Chimeric antigen receptor (CAR) that targets receptor-type tyrosine phosphatase c (CD45) and its use to related applications

Abstract

This application relates to the field of hematologic malignancies, autoimmune, inflammatory and fibrotic diseases, HIV as well as conditioning therapies for hematopoietic stem cell transplant (HSC-T), specifically to a chimeric antigen receptor that specifically binds CD45 and its use for treatment, such as to treat a malignancy, non-malignant or infectious diseases of the hematopoietic system in a subject.

Description

[0001] CHIMERIC ANTIGEN RECEPTOR (CAR) THAT TARGETS RECEPTOR-TYPE TYROSINE PHOSPHATASE C (CD45) AND ITS USE TO RELATED APPLICATIONS

[0002] This application relates to the field of hematologic malignancies, autoimmune, inflammatory and fibrotic diseases, HIV as well as conditioning therapies for hematopoietic stem cell transplant (HSC-T), specifically to a chimeric antigen receptor that specifically binds CD45 and its use for treatment, such as to treat a malignancy, non-malignant or infectious diseases of the hematopoietic system in a subject.

[0003] BACKGROUND OF THE INVENTION

[0004] CD45, also known as leukocyte common antigen (LCA), Ly-5 or protein tyrosine phosphatase receptor type C (PTPRC), is an enzyme encoded by the PTPRC gene (Kaplan et al., PNAS 87:7000- 7004 (1990)). CD45 is a member of the family of receptor-type protein tyrosine phosphatases (RPTPs), which includes signaling molecules that regulate a variety of cellular processes including cell proliferation, differentiation, adhesion, and oncogenic transformation. CD45 contains an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains. CD45 is a type I transmembrane protein that is present in various isoforms on most differentiated hematopoietic cells (Holmes, Immunology 7:145-55 (2006), except e.g. erythrocytes and platelets. CD45 has been shown to be a regulatorof T- and B-cell antigen receptor signaling. It functions through either direct interaction with components of the antigen receptor complexes via its extracellular domain, or by activating various Src family kinases (SFK), such as Lek, required for the antigen receptor signaling via its cytoplasmic domain. CD45 also suppresses JAK kinases, and thus functions as a negative regulator of cytokine receptor signaling.

[0005] CD45 is present on the surface of all nucleated hematopoietic cells, including hematopoietic stem and progenitor cells (HSPC), leukocytes, myeloid cells and osteoclasts, which are of hematopoietic origin (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations within CD45 in humans are associated with severe immunodeficiency. This is primarily due to the absence of CD45 on T cells, where it is typically abundant and required to modulate SFK activity during antigen responses. CD45- deficient (CD45- / -) mouse bone marrow contains normal numbers of hematopoietic cells, but the most primitive HSCs are reduced in number, and their mobilization in response to G-CSF is impaired. In part, this defect is intrinsic to the HSC; without CD45-mediated downregulation of SFK activity, integrin-mediated adhesion is high, and HSCs are more likely to remain in the stem cell niche. CD45- / - HSCs are also deficient in G-CSF-stimulated mobilization and homing to the chemokine CXCL12 / SDF-1 , which negatively affects cell engraftment following transplantation. These deficiencies can be restored by supplementation with SFK inhibitors, indicating that this role is usually performed by CD45. Likewise, CD45- / - recipients also show deficient engraftment and subsequent mobilization of normal HSCs, indicating a role for CD45 in the stem cell niche, as well as in the HSC (Shivtiel et al, J Exp Med 205:2381 (2008)). As CD45 is expressed, for example, on HSCs and leukocytes, it presents a target for therapies including conditioning therapies (Gao et al., Blood Adv. 201924;3(18):2700-2711 ; Yeung et al., MolTher. 20245;32(6):1672-1686), immune reset (Gustafsson et al., bioRxiv [Preprint], 2023 Sep 7:2023.09.05.556397), and treatment of malignant blood diseases (Wellhausen N. et al., Sci Transl Med. 202320;15(714):eadi1145; Garaude S. et al., Nature. 2024;630(8017):728-735). lomab-B is a targeted radiotherapy based on anti-CD45 antibody clone BC8 and used for hematopoietic stem cell transplantation (HSC-T) in hematologic malignancies by depleting blood cancer as well as immune and hematopoietic stem cells, which all express CD45. lomab-ACT is a derivative of lomab-B and applied in lymphodepletion for cellulartherapies or for reduced intensity conditioning prior to gene therapies. While lomab-B is in pivotal phase 3 trials in acute myeloid leukemia (AM L), lomab-ACT is evaluated in a clinical phase 1 .

[0006] Preclinically several groups have shown the applicability of CD45 targeted therapies especially in the context of hematologic malignancies. Garaude et al (Nature. 2024;630(8017):728-735) have shown that a CD45 antibody drug conjugate can eradicate human cell line-derived as well as patient derived AML xenografts in mice. Similar to that a CD45-directed CAR T cell product based on antibody clone BC8 eradicated MOLM-14 cell line based tumors in mice (Wellhausen et al., Sci Transl Med. 202320;15(714):eadi 1145).

[0007] As CD45 is expressed on essentially all nucleated hematopoietic cells it is also broadly expressed in hematologic malignancies. High rates of CD45 positivity are shown for essentially all B-cell lymphoid malignancies (Fig. 1A), T-cell lymphoproliferative disorders (Gorczyca et al., Cytometry (Clinical Cytometry) 50:177-190 (2002)), as well as for all myeloid malignancies such as AML and MDS (Fig. 1 b), BPDCN (blastic plasmacytoid dendritic cell neoplasm; Wang et al., Haematologica. 2021 Apr 1 ; 106(4): 1047-1055) or CMML (chronic myelomonocytic leukemia; Eisenwort et al., Leukemia. 2021 Nov 1 ; 35(11 ): 3176-3187). For many heme malignancies, CD45 negativity is the exception (Jha et al. Neurol India 2015;63:276-9; Yamamoto et al., Rinsho Ketsueki. 2017;58(8):938-941 ). Due to its broad expression in the hematologic system, CD45 targeted therapies can also be applied in non-malignant heme diseases such as autoimmune, inflammatory and fibrotic diseases as well as primary immune deficiencies associated with auto-reactive B and T cells.

[0008] SUMMARY OF THE INVENTION

[0009] In certain embodiments, the present disclosure relates to a chimeric antigen receptor (CAR) comprising a) an extracellular binding domain specific for CD45, b) a transmembrane domain, c) at least one co-stimulatory domain, and d) a T cell receptor signaling domain, wherein said extracellular binding domain specific for CD45 comprises a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 2, or functional equivalents thereof.

[0010] In certain embodiments, the present disclosure relates to a chimeric antigen receptor (CAR) comprising a) an extracellular binding domain specific for CD45, b) a transmembrane domain, c) at least one co-stimulatory domain, and d) a T cell receptor signaling domain, wherein said extracellular binding domain specific for CD45 comprises (i) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 2, (ii) a variable heavy chain of SEQ ID No.

[0011] 1 and a variable light chain of SEQ ID No. 163, (iii) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 164, (iv) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 165, or (v) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 166, or functional equivalents thereof.

[0012] In certain embodiments, the present disclosure relates to a chimeric antigen receptor (CAR) comprising a) an extracellular binding domain specific for CD45, b) a transmembrane domain, c) at least one co-stimulatory domain, and d) a T cell receptor signaling domain, wherein said extracellular binding domain specific for CD45 comprises (i) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 2, (ii) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 163, (iii) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 164, (iv) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 165, or (v) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 166.

[0013] In certain embodiments, said chimeric antigen receptor (CAR) further comprises a spacer between said extracellular binding domain specific for CD45 and said transmembrane domain. In certain embodiments, said spacer is or is derived from CD8a or human lgG1 hinge and Fc region. In certain embodiments, said spacer is or is derived from CD8a or human lgG1 hinge. In certain embodiments, said spacer comprises an amino acid sequence selected from SEQ ID. No. 47 or 53- 57.

[0014] In certain embodiments, the variable heavy chain of said extracellular binding domain specific for CD45 of said chimeric antigen receptor (CAR) is N-terminal to the variable light chain. In certain embodiments, said extracellular binding domain specific for CD45 comprises an amino acid sequence selected from SEQ ID No. 33, 35, 37, 39, 41 and 43.

[0015] In certain embodiments, the variable light chain of said extracellular binding domain specific for CD45 of said chimeric antigen receptor (CAR) is N-terminal to the variable heavy chain. In certain embodiments, said extracellular binding domain specific for CD45 comprises an amino acid sequence of SEQ ID No. 32, 34, 36, 38 and 40.

[0016] In certain embodiments, the variable heavy chain and the variable light chain of said extracellular binding domain specific for CD45 are separated by a linker. In certain embodiments, said linker is a glycine-serine linker, preferably a (GGGGS)3-linker (SEQ ID No. 44) or a (GGGGS)4-linker (SEQ ID No. 45). In other embodiments said linker is a GGGSGGSGEPPEGGSG-linker (SEQ ID No. 153), a GGGSGGSGGEPPEGGSGG-linker (SEQ ID No. 154), a GGGSGGSGEPPKGGSG-linker (SEQ ID No. 155), or a GGGSGGSGGEPPKGGSGG-linker (SEQ ID No. 156).

[0017] In certain embodiments, said transmembrane domain of said chimeric antigen receptor (CAR) is or is derived from CD8a or NKG2D. In certain embodiments, said transmembrane domain is or comprises an amino acid sequence selected from SEQ ID No. 59-61 . In certain embodiments, said chimeric antigen receptor (CAR) has one co-stimulatory domain. In certain embodiments, said co-stimulatory domain is selected from 4-1 BB, CD28 and 2B4.

[0018] In certain embodiments, said chimeric antigen receptor (CAR) has two co-stimulatory domains. In certain embodiments, said co-stimulatory domains are 4-1 BB and CD28, 4-1 BB and 2B4, or CD28 and 2B4.

[0019] In certain embodiments, said T cell receptor signaling domain of said chimeric antigen receptor (CAR) is CD3 zeta domain.

[0020] In certain embodiments, said chimeric antigen receptor (CAR) comprises the amino acid sequence of SEQ ID No. 106, 107, 108 or 109.

[0021] In certain embodiments, the present disclosure provides a nucleic acid encoding aforementioned chimeric antigen receptors (CARs).

[0022] In certain embodiments, the present disclosure provides a vector comprising aforementioned nucleic acid.

[0023] In certain embodiments, the present disclosure provides a host cell comprising aforementioned nucleic acid or aforementioned vector, or which expresses aforementioned chimeric antigen receptors (CARs). In certain embodiments, said host cell is a T cell or a NK cell.

[0024] In certain embodiments, the present disclosure aforementioned chimeric antigen receptors (CARs or aforementioned host cells for use in medicine.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figuresl A and B: RNA expression data (log2 expression) of PTPRC in samples from blood cancer patients as analyzed with Bloodspot database and web interface (https: / / www.fobinf.com / ). (A) across leukemias and myelodysblastic syndromes in the leukemia MILE study (Haferlach, T. et al. Journal of Clinical Oncology 28, 2529-2537 (2010)), and (B) across AML and MDS subtypes in 200 clinically annotated adult cases of de novo AML (N Engl J Med. 201330;368(22):2059-74).

[0027] Figure 2 shows RNA expression data of PTPRC in samples from autoimmune disease patients as analyzed with the ADEx database and web interface (https: / / adex.genyo.es / ). Panel A: Illumina HumanHT-12 V4.0 PTPRC expression from 187 healthy and 325 systemic lupus erythematosus (SLE) samples, Panel B: Illumina HumanWG-6 v3.0 PTPRC expression from 32 healthy and 190 Sjoegren’s syndrome (SjS) samples, Panel C: Affymetrix HT_HG-U133_Plus_PM PTPRC expression from 30 healthy and 493 rheumatoid arthritis (RA) samples, Panel D: Illumina HumanHT-12 V4.0 PTPRC expression from 45 healthy and 116 SLE samples, Panel E: Illumina HiSeq 2500 (Homo sapiens) PTPRC expression from 15 healthy and 57 systemic sclerosis (SSc) samples.

[0028] Figures 3A-F show the expression of CD45 CAR on Jurkat NFAT luciferase reporter cells. eGFP expression (expressed from a 2A-containing bicistronic expression cassette) after transduction of CD45 and control CAR constructs in Jurkat NFAT luciferase CD45 shielded as well as CD45 knock- out (KO) reporter cells as quantified by flow cytometry (GFP+ in percent).

[0029] A: #1169: CD8a:scFV_Mornay-HC-LC-3xGS:CD8a_Hinge:4-1 BB:CD3Zeta:T2A:EGFP (Mornay-C- 8S-bbz)

[0030] B: #2238: CD8a:scFV_Hollandaise-LC-HC-3xGS:CD8a_Hinge:4-1 BB:CD3_Zeta:T2A:EGFP

[0031] (Hollandaise-C-8S-bbz)

[0032] C: #2204: CD8a:scFV_ Valentina-HC-LC-4xGS:CD8a_Hinge:4-1 BB:CD3_Zeta:T2A:EGFP

[0033] (Valentina-C-8S-bbz)

[0034] D: #2244: CD8a:scFV_Sriracha-LC-HC-4xGS:CD8a_Hinge:4-1 BB:CD3_Zeta:T2A:EGFP (Sriracha- C-8S-bbz)

[0035] E: #2256: CD8a:scFV_CD33-3xGS:CD8a_Hinge:4-1 BB:CD3_Zeta:T2A:EGFP(CD33-C-8S-bbz)

[0036] F: #2249: CD8a:scFV_Ref001_3xGS:CD8a_Hinge:4-1 BB:CD3_Zeta:T2A:EGFP (Ref001-C-8S-bbz) Upper panel: Jurkat NFAT CD45 shielded reporter cells; lower panel: Jurkat NFAT CD45 knock-out (KO) reporter cells.

[0037] Figure 4. Representative histograms (A) and mean fluoresence intensity quantification (B) of EGFP and anti-G4S-PE signal in CD45 shielded (BE) or KO Jurkat NFAT cells transduced with indicated CAR constructs.

[0038] Figure 5. Luminescence signal detected in CD45 shielded (BE) Jurkat NFAT cells expressing indicated CD45 CARs co-cultured with CD45 wt (A), shielded (BE) (B) or KO (C) Molm-14 cells for 24 hours at 5:1 (Effector:Target) cell ratio. Same experimental set up was performed with CD45 wt (D), shielded (BE) (E) or KO (F) Jurkat cells as target cells for 6 hours at 5:1 (Effector:Target) cell ratio. Figure 6. Luminescence signal detected in CD45 wt, shielded (BE) or KO Jurkat NFAT cells expressing indicated CD33 or Ref001 isotype control CAR constructs, co-cultured with CD45 wt or shielded (BE) Molm-14 cells for 24 hours at 3:1 (Effector:Target) cell ratio.

[0039] Figure 7. Representative flow cytometry histograms of EGFP signal in CD45 shielded Jurkat NFAT cells transduced with indicated CD45 CARs, compared to untransduced Jurkat NFAT cells (Jurkat NFATwt).

[0040] Figure 8. Representative flow cytometry plots of EGFP, anti-G4S and CD45-Fc signal in CD45 shielded Jurkat NFAT cells transduced with indicated CD45 CARs, compared to untransduced Jurkat NFAT cells.

[0041] Figure 9. Quantification of mean fluorescence intensity of CD45-Fc (A), anti-G4S (B) and eGFP (C) signal in CD45 shielded Jurkat NFAT cells transduced with indicated CD45 CARs, compared to untransduced Jurkat NFAT cells, shown in Figure 8.

[0042] Figure 10. Luminescence signal detected in CD45 shielded Jurkat NFAT cells expressing indicated CD45 CARs, co-cultured with CD45 wt or shielded (BE) Jurkat cells for 6 (A) and 24 (B) hours at 3:1 (Effector:Target) cell ratio. Same experimental set up as in (A) was used with CD45 wt or shielded (BE) Molm-14 cells as target cells for 6 (C) and 24 (D) hours at 3:1 (Effector:Target) cell ratio.

[0043] Figure 11 . Flow cytometry analysis of primary T cells phenotype, activation and exhaustion status after CD45 editing and CAR expression 7 days after base editing and CAR transduction. T cells were stained with antibodies against CD3, CD197, CD45RA, CD69, CD25, PD1 and LAG3 and analyzed by flow cytometry. A. Quantification of the percentage of Central Memory (CM, CD197+CD45RA-), Naive (CD197+CD45RA+), Effector Memory (EM, D197-CD45RA-) and Terminally Differentiated Effector Memory Re-expressing (EMRA, CD197-CD45RA+) cells within the CD3+ T cell population. B. Quantification of the percentage of CD3+CD69+, CD3+CD25+ (markers of activation) and CD3+PD1 + and CD3+LAG3+ (markers of exhaustion) T cells..

[0044] Figure 12. Flow cytometry analysis of CAR expression and CD45 editing in primary T cells. 7 days after CAR transduction and CD45 editing, T cells were stained with two anti-CD45 antibodies, one labelled with APC (which does not bind CD45 shielded variant) and one with BV711 (which binds both CD45 wt and shielded variants), and in a separate staining with an anti-G4S antibody labelled with APC. Representative flow plots are shown in (A). Quantification of percentage of positive cells and mean fluorescence intensity of EGFP and G4S-APC signals are shown in (B), (C), (D) and (E).

