAGCAAGT

ATATCATGCAAGTCAAGCCAATCCTTGCTTGATAGTGATGGCAAGACATACCTTAACTGGC

TCCAACAAAGGCCGGGCCAAAGTCCTCGACGACTTATATATCTTGTTTCTAAGCTGGACA

GCGGTGTACCTGACCGCTTCAGTGGCTCAGGAAGTGGGACCGACTTTACGCTGAAGATT

AGCCGCGTGGAAGCGGAAGACGTGGGCGTTTATCATTGTTGGCAGGGAACCCACTTTCC

TTACACCTTTGGGGGAGGCACAAAAGTGGAGATTAAG

SEQ ID NO: 54 M2 VH

GATGTACAACTGCAGGAAAGTGGACCAGGTTTGGTCAAAAACTCACAGTCTCTCAGCTTG

ACCTGTACCGTCACAGGATATTCTATAACTAGTGACTACGCATGGAATTGGATACGACAAT

TTCCCGGCAATAAACTTGAATGGATGGGATATATCTCCTATTCCGGCTCAACTAGTTACAA

CCCTTCACTGAAATCTAGAATCTCAATAACTCGGGACACAAGTAAAAACCAGTTTTTTTTG

CAACTGAACTCAGTTACTCCTGAGGATACCGCCACCTATTATTGCGCTAGAGATCCCCCG

TATTACTACGGGTCCAATGGTACCTGGGGGCAGGGTACATCTGTGACAGTGTCTTCC SEQ ID NO: 55 M2 VL

GATGTCGTAATGACCCAAACCCCTTTGACGCTCTCTGTTACAATTGGACAGCCCGCGAGT

ATTAGTTGCAAGAGTAGCCAGAGTTTGCTCGACAGTGATGGAAAAACGTACTTGAACTGG

CTCCTTCAACGGCCAGGCCAAAGCCCGAAAAGGCTCATATATCTGGTAAGCAAGCTGGA

CAGTGGAGTTCCCGATAGGTTCACCGGATCAGGTTCAGGTACTGATTTTACCTTGAAAATT

TCCCGAGTGGAAGCCGAGGATTTGGGCGTGTATCACTGTTGGCAGGGCACCCATTTTCC

TTACACTTTTGGAGGTGGGACAAAGCTCGAGATAAAG

SEQ ID NO: 56 Complete Single chain variable fragment AA sequence including signal sequence, serine glycine linker and murine lgG2a Fc domain (RPG-4)

METDTLLLWV LLLWVDGSTG DVQLQESGPG LVKPSQSLSL TCTVTGYSIT

SDYAWNWIRQ 60

FPGNKLEWMG YIDYSGNTNY NPSLKSRISI TRDTSKNQFF LQLSSVTTED

TGTYYCSRGI 120

TGYWGRGTTL TVSSGGGGSG GGGSGGGGSQ AWTQESALT TSPGETVTLT

CRSSTGAITT 180

SNYANWVQEK SDHLFTGLIG GAKNRAPGVP ARFSGSLIGD KAALTITGAQ

TEDEAIYFCA 240

LWYSNHWVFG GGTKLTVLSD PRGPTIKPCP PCKCPAPNLL GGPSVFIFPP

KIKDVLMISL 300

SPIVTCVVVD VSEDDPDVQI SWFVNNVEVH TAQTQTHRED YNSTLRWSA

LPIQHQDWMS 360

GKEFKCKVNN KDLPAPIERT ISKPKGSVRA PQVYVLPPPE EEMTKKQVTL

TCMVTDFMPE 420

DIYVEWTNNG KTELNYKNTE PVLDSDGSYF MYSKLRVEKK NWVERNSYSC

SVVHEGLHNH 480

HTTKSFSRTP GK 492

SEQ ID NO: 57 DNA sequence of SEQ ID NO: 56 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtcgacgg cagcaccgga 60 gacgtacagc ttcaggaaag tggccccggt ctcgtcaagc caagccagag cctttcactt 120 acctgcacag ttacgggcta ttcaatcacg tccgactacg cttggaattg gataagacaa 180 tttccaggta ataaactgga gtggatgggc tacatcgact actctggaaa cactaattac 240 aatcctagcc tcaaaagcag aatatctatt actagggata cgtccaaaaa ccagtttttt 300 cttcaactca gttcagtgac gacggaagat accgggacgt attactgctc tcggggaatt 360 accggatatt ggggacgagg cactaccctg accgtgtcaa gcggcggagg cggaagtggc 420 ggagggggat caggcggggg aggatctcaa gcggtagtta cgcaagaatc cgcgttgacc 480 acgagccctg gcgaaaccgt gacgctcacc tgccgatctt ctacgggcgc aattaccacc 540 agtaattacg caaattgggt acaagagaaa tctgatcatt tgttcacggg attgatcgga 600 ggagcgaaga accgggcccc tggcgtacca gcccgctttt ctggcagtct tataggagac 660 aaggctgctc tcactattac gggtgcccag acggaggacg aagcaatcta tttttgcgca 720 ctctggtatt caaatcattg ggtctttgga ggtgggacca aactgaccgt cctttcggat 780 cccagagggc ccacaatcaa gccctgtcct ccatgcaaat gcccagcacc taacctcttg 840 ggtggaccat ccgtcttcat cttccctcca aagatcaagg atgtactcat gatctccctg 900 agccccatag tcacatgtgt ggtggtggat gtgagcgagg atgacccaga tgtccagatc 960 agctggtttg tgaacaacgt ggaagtacac acagctcaga cacaaaccca tagagaggat 1020 tacaacagta ctctccgggt ggtcagtgcc ctccccatcc agcaccagga ctggatgagt 1080 ggcaaggagt tcaaatgcaa ggtcaacaac aaagacctcc cagcgcccat cgagagaacc 1140 atctcaaaac ccaaagggtc agtaagagct ccacaggtat atgtcttgcc tccaccagaa 1200 gaagagatga ctaagaaaca ggtcactctg acctgcatgg tcacagactt catgcctgaa 1260 gacatttacg tggagtggac caacaacggg aaaacagagc taaactacaa gaacactgaa 1320 ccagtcctgg actctgatgg ttcttacttc atgtacagca agctgagagt ggaaaagaag 1380 aactgggtgg aaagaaatag ctactcctgt tcagtggtcc acgagggtct gcacaatcac 1440 cacacgacta agagcttctc ccggactccg ggtaaatga 1479

