A bispecific chimeric antigen receptor that binds CD19 and CD22

Abstract

Provided herein are bispecific chimeric antigen receptors (CARs) capable of binding to both CD19 and CD22, and immune cells expressing same, as well as therapeutic uses of such immune cells (e.g., CAR-T cells) to eliminate diseased cells, such as cancer cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 140,752, filed January 22, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Chimeric antigen receptor (CAR-T) T cells are genetically engineered T cells that express an artificial T cell receptor for use in immunotherapy. The artificial T cell receptor (known as a chimeric antigen receptor) can specifically bind to disease cell antigens, such as cancer antigens. Upon binding to disease cells, the CAR-T cells are activated and eliminate the disease cells.

[0003] Although CAR-T cell therapy has demonstrated efficacy in the treatment of some hematological cancers, the efficacy of the treatment may be affected by various factors, such as tumor antigen avoidance, for example, the expression level of the tumor antigen may be reduced to a level where CAR-T cells cannot participate and mediate cytotoxic activity.In some cases, tumor cells may avoid killing by expressing an alternative form of the target antigen that lacks the binding epitope for CAR.In other cases, tumor cells may avoid killing by switching to a genetically related but phenotypically distinct disease (so-called lineage switch). It is therefore of great interest to develop improved CAR-T approaches to address such challenges. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the development of an anti-CD19 / CD22 bispecific chimeric antigen receptor (CAR) that has excellent antigen-binding affinity and specificity and excellent anti-tumor efficacy as observed in animal models.

[0005] Accordingly, provided herein are anti-CD19 / CD22 bispecific CARs, nucleic acids encoding same, host cells, such as immune cells, that express the bispecific CARs, and their therapeutic uses.

[0006] In some aspects, the disclosure features a bispecific chimeric antigen receptor (CAR) specific for CD19 and CD22, comprising a first antigen-binding portion specific for CD19, a second antigen-binding portion specific for CD22, a costimulatory signaling domain, and a cytoplasmic signaling domain. The first antigen-binding portion may comprise the same heavy chain complementarity determining regions (CDRs) and / or the same light chain CDRs as reference antibody EPC-001-1, which binds to CD19. The second antigen-binding portion may comprise the same heavy chain CDRs and / or the same light chain CDRs as reference antibody EPC-001-2, EPC-001-3, or EPC-001-4, each of which binds to CD22.

[0007] In some embodiments, the first antigen-binding portion comprises the same heavy chain variable region (V H ) and the same light chain variable region (V L Alternatively, or in addition, the second antigen-binding portion may comprise the same heavy chain variable region (V H ) and the same light chain variable region (V L ).

[0008] In some embodiments, the first antigen-binding portion, the second antigen-binding portion, or both, can be a single chain variable fragment (scFv). For example, the first antigen-binding portion is an scFv comprising the amino acid sequence of SEQ ID NO: 9. Alternatively, or in addition, the second antigen-binding portion is an scFv comprising the amino acid sequence of SEQ ID NO: 18, 27, or 36.

[0009] In any of the CAR constructs disclosed herein, the costimulatory signaling domain can be derived from a costimulatory molecule selected from CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. In some embodiments, the cytoplasmic signaling domain is derived from CD3ζ. Any of the CARs disclosed herein can further comprise a hinge domain and a transmembrane domain. In some cases, the hinge and transmembrane domain can be located in the antigen binding portion and the costimulatory signaling domain.

[0010] In some examples, the bispecific CAR comprises a fusion polypeptide comprising, from N-terminus to C-terminus, (i) a first antigen-binding moiety, (ii) a second antigen-binding moiety, (iii) a costimulatory signaling domain, and (iv) a cytoplasmic signaling domain. In other examples, the bispecific CAR comprises a fusion polypeptide comprising, from N-terminus to C-terminus, (i) a second antigen-binding moiety, (ii) a first antigen-binding moiety, (iii) a costimulatory signaling domain, and (iv) a cytoplasmic signaling domain.

[0011] In some cases, the bispecific CAR may further comprise a peptide linker connecting the first and second antigen binding moieties. Exemplary peptide linkers include, but are not limited to, GGGGS (SEQ ID NO: 38), GGGGSGGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGS (SEQ ID NO: 40), or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41).

[0012] In some embodiments, a bispecific CAR disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 48-53. In some examples, a bispecific CAR disclosed herein may comprise the amino acid sequence of any one of SEQ ID NOs: 55-60 and 63-66.