[0045] Figure 13. A. Flow cytometry analysis of CAR expression and CD45 editing in primary T cells. 7 days after CAR transduction and CD45 editing, T cells were stained with an anti-G4S antibody labelled with APC and a CD45 protein carrying a llama lgG2b Fc tag detected with an anti-llama IgG secondary antibody labelled with PE (Abeam) and analyzed by flow cytometry. Representative flow plots are shown. B. T cells were stained with two anti-CD45 antibodies, one labelled with APC (which does not bind CD45 shielded variant) and one with BV711 (which binds both CD45 wt and shielded variants) to determine CD45 editing efficiency. Representative flow plots are shown.

[0046] Figure 14. Percentage of killing of CD45 wt or shielded (BE) target cells (Jurkat or CCRF-CEM) by CD45 CAR-expressing effector T cells after 24 (A, C) and 48 (B, D) hours of incubation. Different ratios of effector vs target cells (fixed at 1 ) were tested. Target cells express Luciferase and luminescence signal was used to calculate the percentage of cell killing as follows: % specific cell ki lli ng= 100-( 100* (tested samples / untransduced control samples).

[0047] Figure 15. Percentage of killing of CD45 wt or shielded (BE) CCRF-CEM target cells by CD45 CAR- expressing effector T cells after 24 (A) and 48 (B) hours of incubation. Different ratios of effector vs target cells (fixed at 1) were tested. Target cells express luciferase and luminescence signal was used to calculate the percentage of cell killing as follows: % specific cell killing= 100-( 100* (tested samples / untransduced control samples).

[0048] Figure 16. A. Concentration of IFNg (pg / mL) measured in supernatants of co-cultures of CD45 CAR- expressing T cells (effector cells) and CD45 wt CCRF-CEM (target cells) at indicated Effector:Target (E:T) cell ratios after 24 and 48 hours of incubation. Dotted line indicates IFNg levels of control conditions containing untransduced T cells as effector cells at the highest E:T ratio tested (6:1 ). B. Concentration of IFNg (pg / mL) measured in supernatants of co-cultures of CD45 CAR-expressing T cells (effector cells) and CD45 wt or shielded (BE) CCRF-CEM (target cells) at 6:1 E:T cell ratio after 48 hours of incubation. Dotted line indicates IFNg levels of control conditions containing untransduced T cells as effector cells. DESCRIPTION OF THE INVENTION

[0049] Definitions

[0050] The term “antibody” refers to an immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as CD45. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0051] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi-specific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols.1 -2, 2nd Ed., Springer Press, 2010).

[0052] A “single-chain antibody” or “scFv” refers to a genetically engineered molecule containing the VH and VL domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, I Drugs, 13:543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in an scFv, is typically not decisive for scFvs. Thus, scFvs with both possible arrangements (VH-domain-linker domain-VL-domain; VL-domain-linker domain- VH- domain) may be used. Similarly, various linker lengths may be used (e.g., but not limited to 15 or 20 amino acids).

[0053] In a “dsFv” the VH and VL of an scFv have been mutated to introduce a disulfide bond to stabilize the association of the chains (e.g., Reiter et al., Protein Eng. 1995 8(12):1323-31 ). Diabodies also are included, which are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994). ScFvs can also be stabilized by the introduction of charges and thus salt bridges in the variable and linker regions instead of cysteines.

[0054] Antibodies also include genetically engineered forms such as chimeric or humanized antibodies and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman and Co., New York, 1997.

[0055] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). In several embodiments, the VH and VL combine to specifically bind the antigen. In additional embodiments, only the VH is required. For example, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996).

[0056] References to "VL" or "VH" refer to the variable domain of an antibody light chain or the variable heavy chain, respectively, including that of an Fv, scFv, dsFv or Fab.

[0057] The VH and VL contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991 ). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0058] The “CDRs” or “complementarity-determining regions” are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well- known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 ;"Kabat" numbering scheme), Al-Lazikani et al., (JMB 273,927-948, 1997;"Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003;"IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1 , CDR2, and CDR3 (from the N-terminus to C-terminus) and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1 , LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1 , HCDR2, and HCDR3.

[0059] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Harlow and Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).)

[0060] A "humanized" antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, rabbit, chicken or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a "donor," and the human antibody or antigen binding fragment providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90 percent, such as about 95 percent or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences. A "chimeric antibody" is an antibody which includes sequences derived from two different antibodies and are typically of different species. In some embodiments, a chimeric antibody can include the VH and VL regions of a rodent, rabbit or chicken monoclonal antibody (such as anti-CD45 clone BC8) and human constant regions, such as human IgG 1 regions or Fc silenced human lgG1 regions.

[0061] A" fully human antibody" or "human antibody" is an antibody, which includes sequences from (or derived from) the human genome and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0062] The term “epitope” refers to the antigenic determinant of a binding domain, such as an antibody ora fragment thereof. Epitopes are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide. In some examples a disclosed antibody specifically binds to an epitope on CD45. Changing, modifying or mutating one or more amino acids in an antigenic epitope can result in the loss of specific binding of a monoclonal antibody or a similar binding entity.

[0063] When referring to an antibody or antigen binding fragment the terms “specifically binds”, “specific for”, or the like, refer to a binding reaction which determines the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as CD45) and does not bind in a significant amount to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art. With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a KD of less than about 10-7 Molar, such as less than about 10-8 Molar, 10-9, or even less than about 10-10 Molar.

[0064] The term “KD“ refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex. The antibodies disclosed herein specifically bind to a defined target (or multiple targets, in the case of a bispecific antibody). Thus, an antibody that specifically binds to an epitope on CD45 is an antibody that binds substantially to CD45, including cells or tissue expressing CD45 substrate to which the CD45 is attached, or CD45 in a biological specimen. An antibody that specifically binds to an epitope on CD45 might not bind to variant CD45, where the epitope is modified or mutated by a single or several amino acid modifications. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody or conjugate including an antibody (such as an antibody that specifically binds CD45 or conjugate including such antibody) and a non-target (such as a cell that does not express CD45). Typically, specific binding results in a much stronger association between the antibody and protein or cells bearing the antigen than between the antibody and protein or cells lacking the antigen. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats or real-time interaction measurements are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. These formats can be ELISA, flow cytometry, surface plasmon resonance (SPR), biolayer interferometry (BLI) or many others.

[0065] A "chimeric antigen receptor” or “CAR” refers to an engineered T cell receptor having an extracellular binding domain, for example an extracellular antibody-derived targeting domain (such as an scFv) joined to one or more intracellular signaling domains of a T cell receptor.

[0066] A "chimeric antigen receptor T cell" or “CAR T-cell” refers to a T cell expressing a CAR and which has an antigen specificity determined by the extracellular binding domain, such as an antibody- derived targeting domain.

[0067] A "chimeric antigen receptor NK cell" or “CAR NK-cell” refers to an NK cell expressing a CAR and which has an antigen specificity determined by the extracellular binding domain, such as an antibody-derived targeting domain.

[0068] The term “T cell” refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. They are called T cells because they mature in the thymus from thymocytes. Generally, mature T cells express CD3. The terms “natural killer cells” and “NK cells” refer to innate lymphoid cells that are large granular lymphocytes (LGL) and are differentiated from the common lymphoid progenitor- generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells do not express T-cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD16 (FcgammaRIII) and CD56 in humans, NK1 .1 or NK1 .2 in C57BL / 6 mice. NKp46 cell surface marker is expressed in humans, several strains of mice and in monkey species.

[0069] The terms “CD45” and “PTPRC” refer to a protein also known as Leukocyte common antigen (L- CA) or T200. Human CD45 has the following amino acid sequence (UniProt P08575-3, defined as canonical sequence) p p p g least four polypeptide chains, which are non-covalently associated with the T cell receptors on the surface of T cells. The four polypeptide chains include two CD3-epsilon chains, a CD3-delta chain and a CD3-gamma chain. CD3 is present on both helperT cells and cytotoxic T cells.

[0070] The terms “costimulatory signaling domain” or “costimulatory domain” as used herein refer to the domain of a costimulatory molecule or costimulatory receptor responsible for mediating a costimulatory response by the T cell. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof. Exemplary costimulatory signaling domains include 4-IBB (CD137), BAFFR, 0X40, CD27, CD28, ICOS, CD40, 2B4, GITR, HVEM, 0X40, RELT, TACI, TROY, and TWEAK.

[0071] The terms “T cell receptor signaling domain” or “TCR signaling domain” as used herein refer to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the TCR signaling domain contains a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, FcR common gamma chain (FCRG or FCRgamma or CD132), FER1 (Fc Epsilon receptor 1 ), CD79a, CD79b, Fcgamma Rlla, Fcgamma Rllla, DAP10, and DAP12. A preferred TCR signaling domain is a TCR signaling domain selected from CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon. A particularly preferred TCR signaling domain CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon.

[0072] The term “polypeptide” refers to a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some embodiments, the polypeptide is a disclosed antibody or a fragment thereof.

[0073] The term “amino acid substitution” or “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made as long as the final construct possesses the desired characteristics, e.g., reduced or increased binding to an Fc receptor or reduced or increased binding of an antibody to an epitope. Amino acid sequence deletions and insertions include N-and / or C-terminal deletions and insertions of amino acid residues. Particular amino acid mutations are amino acid substitutions. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid residue by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution of glycine at position 237 of the antibody Fc region to alanine can be indicated as 237A, G237, G237A, or Gly237Ala.

[0074] “Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, a CD45-specific antibody can include up to 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for CD45. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0075] Furthermore, one of ordinary skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5 percent, in some embodiments less than 1 percent) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

[0076] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0077] 1 ) Serine (S), Threonine (T);

[0078] 2) Aspartic acid (D), Glutamic acid (E);

[0079] 3) Asparagine (N), Glutamine (Q);

[0080] 4) Arginine (R), Lysine (K);

[0081] 5) Isoleucine (I), Leucine (L), Alanine (A), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0082] Non-conservative substitutions are those that reduce an activity or function of the CD45-specific antibody, such as the ability to specifically bind to CD45. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest. In certain cases, it might be desired to reduce the binding strength of an antibody to the target. In such cases, the introduction of non-conservative substitutions can also be beneficial.

[0083] The term “nucleic acid” refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T." Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a single-stranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand;" sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences;" sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0084] The term "cDNA" refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0085] Recombinant: A “recombinant nucleic acid” is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A “recombinant protein” or a “recombinant polypeptide” is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several embodiments, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

[0086] The similarity between amino acid sequences is expressed in terms of the “sequence similarity” or “sequence homology” between the sequences. Sequence homology is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2:482, 1981 ; Needleman and Wunsch, J. Mol. Biol.48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet.6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0087] Homologs and variants of a VL or a VH of an antibody, or a fragment thereof, that specifically bind a polypeptide are typically characterized by possession of at least about 75 percent, for example at least about 80 percent, 85 percent, 90 percent, 91 percent, 92 percent, 93 percent, 94 percent, 95 percent, 96 percent, 97 percent, 98 percent or 99 percent sequence identity counted over the full-length alignment with the amino acid sequence of interest. Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 98 percent, or at least 99 percent sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80 percent sequence identity over short windows of 10-20 amino acids and may possess sequence identities of at least 85 percent or at least 90 percent or 95 percent depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.

[0088] The terms "encoding", “encode”, and the like, refer to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double- stranded forms of DNA.

[0089] A nucleic acid molecule encoding a protein can be “codon optimized” forexpression of the protein in a particular organism by including the codon most likely to encode a particular amino acid with the amino acid sequence. Codon usage bias is the differences in the frequency of occurrence of synonymous codons (encoding the same amino acid) in coding DNA. A codon is a series of three nucleotides (a triplet) that encodes a specific amino acid residue in a polypeptide chain or for the termination of translation. There are 20 different naturally occurring amino acids, but 64 different codons (61 codons encoding for amino acids plus 3 stop codons). Thus, there is degeneracy because one amino acid can be encoded by more than one codon. A nucleic acid sequence can be optimized for expression in a particular organism (such as a human) by evaluating the codon usage bias in that organism and selecting the codon most likely to encode a particular amino acid. Multivariate statistical methods, such as correspondence analysis and principal component analysis, are widely used to analyze variations in codon usage. Computer programs are available to implement the statistical analyses related to codon usage, such as Codon W, GCUA, and INCA.

[0090] In the context of the present disclosure, a "degenerate variant" refers to a polynucleotide encoding a protein (for example, a CAR that specifically recognizes CD45) that includes a sequence that is degenerate as a result of the genetic code. There are twenty natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the CAR encoded by the nucleotide sequence is unchanged.

[0091] The term “expression” refers to the transcription or translation of a nucleic acid sequence. For example, a gene can be expressed when its DNA is transcribed into an RNAor RNA fragment, which in some examples is processed to become mRNA. A gene may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence.

[0092] Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0093] The term “expression control sequence” refers to a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter. A “promoter” is the minimal sequence sufficient to direct transcription. Also included are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (such as metallothionein, CD8a p, EF-1a or hPGK promoter), from mammalian viruses (such as the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter or the CMV promoter) or synthetic promoters (such as RPBSA) can be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences. A polynucleotide can be inserted into an expression vector that contains a promoter sequence, which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

[0094] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0095] The term “isolated” refers to a biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent pure. A “transformed” cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the terms “transduction” and “transformation” encompass all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, the use of plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0096] The term “linker” refers to a bi-functional, structurally flexible molecule that can be used to link two molecules into one contiguous molecule, for example, to link an antibody fragment to a spacer or transmembrane domain. In some cases, a linker is a peptide within an antigen binding fragment (such as an Fv fragment) which serves to indirectly bond the VH and VL.

[0097] The terms "conjugating,", "joining,", "bonding" or "linking" refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently a polypeptide, such as an scFv to a spacer or a transmembrane domain. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. "Chemical means" refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0098] A first nucleic acid sequence is “operably linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0099] The terms “detect”, “detecting”, and the like, refer to the method or process of identifying the existence, presence, or fact of something, such as the existence of a malignancy, such as a lymphoid malignancy, or an HIV infection. General methods of detecting are known to the skilled artisan and may be supplemented with the protocols and reagents disclosed herein.

[0100] A "detectable marker” refers to a detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non- limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). In one example, a "labeled antibody" refers to incorporation of another molecule in the antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.

[0101] The term “drug” refers to any compound used to treat, ameliorate or prevent a disease or condition in a subject. In some embodiments herein, the drug is a CAR T or CAR NK cell.

[0102] The term “subject” refers to a living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In an example, a subject is a human. In a particular example, the subject is a pediatric subject, such as a human child age 2-5 years old. In an additional example, a subject is selected that has a lymphoid or myeloid malignancy or is at risk of having a lymphoid or myeloid malignancy. In another example, a subject has a non-malignant hematologic disease, which can be an acquired or a genetic (primary) disease.

[0103] The term “therapeutically effective amount” refers to the amount of an agent (such as a T cells and / or NK cells expressing a CAR) that alone, or together with one or more additional agents, induces the desired response, such as, for example treatment of a CD45-positive cancer, in a subject. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations that has been shown to achieve a desired in vitro effect. Ideally, a therapeutically effective amount provides a therapeutic effect without causing a substantial cytotoxic effect in the subject.

[0104] In one example, a desired response is to decrease the size, volume, or number (such as metastases) of CD45-positive cancer cells in a subject, and / or neoplastic lesions or the number of malignant, auto-reactive or activated cells in blood, bone marrow or tissue in a subject, or to decrease the number of hematopoietic stem cells in the bone marrow or to reduce the number or virus-infected cells in a subject. For example, the agent can decrease the size, volume, or number of CD45-positive cells in a subject by a desired amount, for example by at least 5 percent, at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 50 percent, at least 75 percent, at least 90 percent, or at least 95 percent as compared to a response in the absence of the agent. Several preparations disclosed herein are administered in therapeutically effective amounts. A therapeutically effective amount that is administered to a human or veterinary subject will vary depending upon a number of factors associated with that subject, for example the overall health of the subject. A therapeutically effective amount can be determined by varying the dosage and measuring the resulting therapeutic response, such as the regression of a CD45-positive lymphoid malignancy. Therapeutically effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays. The disclosed agents can be administered in a single dose, or in several doses, as needed to obtain the desired response. However, the therapeutically effective amount of can be dependent on the source applied, the subject being treated, the severity and type of the condition being treated, and the manner of administration.

[0105] A therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a therapeutic response. For example, a therapeutically effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

[0106] "Preventing" a disease refers to inhibiting the full development of a disease.

[0107] "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as a malignancy.

[0108] The terms “acute myeloid leukemia” and “AML” refer to a cancer of the myeloid line of bone marrow and / or blood hematopoietic cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cell production.

[0109] The terms “acute lymphoblastic leukemia” and “ALL” refer to a cancer of the lymphoid line of blood cells characterized by the development of large numbers of immature lymphocytes. It can affect B lymphocytes (B-ALL) and T lymphocytes (T-ALL).