SEQ ID NO: 58 CAR sequence of the above binder including 4-1 BB, CD3z and RQR8

MGTSLLCWMA LCLLGADHAD ACPYSNPSLC SGGGGSELPT QGTFSNVSTN

VSPAKPTTTA 60

CPYSNPSLCS GGGGSPAPRP PTPAPTIASQ PLSLRPEACR PAAGGAVHTR

GLDFACDIYI 120

WAPLAGTCGV LLLSLVITLY CNHRNRRRVC KCPRPWRAE GRGSLLTCGD

VEENPGPMET 180

DTLLLWVLLL WVDGSTGDVQ LQESGPGLVK PSQSLSLTCT VTGYSITSDY

AWNWIRQFPG 240 NKLEWMGYID YSGNTNYNPS LKSRISITRD TSKNQFFLQL SSVTTEDTGT

YYCSRGITGY 300

WGRGTTLTVS SGGGGSGGGG SGGGGSQAW TQESALTTSP GETVTLTCRS STGAITTSNY 360

ANWVQEKSDH LFTGLIGGAK NRAPGVPARF SGSLIGDKAA LTITGAQTED

EAIYFCALWY 420

SNHWVFGGGT KLTVLSDPTT TPAPRPPTPA PTIASQPLSL RPEACRPAAG

GAVHTRGLDF 480

ACDIYIWAPL AGTCGVLLLS LVITLYCKRG RKKLLYIFKQ PFMRPVQTTQ

EEDGCSCRFP 540

EEEEGGCELR VKFSRSADAP AYQQGQNQLY NELNLGRREE YDVLDKRRGR

DPEMGGKPRR 600

KNPQEGLYNE LQKDKMAEAY SEIGMKGERR RGKGHDGLYQ GLSTATKDTY

DALHMQALPP 660

R 661

SEQ ID NO: 59 DNA sequence of SEQ ID NO: 58 atgggcacca gcctgctgtg ctggatggcc ctgtgcctgc tgggcgccga ccacgccgat 60 gcctgcccct acagcaaccc cagcctgtgc agcggaggcg gcggcagcga gctgcccacc 120 cagggcacct tctccaacgt gtccaccaac gtgagcccag ccaagcccac caccaccgcc 180 tgtccttatt ccaatccttc cctgtgtagc ggagggggag gcagcccagc ccccagacct 240 cccaccccag cccccaccat cgccagccag cctctgagcc tgagacccga ggcctgccgc 300 ccagccgccg gcggcgccgt gcacaccaga ggcctggatt tcgcctgcga tatctacatc 360 tgggccccac tggccggcac ctgtggcgtg ctgctgctga gcctggtgat caccctgtac 420 tgcaaccacc gcaaccgcag gcgcgtgtgc aagtgcccca ggcccgtggt gagagccgag 480 ggcagaggca gcctgctgac ctgcggcgac gtggaggaga acccaggccc catggagacc 540 gacaccctgc tgctgtgggt gctgctgctg tgggtcgacg gcagcaccgg agacgtacag 600 cttcaggaaa gtggccccgg tctcgtcaag ccaagccaga gcctttcact tacctgcaca 660 gttacgggct attcaatcac gtccgactac gcttggaatt ggataagaca atttccaggt 720 aataaactgg agtggatggg ctacatcgac tactctggaa acactaatta caatcctagc 780 ctcaaaagca gaatatctat tactagggat acgtccaaaa accagttttt tcttcaactc 840 agttcagtga cgacggaaga taccgggacg tattactgct ctcggggaat taccggatat 900 tggggacgag gcactaccct gaccgtgtca agcggcggag gcggaagtgg cggaggggga 960 tcaggcgggg gaggatctca agcggtagtt acgcaagaat ccgcgttgac cacgagccct 1020 ggcgaaaccg tgacgctcac ctgccgatct tctacgggcg caattaccac cagtaattac 1080 gcaaattggg tacaagagaa atctgatcat ttgttcacgg gattgatcgg aggagcgaag 1140 aaccgggccc ctggcgtacc agcccgcttt tctggcagtc ttataggaga caaggctgct 1200 ctcactatta cgggtgccca gacggaggac gaagcaatct atttttgcgc actctggtat 1260 tcaaatcatt gggtctttgg aggtgggacc aaactgaccg tcctttcgga tcccaccacc 1320 accccagccc cacggccacc tacccctgcc ccaaccatcg ccagccagcc cctgagcctg 1380 cggcctgaag cctgcaggcc tgccgccgga ggagccgtgc acacaagggg cctggacttc 1440 gcctgcgaca tctatatctg ggcccccctg gccgggacat gcggggtgct gctgctgtcc 1500 ctggtgatta cactgtattg caaacggggc cggaagaagc tgctgtacat cttcaagcag 1560 cccttcatgc ggcccgtgca gaccacccag gaggaggacg gctgcagctg ccggttcccc 1620 gaggaagagg aaggcggctg cgagctgcgg gtgaagttca gccggagcgc cgacgcccca 1680 gcctaccagc agggccagaa ccagctgtac aacgagctga acctgggacg gcgggaggag 1740 tacgacgtgc tggacaagcg gcggggacgg gaccccgaga tgggcggcaa gcctcgccgg 1800 aagaatcccc aggagggcct gtacaacgag ctgcagaagg acaagatggc cgaggcctac 1860 agcgagatcg gcatgaaggg cgagcggcgc cggggcaagg gccacgacgg cctgtaccag 1920 ggcctgagca ccgccaccaa ggacacctac gacgccctgc acatgcaggc cctgccaccc 1980 cggtga 1986

SEQ ID NO: 60 Complete Single chain variable fragment AA sequence including signal sequence, serine glycine linker and murine lgG2a Fc domain (RPG-11)

METDTLLLWV LLLWVDGSTG QVQLQQSGAE LVKPGSSVKI SCKASGYTFT RNFIHWIKQQ 60

PGNGLEWIGW IFPGDGDTEY NQKFNGKATL TADKSSSTAY MQLSSLTSED SAVYFCARGN 120

YNYEYFDYWG QGVMVTVSSG GGGSGGGGSG GGGSDIQMTQ SPASLSASLG ETVSIECLAS 180

EDIYSYLAWY QQKPGKSPQL LIFAANRLQD GVPSRFSGSG SGTQFSLKIS

GMQPEDEGDY 240 FCLQGSKFPY TFGPGTKLEL NSDPRGPTIK PCPPCKCPAP NLLGGPSVFI FPPKIKDVLM 300

ISLSPIVTCV VVDVSEDDPD VQISWFVNNV EVHTAQTQTH REDYNSTLRV VSALPIQHQD 360

WMSGKEFKCK VNNKDLPAPI ERTISKPKGS VRAPQVYVLP PPEEEMTKKQ VTLTCMVTDF 420

MPEDIYVEWT NNGKTELNYK NTEPVLDSDG SYFMYSKLRV EKKNWVERNS YSCSVVHEGL 480

HNHHTTKSFS RTPGK 495

SEQ ID NO: 61 DNA sequence of SEQ ID NO: 60 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtcgacgg cagcaccgga 60 caggtacagc tgcagcaatc tggggctgaa ctggtgaagc ctgggtcctc agtgaaaatt 120 tcctgcaagg cttctggcta caccttcacc cgtaacttta tacactggat aaaacagcag 180 cctggaaatg gccttgagtg gattgggtgg atttttcctg gagatggtga tacagagtac 240 aatcaaaagt tcaatgggaa ggcaacactc actgcagaca aatcgtccag cacagcctat 300 atgcagctca gcagcctgac atctgaggac tctgcagtct atttctgtgc aagaggaaat 360 tacaactacg agtactttga ttactggggc caaggagtca tggtcacagt ctccagcggc 420 ggaggcggaa gtggcggagg gggatcaggc gggggaggat ctgacatcca gatgacacag 480 tctccggctt ccctgtctgc atctctggga gaaactgtct ccatcgagtg tctagcaagt 540 gaggacattt acagttattt agcatggtat caacagaagc cagggaaatc tcctcagctc 600 ctgatctttg ctgcaaatag gttgcaagat ggggtcccat cacggttcag tggcagtgga 660 tctggcacac agttttctct caagatcagc ggcatgcaac ctgaagatga aggggattat 720 ttctgtctac agggttccaa gtttccgtac acctttggac ctgggaccaa gctggaactg 780 aactcggatc ccagagggcc cacaatcaag ccctgtcctc catgcaaatg cccagcacct 840 aacctcttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg 900 atctccctga gccccatagt cacatgtgtg gtggtggatg tgagcgagga tgacccagat 960 gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac acaaacccat 1020 agagaggatt acaacagtac tctccgggtg gtcagtgccc tccccatcca gcaccaggac 1080 tggatgagtg gcaaggagtt caaatgcaag gtcaacaaca aagacctccc agcgcccatc 1140 gagagaacca tctcaaaacc caaagggtca gtaagagctc cacaggtata tgtcttgcct 1200 ccaccagaag aagagatgac taagaaacag gtcactctga cctgcatggt cacagacttc 1260 atgcctgaag acatttacgt ggagtggacc aacaacggga aaacagagct aaactacaag 1320 aacactgaac cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctgagagtg 1380 gaaaagaaga actgggtgga aagaaatagc tactcctgtt cagtggtcca cgagggtctg 1440 cacaatcacc acacgactaa gagcttctcc cggactccgg gtaaatga 1488

SEQ ID NO: 62 Complete Single chain variable fragment AA sequence including signal sequence, serine glycine linker and murine lgG2a Fc domain (H1)

METDTLLLWV LLLWVDGSTG QVQLVQSGAE VKKPGASVKV SCKASGYSFT GYYIHWVRQA 60

PGQELGWMGY ISCYNGASSY NQKFKGRVTM TVDTSISTAY TELSSLRSED TATYYCASSM 120

DYWGQGTLVT VSSGGGGSGG GGSGGGGSDV VMTQSPLSLP VTLGQPASIS

CKSSQSLLDS 180

DGKTYLNWLQ QRPGQSPRRL IYLVSKLDSG VPDRFSGSGS GTDFTLKISR VEAEDVGVYH 240

CWQGTHFPYT FGGGTKVEIK SDPRGPTIKP CPPCKCPAPN LLGGPSVFIF PPKIKDVLMI 300

SLSPIVTCVV VDVSEDDPDV QISWFVNNVE VHTAQTQTHR EDYNSTLRW

SALPIQHQDW 360

MSGKEFKCKV NNKDLPAPIE RTISKPKGSV RAPQVYVLPP PEEEMTKKQV

TLTCMVTDFM 420

PEDIYVEWTN NGKTELNYKN TEPVLDSDGS YFMYSKLRVE KKNWVERNSY

SCSVVHEGLH 480

NHHTTKSFSR TPGK 494

SEQ ID NO: 63 DNA sequence of SEQ ID NO: 62 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtcgacgg cagcaccgga 60 caagtccagc ttgtccaatc cggggcggag gtgaagaagc cgggtgcgag tgtcaaggtt 120 agttgtaagg cgagtggcta ctcattcacg ggatattata ttcactgggt gagacaggcg 180 ccagggcagg agctcgggtg gatgggttat atatcatgct ataacggcgc ctccagctac 240 aatcaaaagt tcaaaggaag ggttacgatg acggtagaca ccagtatctc cacggcttac 300 actgaacttt caagtttgcg gagtgaggat acggcgacct attactgcgc ttccagtatg 360 gactattggg gtcaaggcac actggtgaca gttagcagtg gcggaggcgg aagtggcgga 420 gggggatcag gcgggggagg atctgacgta gttatgacac aatccccact cagcctcccg 480 gtaaccctgg gtcaaccagc aagtatatca tgcaagtcaa gccaatcctt gcttgatagt 540 gatggcaaga cataccttaa ctggctccaa caaaggccgg gccaaagtcc tcgacgactt 600 atatatcttg tttctaagct ggacagcggt gtacctgacc gcttcagtgg ctcaggaagt 660 gggaccgact ttacgctgaa gattagccgc gtggaagcgg aagacgtggg cgtttatcat 720 tgttggcagg gaacccactt tccttacacc tttgggggag gcacaaaagt ggagattaag 780 tcggatccca gagggcccac aatcaagccc tgtcctccat gcaaatgccc agcacctaac 840 ctcttgggtg gaccatccgt cttcatcttc cctccaaaga tcaaggatgt actcatgatc 900 tccctgagcc ccatagtcac atgtgtggtg gtggatgtga gcgaggatga cccagatgtc 960 cagatcagct ggtttgtgaa caacgtggaa gtacacacag ctcagacaca aacccataga 1020 gaggattaca acagtactct ccgggtggtc agtgccctcc ccatccagca ccaggactgg 1080 atgagtggca aggagttcaa atgcaaggtc aacaacaaag acctcccagc gcccatcgag 1140 agaaccatct caaaacccaa agggtcagta agagctccac aggtatatgt cttgcctcca 1200 ccagaagaag agatgactaa gaaacaggtc actctgacct gcatggtcac agacttcatg 1260 cctgaagaca tttacgtgga gtggaccaac aacgggaaaa cagagctaaa ctacaagaac 1320 actgaaccag tcctggactc tgatggttct tacttcatgt acagcaagct gagagtggaa 1380 aagaagaact gggtggaaag aaatagctac tcctgttcag tggtccacga gggtctgcac 1440 aatcaccaca cgactaagag cttctcccgg actccgggta aatga 1485