[0013] In another aspect, provided herein is a nucleic acid or set of nucleic acids that collectively encode any of the bispecific CARs disclosed herein. For example, the nucleic acid can comprise a nucleotide sequence encoding a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 48-53. In a particular example, the nucleic acid can comprise a nucleotide sequence encoding a CAR comprising the amino acid sequence of any one of SEQ ID NOs: 55-60 and 63-67.

[0014] In some cases, the nucleic acid or set of nucleic acids may further comprise (i) a nucleotide sequence encoding a truncated epidermal growth factor receptor (EGFR) domain, which may comprise the extracellular and transmembrane domains of EGFR, and (ii) a nucleotide sequence encoding a self-cleaving peptide located between the nucleotide sequence encoding the bispecific CAR and the nucleotide sequence encoding the truncated EGFR domain. In some examples, the truncated EGFR domain comprises the amino acid sequence of SEQ ID NO:68.

[0015] Any of the nucleic acids or sets of nucleic acids can be an expression vector. In some examples, the expression vector can be a viral vector.

[0016] In yet another aspect, the present disclosure features an engineered immune cell that expresses any of the bispecific CARs disclosed herein. Such engineered immune cells can include any of the nucleic acids encoding the bispecific CARs disclosed herein. In some examples, the engineered immune cell is a T cell. In some examples, the engineered immune cell is a NK cell. In other examples, the engineered immune cell can be a macrophage.

[0017] Also within the scope of this disclosure are anti-CD19 CARs and anti-CD22 CARs, the nucleic acids encoding them, and engineered immune cells (e.g., T cells) that express them.

[0018] In some embodiments, the anti-CD19 CAR may comprise an extracellular antigen binding domain that binds to CD19, a costimulatory signaling domain, and a cytoplasmic signaling domain. The extracellular antigen binding domain may be an anti-CD19 single chain variable fragment (scFv) that comprises the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as the anti-CD19 antibody EPC-001-1. In some examples, the anti-CD19 scFv comprises the same heavy chain variable domain and the same light chain variable domain as the anti-CD19 antibody EPC-001-1. In one example, the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 9. In one particular example, the anti-CD19 CAR may comprise the amino acid sequence of SEQ ID NO: 62.

[0019] In some embodiments, the anti-CD22 chimeric antigen receptor (CAR) may comprise an extracellular antigen binding domain that binds to CD22, a costimulatory signaling domain, and a cytoplasmic signaling domain. In some examples, the extracellular antigen binding domain may be an anti-CD22 single chain variable fragment (scFv) that comprises the same heavy chain complementarity determining regions (CDRs) and / or the same light chain CDRs as the anti-CD22 antibody EPC-001-2. For example, the anti-CD22 scFv may comprise the same heavy chain variable domain and / or the same light chain variable domain as the anti-CD22 antibody EPC-001-2. In some examples, the extracellular antigen binding domain may be an anti-CD22 single chain variable fragment (scFv) that comprises the same heavy chain complementarity determining regions (CDRs) and / or the same light chain CDRs as the anti-CD22 antibody EPC-001-3. For example, the anti-CD22 scFv may comprise the same heavy chain variable domain and / or the same light chain variable domain as the anti-CD22 antibody EPC-001-3. In some examples, the extracellular antigen-binding domain can be an anti-CD22 single chain variable fragment (scFv) that contains the same heavy chain complementarity determining regions (CDRs) and / or the same light chain CDRs as an anti-CD22 antibody or EPC-001-4. For example, the anti-CD22 scFv can contain the same heavy chain variable domain and / or the same light chain variable domain as the anti-CD22 antibody EPC-001-4. In some examples, the anti-CD22 scFv comprises the amino acid sequence of SEQ ID NO: 18, 27, or 36. In a particular example, the anti-CD22 CAR can comprise the amino acid sequence of SEQ ID NO: 61.

[0020] In addition, the disclosure features a method for eliminating unwanted cells in a subject, comprising administering to a subject in need thereof an effective amount of an engineered immune cell as disclosed herein that expresses an anti-CD19 CAR, an anti-CD22 CAR, or an anti-CD19 / CD22 CAR, or a pharmaceutical composition comprising the same, as disclosed herein. In some cases, the unwanted cells are cancer cells.

[0021] In some embodiments, the subject is a human cancer patient. For example, the subject is a patient with CD19 + and / or CD22 + The cancer cell may be a human cancer patient comprising the cancer cell. In some cases, the human cancer patient may have a hematopoietic malignancy, such as a T cell malignancy or a B cell malignancy.