[0110] The terms “blastic plasmacytoid dendritic cell neoplasm” and “BPDCN” refer to a rare hematologic malignancy, which derives from the precursors of plasmacytoid dendritic cells. The terms “chronic myelomonocytic leukemia” and “CMML” refer to a cancerous disease which starts when a blood-forming hematopoietic stem cell in the bone marrow acquires mutations. This results in abnormal blood cell production and an overproduction of blasts and immature monocytes.

[0111] The terms “myelodysblastic syndrome” and “MDS” refer to a group of cancers in which immature blood cells in the bone marrow do not mature, and as a result, they do not develop into differentiated cells and accumulate due to over-production. Some people with MDS go on to develop acute myeloid leukemia (AML).

[0112] Hematological malignancies may derive from either of the two major blood cell lineages: myeloid and lymphoid cell lines. The myeloid cell line includes granulocytes, erythrocytes, thrombocytes, macrophages and mast cells, such diseases being refer to as “myeloid malignancies”; the lymphoid cell line produces B, T, NK and plasma cells. Lymphoma, lymphocytic leukemia, and myeloma are from the lymphoid line, such diseases being refer to as “lymphoid malignancies”.

[0113] The term “administer”, “administration”, and the like, as used herein, refer to a method of delivering agents, compounds, or compositions to the desired site of biological action. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

[0114] The term “combination therapy” refers to the administration of more than one agent to for the treatment of a diseases or disorder. The term “combination chemotherapy” refers to the administration of more than one agent to for the treatment of cancer.

[0115] The term “chemotherapeutic agent” refers to any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. For example, chemotherapeutic agents can be useful for the treatment of cancer, such as T-ALL or B-ALL. Particular examples of chemotherapeutic agents that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. In one embodiment, a chemotherapeutic agent is a radioactive compound. One of skill in the art can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.17 in Abeloff, Clinical Oncology 2nd ed., (C) 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, HJ. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993; Chabner and Longo, Cancer Chemotherapy and Biotherapy: Principles and Practice (4th ed.). Philadelphia: Lippincott Willians and Wilkins, 2005; Skeel, Handbook of Cancer Chemotherapy (6th ed.). Lippincott Williams and Wilkins, 2003).

[0116] The term “tumor” refers to an abnormal growth of cells, which can be benign or malignant (a malignancy). Cancer is a malignant tumor (a malignancy), which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease" refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.

[0117] The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and / or can metastasize is referred to as "malignant."

[0118] Examples of hematological lymphoid tumors include but are not limited to leukemias, including acute leukemias (such as 11q23-positive acute leukemia, acute lymphocytic leukemia), chronic leukemias (such as chronic lymphocytic leukemia), lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and hairy cell leukemia. In specific non-limiting examples, the lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin’s lymphoma, or a diffuse large B cell lymphoma.

[0119] Examples of hematological myeloid tumors include but are not limited to chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), polycythemia vera (PV) , primary myelofibrosis (PMF), Essential thrombocythemia (ET), chronic eosinophilic leukemia, myeloproliferative neoplasm (MPN), mastocytosis, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), Juvenile myelomonocytic leukemia (JMML), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), myeloid sarcoma, myeloid proliferations related to Down syndrome, transient abnormal myelopoiesis (TAM), acute myelomonocytic leukemia, acute monoblastic / monocytic leukemia, pure erythroid leukemia, acute megakaryoblastic leukemia, acute basophilic leukemia, acute panmyelosis with myelofibrosis and blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0120] Examples of non-malignant, acquired autoimmune and inflammatory hematologic diseases include but are not limited to rheumatoid arthritis (RA), psoriatic arthritis (PA), undifferentiated arthritis, multiple sclerosis (MS), systemic sclerosis (ScS, scleroderma), systemic lupus erythematosus (SLE), Sjoegren’s syndrome (SjS), myasthenia gravis, Guillain-Barre syndrome, vasculitis, ANCA-positive vasculitis, IgA vasculitis, (poly)myositis, polymyalgia rheumatica, granulomatosis, Behcet disease, pemphigus vulgaris, ulcerative colitis (UC), Crohn’s disease (CD), atherosclerosis and giant cell arteritis.

[0121] Examples of non-malignant, genetic / primary immune-deficiencies, some of which can be associated with autoimmunity and / or require hematopoietic stem cell transplantation, include but are not limited to severe combined immunodeficiency (SCID), primary immune deficiency, Omenn syndrome, autoimmune lympho-proliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1 ), BENTA disease, Caspase eight deficiency state (CEDS), CTLA-4 deficiency, common variable immunodeficiency (CVID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, chronic granulomatous disease (CGD), selective IgA deficiency, hemophagocytic lymphohistiocytosis, Wiskott-Aldrich Syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lympho-proliferative disease (XLP), XMEN disease and many others (e.g., https: / / primaryimmune.org / understanding-primary-immunodeficiency / types-of-pi).

[0122] Examples of fibrotic diseases include but are not limited to kidney fibrosis, liver / hepatic fibrosis, lung / pulmonary fibrosis, fibrosis of the skin, systemic sclerosis (SSc), fibrosis of the heart / cardiac fibrosis, pulmonary arterial hypertension (PAH), myelofibrosis, idiopathic pulmonary fibrosis (IPF), interstitial lung disease, hypertrophic cardiomyopathy or pulmonary hypertension.

[0123] Examples of infectious diseases include but are not limited to HIV.

[0124] Chimeric antigen receptors

[0125] The present disclosure provides novel chimeric antigen receptors that are specific for CD45. Despite numerous anti-CD45 antibodies being available in the prior art, it remains a challenge to selected the correct anti-CD45 antibody, convert the molecule into a format usable in a CAR context and design the overall architecture of the CAR In the broadest sense, the present disclosure provides a chimeric antigen receptor (CAR) comprising a) an extracellular binding domain specific for CD45, b) a transmembrane domain, c) at least one co-stimulatory domain, and d) a T cell receptor signaling domain.

[0126] A. Extracellular domain

[0127] The chimeric antigen receptors of the present disclosure contain an extracellular binding domain specific for CD45. In certain embodiments, the chimeric antigen receptors of the present disclosure contain an extracellular binding domain specific for human CD45. In certain embodiments, the chimeric antigen receptors of the present disclosure contain an extracellular binding domain specific for a polypeptide comprising the amino acid sequence of SEQ ID No. 9. CD45. In certain embodiments, the chimeric antigen receptors of the present disclosure contain an extracellular binding domain specific for a polypeptide consisting of the amino acid sequence of SEQ ID No. 9. CD45.

[0128] In certain embodiments, the chimeric antigen receptors of the present disclosure contain an extracellular binding domain specific for CD45, wherein said extracellular binding domain is an antibody or an antibody fragment. In preferred embodiments, said extracellular binding domain is an antibody fragment. In other preferred embodiments, said extracellular binding domain is an scFv. In other preferred embodiments, said extracellular binding domain is a dsFv. In other preferred embodiments, said extracellular binding domain is an scFv stabilized by salt bridges.

[0129] Among the antibodies tested in the present disclosure are two prior art antibodies, known as antibodies Jelly and Kiebitz (WO2024 / 133890).

[0130] Therefore, in certain embodiments, the extracellular binding domain specific for CD45 comprises a VH and a VL of antibody Jelly, or a functional equivalent thereof. Antibody Jelly has a VH of the following amino acid sequence: and a VL of the following amino acid sequence:

[0131] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VL with an amino acid sequence of SEQ ID No. 2.

[0132] In certain embodiments, the extracellular binding domain specific for CD45 has VH consisting of an amino acid sequence of SEQ ID No. 1 and the VL of the amino acid sequence of SEQ ID No. 2.

[0133] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH having a HCDR1 of amino acid sequence NYDMS (SEQ ID No. 3), a HCDR2 of amino acid sequence YISSGGVSTYYPDTVKG (SEQ ID No. 4) and a HCDR3 of amino acid sequence RYDVWWYFDV (SEQ ID No. 5), and a VL having a LCDR1 of amino acid sequence RSSQSIVHSQGNTYLE (SEQ ID No. 6), a LCDR2 of amino acid sequence KVSNRAS (SEQ ID No. 7) and a LCDR3 of amino acid sequence FQGSHVPMYT (SEQ ID No. 8).

[0134] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH and a VL of antibody Kiebitz, ora functional equivalent thereof. Antibody Kiebitz has a VH of the following amino acid sequence: and a VL of the following amino acid sequence:

[0135] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VLwith an amino acid sequence of SEQ ID No. 10.

[0136] In certain embodiments, the extracellular binding domain specific for CD45 has a VH consisting of the amino acid sequence of SEQ ID No. 1 and a VL consisting of the amino acid sequence of SEQ ID No. 10.

[0137] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH having a HCDR1 of amino acid sequence NYDMS (SEQ ID No. 3), a HCDR2 of amino acid sequence YISSGGVSTYYPDTVKG (SEQ ID No. 4) and a HCDR3 of amino acid sequence RYDVWWYFDV (SEQ ID No. 5), and a VL having a LCDR1 of amino acid sequence RSSQSIVHSNAKTYLE (SEQ ID No. 11), a LCDR2 of amino acid sequence KVSNLFS (SEQ ID No. 12) and a LCDR3 of amino acid sequence FQGSHVPMYT (SEQ ID No. 8).

[0138] In certain embodiments, the extracellular binding domain specific for CD45 has a VH of the following amino acid sequence: and a VL of the following amino acid sequence:

[0139] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VL with an amino acid sequence of SEQ ID No. 163.

[0140] In certain embodiments, the extracellular binding domain specific for CD45 has VH consisting of an amino acid sequence of SEQ ID No. 1 and the VL of the amino acid sequence of SEQ ID No. 163.

[0141] In certain embodiments, the extracellular binding domain specific for CD45 has a VH of the following amino acid sequence: and a VL of the following amino acid sequence: In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VL with an amino acid sequence of SEQ ID No. 164.

[0142] In certain embodiments, the extracellular binding domain specific for CD45 has VH consisting of an amino acid sequence of SEQ ID No. 1 and the VL of the amino acid sequence of SEQ ID No. 164.

[0143] In certain embodiments, the extracellular binding domain specific for CD45 has a VH of the following amino acid sequence: and a VL of the following amino acid sequence:

[0144] In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VL with an amino acid sequence of SEQ ID No. 165.

[0145] In certain embodiments, the extracellular binding domain specific for CD45 has VH consisting of an amino acid sequence of SEQ ID No. 1 and the VL of the amino acid sequence of SEQ ID No. 165.

[0146] In certain embodiments, the extracellular binding domain specific for CD45 has a VH of the following amino acid sequence: and a VL of the following amino acid sequence: In certain embodiments, the extracellular binding domain specific for CD45 comprises a VH with an amino acid sequence of SEQ ID No. 1 and a VL with an amino acid sequence of SEQ ID No. 166.

[0147] In certain embodiments, the extracellular binding domain specific for CD45 has VH consisting of an amino acid sequence of SEQ ID No. 1 and the VL of the amino acid sequence of SEQ ID No. 166.

[0148] Antibodies that are converted into scFv format to be used in the context of chimeric antigen receptors may have various architectures. For example, the VH may be N-terminal of the VL. Alternatively, the VL may be N-terminal of the VH. Also, the VH and VL may be separated by linkers of different length and nature.

[0149] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising an amino acid sequence selected from any one of SEQ ID No.’s 13-43. In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising an amino acid sequence selected from any one of SEQ ID No.’s 32-43.

[0150] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 32.

[0151] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 33.

[0152] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 34.

[0153] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 35.

[0154] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 36.

[0155] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 37.

[0156] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 38. In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 39.

[0157] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 40.

[0158] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 41.

[0159] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 42.

[0160] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv comprising the amino acid sequence of SEQ ID No. 43.

[0161] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of an amino acid sequence selected from any one of SEQ ID No.’s 13-43. In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of an amino acid sequence selected from any one of SEQ ID No.’s 32-43.

[0162] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 32.

[0163] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 33.

[0164] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 34.

[0165] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 35.

[0166] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 36.

[0167] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 37. In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 38.

[0168] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 39.

[0169] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 40.

[0170] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 41 .

[0171] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 42.

[0172] In certain embodiments, the extracellular binding domain specific for CD45 comprises an scFv consisting of the amino acid sequence of SEQ ID No. 43.

[0173] The VH and the VL of the scFv can be operatively linked to one another by a flexible linker. One type of linker are glycine-serine linkers, such as a (GGGGS)3-linker (SEQ ID No. 44) or a(GGGGS)4-linker (SEQ ID No. 45). Such linkers ensure that the VL and VH sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker (see, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering, Vols.1-2, 2nd Ed., Springer Press, 2010; Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold Spring Harbor Laboratory, New York, 2013).

[0174] Therefore, in certain embodiments the binding domain specific for CD45 comprises an scFv comprising a VH and a VL joined by a 15 amino acid long peptide linker. In certain embodiments, said 15 amino acid long peptide linker is a (GGGGS)3-linker (SEQ ID No. 44). In certain embodiments the binding domain specific for CD45 comprises an scFv comprising a VH and a VL joined by a 20 amino acid long peptide linker. In certain embodiments, said 20 amino acid long peptide linker is (GGGGS)4-linker (SEQ ID No. 45).

[0175] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from

[0176] N- to the C-terminus: a VH, a (GGGGS)3-linker (SEQ ID No. 44), and a VL. In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VL, a (GGGGS)3-linker (SEQ ID No. 44), and a VH.

[0177] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a (GGGGS)4-linker (SEQ ID No. 45), and a VL.

[0178] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VL, a (GGGGS)4-linker (SEQ ID No. 45), and a VH.

[0179] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGEPPEGGSG-linker (SEQ ID No. 153), and a VL.

[0180] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGGEPPEGGSGG-linker (SEQ ID No. 154), and a VL.

[0181] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGEPPKGGSG-linker (SEQ ID No. 155), and a VL.

[0182] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGGEPPKGGSGG-linker (SEQ ID No. 156), and a VL.

[0183] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGCPPCGGSG-linker (SEQ ID No. 157), and a VL.

[0184] In certain embodiments the binding domain specific for CD45 comprises an scFv comprising from N- to the C-terminus: a VH, a GGGSGGSGGCPPCGSGGG-linker (SEQ ID No. 158), and a VL.

[0185] The VLand VH can be in any order, such that either the VL or the VH is at the N-terminus of the scFv. In some specific non-limiting examples, the VL is at the N-terminus. In other specific non-limiting examples, the VH is at the N-terminus.

[0186] The CAR can include a signal peptide sequence, e.g., N-terminal to the antigen binding domain. The signal peptide sequence can include any suitable signal peptide sequence. In an embodiment, the signal peptide sequence is the human CD8a signal sequence, such as an amino acid sequence including or consisting of MALPVTALLLPLALLLHAARP (SEQ ID NO : 46 ) .

[0187] However, other signal sequences known in the art can be utilized. While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary in order for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence may be cleaved off from the CAR. Accordingly, in some embodiments, the CAR lacks a signal peptide sequence.

[0188] Between the extracellular binding domain specific for CD45, such as an scFv, and the transmembrane domain of the CAR, there can be a spacer domain comprising a polypeptide sequence. In certain embodiments, said spacer domain is an immunoglobulin domain, such as a human immunoglobulin sequence.

[0189] In certain embodiments, the immunoglobulin domain comprises an immunoglobulin hinge, a CH2 and a CH3 region (hinge-CH2-CH3 or hinge-Fc) from human immunoglobulin G1 (lgG1 ). In certain embodiments, the immunoglobulin domain comprises an immunoglobulin hinge, a CH2 and a CH3 region (hinge-CH2-CH3 or hinge-Fc) from human immunoglobulin G1 (lgG1) modified with three amino acid mutations (LALA-PA) to prevent binding to Fc receptors (CH2-CH3 LALA-PA). Alternative immunoglobulin spacers include CH2-CH3 regions from human IgG 1 with alternative mutations to prevent binding to Fc receptors, including but not limited to ‘AEASS’, ‘LALA’ or aglycosylated variants. Further immunoglobulin spacers include hinge-CH2-CH3 regions from human lgG2 or lgG4with orwithout mutations to prevent bindingto Fc receptors. In this regard, the spacer domain can include an immunoglobulin domain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 47, comprising or consisting of human IgG 1 CH2-CH3 domains including ‘LALA-PA’ mutations, which are shown underlined:

[0190] An immunoglobulin hinge is N-terminal to the CH2-CH3 domains. This hinge region can be from wildtype human lgG1 (SEQ ID NO: 48), or from engineered human lgG1 with C220S (SEQ ID NO: 49), C220A mutations (SEQ ID NO: 50), or a shortened human IgG 1 hinge with (SEQ ID NO: 51 ) or without TGGG linker (SEQ ID NO: 52):

[0191] The following Fc silent IgG 1 hinge-CH2-CH3 domains may be used in the context of the CAR of the present disclosure:

[0192] Fc silent human lgG1 C220S hinge-CH2-CH3 domain

[0193] Fc silent human lgG1 C220A hinge-CH2-CH3 domain

[0194] Fc silent human lgG1 TGGG hinge-CH2-CH3 domain

[0195] Fc silent human lgG1 short hinge-CH2-CH3 domain

[0196] In certain embodiments, the spacer domain includes an unstructured part from the extracellular region of human CD8a (‘CD8a spacer’; SEQ ID NO: 53). In this regard, the spacer domain can include CD8a amino acids 138-181 comprising or consisting of the amino acid sequence set forth as: Without being bound to a particular theory, it is believed that a CD8a-based or a hinge-CH2-CH3 spacer moves the antigen binding domain of the CAR away from the membrane of CAR-expressing cells and may therefore allowthe formation of a more active immunological synapse. However, the spacer domain may not be necessary.