SEQ ID NO: 64 CAR sequence of the above binder including 4-1 BB, CD3z and RQR8

MGTSLLCWMA LCLLGADHAD ACPYSNPSLC SGGGGSELPT QGTFSNVSTN VSPAKPTTTA 60

CPYSNPSLCS GGGGSPAPRP PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDIYI 120

WAPLAGTCGV LLLSLVITLY CNHRNRRRVC KCPRPWRAE GRGSLLTCGD VEENPGPMET 180 DTLLLWVLLL WVDGSTGQVQ LVQSGAEVKK PGASVKVSCK ASGYSFTGYY

IHWVRQAPGQ 240

ELGWMGYISC YNGASSYNQK FKGRVTMTVD TSISTAYTEL SSLRSEDTAT

YYCASSMDYW 300

GQGTLVTVSS GGGGSGGGGS GGGGSDWMT QSPLSLPVTL GQPASISCKS

SQSLLDSDGK 360

TYLNWLQQRP GQSPRRLIYL VSKLDSGVPD RFSGSGSGTD FTLKISRVEA

EDVGVYHCWQ 420

GTHFPYTFGG GTKVEIKSDP TTTPAPRPPT PAPTIASQPL SLRPEACRPA

AGGAVHTRGL 480

DFACDIYIWA PLAGTCGVLL LSLVITLYCK RGRKKLLYIF KQPFMRPVQT

TQEEDGCSCR 540

FPEEEEGGCE LRVKFSRSAD APAYQQGQNQ LYNELNLGRR EEYDVLDKRR

GRDPEMGGKP 600

RRKNPQEGLY NELQKDKMAE AYSEIGMKGE RRRGKGHDGL YQGLSTATKD

TYDALHMQAL 660

PPR 663

SEQ ID NO: 65 DNA sequence of SEQ ID NO: 64 atgggcacca gcctgctgtg ctggatggcc ctgtgcctgc tgggcgccga ccacgccgat 60 gcctgcccct acagcaaccc cagcctgtgc agcggaggcg gcggcagcga gctgcccacc 120 cagggcacct tctccaacgt gtccaccaac gtgagcccag ccaagcccac caccaccgcc 180 tgtccttatt ccaatccttc cctgtgtagc ggagggggag gcagcccagc ccccagacct 240 cccaccccag cccccaccat cgccagccag cctctgagcc tgagacccga ggcctgccgc 300 ccagccgccg gcggcgccgt gcacaccaga ggcctggatt tcgcctgcga tatctacatc 360 tgggccccac tggccggcac ctgtggcgtg ctgctgctga gcctggtgat caccctgtac 420 tgcaaccacc gcaaccgcag gcgcgtgtgc aagtgcccca ggcccgtggt gagagccgag 480 ggcagaggca gcctgctgac ctgcggcgac gtggaggaga acccaggccc catggagacc 540 gacaccctgc tgctgtgggt gctgctgctg tgggtcgacg gcagcaccgg acaagtccag 600 cttgtccaat ccggggcgga ggtgaagaag ccgggtgcga gtgtcaaggt tagttgtaag 660 gcgagtggct actcattcac gggatattat attcactggg tgagacaggc gccagggcag 720 gagctcgggt ggatgggtta tatatcatgc tataacggcg cctccagcta caatcaaaag 780 ttcaaaggaa gggttacgat gacggtagac accagtatct ccacggctta cactgaactt 840 tcaagtttgc ggagtgagga tacggcgacc tattactgcg cttccagtat ggactattgg 900 ggtcaaggca cactggtgac agttagcagt ggcggaggcg gaagtggcgg agggggatca 960 ggcgggggag gatctgacgt agttatgaca caatccccac tcagcctccc ggtaaccctg 1020 ggtcaaccag caagtatatc atgcaagtca agccaatcct tgcttgatag tgatggcaag 1080 acatacctta actggctcca acaaaggccg ggccaaagtc ctcgacgact tatatatctt 1140 gtttctaagc tggacagcgg tgtacctgac cgcttcagtg gctcaggaag tgggaccgac 1200 tttacgctga agattagccg cgtggaagcg gaagacgtgg gcgtttatca ttgttggcag 1260 ggaacccact ttccttacac ctttggggga ggcacaaaag tggagattaa gtcggatccc 1320 accaccaccc cagccccacg gccacctacc cctgccccaa ccatcgccag ccagcccctg 1380 agcctgcggc ctgaagcctg caggcctgcc gccggaggag ccgtgcacac aaggggcctg 1440 gacttcgcct gcgacatcta tatctgggcc cccctggccg ggacatgcgg ggtgctgctg 1500 ctgtccctgg tgattacact gtattgcaaa cggggccgga agaagctgct gtacatcttc 1560 aagcagccct tcatgcggcc cgtgcagacc acccaggagg aggacggctg cagctgccgg 1620 ttccccgagg aagaggaagg cggctgcgag ctgcgggtga agttcagccg gagcgccgac 1680 gccccagcct accagcaggg ccagaaccag ctgtacaacg agctgaacct gggacggcgg 1740 gaggagtacg acgtgctgga caagcggcgg ggacgggacc ccgagatggg cggcaagcct 1800 cgccggaaga atccccagga gggcctgtac aacgagctgc agaaggacaa gatggccgag 1860 gcctacagcg agatcggcat gaagggcgag cggcgccggg gcaagggcca cgacggcctg 1920 taccagggcc tgagcaccgc caccaaggac acctacgacg ccctgcacat gcaggccctg 1980 ccaccccggt ga 1992

SEQ ID NO: 66 Complete Single chain variable fragment AA sequence including signal sequence, serine glycine linker and murine lgG2a Fc domain (M2)

METDTLLLWV LLLWVDGSTG DVQLQESGPG LVKNSQSLSL TCTVTGYSIT SDYAWNWIRQ 60

FPGNKLEWMG YISYSGSTSY NPSLKSRISI TRDTSKNQFF LQLNSVTPED

TATYYCARDP 120 PYYYGSNGTW GQGTSVTVSS GGGGSGGGGS GGGGSDVVMT QTPLTLSVTI GQPASISCKS 180

SQSLLDSDGK TYLNWLLQRP GQSPKRLIYL VSKLDSGVPD RFTGSGSGTD FTLKISRVEA 240

EDLGVYHCWQ GTHFPYTFGG GTKLEIKSDP RGPTIKPCPP CKCPAPNLLG GPSVFIFPPK 300

IKDVLMISLS PIVTCVVVDV SEDDPDVQIS WFVNNVEVHT AQTQTHREDY NSTLRVVSAL 360

PIQHQDWMSG KEFKCKVNNK DLPAPIERTI SKPKGSVRAP QVYVLPPPEE EMTKKQVTLT 420

CMVTDFMPED IYVEWTNNGK TELNYKNTEP VLDSDGSYFM YSKLRVEKKN WVERNSYSCS 480

VVHEGLHNHH TTKSFSRTPG K 501

SEQ ID NO: 67 DNA sequence of SEQ ID NO: 66 atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtcgacgg cagcaccgga 60 gatgtacaac tgcaggaaag tggaccaggt ttggtcaaaa actcacagtc tctcagcttg 120 acctgtaccg tcacaggata ttctataact agtgactacg catggaattg gatacgacaa 180 tttcccggca ataaacttga atggatggga tatatctcct attccggctc aactagttac 240 aacccttcac tgaaatctag aatctcaata actcgggaca caagtaaaaa ccagtttttt 300 ttgcaactga actcagttac tcctgaggat accgccacct attattgcgc tagagatccc 360 ccgtattact acgggtccaa tggtacctgg gggcagggta catctgtgac agtgtcttcc 420 ggcggaggcg gaagtggcgg agggggatca ggcgggggag gatctgatgt cgtaatgacc 480 caaacccctt tgacgctctc tgttacaatt ggacagcccg cgagtattag ttgcaagagt 540 agccagagtt tgctcgacag tgatggaaaa acgtacttga actggctcct tcaacggcca 600 ggccaaagcc cgaaaaggct catatatctg gtaagcaagc tggacagtgg agttcccgat 660 aggttcaccg gatcaggttc aggtactgat tttaccttga aaatttcccg agtggaagcc 720 gaggatttgg gcgtgtatca ctgttggcag ggcacccatt ttccttacac ttttggaggt 780 gggacaaagc tcgagataaa gtcggatccc agagggccca caatcaagcc ctgtcctcca 840 tgcaaatgcc cagcacctaa cctcttgggt ggaccatccg tcttcatctt ccctccaaag 900 atcaaggatg tactcatgat ctccctgagc cccatagtca catgtgtggt ggtggatgtg 960 agcgaggatg acccagatgt ccagatcagc tggtttgtga acaacgtgga agtacacaca 1020 gctcagacac aaacccatag agaggattac aacagtactc tccgggtggt cagtgccctc 1080 cccatccagc accaggactg gatgagtggc aaggagttca aatgcaaggt caacaacaaa 1140 gacctcccag cgcccatcga gagaaccatc tcaaaaccca aagggtcagt aagagctcca 1200 caggtatatg tcttgcctcc accagaagaa gagatgacta agaaacaggt cactctgacc 1260 tgcatggtca cagacttcat gcctgaagac atttacgtgg agtggaccaa caacgggaaa 1320 acagagctaa actacaagaa cactgaacca gtcctggact ctgatggttc ttacttcatg 1380 tacagcaagc tgagagtgga aaagaagaac tgggtggaaa gaaatagcta ctcctgttca 1440 gtggtccacg agggtctgca caatcaccac acgactaaga gcttctcccg gactccgggt 1500 aaatga 1506