[0197] B. Transmembrane domain

[0198] The chimeric antigen receptors of the present disclosure contain a transmembrane domain specific for CD45. The transmembrane domain of the CAR is genetically fused to the extracellular domain.

[0199] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be from any membrane-bound or transmembrane protein. Exemplary transmembrane domains for use in the disclosed CARs include the transmembrane region(s) of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134 (0X40), CD137, CD154, CD278 (ICOS) and CD314 (NKG2D). Alternatively, the transmembrane domain can be a synthetic transmembrane domain, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In several embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular T cell signaling domain and / or T cell costimulatory domain of the CAR. Such short oligo- or polypeptide linker sequences can include or consist of parts of glycine-serine rich portions of linker sequences described above and listed in SEQ ID NOs: 44 and 45 or consist of the four amino acids NHRN (SEQ ID NO: 58).

[0200] In some embodiments, the transmembrane domain comprises the transmembrane domain of a T cell receptor, such as a CD8a transmembrane domain. Thus, in certain embodiments the CAR comprises a CD8a transmembrane domain including or consisting of the following amino acid sequence:

[0201] In other embodiments, the CAR comprises a CD8a transmembrane domain with four hydrophilic amino acid residues at the C-terminus including or consisting of the following amino acid sequence: In other embodiments, the transmembrane domain comprises the transmembrane domain of a T cell costimulatory molecule, such as CD137 or CD28 or of an NK cell stimulatory molecule such as human NKG2D. Thus, in certain embodiments the CAR comprises a human NKG2D transmembrane domain including or consisting of the following amino acid sequence:

[0202] C. Intracellular domain

[0203] The intracellular regions of the CARs of the present disclosure comprise at least one co-stimulatory domain and a T cell receptor signaling domain. Exemplary co-stimulatory domains and T cell receptor signaling domains are provided herein, and are known to the person of ordinary skill in the art.

[0204] In many cases it is not necessary to use the entire polypeptide sequence of the wildtype proteins comprising the co-stimulatory domains and T cell receptor signaling domain. To the extent that a truncated portion of these domains is used, such truncated portion may be used in place of the intact full-length polypeptide, as long as the respective domains are functional and, for example, transduces the relevant T cell effector function signal. These domains can act alone or in combinations to initiate signal transduction following antigen receptor engagement. Derivatives or variant of the foregoing, as well as synthetic sequence with the same functional capabilities can also be employed.

[0205] T cell receptor signaling domains regulate primary activation of the T cell receptor complex either in a stimulatory way, or in an inhibitory way. The disclosed CARs can include primary cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of ITAM containing primary cytoplasmic signaling sequences that can be included in a disclosed CAR include those from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In several embodiments, the cytoplasmic signaling molecule in the CAR includes an intracellularT cell signaling domain from CD3 zeta.

[0206] The intracellular region of the CAR of the present disclosure can contain the ITAM containing primary cytoplasmic signaling domain (such as CD3-zeta) by itself, or combined with any other desired cytoplasmic domain(s) useful in the context of a CAR. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and one or more intracellular co- stimulatory signaling domains. Such co-stimulatory signaling domains include, but are not limited to, a CD28, a 4-1 BB (CD137) and / or a CD244 (2B4) domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of one or more costimulatory molecules. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1 BB (CD137), 0X40 (CD134), CD30, CD40, PD-1 , ICOS, lymphocyte function- associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD244 (2B4) and B7-H3.

[0207] In certain embodiments, the co-stimulatory domain is a 4-1 BB costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 62). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 62. In certain embodiments, the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO: 62.

[0208] In certain embodiments, the co-stimulatory domain is a CD28 costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 63). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 63. In certain embodiments, the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO: 63.

[0209] In certain embodiments, the co-stimulatory domain is a CD28-41 BB dual costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCEL (SEQ ID NO: 64). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 64. In certain embodiments, the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO: 64.

[0210] In certain embodiments, the co-stimulatory domain is a 41 BB-CD28 dual costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRSKRSRLLHSDYMNMTPRRPGPTRKHY QPYAPPRDFAAYRS (SEQ ID NO: 112). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 112. In certain embodiments, the co- stimulatory domain consists of the amino acid sequence of SEQ ID NO: 112. In certain embodiments, the co-stimulatory domain is a 2B4 costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQP SRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 65). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 65. In certain embodiments, the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO: 65.

[0211] In certain embodiments, the co-stimulatory domain is a 4-1 BB-2B4 dual costimulatory domain. In certain embodiments, the co-stimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELWRRKRKEKQSETSPKEFLTIYEDVKDLKT RRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPK AQNPARLSRKELENFDVYS (SEQ ID NO: 66). In certain embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID No. 66. In certain embodiments, the co-stimulatory domain consists of the amino acid sequence of SEQ ID NO: 66.

[0212] In certain embodiments, the T cell receptor signaling domain is a humanCD3 zeta signaling domain. In certain embodiments, the T cell receptor signaling domain comprises the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 67) In certain embodiments, the T cell receptor signaling domain comprises the amino acid sequence of SEQ ID No. 67. In certain embodiments, the T cell receptor signaling domain consists of the amino acid sequence of SEQ ID NO: 67.

[0213] The cytoplasmatic co-stimulatory and T cell receptor signaling domains can be linked to each other in a random or a specific order. In certain embodiments, the 4-1 BB domain is N-terminalof the CD3 zeta domain. In certain embodiments, the 2B4 domain is N-terminal of the CD3 zeta domain. In certain embodiments, the dual CD28-4-1 BB or 4-1 BB-CD28 domain is N-terminal of the CD3 zeta domain. Optionally, a short polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage between such domains. A glycine-serine doublet provides a particularly suitable linker. A linker may also be employed between the transmembrane domain and the signaling domains of the CAR. D. Additional Description

[0214] As already described herein above the arrangement of the domains of the CARs of the presentation has some flexibility.

[0215] In some embodiments, the order of the domains starting from the N-terminus is: signal sequence

[0216] - VL- linker - VH - spacer - transmembrane domain - human 4-1 BB signaling molecule - human CD3 zeta signaling molecule, C-terminus.

[0217] In some embodiments, the order of the domains starting from the N-terminus is: signal sequence

[0218] - VH - linker - VL - spacer - transmembrane domain - human 4-1 BB signaling molecule - human CD3 zeta signaling molecule, C-terminus.

[0219] An exemplary second generation CAR of the present disclosure containing one costimulatory domain has the following amino acid sequence (signal sequence in bold, VL is underlined, the linker is shown in italics, VH is bold and underlined, the human CD8 hinge and transmembrane domain is highlighted, the human 4-1 BB costimulatory domain is in italics and underlined, the human CD3 zeta signaling domain is shown in plain text):

[0220] An exemplary third generation CAR of the present disclosure containing two costimulatory domains has the following amino acid sequence (signal sequence in bold, VL is underlined, the linker is shown in italics, VH is bold and underlined, the human CD8 hinge and transmembrane domain is highlighted, the human CD28 costimulatory domain is in italics and bold, the human 4- 1 BB costimulatory domain is in italics and underlined, the human CD3 zeta signaling domain is shown in plain text):

[0221]

[0222] Also provided are functional portions of any of the CARs described herein. The term "functional portion" when used in reference to a CAR refers to any part or fragment of a CAR, which part or fragment retains the biological activity of the CAR of which it is a part or derived from (the parent CAR), and thus can be used to target T cells and / or natural killer cells to CD45. Functional portions encompass, for example, those parts of a CAR that retain the ability to recognize target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CAR. In reference to the parent CAR, the functional portion can comprise, for instance, about 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, or more, of the parent CAR. The CAR or functional portion thereof, can include additional amino acids at the amino or carboxy terminus, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR. In some examples, the additional amino acids do not interfere with the biological function of the CAR or functional portion, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. In other examples, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CAR.

[0223] Also provided are functional variants of the CARs described herein, which have substantial or significant sequence identity or similarity to a parent CAR, which functional variant retains the biological activity of the CAR of which it is a variant. As modulation, e.g. increase or reduction of the binding affinity to the target, can fundamentally change the activity and persistence of a CAR, functional variants encompass, for example, those variants of the CAR described herein (the parent CAR) that retain the ability to recognize target cells to a similar extent, the same extent, or to a higher extent. As reduced / low affinity CARs can have superior in vivo efficacy than high affinity CARs (Olson MLet al., Leukemia volume 36, pagesl 943-1946, 2022) this can also be CARs binding the target to a lower extent than the parent CAR. In reference to the parent CAR, the functional variant can, for instance, be at least about 30 percent, about 50 percent, about 75 percent, about 80 percent, about 85 percent, about 90 percent, about 91 percent, about 92 percent, about 93 percent, about 94 percent, about 95 percent, about 96 percent), about 97 percent, about 98 percent, about 99 percent or more identical in amino acid sequence to the parent CAR.

[0224] Afunctional variant can, for example, comprise the amino acid sequence of the parent CAR with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent CAR with at least one non- conservative amino acid substitution. In this case, the conservative or non-conservative amino acid substitution does not inhibit the biological activity of the functional variant. The conservative or non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent CAR. The conservative or non-conservative amino acid substitution may change (increase or reduce) the binding affinity of the functional variant, such that the biological activity of the functional variant is increased and / or exhaustion is reduced and / or in vivo expansion is increased as compared to the parent CAR. The conservative or non-conservative amino acid substitution may change (increase or reduce) the binding affinity of the functional variant, such that the in vivo persistence of the functional variant is increased as compared to the parent CAR. The CAR can also include up to ten conservative amino acid substitutions, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions, provided the binding of the molecule is not lost. Substitutions can be made, for example, in the VH region, VL region, linker region, spacers, and / or the signal sequence.

[0225] A functional variant can also, for example, comprise the amino acid sequence of the parent CAR with insertions or deletions of amino acids. For example, the amino acid deletion or insertion may change (increase or reduce) the binding affinity of the functional variant, such that the biological activity of the functional variant is increased and / or exhaustion is reduced and / or in vivo expansion is increased as compared to the parent CAR. The amino acid deletion or insertion may change (increase or reduce) the binding affinity of the functional variant, such that the in vivo persistence of the functional variant is increased as compared to the parent CAR. For example, the linker length in the scFv can influence binding and expression and thus the CAR activity. Accordingly, the linker composition in the scFv can influence binding and expression and thus the CAR activity.

[0226] The CARs (including functional portions and functional variants of the invention) can include synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4- aminophenylalanine, 4- nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, beta -phenylserine beta -hydroxyphenylalanine, phenylglycine, a - naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1 ,2,3,4- tetrahydroisoquinoline- 3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N'.N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a- aminocyclopentane carboxylic acid, a- aminocyclohexane carboxylic acid, oc- aminocycloheptane carboxylic acid, -(2-amino-2- norbornane)-carboxylic acid, gamma - diaminobutyric acid, a, beta -diaminopropionic acid, homophenylalanine, and a-tert-butylglycine. The CARs (including functional portions and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N- acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0227] Methods of generating chimeric antigen receptors, immune cells including such receptors, and their use (e.g., for treatment of cancer) are known in the art and further described herein (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; Till et al., 2008, Blood, 112:2261 -2271 ; Park et al., Trends BiotechnoL, 29:550-557, 2011 ; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Publication Nos. WC2012 / 079000, WC2013 / 126726; and U.S. Publication No.2012 / 0213783, each of which is incorporated by reference herein in its entirety). For example, a nucleic acid molecule encoding a disclosed chimeric antigen binding receptor can be included in an expression vector (such as a lentiviral vector) for expression in a host cell, such as a T or an NK cell, to make the disclosed CAR. In some embodiments, methods of using the chimeric antigen receptor include isolating T or NK cells from a subject, transforming or transducing the T or NK cells with an expression vector (such as a lentiviral vector or a gamma retroviral vector) encoding the chimeric antigen receptor, and administering the engineered T or NK cells expressing the chimeric antigen receptor to the subject for treatment, for example for treatment of a CD45-positive cancer in the subject.

[0228] Chimeric antigen receptors (CARs) are disclosed that are artificially constructed chimeric proteins including an extracellular antigen binding domain (e.g., single chain variable fragment, scFv) that specifically binds to CD45, linked via a spacer to a transmembrane domain, and linked to one or more intracellular T cell signaling domains. Characteristics of the disclosed CARs include their ability to redirect T cell specificity and reactivity towards wildtype human CD45 expressing cells in a non-HLA-restricted manner. The non-H LA-restricted CD45 recognition gives T or NK cells expressing a disclosed CAR the ability to recognize antigen independent of antigen processing and / or presentation by HLA molecules. The intracellular T cell signaling domains can include, for example, a T cell receptor signaling domain and one or more T cell costimulatory signaling domains. The T cell receptor signaling domain refers to a portion of the CAR comprising the intracellular domain of a T cell receptor, such as the intracellular portion of the CD3 zeta protein. The costimulatory signaling domains refer to a portion of the CAR comprising the intracellular domains of one or more costimulatory molecules, which are cell surface molecules other than an antigen receptor or their ligands that are required for an efficient response of lymphocytes to antigen, such as CD28, 4-1 BB and / ora CD244 (2B4). Details of the CAR are disclosed in further detail below.

[0229] The CAR Hollandaise-C-8S-BBz has the following amino acid sequence:

[0230] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 106. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 106. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0231] The CAR Mornay-C-8S-BBz has the following amino acid sequence: In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 107. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 107. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0232] The CAR Sriracha -C-8S-BBz has the following amino acid sequence:

[0233] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 108. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 108. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0234] The CAR Valentina -C-8S-BBz has the following amino acid sequence:

[0235] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 109. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 109. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0236] The CAR Hollandaise-l-8L-BBz has the following amino acid sequence:

[0237] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 82. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 82. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0238] The CAR Mornay-l-8L-BBz has the following amino acid sequence:

[0239] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 83. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 83. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0240] The CAR Hollandaise-C-8L-BBz has the following amino acid sequence:

[0241] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 84. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 84. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0242] The CAR Mornay-C-8L-BBz has the following amino acid sequence: In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 85. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 85. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0243] The CAR Hollandaise-l-8L-28BBz has the following amino acid sequence:

[0244] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 113. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 113. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0245] The CAR Mornay-l-8L-28BBz has the following amino acid sequence:

[0246] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 114. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 114. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0247] The CAR Hollandaise-C-8L-28BBz has the following amino acid sequence:

[0248] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 115. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 115. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0249] The CAR Mornay-C-8L-28BBz has the following amino acid sequence:

[0250] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 116. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 116. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0251] The CAR Hollandaise-C-8L-2B4z has the following amino acid sequence:

[0252] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 117. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 117. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0253] The CAR Mornay-C-8L-2B4z has the following amino acid sequence:

[0254] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 118. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 118. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0255] The CAR Hollandaise C-NK-2B4z has the following amino acid sequence:

[0256] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 119. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 119. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0257] The CAR Mornay-C-NK-2B4z has the following amino acid sequence:

[0258] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 120. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 120. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0259] The CAR Hollandaise-l-NK-2B4z has the following amino acid sequence:

[0260] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 121. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 121. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0261] The CAR Mornay-l-NK-2B4z has the following amino acid sequence:

[0262]

[0263] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 122. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 122. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0264] The CAR Hollandaise-C-8L-BB2B4z has the following amino acid sequence:

[0265] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 123. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 123. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0266] The CAR Mornay- C-NK-BB2B4zhas the following amino acid sequence:

[0267]

[0268] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 124. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 124. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0269] The CAR Hollandaise-l-8L-BB2B4z has the following amino acid sequence:

[0270] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 125. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 125. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0271] The CAR Mornay-l-8L-BB2B4z has the following amino acid sequence:

[0272] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 126. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 126. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0273] The CAR Hollandaise-C-NK-BB2 has the following amino acid sequence: In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 127. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 127. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0274] The CAR Mornay-C-NK-BB2 has the following amino acid sequence:

[0275] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 128. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 128. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0276] The CAR Hollandaise-l-NK-BB2B4z has the following amino acid sequence:

[0277] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 129. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 129. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0278] The CAR Mornay-l-NK-BB2B4z has the following amino acid sequence:

[0279] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 130. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 130. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0280] The CAR Hollandaise-l-8S-BBz has the following amino acid sequence:

[0281]

[0282] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 131. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 131 . In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0283] The CAR Mornay-l-8S-BBz has the following amino acid sequence:

[0284] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 132. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 132. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0285] The CAR Hollandaise-l-8S-28BBz has the following amino acid sequence:

[0286]

[0287] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 133. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 133. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0288] The CAR Mornay-l-8S-28BBz has the following amino acid sequence:

[0289] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 134. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 134. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0290] The CAR Hollandaise-C-8S-28BBz has the following amino acid sequence:

[0291] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 135. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 135. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0292] The CAR Mornay-C-8S-28BBz has the following amino acid sequence:

[0293] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 134. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 136. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) . The CAR Hollandaise-C-8S-2B4z has the following amino acid sequence:

[0294] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 137. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 137. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0295] The CAR Mornay-C-8S-2B4z has the following amino acid sequence:

[0296] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 138. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 138. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) . The CAR Hollandaise-l-8S-2B4z has the following amino acid sequence:

[0297] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 139. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 139. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0298] The CAR Mornay-l-8S-2B4z has the following amino acid sequence: In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 140. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 140. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0299] The CAR Hollandaise-C-8S-BB2B4z has the following amino acid sequence:

[0300] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 141. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 141. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0301] The CAR Mornay-C-8S-BB2B4z has the following amino acid sequence:

[0302] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 142. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 142. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0303] The CAR Hollandaise-l-8S-BB2B4z has the following amino acid sequence:

[0304] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 143. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 143. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0305] The CAR Mornay-l-8S-BB2B4z has the following amino acid sequence:

[0306]

[0307] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 144. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 144. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP (SEQ ID NO : 46) .

[0308] The CAR Hollandaise-l-8L-BB28z has the following amino acid sequence:

[0309] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 145. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 145. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP (SEQ ID NO : 46) .

[0310] The CAR Mornay-l-8L-BB28z has the following amino acid sequence:

[0311]

[0312] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 146. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 146. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0313] The CAR Hollandaise-C-8L-BB28z has the following amino acid sequence:

[0314] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 147. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 147. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0315] The CAR Mornay-C-8L-BB28z has the following amino acid sequence:

[0316]

[0317] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 148. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 148. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0318] The CAR Hollandaise-l-8S-BB28z has the following amino acid sequence:

[0319] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 149. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 149. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0320] The CAR Mornay-l-8S-BB28z has the following amino acid sequence:

[0321]

[0322] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 150. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 150. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0323] The CAR Hollandaise-C-8S-BB28z has the following amino acid sequence:

[0324] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 151. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 151 . In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0325] The CAR Mornay-C-8S-BB28z has the following amino acid sequence:

[0326]

[0327] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 152. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 152. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0328] The CAR Agrodolce l-8S-BBz has the following amino acid sequence:

[0329] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 159. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 159. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) . The CAR Agrodolce C-8S-BBz has the following amino acid sequence:

[0330] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 160. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 160. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0331] The CAR Tkemali l-8S-BBz has the following amino acid sequence:

[0332] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 161. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 161 . In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0333] The CAR Tkemali C-8S-BBz has the following amino acid sequence:

[0334] In certain embodiments, the present application provides a chimeric antigen receptor comprising the amino acid sequence of SEQ ID No. 162. In certain embodiments, the present application provides a chimeric antigen receptor consisting of the amino acid sequence of SEQ ID No. 162. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide sequence, preferably a signal peptide sequence according to amino acids MALPVTALLLPLALLLHAARP ( SEQ ID NO : 46) .

[0335] As a control CAR, a CAR with an scFv against chicken lysozyme was used. This CAR, RefOOl -C-8S- BBz, has the following amino acid sequence:

[0336] Also as a control CAR, a CAR with an scFv against CD33 was used. This CAR, CD33-C-8S-BBZ, has the following amino acid sequence:

[0337]

[0338] Polynucleotides, vectors and host cells

[0339] Nucleic acid molecules encoding the CARs of the present disclosure are also provided by the present disclosure.

[0340] Nucleic acids encoding these molecules can readily be produced by one of skill in the art, using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several embodiments, a nucleic acid molecule can encode a CAR including the VH, the VL, or both the VH and VL of a monoclonal antibody that specifically binds CD45, such as, but not limited to, the antibodies Jelly and Kiebitz or an antigen binding fragment thereof. In several embodiments, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell, for example a T or NK cell) to produce the CAR.

[0341] In some embodiments, the complete nucleic acid sequence encoding the CAR is codon optimized for expression in human cells, such as human T cells or natural killer (NK) cells. In additional embodiments, a nucleic acid sequence encoding one or more components of the CAR (VH, VL, signal sequence, human spacer (hinge-CH2-CH3 or CD8a) and transmembrane domain (CD8a or NKG2D), human costimulatory domains, human CD3 zeta signaling molecule) can be codon optimized for expression in human cells.

[0342] Thus, in some embodiments, the nucleic acid sequence includes one or more codon optimized nucleic acid sequences.

[0343] Nucleic acid sequences encoding the antibodies, antibody binding fragments, CARs and conjugates that specifically bind CD45 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol.68:90-99, 1979; the phosphodiester method of Brown et al., Meth. Enzymol.68:109-151, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981 ; the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862, 1981 , for example, using an automated synthesizer as described in, for example, Needham-VanDevanter et al., Nucl. Acids Res.12:6159- 6168, 1984; and the solid support method of U.S. Patent No.4,458,066. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0344] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In: Current Protocols in Molecular Biology, John Wiley and Sons, New York, 2013).

[0345] Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill. Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation, site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps. In addition to recombinant methods, the CARs of the present disclosure can also be constructed in whole or in part using standard peptide synthesis well known in the art.

[0346] The nucleic acid molecule encoding the chimeric antigen binding receptor can be operably linked to a promoter. The nucleic acid molecule encoding the CAR can be included in a vector (such as a lentiviral vector or gamma retroviral vector) for expression in a host cell. Exemplary cells are mammalian cells, and include a T cell, such as a cytotoxic T lymphocyte (CTL) ora regulatory T cell, and a NK cell. In specific non-limiting examples, the cell is a T cell, such as a CD3+ T cell. The CD3+ T cell can be a CD4+ or a CD8+ T cell. In other specific non-limiting examples, the cell is a NK cell. Methods of generating nucleic acid molecules encoding chimeric antigen receptors and T cells (or NK cells) including such receptors are known in the art (see, e.g., Brentjens et al., 2010, Molecular Therapy, 18:4, 666-668; Morgan et al., 2010, Molecular Therapy, published online February 23, 2010, pages 1 -9; Till et al., 2008, Blood, 112:2261 -2271 ; Park et al., Trends BiotechnoL, 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT Pub. WO2012 / 079000, WO2013 / 126726; and U.S. Pub. 2012 / 0213783,

[0347] The expression of nucleic acids encoding the antibodies and antigen binding fragments described herein can be achieved by operably linking the DNA encoding the CAR to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus (CMV) promoter, EF-1a promoter, hPGK (phosphoglycerate kinase), a human T cell lymphotrophic virus promoter (HTLV)-1 or a synthetic RPBSA promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

[0348] To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation (internal ribosomal binding sequences), and a transcription / translation terminator. For eukaryotic cells, the control sequences can include a promoter and / or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40, cytomegalovirus or a synthetic promoter, and a polyadenylation sequence, and can further include splice donor and / or acceptor sequences (for example, CMV and / or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by well-known methods such as calcium phosphate treatment, electroporation, transduction, lipofection or LNP transfection of mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes. When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or transduction with virus vectors may be used. Eukaryotic cells can also be co- transformed with polynucleotide sequences encoding the CAR, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40), a lentivirus or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011 ).

[0349] In some embodiments, a viral vector is utilized forexpression of the CAR. Viral vectors include, but are not limited to simian virus 40, adenoviruses, adeno-associated virus (AAV), lentiviral vectors, and retroviruses, such as gamma retroviruses. Retroviral vectors provide a highly efficient method for gene transfer into eukaryotic cells. Moreover, retroviral integration takes place in a controlled fashion and results in the stable integration of one or a few copies of the new genetic information per cell. Without being bound by theory, lentiviral vectors have the advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non- proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. The use of lentiviral vectors to express a CAR is known in the art and is disclosed for example in U.S. Application No. 2014 / 0050708.

[0350] In some embodiments, host cells are produced for introduction into a subject of interest. The host cell can be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC), a purified T cell, or a purified NK cell. The T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal (such as a human patient to which the CAR-T cell will later be administered). If obtained from a mammalian subject, such as a human subject, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, iPSCs or other tissues or fluids. T cells can also be enriched for or purified. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD3+ cells, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD3+ T cell, such as a CD8+ T cell or a CD4+ T cell. In alternative embodiments, the cell can be an NK cells, such as an NK, NKT or iNK-T cell obtained from the same subject to which the CAR-NK cell will later be administered. Accordingly, if obtained from a mammalian subject, such as a human subject, the NK cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, iPSCs or other tissues or fluids. T cells can also be enriched for or purified.

[0351] In some embodiment the CAR can also be expressed by in vivo transfection or transduction of hematopoietic cells, eg, as T and / or NK cells through direct infusion of nanoparticle-formulated nucleic acids or engineered viral vectors. In some embodiments, in vivo transfection can be done using RNA or DNA encoding the CAR formulated in targeted or untargeted lipid nanoparticles (LNPs) of different compositions. In vivo transduction can be done by using virus or viral particles using endogenous or engineered cell-type specificity for targeting and delivering RNA or DNA encoding the CAR.

[0352] Also provided is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., aT cell), which does not comprise any recombinant expression vector, ora cell other than a T or NK cell, e.g., a B cell, a macrophage, a neutrophil, a monocyte, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the recombinant expression vector encoding the CAR. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein. The T cells can be CD3+ T cells, such as CD8+ T cell or a CD4+ T cells. In some embodiments, the T cells are transformed with Epstein Barr virus, see Savoldo et al., Blood 110: 2620-2630, 2007, incorporated herein by reference. In other embodiments, the cells are heterologous to a recipient (see below), and are deleted fora HLA class I and / or T cell receptor, so they do not provoke a graft versus host disease (GVHD) or host versus graft reaction. The cells can also be NK, NKTor iNKT cells. The cells can be autologous to a recipient or allogeneic. These populations are of use in any of the methods disclosed herein.

[0353] Medical use, methods of treatment and pharmaceutical compositions

[0354] Disclosed herein are methods for treating a tumor, wherein the cells of the tumor express CD45, specifically they produce the mRNA that encodes CD45 and / or CD45 protein. In some embodiments, the tumor is a malignancy, such as a lymphoid malignancy. The lymphoid malignancy can be adult T cell leukemia, cutaneous T cell lymphoma, anaplastic large cell lymphoma, Hodgkin’s lymphoma, or diffuse large B cell lymphoma. The adult T cell leukemia can be chronic, acute, smoldering or lymphomatous type. The cutaneous T cell lymphoma can be mycosis fungoides, pagetoid reticulosis, Sezary syndrome, granulomatous slack skin, lymphomatoid papulosis, pityriasis lichenoides chronica, pityriasis lichenoides et varioliformis acuta, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, non- mycosis fungoides CD30- cutaneous large T-cell lymphoma, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, angiocentric lymphoma, or blastic NK-cell lymphoma. In specific non-limiting examples, the cutaneous T cell lymphoma is peripheral T-cell lymphoma (PTCL) / cutaneous T-cell lymphoma (CTCL), or mycosis fungoides (MF) / Sezary syndrome (SS).

[0355] In other embodiments, the tumor is a myeloid malignancy. The myeloid malignancies are categorized into five primary types, namely acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN; e.g., chronic myeloid leukemia (CML), polycythemia vera, essential thrombocythemia, primary myelofibrosis, and mastocytosis), myelodysplastic and myeloproliferative (MDS / MPN) neoplasms, and myeloid neoplasms associated with eosinophilia and abnormalities of growth factor receptors derived from platelets or fibroblasts.

[0356] The method includes administering to the subject a therapeutically effective amount of the pharmaceutical composition including a vector, such as a lentiviral vector encoding the CAR, and / or administering a therapeutically effective amount of a pharmaceutical composition comprising cells, such as T cells and / or NK cells, that express the CAR and will deplete CD45 positive cells such as malignant hematologic cells. Subjects in need thereof may subsequently undergo standard treatment with chemotherapy or surgery. Non-clinical proof of concept for this concept using CD45-directed CAR T-cells in hematologic malignancy was shown by Wellhausen N. et al., Sci Transl Med 20;15(714), 2023 as well as Harfmann M et al., Cancers 16(2):334, 2024.

[0357] Human Immunodeficiency Virus (HIV) infection, and the resulting Acquired Immunodeficiency Syndrome (AIDS), remain threats to global public health, despite extensive efforts to develop anti- HIV therapeutic agents. Virus that persists despite suppressive combination antiretroviral treatment (cART) remains an obstacle to definitive treatment of HIV infection. Infected T follicular helper cells (Tfh), sheltered in the immune privileged site of B cell follicles of secondary lymphoid tissues, as well as memory and naive resting CD4 T cells as well as myeloid cells such as monocytes and macrophages represent an important source of such residual virus, at least in part because effector CD8+ T cells able to clear infected cells from other sites are unable to efficiently access all such cells. Methods are disclosed herein for treating a subject, such as a subject with an HIV infection. The methods include administering to the subject a therapeutically effective amount of T cells and / or NK cells that express a CAR.

[0358] In other embodiments, disclosed are methods of treating inflammatory, autoimmune and fibrotic diseases by administering to the subject a therapeutically effective amount of the pharmaceutical composition including a vector, such as a lentiviral vector encoding the CAR, and / or administering a therapeutically effective amount of a pharmaceutical composition comprising cells, such as T cells and / or NK cells, that express the CAR. In general, variants of CD45 are associated with altered immune function eg in auto-immune diseases such as multiple sclerosis, rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, Crohn’s disease, ulcerative colitis or psoriasis vulgaris (reviewed in Rheinlaender A., Immunology Letter 196, 22-32, 2018). Furthermore, as a marker of lymphoid cells CD45 is highly expressed on auto-reactive B and T-cells in auto-immune diseases. Consistent CD45 expression is also detected on monocytes, macrophages, neutrophils, basophils and other hematopoietic cell types (https: / / www.proteinatlas.org / ENSG00000081237- PTPRC / immune+cell) involved in inflammation, autoimmunity and fibrosis. Accordingly, a CD45 targeted therapy, in this case an antibody drug conjugate (ADC) can deplete tissue-resident myeloid cells. Moreover, it was shown that the replacement of tissue-resident myeloid cells using a CD45-ADC can result in a measurable therapeutic effect in clonal hematopoiesis-driven atherosclerotic disease (Gustafsson K. et al., Blood Adv. 7(22): 6964-6973, 2023). Along the same lines, transplantation after CD45-ADC can correct Rag1 immunodeficiency in mice (Paia F. et al., J Allergy Clin Immunol 153(1 ):341 -348, 2024) and there is also reason to believe that associated autoimmunity can be alleviated as well.

[0359] In other embodiments, disclosed are methods of CD45 targeted conditioning for hematopoietic stem cell transplant by administering to the subject a therapeutically effective amount of the pharmaceutical composition including a vector, such as a lentiviral vector encoding the CAR, and / or administering a therapeutically effective amount of a pharmaceutical composition comprising cells, such as T cells and / or NK cells, that express the CAR. Proof of concept was shown with CD45-directed ADCs and radioligand conjugates in humans, non-human primates and mice. The CD45 targeted compounds completely ablated human HSCs without major toxicity and enabled successful engraftment of gene-modified autologous and allogeneic human HSCs (Yeung J., et al., Mol Ther 5;32(6):1672-1686, 2024; Saha A. et al., Blood. 23;143(21 ):2201 -2216, 2024; Gustafsson K. et al., Blood Adv. 7(22): 6964-6973, 2023; Paia F. et al., J Allergy Clin Immunol 153(1 ):341 -348, 2024; Dawicki W. et al., Oncotarget 11 (39) :3571 -3581 , 2020; Cassaday RD et al., Clin Cancer Res 25(23):6932-6938, 2019).

[0360] In some embodiments, disclosed is a method of treating an HIV infection, by administering to the subject a therapeutically effective amount of the pharmaceutical composition including a vector, such as a lentiviral vector encoding the CAR, and / or administering a therapeutically effective amount of a pharmaceutical composition comprising cells, such as T cells and / or NK cells, that express the CAR. Subjects in need thereof may subsequently undergo standard treatment with anti-viral agents (for an HIV infection). Administration of the T cells that express the CAR, as disclosed herein, will increase the ability of a subject to eliminate residual HIV, such as to in the B cell follicles, to reduce or eliminate residual virus.

[0361] As shown in two independent publications (Wellhausen et al., Sci Transl Med. 2023 20;15(714):eadi1145; Garaude et al., Nature. 2024;630(8017):728-735) single amino acid mutations can be introduced into the human CD45 extracellular domain. These mutations prevent the binding of corresponding antibodies targeting human CD45. Accordingly, CAR T or NK cells based on antibody Jelly and variants thereof will bind and deplete cells expressing the wildtype isoform of CD45 but not cells expressing another isoform of CD45. This second isoform of CD45, which was shown to retain expression and function of CD45 can be used to protect CD45+ healthy HSCs, HSPCs and differentiated immune cells. As CAR T-cells also express CD45, they also need to either harbor a CD45 knock-out, or even better, a CD45 mutation preventing fratricide while retaining CD45 expression and function (Wellhausen N. etal., Sci Transl Med 20; 15(714), 2023 and Harfmann M et al., Cancers 16(2):334, 2024). The chimeric antigen receptors of the present disclosure can be used in conjunction with respective treatment regimen.

[0362] Also disclosed herein, is the use of the chimeric antigen receptors of the present disclosure in medicine. Such use in medicine includes the preventions or treatment of the diseases and disorders recited herein above.