SEQ ID NO: 68 CAR sequence of the above binder including 4-1 BB, CD3z and RQR8

MGTSLLCWMA LCLLGADHAD ACPYSNPSLC SGGGGSELPT QGTFSNVSTN VSPAKPTTTA 60

CPYSNPSLCS GGGGSPAPRP PTPAPTIASQ PLSLRPEACR PAAGGAVHTR GLDFACDIYI 120

WAPLAGTCGV LLLSLVITLY CNHRNRRRVC KCPRPWRAE GRGSLLTCGD VEENPGPMET 180

DTLLLWVLLL WVDGSTGDVQ LQESGPGLVK NSQSLSLTCT VTGYSITSDY

AWNWIRQFPG 240

NKLEWMGYIS YSGSTSYNPS LKSRISITRD TSKNQFFLQL NSVTPEDTAT

YYCARDPPYY 300

YGSNGTWGQG TSVTVSSGGG GSGGGGSGGG GSDWMTQTP LTLSVTIGQP ASISCKSSQS 360

LLDSDGKTYL NWLLQRPGQS PKRLIYLVSK LDSGVPDRFT GSGSGTDFTL KISRVEAEDL 420

GVYHCWQGTH FPYTFGGGTK LEIKSDPTTT PAPRPPTPAP TIASQPLSLR PEACRPAAGG 480

AVHTRGLDFA CDIYIWAPLA GTCGVLLLSL VITLYCKRGR KKLLYIFKQP

FMRPVQTTQE 540

EDGCSCRFPE EEEGGCELRV KFSRSADAPA YQQGQNQLYN ELNLGRREEY DVLDKRRGRD 600 PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS EIGMKGERRR GKGHDGLYQG

LSTATKDTYD 660

ALHMQALPPR 670

SEQ ID NO 69 DNA sequence of SEQ ID NO: 68 atgggcacca gcctgctgtg ctggatggcc ctgtgcctgc tgggcgccga ccacgccgat 60 gcctgcccct acagcaaccc cagcctgtgc agcggaggcg gcggcagcga gctgcccacc 120 cagggcacct tctccaacgt gtccaccaac gtgagcccag ccaagcccac caccaccgcc 180 tgtccttatt ccaatccttc cctgtgtagc ggagggggag gcagcccagc ccccagacct 240 cccaccccag cccccaccat cgccagccag cctctgagcc tgagacccga ggcctgccgc 300 ccagccgccg gcggcgccgt gcacaccaga ggcctggatt tcgcctgcga tatctacatc 360 tgggccccac tggccggcac ctgtggcgtg ctgctgctga gcctggtgat caccctgtac 420 tgcaaccacc gcaaccgcag gcgcgtgtgc aagtgcccca ggcccgtggt gagagccgag 480 ggcagaggca gcctgctgac ctgcggcgac gtggaggaga acccaggccc catggagacc 540 gacaccctgc tgctgtgggt gctgctgctg tgggtcgacg gcagcaccgg agatgtacaa 600 ctgcaggaaa gtggaccagg tttggtcaaa aactcacagt ctctcagctt gacctgtacc 660 gtcacaggat attctataac tagtgactac gcatggaatt ggatacgaca atttcccggc 720 aataaacttg aatggatggg atatatctcc tattccggct caactagtta caacccttca 780 ctgaaatcta gaatctcaat aactcgggac acaagtaaaa accagttttt tttgcaactg 840 aactcagtta ctcctgagga taccgccacc tattattgcg ctagagatcc cccgtattac 900 tacgggtcca atggtacctg ggggcagggt acatctgtga cagtgtcttc cggcggaggc 960 ggaagtggcg gagggggatc aggcggggga ggatctgatg tcgtaatgac ccaaacccct 1020 ttgacgctct ctgttacaat tggacagccc gcgagtatta gttgcaagag tagccagagt 1080 ttgctcgaca gtgatggaaa aacgtacttg aactggctcc ttcaacggcc aggccaaagc 1140 ccgaaaaggc tcatatatct ggtaagcaag ctggacagtg gagttcccga taggttcacc 1200 ggatcaggtt caggtactga ttttaccttg aaaatttccc gagtggaagc cgaggatttg 1260 ggcgtgtatc actgttggca gggcacccat tttccttaca cttttggagg tgggacaaag 1320 ctcgagataa agtcggatcc caccaccacc ccagccccac ggccacctac ccctgcccca 1380 accatcgcca gccagcccct gagcctgcgg cctgaagcct gcaggcctgc cgccggagga 1440 gccgtgcaca caaggggcct ggacttcgcc tgcgacatct atatctgggc ccccctggcc 1500 gggacatgcg gggtgctgct gctgtccctg gtgattacac tgtattgcaa acggggccgg 1560 aagaagctgc tgtacatctt caagcagccc ttcatgcggc ccgtgcagac cacccaggag 1620 gaggacggct gcagctgccg gttccccgag gaagaggaag gcggctgcga gctgcgggtg 1680 aagttcagcc ggagcgccga cgccccagcc taccagcagg gccagaacca gctgtacaac 1740 gagctgaacc tgggacggcg ggaggagtac gacgtgctgg acaagcggcg gggacgggac 1800 cccgagatgg gcggcaagcc tcgccggaag aatccccagg agggcctgta caacgagctg 1860 cagaaggaca agatggccga ggcctacagc gagatcggca tgaagggcga gcggcgccgg 1920 ggcaagggcc acgacggcct gtaccagggc ctgagcaccg ccaccaagga cacctacgac 1980 gccctgcaca tgcaggccct gccaccccgg tga 2013

Table 6

Sequences in scfv format with signal peptide and using serine glycine linker

Human VH1-VL1

SEQ ID NO: 81

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAACTGCAAGAGAGCGGACCAGGGTTGGTAAAGCCCTCACAAACTCTCTCC

TTGACTTGCACTGTTACGGGTTACAGTATAACTTCAGACTATGCGTGGAACTGGATAAGAC

AACCGCCAGGTAAAGGACTTGAATGGATGGGTTACATTGATTATTCTGGTAACACAAACTA

CAACCCTTCACTGAAAAGCCGAATTACCATCTCTCGAGACACGTCAAAAAACCAGTTCAG

CCTGAAGCTGAGTAGCGTGACTGCTGCTGACACAGCGGTTTATTACTGCAGCCGAGGTAT

CACGGGCTACTGGGGACAGGGAACGACGGTTACAGTAAGTAGCggcggaggcggaagtggcgga gggggatcaggcgggggaggatctCAGGCCGTAGTAACACAGGAGCCCAGCCTCACCGTGTCACC

CGGTGGAACGGTTACGCTGACCTGTGCATCATCAACGGGAGCCATAACTACATCTAACTA

TGCGAACTGGTTCCAAGAAAAACCAGGACAAGCCTTTAGGGGGTTGATAGGTGGCGCTA

AAAATCGAGCTCCCTGGGTCCCAGCGCGGTTTAGCGGGAGTCTGATCGGGGACAAAGCC

GCGCTGACACTGTCTGGGGTACAACCAGAAGATGAAGCTATATACTTTTGTGCGCTTTGG

TACAGCAATCACTGGGTATTCGGCGGCGGAACCAAACTGACGGTTTTGGGGtcg

SEQ ID NO: 82 METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN

WFQEKPGQAFRGLIGGAKNRAPWVPARFSGSLIGDKAALTLSGVQPEDEAIYFCALWYSNH

WVFGGGTKLTVLGS

Human VH1-VL2

SEQ ID NO: 83

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAACTGCAAGAGAGCGGACCAGGGTTGGTAAAGCCCTCACAAACTCTCTCC

TTGACTTGCACTGTTACGGGTTACAGTATAACTTCAGACTATGCGTGGAACTGGATAAGAC

AACCGCCAGGTAAAGGACTTGAATGGATGGGTTACATTGATTATTCTGGTAACACAAACTA

CAACCCTTCACTGAAAAGCCGAATTACCATCTCTCGAGACACGTCAAAAAACCAGTTCAG

CCTGAAGCTGAGTAGCGTGACTGCTGCTGACACAGCGGTTTATTACTGCAGCCGAGGTAT

CACGGGCTACTGGGGACAGGGAACGACGGTTACAGTAAGTAGCggcggaggcggaagtggcgga gggggatcaggcgggggaggatctCAAGCTGTCGTTACTCAAGAACCGAGCCTTACAGTCAGTCCA

GGAGGAACAGTTACACTGACCTGTGCAAGCAGTACGGGAGCTATCACTACCAGCAATTAT

GCTAACTGGTTTCAGCAGAAACCTGGACAAGCCTTCCGGGGTTTGATAGGGGGTGCGAA

GAATAAAGCATCCTGGACGCCCGCTCGATTCTCAGGATCACTCCTGGGCGACAAAGCTG

CCCTCACTTTGTCAGGCGTCCAGCCTGAAGACGAGGCCGAGTACTATTGTGCTTTGTGGT

ACAGCAACCATTGGGTGTTTGGTGGCGGTACAAAGCTGACTGTTCTTGGCtcg

SEQ ID NO: 84

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN

WFQQKPGQAFRGLIGGAKNKASWTPARFSGSLLGDKAALTLSGVQPEDEAEYYCALWYSNH

WVFGGGTKLTVLGS

Human VH1-VL3

SEQ ID NO: 85

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAACTGCAAGAGAGCGGACCAGGGTTGGTAAAGCCCTCACAAACTCTCTCC

TTGACTTGCACTGTTACGGGTTACAGTATAACTTCAGACTATGCGTGGAACTGGATAAGAC

AACCGCCAGGTAAAGGACTTGAATGGATGGGTTACATTGATTATTCTGGTAACACAAACTA CAACCCTTCACTGAAAAGCCGAATTACCATCTCTCGAGACACGTCAAAAAACCAGTTCAG