[0363] Pharmaceutical compositions can include a CAR-expressing cell, e.g., a plurality of CAR- expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. The CAR-expressing cells can be T cells, such as CD3+ T cells, such as CD4+ and / or CD8+ T cells, and / or NK cells, but also monocytes or macrophages. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0364] The cells can be autologous to the recipient. However, the cells can also be heterologous (allogeneic). In some embodiments, the cells are T cells, such as T cells transformed with Epstein Barr virus, see Savoldo et al., Blood 110: 2620-2630, 2007, incorporated herein by reference. In other embodiments, the cells are heterologous (allogeneic) to a recipient (see below) and are deleted for an HLA class I and / or T cell receptor, so they do not provoke a graft versus host disease (GVHD) or host versus graft reaction. In some embodiments the cells can be autologous or allogeneic and have been knocked-out for immune checkpoint proteins such as PD-1 , LAG-3, TIM- 3, CTLA-4, BTLA, HVEM, LAIR-1 , LAI R-2, TGFBR-1 , TGFBR-2, 2B4 or CD5 and combinations thereof. In some embodiments the cells can be autologous or allogeneic and have been knocked-out for CD52 to allow for lymphodepletion with anti-CD52 agents without depleting the CAR cells.

[0365] With regard to the cells, a variety of aqueous carriers can be used, for example, buffered saline and the like, for introducing the cells. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

[0366] In one embodiment, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, such as endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.

[0367] The precise amount of the composition to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells (and / or NK or other hematopoietic cells) described herein may be administered at a dosage of 104to 109cells / kg body weight, such as 105to 106cells / kg body weight, including all integer values within those ranges. Exemplary doses are 106cells / kg to about 1 X 108cells / kg, such as from about 5 X 106cells / kg to about 7.5 X 107cells / kg, such as at about 2.5 X

[0368] 107cells / kg, or at about 5.0 X 107cells / kg.

[0369] A composition can be administered once or multiple times, such as 2, 3,4, 5, 6, 7, 8, 9, or 10 times at these dosages. The composition can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The compositions can be administered daily, weekly, bimonthly or monthly. In some non- limiting examples, the composition is formulated for intravenous administration and is administered multiple times. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject’s disease, although appropriate dosages may be determined by clinical trials.

[0370] In one embodiment, the CAR is introduced into cells, such T cells or NK cells, and the subject receives an initial administration of cells, and one or more subsequent administrations of the cells, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the CAR cells are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the CAR cells of the invention are administered per week. In one embodiment, the subject receives more than one administration of the CART cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to as a cycle), followed by a week of no CAR cells administrations, and then one or more additional administration of the CAR cells (e.g., more than one administration of the CAR T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR cells are administered every other day for 3 administrations perweek. In another embodiment, the CAR cells are administered for at least two, three, four, five, six, seven, eight or more weeks. The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

[0371] In some embodiments, CAR-modified T cells are able to replicate in vivo resulting in long- term persistence that can lead to sustained tumor control. In various aspects, the T cells administered to the subject, or the progeny of these cells, persist in the subject for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, or for years after administration of the T cell to the subject. In other embodiments, the cells and their progeny are present for less than six months, five month, four months, three months two months, or one month, e.g., three weeks, two weeks, one week, after administration of the T cell to the subject.

[0372] The administration of the subject compositions may be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The disclosed compositions can be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the compositions are administered to a patient by intradermal or subcutaneous injection. In other embodiments, the compositions of the present invention are administered by i.v. injection. The compositions can also be injected directly into a tumor or lymph node.

[0373] In some embodiments, subjects can undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and / or isolate the cells of interest, e.g., T cells and or NK cells. These cell isolates may be expanded by methods known in the art and treated such that one or more CAR constructs can be introduced, thereby creating an autologous cell that express the CAR. In one aspect, CAR expressing cells are generated using lentiviral viral vectors.

[0374] In some embodiments, the T and / or NK cells are autologous. In other embodiments, the T cells and / or NK cells are allogeneic. The T cells and / or NK cells are then introduced into the subject, as disclosed above. In one embodiment, the cells transiently express the vector for 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 days after transduction. In one non-limiting example, the vector is transduced into the T cell by electroporation.

[0375] In some embodiments, a subject is administered a therapeutically effective amount of T cells and / or NK cells expressing the disclosed CAR. In particular embodiments (see U.S. Published Application No. US20140271635 A1), prior to expansion and genetic modification, a source of T cells is obtained from a subject.

[0376] 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 other embodiments, any number of T cell lines or NK cell lines available in the art, may be used. In some non-limiting examples, T cells and / or NK 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, or the cells can be obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, NK cells, other nucleated white blood cells, red blood cells, and platelets. In some specific non-limiting examples, the cells are autologous.

[0377] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubation with anti- CD3 / anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells, see U.S. Published Application No. US20140271635 A1. In a non-limiting example, the time period is about 30 minutes. In other non- limiting examples, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In further non-limiting examples, the time period is at least 1 , 2, 3, 4, 5, 6 hours, 10 to 24 hours, 24 hours or longer. Longer incubation times can be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolation from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cellscan be preferentially selected fororagainst at culture initiation or at other desired time points. Multiple rounds of selection can also be used.

[0378] 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 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, CD11 b, CD16, HLA-DR, and CD8. A T cell population can be selected that expresses one or more cytokines. Methods for screening for cell expression are disclosed in PCT Publication No. WO 2013 / 126712. For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. Into ensure maximum contact of cells and beads. In some embodiments, a concentration of 1 billion cells / ml is used. In further embodiments, greater than 100 million cells / ml is used. In other embodiments, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, 50, 65, 70, 75, 80, 85, 90, 95, or 100 million cells / ml is used. Without being bound by theory, using high concentrations can result in increased cell yield, cell activation, and cell expansion. Lower concentrations of cells can also be used.

[0379] Without being bound by theory, significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In some embodiments, the concentration of cells used is 5x106 / ml. In other embodiments, the concentration used can be from about 1x105 / ml to 1x106 / ml, and any integer value in between.

[0380] Cells can be incubated on a rotator for varying lengths of time at varying speeds at either 2- 10 degrees centigrade or at room temperature. T cells for stimulation can also be frozen after a washing step. Without being bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20 percent DMSO and 8 percent human serum albumin, or culture media containing 10 percent Dextran 40 and 5 percent Dextrose, 20 percent Human Serum Albumin and 7.5 percent DMSO, or 31.25 percent Plasmalyte- A, 31.25 percent Dextrose 5 percent, 0.45 percent NaCl, 10 percent Dextran 40 and 5 percent Dextrose, 20 percent Human Serum Albumin, and 7.5 percent DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to-80 degrees centigrade at a rate of 1 degrees per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at-20 degrees centigrade or in liquid nitrogen, see U.S. Publication No. US-2014- 0271635 A1.

[0381] Blood samples or apheresis product can be collected from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such asT cells, isolated and frozen for later use inT cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti- CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to use. Blood samples orapheresis product can be collected from a subject when needed, and notfrozen.

[0382] T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos.6, 352, 694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041 ; and U.S. Patent Application Publication No.20060121005.

[0383] T cells can be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In some non-limiting examples, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD28 antibody immobilized on a surface, or an anti-CD3 / anti-CD28 combination, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti- CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et aL, Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.190(9):13191328, 1999; Garland et al., J. Immunol. Meth.227(1 -2):53-63, 1999).

[0384] Once a CD45 CAR is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models.

[0385] Isolated immune cells expressing a CAR, such as T cells, for example CD3+ T cells such as CD4+ and / or CD8+ T cells, and / or NK cells, can be administered in a pharmaceutically acceptable carrier, such as buffered saline or another medium suitable for administration to a subject. The cells can be administered in conjunction with other cells, or in the absence of other cells. In one embodiment, compositions containing isolated populations of cells can also contain one or more additional pharmaceutical agents, such as one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti-tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), or non-steroidal anti-inflammatory agents such as acetylsalicylic acid, ibuprofen or naproxen sodium), cytokines (for example, interleukin-2), or a vaccine. Many chemotherapeutic agents are presently known in the art. In one embodiment, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, e.g. anti-androgens, and anti-angiogenesis agents.

[0386] In other embodiments, a subject is administered the DNA or RNA encoding the CAR, to provide in vivo production. Immunization by nucleic acid constructs is well known in the art and taught, for example, in U.S. Patent No.5,643,578, and U.S. Patent No.5, 593, 972 and U.S. Patent No. 5,817,637. U.S. Patent No.5, 880, 103 describes several methods of delivery of nucleic acids encoding to an organism. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antibody binding fragments thereof, by one of ordinary skill in the art. Such methods can also include the delivery by virus or virus-like particles.

[0387] One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antibody binding fragments thereof, can be placed under the control of a promoter to increase expression.

[0388] In another approach to using nucleic acids, a disclosed CAR can also be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adenovirus, adeno- associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors or gutless viral particles thereof can be used to express the CAR. For example, vaccinia vectors and methods useful protocols are described in U.S. Patent No.4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed antibodies (see Stover, Nature 351 :456- 460, 1991). In a specific non-limiting example, the vector is a lentiviral vector.

[0389] In one embodiment, a nucleic acid encoding a disclosed CAR, is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced by a device such as Bio-Rad’s HELIOS Gene Gun. The nucleic acids can be "naked," consisting of plasmids under control of a strong promoter. The nucleic acid can be RNA; RNA encoding the CAR can be directly administered to the cells. In some embodiments, the cells are NK cells orT cells.

[0390] For the treatment of malignancy, the method can also include administering to the subject a therapeutically effective amount of an additional chemotherapeutic agent, surgery or radiation. In some embodiments, the malignancy is a lymphoid or a myeloid malignancy. The subject can also have a solid tumor.

[0391] In further embodiments for the treatment of malignancies, a CAR-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH or other anti-CD52 antibodies, anti-thymocyte globulin (ATG) or other mono- or polyclonal anti-lymphocyte preparations, anti-CD3 antibodies or other antibody therapies and antibody-drug conjugates, fludarabine, cyclophosphamide, bendamustine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, irradiation, immune checkpoint inhibitors, or peptide vaccine. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)), a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, tositumomab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide) or pro-apoptotic agents such as venetoclax and related molecules.

[0392] For the treatment of an HIV infection, the subject can be administered an antiretroviral agent. Antiretroviral drugs are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits. The disclosed antibodies can be administered in conjunction with Nucleoside and nucleotide reverse transcriptase inhibitors (nRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors, entry inhibitors (orfusion inhibitors), maturation inhibitors, or a broad spectrum inhibitors, such as natural antivirals. Exemplary agents include lopinavir, ritonavir, zidovudine, lamivudine, tenofovir, emtricitabine and efavirenz.

[0393] In some embodiments, the subject can be administered an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD-1), can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD-1 , PD-L1 , CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1 , CD160 / HVEM, 2B4 and TGFR-beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., a siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the CAR- expressing cell. In an embodiment the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a CAR-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the CAR. In one embodiment, the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-1 , PD-L1 , PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101 and marketed as YERVOY(R); Bristol-Myers Squibb; Tremelimumab (lgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).). In an embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3.

[0394] Kits

[0395] Kits are also provided. For example, kits for treating a subject with a cancer that expresses CD45, or for HIV. The kits will typically include a disclosed nucleic acid encoding a CAR, T cell expressing a CAR or compositions including such molecules. The kit can include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container typically holds a composition including one or more of the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, or compositions. In several embodiments the container may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). A label or package insert indicates that the composition is used for treating the particular condition.

[0396] The label or package insert typically will further include instructions for use of the antibodies, antigen binding fragments, conjugates, nucleic acid molecules, or compositions included in the kit. The package insert typically includes instructions customarily included in commercial packages of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. The instructional materials may be written, in an electronic form (such as a computer diskette or compact disk) or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.

[0397] EXAMPLES

[0398] Example 1 : Production and testing of anti-CD45 antibodies in scFv format

[0399] Numerous anti-CD45 antibodies are known in the art. See for example WO2024 / 133890. Several of these antibodies were tested in scFv format, either in VH-VL or in VL-VH orientation and with various linkers.

[0400] The amino acid sequences of the human IgG 1 hinge-CH2-CH3 spacer regions (hinge-Fc) are based on the IGHG1_HUMAN sequence (Uniprot P01857), with point mutations L234A, L235A, and P329A (‘LALA-PA’) introduced to avoid Fc receptor and complement binding (human Fc silent IgG 1 s hinge- CH2-CH3: SEQ ID NO: 47; human lgG1 hinge region only: SEQ ID NO: 48). The human lgG1-based, Fc silent hinge-CH2-CH3 constructs were produced with different engineered hinge variants, such as C220S (SEQ ID NO. 49), C220A (SEQ ID NO. 50), TGGG (SEQ ID NO. 51) and short hinge (SEQ ID NO. 52) in addition to the human wildtype hinge sequence (SEQ ID NO. 48), resulting in the corresponding hinge-CH2-CH3 constructs (SEQ ID NO. 54-57).

[0401] The amino acid sequences of the scFv Hollandaise in combination with certain spacer regions are as follows:

[0402] With a lgG4s hinge-CH2-CH3 domain:

[0403] With a lgG2 hinge lgG4s-CH2-CH3 domain

[0404] With a lgG1s hinge-CH2-CH3 domain

[0405] With a lgG1s C220S hinge-CH2-CH3 domain

[0406] With a lgG1s C220A hinge-CH2-CH3 domain

[0407] With a lgG1s TGGG hinge-CH2-CH3 domain

[0408] With a lgG1s short hinge-CH2-CH3 domain (LALA-PA)

[0409]

[0410] The amino acid sequences of the scFv Mornay in combination with certain spacer regions are as follows:

[0411] With a lgG1s hinge-CH2-CH3 domain (LALA-PA)

[0412] With a lgG1s C220S hinge-CH2-CH3 domain (LALA-PA)

[0413] With a lgG1s C220A hinge-CH2-CH3 domain (LALA-PA)

[0414] With a lgG1s TGGG hinge-CH2-CH3 domain (LALA-PA)

[0415] With a lgG1s short hinge-CH2-CH3 domain (LALA-PA)

[0416] After affinity chromatography (Ni-NTA for scFv-His6 and Protein A for scFv-Fc) expression yields were quantified, and monomer content was quantified by analytical size exclusion chromatography after expression. The thermal stability of each CH2-CH3 region was determined by nanoDSF and used as a measure of stability of the scFvs. Binding affinity was measured by biolayer interferometry (BLI) using the recombinant extracellular domain (ECD) d1 & d2 of human deglycosylated wildtype CD45. Binding specificity was assessed by measuring binding to the recombinant extracellular regions of human FLT3, CD117 or CD123, as well as the single point mutant versions of recombinant extracellular CD45 (d1 & d2) K352E and / or K352G (Garaude S et al., Nature 2024;630(8017): 728-735). Analytical size exclusion chromatography and SDS PAGE / capillary electrophoresis determined monomer content and the correct protein size of the constructs, respectively.

[0417] Sequences of the scFv’s tested are as follows.

[0418] Table 1:

[0419]

[0420] Table 2: Expression yield, aggregation propensity, thermostability, binding affinity and specificity of Jelly-derived scFvs or scFv-Fc constructs

[0421]

[0422] The results show that most of the scFvs tested could not be expressed at all or with sufficient yields, even if the scFv’s contained very similar VH and VL sequences. Only Hollandaise, Mornay, Veloute, Hummus, Knoblauch, Guacamole, Zaziki, Caesar, Sambal Oelek and Tabasco were expressed sufficient amounts of recombinant protein and could therefore be evaluated protein- biochemically. Eight of the ten scFvs showed good expression yields (>50 mg / L) as well as >90% monomer content and good thermostability. Moreover, Sambal Oelek and Tabasco were deselected because they lost binding to CD45. In consequence, of in total 30 scFvs only seven (Hollandaise, Mornay, Veloute, Hummus, Knoblauch, Guacamole and Caesar) retained binding affinity and specificity to wildtype human CD45 below 2 nM , with no binding detectable to the CD45 K352E variant and irrelevant receptor ECDs and were also well expressed, had high monomer content (>90%) and were stable (Tm >54°C).

[0423] Therefore, based on the sequences of the well expressed, stable and monomeric scFvs, which had also fully retained CD45 binding, further derivates were generated and expressed recombinantly. Table 4: Expression yield, aggregation propensity, thermostability, binding affinity and specificity of scFv-Fc constructs with varying linker length and composition

[0424] In these scFvs, the linker connecting VH and VL was engineered (variation in linker length and composition) with the aim to increase the expression yield and stability of the scFv’s. From this, Sriracha, Valentina, Chermoula, Pebre, Tkemali, Agrodolce, Louisiana, Yakisoba and Tsuyu could be expressed, and Guasaca and Ponzu did notyield recombinant protein and were discarded. Four of eight remaining scFvs showed good expression yields (>50 mg / L) as well as >90% monomer content, good thermostability and retained high binding affinity (below 2 nM) and specificity to wildtype CD45, with no binding detectable to the CD45 K352E variant and irrelevant receptor ECDs. Whereas Agrodolce proved exceptionally stable (Tm 57.4°C), Tkemali showed the highest affinity for wildtype CD45 (70 pM) as well as best expression yields. Example 2: Comparison of different spacer regions

[0425] Spacers are important for CAR constructs because they tune the distance of the scFvfrom the cell membrane and therefore can improve the organization of the immunological synapse. Generally, there are CD8-based unstructured spacers, or highly structured IgG-based spacers. One important aspect is to avoid Fc receptor binding of the Ig-based spacer regions in order to prevent secondary crosslinking and activation of the CAR by binding to Fc receptors through the spacer. Therefore, in addition to the lgG1 -based spacer constructs (see Example 1), also two lgG4-based spacer constructs were engineered with established Fc silencing mutations (lgG4: EQN (SEQ ID 68), lgG1 : LALA-PA (SEQ ID 69)). Additionally, an unpaired Cys which is present in the human lgG1 based spacer due to the absence of the light chain, was removed by mutations C220A or C220S, or by modified hinge composition (TGGG and shortened hinge).

[0426] The amino acid sequence of the human lgG4 hinge-CH2-CH3-based spacer region was based on the IGHG4_HUMAN sequence (Uniprot: P01861 ), with point mutations L235E, N297Q and S228P (‘EQN’, SEQ ID No. 68) added to avoid Fab arm exchange as well as prevent Fc receptor and complement binding. It has the following amino acid sequence:

[0427] The amino acid sequence of the human lgG2 / 4 hinge-CH2-CH3-based spacer region was based on the IGHG4_HUMAN sequence (Uniprot: P01861 ), with point mutations L235E, N297Q and S228P (SchlothauerT. et al., Protein Engineering, Design and Selection, 29 (10) 457-466, 2016) added to avoid Fab arm exchange as well as Fc receptor and complement binding plus exchanging the lgG4 hinge region for a human lgG2-derived one. It has the following amino acid sequence:

[0428] Each CH2-CH3 region was genetically fused to the C-terminus of the scFvs Hollandaise and Mornay and expressed in the supernatant of CHO suspension cell cultures. scFv-Fc expression yields, monomer contents, thermal stability, binding affinity and the correct size of the constructs were analyzed as in Example 1. Results for the scFv Mornay are shown in Table 3.

[0429] Table 3 : Expression yield, aggregation propensity, thermostability and Fc receptor binding of the different hinge-CH2-CH3 constructs:

[0430] As indicated, one important aspect is to avoid Fc receptor binding of the Ig-based spacer regions in order to prevent secondary crosslinking and activation of the CAR by binding to Fc receptors through the spacer. The different spacer constructs described above were all analyzed for their binding to human FcgR2A and FcgR3A, and no binding was detected for either of them.

[0431] A second aspect to be considered is the robust, monodisperse expression and good stability of the spacer. Considering expression yield, monomeric purity and thermal stability the human lgG1 hinge-CH2-CH3-based constructs with C220A or C220S mutations are the preferred constructs, over lgG4-based constructs with either lgG2 or lgG4 hinge region. The lgG4 hinge-CH2-CH3 construct additionally results in a decreased thermal stability.

[0432] Example 3: Construction of CD45 chimeric antigen receptors and lentiviral stock preparations

[0433] The sequences of the variable heavy (VH) and variable light (VL) chain regions of selected scFvs of prior examples, which could be expressed, retained binding of human wildtype CD45 and were stable and had a high degree of monomer content (Hollandaise, Mornay, Sriracha, Valentina, Tkemali and Agrodolce), as well as further variants thereof were utilized in the design and generation of the various CD45 CAR constructs.

[0434] The amino acid sequences of CD8a or hinge-CH2-CH3 spacer and CD8a or NKG2D transmembrane domains, CD28, 4-1 BB and / or 2B4 co-stimulatory module, and CD3zeta signaling module were derived from established or clinically tested CAR constructs, respectively. An isotype (Ref001 ; anti-chicken lysozyme) and a CD33-directed CAR were generated as control CARs. All CAR constructs were equipped with a 2A-EGFP unit in order to monitor transduction and mRNA expression rates by EGFP fluorescence.

[0435] For the second-generation CAR constructs with a single co-stimulatory domain, the individual spacers, transmembrane and signaling domain sequences were assembled together with the scFv sequences into transcriptional / translational units. The synthetic DNA fragments were cloned into an expression plasmid, which was eventually co-transfected with lentiviral packaging helper plasmids into human embryonic kidney cells HEK 293T to generate stocks of CD45 CAR-encoding lentiviral particles (VSV-G pseudotyped third-generation lentivirus non-replicative; Vectorbuilder). Following transfection of HEK 293T cells, the culture supernatant was harvested 48 hours later and clarified by PEG concentration. The clarified supernatant was subjected to ultracentrifugation (see website www.vectorbuilder.com). The CD45 CAR lentiviral pellets were dissolved in PBS to achieve a suitable virus concentration and stored at -80°C until use. For QC the lentiviral particles underwent titer measurement, sterility testing for bacteria and fungi, and mycoplasma detection as well as test-transduction (www.vectorbuilder.com).

[0436] Table 4. CAR constructs and quality control of lentiviral particles

[0437] Accordingly, third generation CAR constructs with two consecutive co-stimulatory domains were designed and generated. The amino acid sequences of hinge-CH2-CH3 as well as CD8a spacers, CD8a transmembrane domain, CD28 / 4-1 BB, 4-1 BB / CD28 as well as 4-1 BB / 2B4 co-stimulatory modules, and the CD3zeta signaling module were derived from established and clinically tested second generation CAR constructs, respectively.

[0438] Individual spacers, transmembrane and signaling domain sequences were assembled with co- stimulatory domains CD28 / 4-1 BB, 4-1 BB / CD28 or 4-1 BB / 2B4 together with the scFv sequences into transcriptional / translational units. The synthetic DNA fragments were cloned into an expression plasmid. CD45 CAR-encoding lentiviral particles were produced as described above.

[0439] Both second and third generation lentiviral CAR constructs are additionally equipped with an eGFP reporter that is separated by a 2A peptide. During expression, a short peptide of 17 AA (EGRGSLLTCGDVEENPG, SEQ ID NO. 101 ) remains C-terminally attached to the CD3zeta signaling module.

[0440] Example 4: CD45 base editing of human cells

[0441] NFAT Luciferase Reporter Jurkat cells express human CD45. In order to be able to assess CD45 CAR-mediated signaling through binding to CD45+ tumor cells, the CD45+ NFAT Luciferase Reporter Jurkat Cell Line was base edited to either introduce CD45 shielding edits (K352E / K352G) and generate a CD45 gene. To generate a CD45 knock-out, the donor splice site of exon 8 was mutated causing a frameshift in the reading frame of CD45.

[0442] Along the same lines, CD45+ human MOLM-14 and Jurkat cell lines were gene edited to introduce CD45 shielding edits (K352E / K352G) and generate a CD45 gene knock-out by base editing. Cells were prepared for the electroporation. Jurkat, Jurkat-NFAT or MOLM-14 cells were washed and resuspended in electroporation buffer, such as SE buffer (Lonza). Per condition 1x106-5x106cells were mixed in a 10Opl electroporation cuvette with 5pM of the respective single guide RNA (sgRNA Synthego or Biospring) and 100 pg / ml adenine base editor (ABE) mRNA (TriLink, N1 - Methylpseudouridine, CleanCap). The cell suspension mixes were electroporated with a Lonza 4D Nucleofector (such as program CL-120) following the manufacturer’s recommendations. Immediately after electroporation, pre-warmed medium was added, and the mixes were incubated for 20 minutes at 37°C for cells to recover optimally. Electroporated conditions were then transferred to an appropriate cell culture plate. Medium was renewed every 48 hours.

[0443] For assessment of base editing efficiencies on a genomic level, 4 days post-electroporation, bulk cells of each condition were spun down for 5 min at 2200 RCF. Genomic DNA was extracted from the bulk cells using a DNA extraction kit, according to manufacturer’s instructions (QIAamp DNA Blood Mini Kit). The edited region was PCR amplified and the amplicons were sequenced via Next- generation Amplicon sequencing (150nt paired end sequencing, Illumina MiniSeq) or via Sanger sequencing, using standard protocols. Base editing efficiency, regarding variant generation, knock- out as well as potential bystander events, was assessed for Next-generation sequencing reads via CRISPRESSO2 (Clement et al (2019). Nat Biotech) or from Sanger sequencing reads using EditR (CRISPR J. (2018) 1 :239-250).

[0444] Using the above described method, in all cell lines the intended edits could be installed successfully.

[0445] Example 5: Assessment of CD45 CAR-induced T-cell signaling in NFAT reporter cells

[0446] The NFAT T-cell activation reporter assay consists of a genetically engineered Jurkat T cell line expressing a luciferase reporter driven by an NFAT-response element (NFAT-RE). Lentiviral introduction of an anti-CD45 CAR construct will result in induction of TCR-mediated signaling once the CAR is engaged by wildtype CD45 on the surface of a CD45 expressing cell or immobilized wt CD45 extracellular domain. The resulting luminescence can be detected by adding luciferase assay reagent (Bio-Gio Luciferase Assay System, Promega) and quantitating with a luminometer.

[0447] As the NFAT Jurkat cells express CD45 endogenously CD45 knock-out and variant / shielded K352E / K352G cells were generated as described in the previous example and used for these assays. To assess expression and activation by the various CAR constructs, CD45 shielded and knock-out Jurkat NFAT Luciferase Reporter cells were transduced with the different CD45-CAR, CD33-CAR or Isotype (Ref001 )-CAR lentivirus constructs, all expressing EGFP from the bicistronic expression cassette, at an MOI of 20.

[0448] Five days after transduction, the transduction efficiency was quantified by flow cytometry as percentage of EGFP positive cells within the alive cell population (Table 6; Figure 3; upper panel: Jurkat NFAT CD45 shielded reporter cells; lower panel: Jurkat NFAT CD45 knock-out (KO) reporter cells). Sriracha- and Valentin a -based CAR constructs with the (GGGGS)4linker (SEQ ID 45) resulted in higher percentages of EGFP positive cells (66-76%) than Hollandaise- and Mornay-based CAR constructs with the (GGGGS)3linker (SEQ ID No. 44) (30-44%), but suffered from minor loss of binding affinity (Table 4). Subsequently, transduced cells were sorted to enrich for EGFP+ cells for further characterization and functional testing. Staining with an anti-G4S antibody labelled with the fluorochrome PE (Cell Signaling, dilution 1 :50) was used to detect CAR expression on the cell surface by flow cytometry. As shown in Figure 4, both transduced Jurkat NFAT CD45 shielded (BE) and KO reporter cells showed good correlation between mean fluorescence intensity (MFI) of EGFP signal and anti-G4S staining. In agreement with previous results, Sriracha- and Valentina-based CAR constructs resulted in higher cell surface expression of the CD45 CAR (percentage of G4S positive cells and G4S MFI) than Hollandaise- and Mornay-based CAR constructs (Table 6 and Figure 4), overall demonstrating that these CD45 CAR constructs transduce and express more efficiently.

[0449] To measure the activity of the CD45 CAR constructs, co-cultures of CAR expressing cells and target cells were set up. In brief, CD45 shielded NFAT Jurkat cells expressing the various CD45 CAR constructs were resuspend at 0.1 x 106cells / ml in fresh, pre-warmed growth media (RPMI 1640 Medium with 10%FBS). Human CD45 wt, shielded (BE) or KO target cells (Molm-14 or Jurkat cells) were resuspended at ca 0.02 x 106cells / ml in fresh, pre-warmed growth media. 50 μL of the target cell suspension were dispensed to each of the inner 60 wells of 96-well flat, white-bottom assay plates. Then, 50 μl of the CAR reporter cell suspension were added to each of the inner 60 wells already containing 50 μL target cells or appropriate controls in triplicates. The final volume was 100 μL. Assay plates were covered with a lid and incubated in a humid 37°C, 5% CO2incubator for 6 or 24 hours.

[0450] After incubation, the assay plates were removed from the incubator and equilibrated to room temperature (10-15 min). Then, 100 μL of Bio-Gio Reagent (Promega) were added to the test wells of the assay plates followed by 10 min incubation at room temperature. Luminescence was measured using a luminescence plate reader.

[0451] Luminescence signal for each condition was calculated as fold induction by subtracting the background signal obtained in the CAR expressing cells alone conditions (no target cells) and normalizing it to the isotype control (Ref001 )-CAR expressing cell line. Results are shown in Figure 5 (A-C: Molm-14 as target cells, D-F: Jurkat as target cells).

[0452] Overall, when cells expressing CD45 wt (Molm-14 or Jurkat) were used as target cells, a robust activation of CD45 CAR expressing reporter cells in all conditions was detected, indicating target- dependent activation (Figure 5). Among all the CD45 CAR constructs tested, Mornay and Valentina showed the highest signal in the presence of both Molm-14 and Jurkat cells, indicating that VH-VL orientation (Mornay and Valentina) is preferred over VL-VH orientation (Hollandaise and Sriracha) and therefore relevant for good efficacy. Importantly, when CD45 engineered (shielded or KO) cells were used as target cells, the CD45 CAR activation was highly reduced for all CD45 CAR constructs and close to background levels, demonstrating that the CD45 shielded variant does not bind to and activate the CD45 CAR constructs analyzed.

[0453] Moreover, CD33 CAR expressing reporter cells showed significant activation only in the presence of Molm-14 which are CD33 positive, regardless of the CD45 variant (wt or shielded) expressed, but notwith Jurkat cells (CD33 negative) (Figure 5). Altogether demonstrating that the CD45 CAR tested can bind CD45 wt expressing cells and initiate downstream signaling required forT cell activation, while CD45 shielded expressing cells are protected.

[0454] In summary, the Mornay-and Valentina-based CARs show highest target-specific activation together with low background signaling on CD45 shielded or knock-out target cells and are thus preferred over the Hollandaise- and Sriracha-based CARs.

[0455] CD45 is a phosphatase that regulates CAR-T cell activation by being excluded from the immune synapse, a process that allows downstream kinases to phosphorylate the CAR, initiating signaling. In orderto evaluate whether engineering / shielding of CD45 in effector cells has any impact on CAR binding and / or downstream signalling and T cell activation, the CD33 CAR construct was transduced in CD45 wt, shielded and KO Jurkat NFAT reporter cells. After enrichment of EGFP+ cells, co-cultures of the CAR expressing cells with CD45 wt or shielded Molm-14 as target cells were performed as described above. As shown in Figure 6, robust luminescence signal was measured for CD45 wt and shielded Jurkat NFAT reporter cells expressing CD33 CAR, but very low signal was obtained in conditions with CD45 KO NFAT cells. Overall, demonstrating that CD45 shielded Jurkat NFAT have similar activation potential compared to CD45 wt cells, while knocking out CD45 significantly compromises CAR-induced activation of Jurkat NFAT reporter cells.

[0456] In an additional set of experiments, a second batch of CD45 CAR constructs were also evaluated in Jurkat NFAT CD45 shielded cells for expression and activity. These CARs were based on Mornay scFv as this was the preferred CAR from previous experiments. Next generation CAR constructs contained Tkemali and Agrodolce scFvs, which proved to be either exceptionally stable or showed unusually high anti-CD45 affinity due to charged and stabilized linker regions (Table 4).

[0457] Following the same protocol as above, cells were transduced and analyzed a few days later in a flow cytometer for EGFP expression. Percentage of EGFP+ within the alive cells population was >88% for all the CD45 CAR constructs evaluated (Figure 7). To measure CD45 CAR expression on the cell surface, in addition to anti-G4S staining, cells were also incubated with human CD45 protein carrying a llama lgG2b Fc tag (Aero Biosystems) that was detected with an anti-llama IgG secondary antibody labelled with PE (Abeam) by flow cytometry.

[0458] As expected, negligible anti-G4S staining was observed in CD45 CAR#1898, #1896, #1902 and #1904 since these CAR constructs do not have a classic G4S linker between the heavy and light chains, but rather variations thereof, thus the G4S antibody cannot bind those constructs (Table 7 and Figure 8). However, CD45-Fc binding was successfully detected for all CD45 CAR constructs and a high degree of correlation was observed between mean fluorescence intensity of EGFP and CD45-Fc signals (Figure 8 and 9). Importantly, higher percentage of cells with surface CAR expression was observed for CD45 CAR#1895 and #1900 (75%) compared to other CD45 CARs. CD45 CAR#1900 was clearly the best expressed (highest EGFP, G4S and CD45-Fc MFI) while #1169 showed the lowest expression. See Table 7 and Figure 8 and 9.

[0459] In terms of activity, co-culture assays were run as stated above with Molm-14 and Jurkat cells expressing CD45 wt or shielded (BE) variants. As shown in Figure 10, robust NFAT activation was measured for all CD45 CAR constructs compared to isotype control (Ref001) CAR, with some variability between Molm-14 and Jurkat-based assays. In line with previous experiments, reduced signal was obtained when CD45 shielded cells were used as target cells, supporting the high specificity of the CD45 CAR constructs assessed. In summary, all CD45-CARs evaluated allowed specific and robust activation of Jurkat T reporter cells in the presence of CD45 wt target cells.