CCTGAAGCTGAGTAGCGTGACTGCTGCTGACACAGCGGTTTATTACTGCAGCCGAGGTAT

CACGGGCTACTGGGGACAGGGAACGACGGTTACAGTAAGTAGCggcggaggcggaagtggcgga gggggatcaggcgggggaggatctCAGGCCGTCGTCACACAAGAGCCTTCTTTTTCTGTTAGCCCA

GGTGGAACTGTAACCCTTACGTGCGGCTCATCCACTGGAGCGATCACGACCTCTAATTAC

GCCAATTGGTATCAAGAAACCCCTGGACAAGCTTTTAGGGGACTTATAGGTGGTGCCAAG

AATAGGGCGCCAGGAGTACCCGACAGGTTTAGTGGTAGCTTGATTGGCGATAAGGCTGC

ACTTACAATAACAGGAGCCCAGGCCGATGACGAAAGCATTTATTTTTGTGCGCTTTGGTAT

TCTAATCATTGGGTATTCGGTGGGGGTACGAAGCTTACAGTTCTCGGCtcg

SEQ ID NO: 86

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN

WYQETPGQAFRGLIGGAKNRAPGVPDRFSGSLIGDKAALTITGAQADDESIYFCALWYSNHW

VFGGGTKLTVLGS

Human VH1-VL4

SEQ ID NO: 87

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAACTGCAAGAGAGCGGACCAGGGTTGGTAAAGCCCTCACAAACTCTCTCC

TTGACTTGCACTGTTACGGGTTACAGTATAACTTCAGACTATGCGTGGAACTGGATAAGAC

AACCGCCAGGTAAAGGACTTGAATGGATGGGTTACATTGATTATTCTGGTAACACAAACTA

CAACCCTTCACTGAAAAGCCGAATTACCATCTCTCGAGACACGTCAAAAAACCAGTTCAG

CCTGAAGCTGAGTAGCGTGACTGCTGCTGACACAGCGGTTTATTACTGCAGCCGAGGTAT

CACGGGCTACTGGGGACAGGGAACGACGGTTACAGTAAGTAGCggcggaggcggaagtggcgga gggggatcaggcgggggaggatctCAAGCCGTGGTGACTCAGGAGCCTTCTTTTTCAGTCTCTCCT

GGTGGGACTGTGACCCTCACGTGTGGGAGCAGTACAGGTGCTATTACAACATCTAATTAC

GCTAATTGGTATCAGCAAACACCGGGTCAGGCTTTTCGGGGTCTTATTGGAGGCGCAAAA

AATAGGGCAAGTGGCGTCCCTGATAGATTCTCCGGCAGTCTCCTGGGCGACAAAGCGGC

TCTTACCATAACCGGTGCTCAAGCGGACGATGAGTCCGATTATTACTGCGCCTTGTGGTA

TTCTAATCATTGGGTGTTCGGTGGCGGTACGAAGCTCACAGTCCTGGGTtcg

SEQ ID NO: 88 METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN WYQQTPGQAFRGLIGGAKNRASGVPDRFSGSLLGDKAALTITGAQADDESDYYCALWYSNH WVFGGGTKLTVLGS

Human VH2-VL1

SEQ ID NO: 89

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTTCAGCTCCAGGAATCCGGGCCGGGCTTGGTAAAACCATCTCAGACCCTCTCT

CTGACCTGTACCGTCACAGGTTACAGCATTACGAGCGACTATGCCTGGAATTGGATTAGG CAACCCCCTGGAAAAGGTCTTGAATGGATAGGCTACATAGATTACAGCGGCAATACGAAC TACAACCCCTCCCTTAAATCCCGAGTCACGATAAGTCGCGATACTAGCAAAAATCAGTTTT CACTGAAACTCAGCAGTGTCACAGCGGCCGACACAGCCGTCTACTATTGTAGCCGGGGT ATTACGGGATATTGGGGACAGGGTACAACCGTGACTGTGTCCTCTggcggaggcggaagtggcg gagggggatcaggcgggggaggatctCAGGCCGTAGTAACACAGGAGCCCAGCCTCACCGTGTCAC CCGGTGGAACGGTTACGCTGACCTGTGCATCATCAACGGGAGCCATAACTACATCTAACT ATGCGAACTGGTTCCAAGAAAAACCAGGACAAGCCTTTAGGGGGTTGATAGGTGGCGCT

AAAAATCGAGCTCCCTGGGTCCCAGCGCGGTTTAGCGGGAGTCTGATCGGGGACAAAGC CGCGCTGACACTGTCTGGGGTACAACCAGAAGATGAAGCTATATACTTTTGTGCGCTTTG GTACAGCAATCACTGGGTATTCGGCGGCGGAACCAAACTGACGGTTTTGGGGtcg

SEQ ID NO: 90

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN WFQEKPGQAFRGLIGGAKNRAPWVPARFSGSLIGDKAALTLSGVQPEDEAIYFCALWYSNH WVFGGGTKLTVLGS

Human VH2-VL2

SEQ ID NO: 91

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAGCTCCAGGAATCCGGGCCGGGCTTGGTAAAACCATCTCAGACCCTCTCT

CTGACCTGTACCGTCACAGGTTACAGCATTACGAGCGACTATGCCTGGAATTGGATTAGG CAACCCCCTGGAAAAGGTCTTGAATGGATAGGCTACATAGATTACAGCGGCAATACGAAC TACAACCCCTCCCTTAAATCCCGAGTCACGATAAGTCGCGATACTAGCAAAAATCAGTTTT CACTGAAACTCAGCAGTGTCACAGCGGCCGACACAGCCGTCTACTATTGTAGCCGGGGT ATTACGGGATATTGGGGACAGGGTACAACCGTGACTGTGTCCTCTggcggaggcggaagtggcg gagggggatcaggcgggggaggatctCAAGCTGTCGTTACTCAAGAACCGAGCCTTACAGTCAGTCC AGGAGGAACAGTTACACTGACCTGTGCAAGCAGTACGGGAGCTATCACTACCAGCAATTA TGCTAACTGGTTTCAGCAGAAACCTGGACAAGCCTTCCGGGGTTTGATAGGGGGTGCGA AGAATAAAGCATCCTGGACGCCCGCTCGATTCTCAGGATCACTCCTGGGCGACAAAGCT

GCCCTCACTTTGTCAGGCGTCCAGCCTGAAGACGAGGCCGAGTACTATTGTGCTTTGTG GTACAGCAACCATTGGGTGTTTGGTGGCGGTACAAAGCTGACTGTTCTTGGCtcg

SEQ ID NO: 92

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN

WFQQKPGQAFRGLIGGAKNKASWTPARFSGSLLGDKAALTLSGVQPEDEAEYYCALWYSNH WVFGGGTKLTVLGS

Human VH2-VL3

SEQ ID NO: 93

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAGCTCCAGGAATCCGGGCCGGGCTTGGTAAAACCATCTCAGACCCTCTCT CTGACCTGTACCGTCACAGGTTACAGCATTACGAGCGACTATGCCTGGAATTGGATTAGG CAACCCCCTGGAAAAGGTCTTGAATGGATAGGCTACATAGATTACAGCGGCAATACGAAC TACAACCCCTCCCTTAAATCCCGAGTCACGATAAGTCGCGATACTAGCAAAAATCAGTTTT CACTGAAACTCAGCAGTGTCACAGCGGCCGACACAGCCGTCTACTATTGTAGCCGGGGT ATTACGGGATATTGGGGACAGGGTACAACCGTGACTGTGTCCTCTggcggaggcggaagtggcg gagggggatcaggcgggggaggatctCAGGCCGTCGTCACACAAGAGCCTTCTTTTTCTGTTAGCCC AGGTGGAACTGTAACCCTTACGTGCGGCTCATCCACTGGAGCGATCACGACCTCTAATTA

CGCCAATTGGTATCAAGAAACCCCTGGACAAGCTTTTAGGGGACTTATAGGTGGTGCCAA GAATAGGGCGCCAGGAGTACCCGACAGGTTTAGTGGTAGCTTGATTGGCGATAAGGCTG CACTTACAATAACAGGAGCCCAGGCCGATGACGAAAGCATTTATTTTTGTGCGCTTTGGT ATTCTAATCATTGGGTATTCGGTGGGGGTACGAAGCTTACAGTTCTCGGCtcg SEQ ID NO: 94

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN WYQETPGQAFRGLIGGAKNRAPGVPDRFSGSLIGDKAALTITGAQADDESIYFCALWYSNHW VFGGGTKLTVLGS

Human VH2-VL4

SEQ ID NO: 95

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTTCAGCTCCAGGAATCCGGGCCGGGCTTGGTAAAACCATCTCAGACCCTCTCT CTGACCTGTACCGTCACAGGTTACAGCATTACGAGCGACTATGCCTGGAATTGGATTAGG

CAACCCCCTGGAAAAGGTCTTGAATGGATAGGCTACATAGATTACAGCGGCAATACGAAC TACAACCCCTCCCTTAAATCCCGAGTCACGATAAGTCGCGATACTAGCAAAAATCAGTTTT CACTGAAACTCAGCAGTGTCACAGCGGCCGACACAGCCGTCTACTATTGTAGCCGGGGT ATTACGGGATATTGGGGACAGGGTACAACCGTGACTGTGTCCTCTggcggaggcggaagtggcg gagggggatcaggcgggggaggatctCAAGCCGTGGTGACTCAGGAGCCTTCTTTTTCAGTCTCTCC TGGTGGGACTGTGACCCTCACGTGTGGGAGCAGTACAGGTGCTATTACAACATCTAATTA CGCTAATTGGTATCAGCAAACACCGGGTCAGGCTTTTCGGGGTCTTATTGGAGGCGCAAA AAATAGGGCAAGTGGCGTCCCTGATAGATTCTCCGGCAGTCTCCTGGGCGACAAAGCGG