[0460] Example 6: Assessment of CD45 CAR expression. CAR-induced T-cell signaling and cytotoxicity in primary human T cells

[0461] Lentiviral introduction of the anti-CD45 CAR constructs results in induction of TCR-mediated signaling, T-cell activation and the secretion of cytotoxic proteases and cytokines, once the CAR is engaged by wildtype CD45 on the surface of CD45 expressing cells or an immobilized wildtype CD45 extracellular domain. Accordingly, endogenous CD45 expression on human T cells would lead to fratricide (in trans and / or cis), once a CD45 CAR is expressed. Therefore, wildtype CD45+ cells were base edited to introduce the CD45 shielding variant K352E / G prior transduction with the CD45 CAR constructs.

[0462] T cell isolation and expansion

[0463] Leucocyte buffy coats from anonymous healthy human donors are purchased from the blood donation center Basel (Blutspendezentrum SRK beider Basel, BSZ). Peripheral blood mononuclear cells (PBMCs) are isolated by density centrifugation using SepMate tubes (StemCell Technologies) and the density gradient medium Ficoll-Paque (GE Healthcare) according to the manufacturer’s protocol. Primary human T cells are purified (>96% purity) by magnetic negative selection using an EasySep Human T Cell Isolation Kit (Cat#17951 ; StemCell Technologies) according to the manufacturer’s instructions. If frozen PBMCs are used, T cells are isolated after thawing and cultured in supplemented media without stimulation overnight. T cells are cultured in RPMI-1640 Medium (Sigma-Aldrich) supplemented with 10% heat-inactivated human serum (AB+, male; purchased from BSZ Basel), 10 mM HEPES (Sigma-Aldrich), 2 mM GlutaMAX, 1 mM sodium pyruvate, 0.05 mM 2-mercaptoethanol, 1% MEM non-essential amino acids (100×, all Gibco Life Technologies), and IL-2 150 U / ml (proleukin; University Hospital Basel). The medium and IL-2 are replenished every 2 d, and the cells are kept at a cell density of 1 x 106cells / ml.

[0464] Primary human T cells are isolated from leukopaks (CytoCare) via the TCT program employing the CD4 CD8 ab TS 520 kit, on the CiiniMacs Prodigy (Miltenyi). Alternatively, primary human T cells are isolated from the hCD34+ HSPC negative fraction subsequently to the hCD34+ HSPC isolation process (CiiniMacs Prodigy, TS310 kit, LP-34 program), employing the CiiniMacs Prodigy CD4 CD8 ab TS 520 and TCT program. Another approach includes isolation of primary human T cells from the hCD34+ HSPC negative fraction subsequently to the hCD34+ HSPC isolation process on the CiiniMacs Prodigy (TS310 kit, LP-34 program) according to manufacturer’s recommendations on the autoMacs separator (Miltenyi). Isolated T cells are frozen in CS5 or CS10 (StemCell) or used freshly.

[0465] Activation and expansion

[0466] The T cells are activated in TexMACS (Miltenyi) medium containing TransAct and hematopoietic cytokines (including IL7 and IL15) and expanded for 2-10 days.). During expansion, cell count and viability are measured, and phenotype is assessed via flow cytometry by staining for markers such as CD45, CD3, CD4, CD8, CD69, CD25. T cells are cryopreserved in CS5 or CS10 (StemCell).

[0467] CD45 base editing

[0468] Primary T cells are activated prior to electroporation to introduce the CD45 shielding variant K352E / G or a CD45 knockout. For large scale electroporation, per condition 2x106 - 5x106cells are mixed in a 100ul electroporation cuvette (CC-100) with 5uM of the respective single guide RNA (sgRNA Synthego or Biospring) and 100 ug / ml adenine base editor (ABE) mRNA (TriLink, N1- Methylpseudouridine, CleanCap) such as iSpyMac-ABE8e (Liu 2019; Chatterjee 2020) or SpRY- ABE8e (Walton 2020). The cell suspension mixes are electroporated (expanded T cell program 2) with the GTx nucleofector (Maxcyte) following the manufacturer’s recommendations. Alternatively, for small scale electroporation, T cells are electroporated with a Lonza 4D Nucleofector (such as program CL-120) following the manufacturer’s recommendations. Immediately after electroporation, pre-warmed medium is added, and the mixes are incubated for 20 minutes at 37°C for cells to recover optimally, prior transferring cells to an appropriate culture vessel. Electroporated cells are either expanded for 2-10 days prior cryopreservation or alternatively, are 2h - 24h after electroporation subjected to lentiviral transduction with CAR constructs.

[0469] Genomic DNA is isolated from bulk cells, the edited region is PCR amplified and the amplicons are sequenced via Sanger sequencing (Microsynth) or next-generation sequencing (Illumina Mini-seq). Base editing efficiency, regarding variant generation as well as bystander events, is assessed for Sanger sequencing reads via EditR (Kluesner et al (2018). CRISPR Journal) and for next-generation sequencing via CRISPRESSO2 (Clement et al (2019). Nat Biotech).

[0470] T cell lentiviral transduction with CAR constructs

[0471] Lentiviral transduction of activated T cells was conducted after the introduction of the CD45 shielding edits to minimize fratricide. T cells were cultured for 24h in TexMACS containing TransAct and hematopoietic cytokines, including IL7 and IL15. T cells were transduced with respective lentiviral vectors at MOI between 1 and 20. After 24h of transduction, the culture medium was exchanged, and cells were cultured in G-Rex plates according to manufacturer’s instructions (Wilson Wolf, MN, USA).

[0472] To exclude any negative impact of CD45 editing and / or CD45 CAR transduction on T cell activity, T cell phenotyping (CD197 and CD45RA) as well as activation (CD69 and CD25) and exhaustion (PD1 and LAG3) status were analyzed by measuring the expression of indicated markers using flow cytometry. No significant changes were observed for any of the CD45 CARs evaluated compared to control (CD45 WT and untransduced) T cells. A representative example can be found in Figure 11 . This assessment was always performed before using the CD45 shielded CAR-T cells for downstream cytotoxic assays.

[0473] CAR expression of the various constructs on primary human T cells was quantified by measuring EGFP signal, anti-G4S antibody staining and human CD45 protein binding by flow cytometry as previously explained in Example 5.

[0474] As observed in Jurkat NFAT cells, CAR expression on the cell surface was detected in different experiments for all CD45 CAR constructs tested either by G4S staining or CD45 protein binding or both (Figure 12 and 13A). Consistently, the highest levels of CAR expression were observed for CD45 CAR#1900. Of note, the percentage of shielded T cells was increased for all CD45 CAR constructs compared to untransduced T cells, demonstrating that there was active killing of CD45 wt T cells by shielded CD45 CAR-expressing cells, known as fratricide (Figure 12A and 13B). Cytotoxicity assays

[0475] The functionality of shielded CD45 CAR-expressing cells was further tested in co-culture assays in the presence of CD45 wt or shielded tumor cells expressing Luciferase as target cells. Effector cells (CD45 CAR positive T cells) at different concentrations were co-cultured with a fixed concentration of target cells (Jurkat or CCRF-CEM) to test various Effector:Target cell ratios for 24 and 48 hours. Luciferase signal was measured in a luminometer after 10-15min of incubation with Bio-Gio (Promega).

[0476] Percent specific lysis is determined using a control of target cells co-cultured with untransduced T cells as reference to subtract unspecific cell lysis and quantify only CAR-mediated cell killing. The results from duplicate wells were averaged, and percent lysis was calculated using the following formula:

[0477] % specific cell killing = 100-(100* (tested samples / Untransduced control samples).

[0478] As shown in Figure 14 and 15, CD45 CAR-mediated killing of shielded (BE) Jurkat or CCRF-CEM was highly reduced (never higher than 20%) compared to CD45 wt target cells, which reaches 100% for the highest E:T ratios tested for some CD45 CAR constructs. In agreement with the expression data described above, CD45 CAR#1900 showed the most efficient killing of CD45 wt cells with both Jurkat and CCRF-CEM target cells. Altogether these results demonstrate efficient and specific cell killing of CD45 wt target cells by shielded T cells expressing CD45 CAR.

[0479] Moreover, during the cell killing assays the culture supernatants were harvested at 24 and 48 hours and secreted cytokines were measured using protein quantification immunoassays from Meso Scale Discovery (MSD). In brief, supernatants were diluted 1 in 2 with diluent provided in the kit and the concentration of Interferon gamma (I FNg) was calculated using the MSD discovery workbench tool and standard curves, according to the instructions provided by the manufacturer. As shown in Figure 16, significant production of IFNg was detected in co-cultures containing CD45 CAR- expressing T cells compared to untransduced T cells (dotted line). Of note, levels of IFNg correlated well with CD45 CAR activity and CD45 CAR#1900 produced the highest levels of IFNg. Importantly, IFNg production in co-cultures with CD45 shielded target cells were equal or lower compared to untransduced control conditions (dotted line), indicating once more that CD45 CAR activation is CD45 wt specific and that shielded CD45 cells are protected from CD45 CAR-mediated killing (Figure 16B). In summary, highly functional CD45 CAR constructs were generated and validated in cytotoxic assays which include measurement of cell killing and cytokine release by CD45-CAR expressing T cells. Among all the CD45 CARs tested, Mornay C-8S-BBz is preferrable due to its higher expression and cytotoxic potential in primary T cells, followed by Agrodolce C-8S-BBz and Tkemali C-8S-BBz.

[0480] Example 7: In vivo depletion of human cancer cells by CD45 CAR-modified T cells

[0481] MOLM-14-mCherry-luc cells are base edited to introduce CD45-shielding variants, as described in Example 4. 0.5-1 million CD45-WT or CD45-edited MOLM-14-mCherry-luc cells are inoculated into the tail vein of immunodeficient mice (e.g., strain NBSGW (NOD.Cg-Kit W-41 J Tyr + Prkdc scid Il2rg tmlWjl / ThomJ), NSG (NOD.Cg-Prkdc scid Il2rg tmlWjl / SzJ), NSG-SGM3 (NOD.Cg-Prkdcscid H2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1 Eav / MloySzJ), NSG-SGM3-IL-15 (NOD.Cg-Prkdcscid H2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1 Eav Tg(l L15)1 Sz / J) or NSG-FLT3 (NOD.Cg- Flt3em2Mvw Prkdcscid H2rgtm1WjlTg(FLT3LG)7Sz / SzJ)). Five to ten days after MOLM-14- mCherry-Luc inoculation, the treatment group of mice is injected intraperitoneally with 1 to 10 million CD45 CAR-modified T-cells per animal. Control mice are inoculated with isotype Ref001 CAR or anti-CD33 CAR modified T cells. To monitor tumor growth by in vivo bioluminescence imaging, mice are injected intraperitoneally with 100 pl d-luciferin (BioSynth, L-8220) and are subjected to Newton7.0 imaging (Vilber). Animals experiencing continuous tumor growth are euthanized based on clinical scoring criteria and in vivo bioluminescence signal. Tumor free animals are terminated after a reasonable time without detectable tumor cells. Upon euthanization of the animals tumor growth orclearance as well as CART cell persistence isverified by flow cytometry of the bone marrow, blood and the spleen.

[0482] The administration of CD45 CAR T cells to wild-type CD45 tumor bearing mice results in the reduction of tumor cells and increased survival of the mice.

[0483] Example 8: In vivo depletion of human cancer cells and protection of CD45-shielded hematopoietic cells by CD45 CAR-modified T cells

[0484] Human CD34+ HSPCs are isolated from immobilized leukopaks (CytoCare) via the LP-34 process on the CiiniMacs Prodigy (Miltenyi). The human CD34+ HSPCs are base edited to introduce CD45- shielding variants as described in (Garaude S et al., Nature. 2024;630(8017):728-735). Electroporation-only / mock-edited HSPCs were used as wildtype CD45 control cells. Two days after editing 0.1-1 mio HSPCs are transplanted each into immunodeficient mice (e.g., strain NBSGW (NOD.Cg-Prkdc scid Il2rg tmlWjl / SzJ), NSG-SGM3 (NOD.Cg-Prkdcscid H2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1 Eav / MloySzJ), NSG-SGM3-IL-15 (NOD.Cg-Prkdcscid H2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1 Eav Tg(IL15)1 Sz / J) or NSG-FLT3 (NOD.Cg- Flt3em2Mvw Prkdcscid H2rgtm1WjlTg(FLT3LG)7Sz / SzJ)). About 12-16 weeks after stem cell engraftment animals are additionally injected with one million CD45-WT MOLM-14-mCherry-luc cells into the tail vein. Five days after MOLM-14-mCherry-Luc inoculation, the treatment group of mice is injected intraperitoneally with 1 to 10 million CD45 CAR-modified T-cells per animal. Control mice are inoculated with isotype Ref001 CAR or anti-CD33 CAR modified T cells. To monitor tumor growth by in vivo bioluminescence imaging, mice are injected intraperitoneally with 100 μl d-luciferin (BioSynth, L-8220) and are subjected to Newton7.0 imaging (Vilber). Animals experiencing continuous tumor growth are euthanized based on clinical scoring criteria and in vivo bioluminescence signal. Tumor free animals are terminated after a reasonable time without detectable tumor cells. Upon euthanization tumor growth and shielding of non-tumor cells is verified by flow cytometry and next generation sequencing of bone marrow, blood and splenic cells.

[0485] The administration of CD45 CAR T cells to wild-type CD45 tumor bearing mice with human, CD45 shielded hematopoietic cells results in the reduction of tumor cells and increased survival of the mice. Importantly, CD45 shielded hematopoietic cells are resistant to CAR treatment and persist in the mice.

Claims

CLAIMS1 . A chimeric antigen receptor (CAR) comprising a) an extracellular binding domain specific for CD45, b) a transmembrane domain, c) at least one co-stimulatory domain, and d) a T cell receptor signaling domain, wherein said extracellular binding domain specific for CD45 comprises (i) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 2, (ii) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 163, (iii) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 164, (iv) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 165, or (v) a variable heavy chain of SEQ ID No. 1 and a variable light chain of SEQ ID No. 166.2.The chimeric antigen receptor (CAR) of claim 1, further comprising a spacer between said extracellular binding domain specific for CD45 and said transmembrane domain.

3. The chimeric antigen receptor (CAR) of claim 2, wherein said spacer is or is derived from CD8a or human lgG1 hinge-CH2-CH3, preferably wherein said spacer comprises an amino acid sequence selected from SEQ ID. No. 47 or 53-57.

4. The chimeric antigen receptor (CAR) according to any one of the preceding claims, wherein the variable heavy chain of said extracellular binding domain specific for CD45 is N-terminal to the variable light chain, preferably wherein said extracellular binding domain comprises an amino acid sequence of SEQ ID No. 33, 35, 37, 39, 41 , 93 or 94.

5. The chimeric antigen receptor (CAR) according to any one of claims 1 -3, wherein the variable light chain of said extracellular binding domain specific for CD45 is N-terminal to the variable heavy chain, preferably wherein said extracellular binding domain specific for CD45 comprises an amino acid sequence of SEQ ID No. 32, 34, 36, 38 or 40.

6. The chimeric antigen receptor (CAR) according to any one of the preceding claims, wherein the variable heavy chain and the variable light chain of said extracellular binding domain specific for CD45 are separated by a linker, preferably wherein said linker is a glycine-serine linker, preferably a (GGGGS)3-linker (SEQ ID No. 44) or a (GGGGS)4-linker (SEQ ID No. 45) or aGGGSGGSGEPPEGGSG-linker (SEQ ID No. 153), a GGGSGGSGGEPPEGGSGG-linker (SEQ ID No. 154), a GGGSGGSGEPPKGGSG-linker (SEQ ID No. 155), or a GGGSGGSGGEPPKGGSGG- linker (SEQ ID No. 156).

7. The chimeric antigen receptor (CAR) according to anyone of the preceding claims, wherein said transmembrane domain is or is derived from CD8a or NKG2D, preferably wherein said transmembrane domain is or comprises an amino acid sequence selected from SEQ ID No. 59- 61.

8. The chimeric antigen receptor (CAR) according to anyone of the preceding claims, wherein said chimeric antigen receptor has one co-stimulatory domain, preferably, wherein said co- stimulatory domain is selected from 4-1 BB, CD28 and 2B4.

9. The chimeric antigen receptor (CAR) according to any one of claims 1 -7, wherein said chimeric antigen receptor has two co-stimulatory domains, preferably wherein said co-stimulatory domains are 4-1 BB and CD28, 4-1 BB and 2B4, or CD28 and 2B4.

10. The chimeric antigen receptor (CAR) according to any one of the preceding claims, wherein said T cell receptor signaling domain is CD3 zeta domain.11 . The chimeric antigen receptor (CAR) according to any one of the preceding claims, wherein said chimeric antigen receptor comprises an amino acid sequence the amino acid sequence of SEQ ID No. 106, 107, 108,109, 159, 160, 161 or 162.

12. A nucleic acid encoding a chimeric antigen receptor (CAR) according to any one of claims 1- 11.

13. A vector comprising a nucleic acid according to claim 12.

14. A host cell comprising a nucleic acid according to claim 12 or a vector according to claim 13, or expressing a chimeric antigen receptor (CAR) according to any one of claims 1-11, preferably wherein said host cell is a T cell or a NK cell.

15. A chimeric antigen receptor (CAR) according to any one of claims 1-11 ora host cell according to claim 14 or for use in medicine.

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