CTCTTACCATAACCGGTGCTCAAGCGGACGATGAGTCCGATTATTACTGCGCCTTGTGGT ATTCTAATCATTGGGTGTTCGGTGGCGGTACGAAGCTCACAGTCCTGGGTtcg

SEQ ID NO: 96

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN WYQQTPGQAFRGLIGGAKNRASGVPDRFSGSLLGDKAALTITGAQADDESDYYCALWYSNH WVFGGGTKLTVLGS VH3-VL1

SEQ ID NO: 97

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTACAACTTCAGGAATCAGGTCCGGGGCTTGTTAAGCCCTCCGAAACTCTCAGC CTTACCTGCACCGTGACCGGATATAGCATCACTAGCGACTATGCCTGGAACTGGATACGC CAACCCCCCGGCAAAGGACTGGAATGGATGGGCTATATTGACTACTCAGGCAATACTAAT TATAATCCATCACTGAAATCACGAATCACAATCTCACGCGATACATCAAAGAACCAGTTTA GTTTGAAGCTCAGCAGTGTTACCGCCGCTGATACAGCAGTATATTACTGTTCCCGAGGCA

TAACCGGTTACTGGGGACAAGGGACGACGGTCACGGTTTCAAGTggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAGGCCGTAGTAACACAGGAGCCCAGCCTCACCGTGTCACC CGGTGGAACGGTTACGCTGACCTGTGCATCATCAACGGGAGCCATAACTACATCTAACTA TGCGAACTGGTTCCAAGAAAAACCAGGACAAGCCTTTAGGGGGTTGATAGGTGGCGCTA AAAATCGAGCTCCCTGGGTCCCAGCGCGGTTTAGCGGGAGTCTGATCGGGGACAAAGCC GCGCTGACACTGTCTGGGGTACAACCAGAAGATGAAGCTATATACTTTTGTGCGCTTTGG TACAGCAATCACTGGGTATTCGGCGGCGGAACCAAACTGACGGTTTTGGGGtcg

SEQ ID NO: 98

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN WFQEKPGQAFRGLIGGAKNRAPWVPARFSGSLIGDKAALTLSGVQPEDEAIYFCALWYSNH WVFGGGTKLTVLGS

VH3-VL2

SEQ ID NO: 99

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTACAACTTCAGGAATCAGGTCCGGGGCTTGTTAAGCCCTCCGAAACTCTCAGC CTTACCTGCACCGTGACCGGATATAGCATCACTAGCGACTATGCCTGGAACTGGATACGC CAACCCCCCGGCAAAGGACTGGAATGGATGGGCTATATTGACTACTCAGGCAATACTAAT TATAATCCATCACTGAAATCACGAATCACAATCTCACGCGATACATCAAAGAACCAGTTTA GTTTGAAGCTCAGCAGTGTTACCGCCGCTGATACAGCAGTATATTACTGTTCCCGAGGCA TAACCGGTTACTGGGGACAAGGGACGACGGTCACGGTTTCAAGTggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAAGCTGTCGTTACTCAAGAACCGAGCCTTACAGTCAGTCC

AGGAGGAACAGTTACACTGACCTGTGCAAGCAGTACGGGAGCTATCACTACCAGCAATTA TGCTAACTGGTTTCAGCAGAAACCTGGACAAGCCTTCCGGGGTTTGATAGGGGGTGCGA AGAATAAAGCATCCTGGACGCCCGCTCGATTCTCAGGATCACTCCTGGGCGACAAAGCT GCCCTCACTTTGTCAGGCGTCCAGCCTGAAGACGAGGCCGAGTACTATTGTGCTTTGTG GTACAGCAACCATTGGGTGTTTGGTGGCGGTACAAAGCTGACTGTTCTTGGCtcg

SEQ ID NO: 100

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN WFQQKPGQAFRGLIGGAKNKASWTPARFSGSLLGDKAALTLSGVQPEDEAEYYCALWYSNH WVFGGGTKLTVLGS

VH3-VL3

SEQ ID NO: 101

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTACAACTTCAGGAATCAGGTCCGGGGCTTGTTAAGCCCTCCGAAACTCTCAGC CTTACCTGCACCGTGACCGGATATAGCATCACTAGCGACTATGCCTGGAACTGGATACGC CAACCCCCCGGCAAAGGACTGGAATGGATGGGCTATATTGACTACTCAGGCAATACTAAT TATAATCCATCACTGAAATCACGAATCACAATCTCACGCGATACATCAAAGAACCAGTTTA GTTTGAAGCTCAGCAGTGTTACCGCCGCTGATACAGCAGTATATTACTGTTCCCGAGGCA TAACCGGTTACTGGGGACAAGGGACGACGGTCACGGTTTCAAGTggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAGGCCGTCGTCACACAAGAGCCTTCTTTTTCTGTTAGCCCA

GGTGGAACTGTAACCCTTACGTGCGGCTCATCCACTGGAGCGATCACGACCTCTAATTAC GCCAATTGGTATCAAGAAACCCCTGGACAAGCTTTTAGGGGACTTATAGGTGGTGCCAAG AATAGGGCGCCAGGAGTACCCGACAGGTTTAGTGGTAGCTTGATTGGCGATAAGGCTGC ACTTACAATAACAGGAGCCCAGGCCGATGACGAAAGCATTTATTTTTGTGCGCTTTGGTAT TCTAATCATTGGGTATTCGGTGGGGGTACGAAGCTTACAGTTCTCGGCtcg

SEQ ID NO: 102

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWMGYIDYSGNTNYNPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN

WYQETPGQAFRGLIGGAKNRAPGVPDRFSGSLIGDKAALTITGAQADDESIYFCALWYSNHW VFGGGTKLTVLGS VH3-VL4

SEQ ID NO: 103

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTACAACTTCAGGAATCAGGTCCGGGGCTTGTTAAGCCCTCCGAAACTCTCAGC CTTACCTGCACCGTGACCGGATATAGCATCACTAGCGACTATGCCTGGAACTGGATACGC

CAACCCCCCGGCAAAGGACTGGAATGGATGGGCTATATTGACTACTCAGGCAATACTAAT TATAATCCATCACTGAAATCACGAATCACAATCTCACGCGATACATCAAAGAACCAGTTTA GTTTGAAGCTCAGCAGTGTTACCGCCGCTGATACAGCAGTATATTACTGTTCCCGAGGCA TAACCGGTTACTGGGGACAAGGGACGACGGTCACGGTTTCAAGTggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAAGCCGTGGTGACTCAGGAGCCTTCTTTTTCAGTCTCTCCT GGTGGGACTGTGACCCTCACGTGTGGGAGCAGTACAGGTGCTATTACAACATCTAATTAC GCTAATTGGTATCAGCAAACACCGGGTCAGGCTTTTCGGGGTCTTATTGGAGGCGCAAAA AATAGGGCAAGTGGCGTCCCTGATAGATTCTCCGGCAGTCTCCTGGGCGACAAAGCGGC

TCTTACCATAACCGGTGCTCAAGCGGACGATGAGTCCGATTATTACTGCGCCTTGTGGTA TTCTAATCATTGGGTGTTCGGTGGCGGTACGAAGCTCACAGTCCTGGGTtcg

SEQ ID NO: 104

WYQQTPGQAFRGLIGGAKNRASGVPDRFSGSLLGDKAALTITGAQADDESDYYCALWYSNH WVFGGGTKLTVLGS

VH4-VL1

SEQ ID NO: 105

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAGTTGCAGGAGTCTGGGCCCGGTTTGGTAAAGCCATCCGAAACTCTTAGC CTGACTTGTACTGTAACAGGTTACTCCATTACCTCCGACTACGCTTGGAATTGGATACGG

CAACCGCCGGGGAAAGGCTTGGAATGGATCGGTTATATCGACTATTCAGGAAATACCAAC TACAACCCCAGCCTTAAATCTCGCGTAACTATTTCCCGCGACACCTCCAAAAACCAGTTTT CACTGAAGCTGTCATCCGTGACTGCTGCAGATACGGCGGTTTACTATTGTTCACGCGGCA TTACGGGTTACTGGGGACAGGGGACCACAGTAACCGTATCTTCAggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAGGCCGTAGTAACACAGGAGCCCAGCCTCACCGTGTCACC CGGTGGAACGGTTACGCTGACCTGTGCATCATCAACGGGAGCCATAACTACATCTAACTA TGCGAACTGGTTCCAAGAAAAACCAGGACAAGCCTTTAGGGGGTTGATAGGTGGCGCTA

AAAATCGAGCTCCCTGGGTCCCAGCGCGGTTTAGCGGGAGTCTGATCGGGGACAAAGCC

GCGCTGACACTGTCTGGGGTACAACCAGAAGATGAAGCTATATACTTTTGTGCGCTTTGG

TACAGCAATCACTGGGTATTCGGCGGCGGAACCAAACTGACGGTTTTGGGGtcg

SEQ ID NO: 106

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN

WFQEKPGQAFRGLIGGAKNRAPWVPARFSGSLIGDKAALTLSGVQPEDEAIYFCALWYSNH

WVFGGGTKLTVLGS

VH4-VL2

SEQ ID NO: 107

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

GACAGGTTCAGTTGCAGGAGTCTGGGCCCGGTTTGGTAAAGCCATCCGAAACTCTTAGC

CTGACTTGTACTGTAACAGGTTACTCCATTACCTCCGACTACGCTTGGAATTGGATACGG

CAACCGCCGGGGAAAGGCTTGGAATGGATCGGTTATATCGACTATTCAGGAAATACCAAC

TACAACCCCAGCCTTAAATCTCGCGTAACTATTTCCCGCGACACCTCCAAAAACCAGTTTT

CACTGAAGCTGTCATCCGTGACTGCTGCAGATACGGCGGTTTACTATTGTTCACGCGGCA

TTACGGGTTACTGGGGACAGGGGACCACAGTAACCGTATCTTCAggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAAGCTGTCGTTACTCAAGAACCGAGCCTTACAGTCAGTCC

AGGAGGAACAGTTACACTGACCTGTGCAAGCAGTACGGGAGCTATCACTACCAGCAATTA

TGCTAACTGGTTTCAGCAGAAACCTGGACAAGCCTTCCGGGGTTTGATAGGGGGTGCGA

AGAATAAAGCATCCTGGACGCCCGCTCGATTCTCAGGATCACTCCTGGGCGACAAAGCT

GCCCTCACTTTGTCAGGCGTCCAGCCTGAAGACGAGGCCGAGTACTATTGTGCTTTGTG

GTACAGCAACCATTGGGTGTTTGGTGGCGGTACAAAGCTGACTGTTCTTGGCtcg

SEQ ID NO: 108

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCASSTGAITTSNYAN

WFQQKPGQAFRGLIGGAKNKASWTPARFSGSLLGDKAALTLSGVQPEDEAEYYCALWYSNH

WVFGGGTKLTVLGS VH4-VL3

SEQ ID NO: 109

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG

CAACCGCCGGGGAAAGGCTTGGAATGGATCGGTTATATCGACTATTCAGGAAATACCAAC TACAACCCCAGCCTTAAATCTCGCGTAACTATTTCCCGCGACACCTCCAAAAACCAGTTTT CACTGAAGCTGTCATCCGTGACTGCTGCAGATACGGCGGTTTACTATTGTTCACGCGGCA TTACGGGTTACTGGGGACAGGGGACCACAGTAACCGTATCTTCAggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAGGCCGTCGTCACACAAGAGCCTTCTTTTTCTGTTAGCCCA GGTGGAACTGTAACCCTTACGTGCGGCTCATCCACTGGAGCGATCACGACCTCTAATTAC GCCAATTGGTATCAAGAAACCCCTGGACAAGCTTTTAGGGGACTTATAGGTGGTGCCAAG AATAGGGCGCCAGGAGTACCCGACAGGTTTAGTGGTAGCTTGATTGGCGATAAGGCTGC

ACTTACAATAACAGGAGCCCAGGCCGATGACGAAAGCATTTATTTTTGTGCGCTTTGGTAT TCTAATCATTGGGTATTCGGTGGGGGTACGAAGCTTACAGTTCTCGGCtcg

SEQ ID NO: 110

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN

WYQETPGQAFRGLIGGAKNRAPGVPDRFSGSLIGDKAALTITGAQADDESIYFCALWYSNHW VFGGGTKLTVLGS

VH4-VL4

SEQ ID NO: 111

ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTCGACGGCAGCACCG GACAGGTTCAGTTGCAGGAGTCTGGGCCCGGTTTGGTAAAGCCATCCGAAACTCTTAGC CTGACTTGTACTGTAACAGGTTACTCCATTACCTCCGACTACGCTTGGAATTGGATACGG

CAACCGCCGGGGAAAGGCTTGGAATGGATCGGTTATATCGACTATTCAGGAAATACCAAC TACAACCCCAGCCTTAAATCTCGCGTAACTATTTCCCGCGACACCTCCAAAAACCAGTTTT CACTGAAGCTGTCATCCGTGACTGCTGCAGATACGGCGGTTTACTATTGTTCACGCGGCA TTACGGGTTACTGGGGACAGGGGACCACAGTAACCGTATCTTCAggcggaggcggaagtggcgg agggggatcaggcgggggaggatctCAAGCCGTGGTGACTCAGGAGCCTTCTTTTTCAGTCTCTCCT GGTGGGACTGTGACCCTCACGTGTGGGAGCAGTACAGGTGCTATTACAACATCTAATTAC GCTAATTGGTATCAGCAAACACCGGGTCAGGCTTTTCGGGGTCTTATTGGAGGCGCAAAA

AATAGGGCAAGTGGCGTCCCTGATAGATTCTCCGGCAGTCTCCTGGGCGACAAAGCGGC

TCTTACCATAACCGGTGCTCAAGCGGACGATGAGTCCGATTATTACTGCGCCTTGTGGTA

TTCTAATCATTGGGTGTTCGGTGGCGGTACGAAGCTCACAGTCCTGGGTtcg

SEQ ID NO: 112

METDTLLLWVLLLWVDGSTGQVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPP

GKGLEWIGYIDYSGNTNYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCSRGITGYWG

QGTTVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGSSTGAITTSNYAN

WYQQTPGQAFRGLIGGAKNRASGVPDRFSGSLLGDKAALTITGAQADDESDYYCALWYSNH

WVFGGGTKLTVLGS

References

1. Moulard O, Mehta J, Fryzek J, Olivares R, Iqbal U, Mesa RA. Epidemiology of myelofibrosis, essential thrombocythemia, and polycythemia vera in the European Union. European Journal of Haematology. 2014;92(4):289-297. doi:10.1111/ejh.12256

2. Tefferi A. Primary myelofibrosis: 2021 update on diagnosis, risk-stratification and management. American Journal of Hematology. 2021 ;96(1):145-162. doi:10.1002/ajh.26050

3. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. 2017;129(6):667-679. doi:10.1182/blood-2016-10-695940

4. Rodriguez-Meira A, Norfo R, Wen WX, et al. Deciphering TP53 mutant Cancer Evolution with Single-Cell Multi-Omics. Published online March 29, 2022:2022.03.28.485984. doi:10.1101/2022.03.28.485984

5. Mead AJ, Mullally A. Myeloproliferative neoplasm stem cells. Blood. 2017;129(12):1607- 1616. do i : 10.1182/blood-2016-10-696005

6. Pemmaraju N, Bose P, Rampal R, Gerds AT, Fleischman A, Verstovsek S. Ten years after ruxolitinib approval for myelofibrosis: a review of clinical efficacy. Leukemia & Lymphoma. 2023;0(0):1 -19. doi: 10.1080/10428194.2023.2196593

7. Bose P, Verstovsek S. JAK Inhibition for the Treatment of Myelofibrosis: Limitations and Future Perspectives. Hemasphere. 2020;4(4):e424. doi:10.1097/HS9.0000000000000424

8. Gowin K, Ballen K, Ahn KW, et al. Survival following allogeneic transplant in patients with myelofibrosis. Blood Advances. 2020;4(9):1965-1973. doi:10.1182/bloodadvances.2019001084

9. Holmstrom MO, Ahmad SM, Klausen U, et al. High frequencies of circulating memory T cells specific for calreticulin exon 9 mutations in healthy individuals. Blood Cancer Journal. 2019;9(2):1 -14. doi: 10.1038/s41408-018-0166-4 10. Kyllesbech C, Trier N, Mughal F, et al. Antibodies to calnexin and mutated calreticulin are common in human sera. Current Research in Translational Medicine. 2023;71 (2):103380. doi:10.1016/j. retram.2023.103380

11. Sousos N, Ni Leathlobhair M, Simoglou Karali C, et al. In utero origin of myelofibrosis presenting in adult monozygotic twins. Nat Med. 2022;28(6):1207-1211 . doi:10.1038/s41591- 022-01793-4

12. Psaila B, Wang G, Rodriguez-Meira A, et al. Single-Cell Analyses Reveal Megakaryocyte- Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets. Mol Cell. 2020;78(3):477-492.e8. doi:10.1016/j.molcel.2020.04.008

13. Liu P, Zhao L, Loos F, et al. Immunosuppression by Mutated Calreticulin Released from Malignant Cells. Mol Cell. 2020;77(4):748-760.e9. doi:10.1016/j.molcel.2019.11.004

14. Edahiro Y, Araki M, Komatsu N. Mechanism underlying the development of myeloproliferative neoplasms through mutant calreticulin. Cancer Sci. 2020;111 (8):2682-2688. doi:10.1111/cas.14503

15. Melenhorst JJ, Chen GM, Wang M, et al. Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature. 2022;602(7897):503-509. doi:10.1038/s41586-021- 04390-6

16. Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021 ;11 (4): 1-11 . doi:10.1038/s41408-021 -00459-7

17. Kroger N, Thiele J, Zander A, et al. Rapid regression of bone marrow fibrosis after dose- reduced allogeneic stem cell transplantation in patients with primary myelofibrosis. Experimental Hematology. 2007;35(1 1):1719-1722. doi: 10.1016/j.exphem.2007.08.022

18. Klyuchnikov E, Holler E, Bornhauser M, et al. Donor lymphocyte infusions and second transplantation as salvage treatment for relapsed myelofibrosis after reduced-intensity allografting. Br J Haematol. 2012;159(2):172-181 . doi:10.1111/bjh.12013

19. Khan AO, Rodriguez-Romera A, Reyat JS, Olijnik AA, Colombo M, Wang G, Wen WX, Sousos N, Murphy LC, Grygielska B, Perrella G, Mahony CB, Ling RE, Elliott NE, Karali CS, Stone AP, Kemble S, Cutler EA, Fielding AK, Croft AP, Bassett D, Poologasundarampillai G, Roy A, Gooding S, Rayes J, Machlus KR, Psaila B. Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies. Cancer Discov. 2023 Feb 6;13(2):364-385. doi: 10.1 158/2159-8290.CD-22-0199. PMID: 36351055; PMCID: PMC9900323.

Claims

1. An isolated chimeric antigen receptor (CAR) comprising an antibody or antigen binding portion thereof that specifically binds to mutant calreticulin, wherein said antibody or antigen binding portion thereof comprises a heavy chain variable (VH) region comprising a CDR1 comprising SEQ ID NO. 1 or SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or SEQ ID NO. 11 or a sequence with at least 40% homology thereto and a light chain variable (VL) region comprising a CDR1 comprising SEQ ID NO. 5 or SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or SEQ ID NO. 15 or a sequence with at least 40% homology thereto.

2. The CAR according to claim 1 , wherein the heavy chain variable (VH) region comprises a CDR1 comprising SEQ ID NO. 1 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 2 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 3 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 5 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 6 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 7 or a sequence with at least 40% homology thereto.

3. The CAR according to claim 1 , wherein the heavy chain variable (VH) region comprises a CDR1 comprising SEQ ID NO. 9 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 10 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 11 or a sequence with at least 40% homology thereto and wherein the light chain variable (VL) region comprises a CDR1 comprising SEQ ID NO. 13 or a sequence with at least 40% homology thereto, a CDR2 comprising SEQ ID NO. 14 or a sequence with at least 40% homology thereto and a CDR3 comprising SEQ ID NO. 15 or a sequence with at least 40% homology.

4. The CAR according to claim 2, wherein the heavy chain variable (VH) region comprises a CDR1 comprising a sequence with at least 100% homology of SEQ ID NO 1 and a CDR2 comprising a sequence with at least 82% homology of SEQ ID NO. 2.

5. The CAR according to claim 3, wherein the heavy chain variable (VH) region, comprises a CDR2 comprising a sequence with at least 43% homology of SEQ ID NO. 10 and wherein the light chain variable (VL) region comprises a CDR3 comprising a sequence with at least 67% homology of SEQ ID NO. 15.

6. The CAR according to any one of claims 1 to 5, wherein the VH region comprises SEQ ID NO. 4, 12, 72-75 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto and the VL region comprises SEQ ID NO. 8 or 16, 77-80 or a sequence having at least 75%, 80%, 85%, 90% or 95% sequence identity thereto.

7. The CAR according to a preceding claim comprising a hinge region.

8. The CAR according to claim 7 wherein the hinge region comprises a CD8alpha domain.

9. The CAR according to a preceding claim comprising a transmembrane domain.

10. The CAR according to claim 9, wherein the transmembrane domain is selected from the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, PD-1 , 4- 1 BB, 0X40, ICOS, CTLA-4, LAG3, 2B4, BTLA4, TIM-3, TIGIT, SIRPA, CD28, CD3 epsilon, CD3zeta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

11 . The CAR according to a preceding claim comprising an intracellular signalling domain.

12. The CAR according to claim 11 , wherein said intracellular signalling domain comprises one or more of the following domains: CD28, 0X40, LCK, LAT, FYN, SLP76, ZAP70 and/or CD3zeta endodomain.

13. The CAR according to a preceding claim comprising one or more co-stimulatory domains.

14. The CAR according to claim 13, wherein the costimulatory domain is a signaling region of CD28, CD8, 0X40, 4-1 BB, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1 (CD1 la/CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM1 I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof.

15. The CAR according to a preceding claim comprising a protein encoded by suicide gene.

16. The CAR according to claim 15, wherein the protein encoded by a suicide gene comprises iCasp9, CD20, RapaCasp9 or RQR8.

17. An isolated nucleic acid encoding a CAR according to any one of the preceding claims.

18. An isolated nucleic acid according to claim 17, wherein the VH region comprises SEQ ID NO. 32 or 34 and the VL region comprises SEQ ID NO. 33 or 35 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto.

19. A vector comprising a nucleic acid according to claim 17 or 18.

20. The vector according to claim 19, which is a retroviral vector, a DNA vector, a plasmid, a RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.

21. A host cell comprising a nucleic acid according to claim 17 or 18 or a vector according to claims 19 or 20.

22. The host cell of claim 21 , wherein said host cell is a bacterial, yeast, viral or mammalian cell.

23. An isolated cell or cell population comprising one or more CAR according to any one of the preceding claims, a nucleic acid according to 17 or 18 or a vector according to claims 15 or 16.

24. The isolated cell or cell population according to claim 23, wherein said cell is an immune cell.

25. The isolated cell or cell population according to claim 24, wherein the immune cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR-expressing cell, dendritic cell, or NK-T cell and a regulatory T cell.

26. The isolated cell or cell population according to claim 24, wherein the cell is an autologous T cell.

27. The isolated cell or cell population according to claim 24, wherein the cell is an allogeneic T cell.

28. A pharmaceutical composition comprising a cell or cell population as defined in any of claims 23 to 27 and a pharmaceutical acceptable carrier, excipient or diluent.

29. A method for treating a malignancy comprising administering a cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28.

30. The use of a cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28 for the manufacture of a medicament in the treatment of a malignancy.

31 . A cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28 for use in the treatment of a malignancy.

32. The method according to claim 29, use according to claim 30, or the cell or cell population according to claim 31 , wherein the malignancy is a myeloid malignancy and wherein said myeloid malignancy is a Philadelphia-negative Myeloproliferative Neoplasm (MPN), hairy cell leukemia, Prolymphocytic leukemia, Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, a histiocytic disorder and a mast cell disorder.

33. The method, use or cell or cell population according to claim 32, wherein the MPN is selected from the list comprising acute myeloid leukemia, chronic myeloid leukemia, primary myelofibrosis, secondary myelofibrosis, pre-fibrotic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic neutrophilic leukemia and chronic eosinophilic leukemia.

34. The method according to any of claims 29, 32 or 33, use according to any of claims 30, 32 or 33 or the cell or cell population according to claim 31 , 32 or 33, further comprising the step of measuring mutant calreticulin expression in a sample from said subject.

35. The method according to any of claims 29, 32 to 34, use according to any of claims 30, 32 to 34 or the cell or cell population according to claim 31 , 32 to 34, further comprising at least one further therapy.

36. The method, use or cell or cell population according to claim 35, wherein the further therapy is selected from the list comprising administering an immunotherapy, chemotherapy, cellular therapy, biological agents, cytokine therapy, gene therapy and/or a stem cell transplant.

37. The method, use or cell or cell population according to claim 36, wherein the further therapy is a cytokine therapy and is selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics.

38. The method, use or cell or cell population according to claim 36, wherein the cytokine therapy is Eltrombopag.

39. The method, use or cell or cell population according to claim 35 to 38, wherein the further therapy is administered before, after or at the same time as the cell or cell population.

40. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, the method comprising administering to the subject an effective amount of a cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28.

41. A method of providing an anti- myeloid malignancy immunity in a subject, the method comprising administering to the subject an effective amount of a cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28.

42. An ex vivo method for generating a population of cells for use in adaptive immunotherapy comprising transforming said cell with a nucleic acid encoding a CAR as defined in any of claims 1 to 16, a nucleic acid according to 17 or 18 or a vector according to claims 19 or 20.

43. A kit comprising a CAR as defined in any of claims 1 to 16, a nucleic acid according to 17 or 18 or a vector according to claims 19 or 20 or a cell or cell population according to any of claims 23 to 27.

44. A combination therapy comprising an effective amount of a cell or cell population according to any of claims 23 to 27 or a pharmaceutical composition according to claim 28 and an effective amount of a further therapy is selected from the list comprising an immunotherapy, a chemotherapy, a cytokine therapy, gene therapy and/or a stem cell transplant.

45. The combination therapy according to claim 44, wherein the cytokine therapy is selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics

46. A method of making a population of cells of any one of claims 23 to 27, the method comprising: i. contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells; ii. contacting the population of cells (for example, T cells) with the nucleic acid molecule of claim 17 or 18, thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and

Hi. harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration.

47. A combination of an effective amount of a cell or cell population genetically modified to express a CAR and at least one cytokine selected from the list comprising thrombopoietin, Eltrombopag, Romipostim and thrombopoietin mimetics.

48. The combination according to claim 47, wherein the at least one cytokine comprises Eltrombopag.

49. The combination according to any one of claims 47 to 48 wherein the at least one cytokine is administered before, after or at the same time as the cell or cell population.

50. The combination according to any one of claims 47 to 49, wherein the cell or cell population is an immune cell.

51. The combination according to claim 50, wherein the immune cell is selected from from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), tumor infiltrating lymphocyte (TIL), TCR-expressing cell, dendritic cell, or NK-T cell and a regulatory T cell.

52. The combination according to claim 51 , wherein immune cell is a T cell and the T cell is an autologous T cell or allogeneic T cell.

53. A method for treating a malignancy comprising administering a combination according to any of claims 47 to 52.

54. The use of a combination according to any of claims 47 to 52 for the manufacture of a medicament in the treatment of a malignancy.

55. A combination according to any of claims 47 to 52 for use in the treatment of a malignancy.

56. The method according to claim 53, use according to claim 54, or the combination according to claim 55, wherein the malignancy is a myeloid malignancy and wherein said myeloid malignancy is a Philadelphia-negative Myeloproliferative Neoplasm (MPN), hairy cell leukemia, Prolymphocytic leukemia, Hodgkin lymphoma, Blastic plasmacytoid dendritic cell neoplasm, lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, a histiocytic disorder and a mast cell disorder.

57. The method, use or the combination according to claim 56, wherein the MPN is selected from the list comprising acute myeloid leukemia, chronic myeloid leukemia, primary myelofibrosis, secondary myelofibrosis, pre-fibrotic myelofibrosis, polycythemia vera, essential thrombocythemia, chronic neutrophilic leukemia and chronic eosinophilic leukemia.