[0022] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief description of the drawings]

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.

[0024] [Figure 1] FIG. 1 shows quantification of surface expression of recombinant or endogenous CD19, CD22, or both on K562, Raji, and Nalm6 cells by qFACS. [Figure 2A-2B] Figures 2A and 2B include schematic designs of expression cassettes for expressing scFv antibodies and bispecific chimeric antigen receptors (CARs). Figure 2A shows an exemplary design of a bispecific antibody. Figure 2B shows an exemplary design of an anti-CD19 / CD22 bispecific chimeric antigen receptor (CAR). [Diagram 3] FIG. 1 shows the binding activity of various bispecific antibodies on CD19+ and / or CD22+ cells, including K562 cells (CD19- and CD22-), K562 cells engineered to express CD19 (CD19 K562), K562 cells engineered to express CD22 (CD22 K562), K562 cells engineered to express both CD19 and CD22 (CD19 / CD22 K562), Raji cells (CD19+ and CD22+), and Nalm6 cells (CD19+ and CD22+). [Figure 4A-4B] Figure 4 includes photographs showing expression of an exemplary anti-CD19 / CD22 bispecific chimeric antigen receptor (CAR), EPC-001-19, in immune cells detected by fluorochrome Alexa Fluor® 647-labeled anti-EGFR antibody (EGFR co-expressed with bispecific CAR) or fluorochrome Alexa Fluor® 647-labeled CD22-Fc fusion polypeptide. Figure 4A: Detected by anti-EGFR-AF647. Figure 4B: Detected by anti-human CD22-AF647. [Figure 5A-5D] Figures showing cytotoxic T lymphocyte (CTL) activity of immune cells expressing anti-CD19 / CD22 bispecific CARs are included. Figure 5A: Percentage of killing (E:T=1:1). Left panel: EPC-001-11, center panel: EPC-001-12, right panel: EPC-001-13. Figure 5B: Percentage of killing (E:T=1:1). Left panel: EPC-001-14, center panel: EPC-001-15, right panel: EPC-001-16. Figure 5C: Interferon gamma (IFNγ) secretion (E:T=1:1). Left panel: EPC-001-11, center panel: EPC-001-12, right panel: EPC-001-13. Figure 5D: Interferon gamma (IFNγ) secretion (E:T=1:1). ). Left panel: EPC-001-14, center panel: EPC-001-15, right panel: EPC-001-16. [Figures 6A-6C]Figures showing CTL activity of T cells expressing an exemplary anti-CD19 / CD22 bispecific CAR against target cells at various effector to target cell (E:T) ratios are included. Figure 6A: A diagram showing the level of specific cell lysis of CAR-T cells prepared from PBMCs of donor 1 against K562 cells, CD22 K562 cells, CD19 K562 cells, and CD19 / CD22 K562 cells at the various E:T ratios shown. Figure 6B: A diagram showing CTL activity of CAR-T cells prepared from PBMCs of donor 2 and donor 3 against K562 cells, CD22 K562 cells, CD19 K562 cells, and CD19 / CD22 K562 cells at the various E:T ratios shown. FIG. 6C: IFNγ levels in co-cultures of CAR-T cells prepared from PBMCs of donor 2 and donor 3 with K562 cells, CD22 K562 cells, CD19 K562 cells, and CD19 / CD22 K562 cells at different E:T ratios as indicated. [Figure 7] Includes figures showing the CTL time courses of T cells expressing various anti-CD19 / CD22 bispecific CARs or anti-CD19, anti-CD22 monospecific CARs against K562 cells, CD22 K562 cells, CD19 K562 cells, and CD19 / CD22 K562 cells at various E:T ratios as indicated. [Figure 8] FIG. 1 includes a diagram showing the CTL activity of T cells expressing anti-CD19 / CD22 bispecific CARs with various linkers connecting the anti-CD19 and anti-CD22 binding moieties in the CAR construct. [Figure 9] Includes a diagram showing CAR-T cell proliferation upon engagement with target cells (E:T=1:1). [Figure 10A-10B] Figures showing in vitro persistence of CAR-T cells upon multiple rounds of target cell challenge are included. Figure 10A: Percentage of killing (E:T=1:1). Left panel: Target cell challenge 1 at 48 hours, center panel: Target cell re-challenge 2 at 120 hours, right panel: Target cell re-challenge 3 at 192 hours. Figure 10B: IFNγ secretion (E:T=1:1). Left panel: Target cell challenge 1 at 48 hours, center panel: Target cell re-challenge 2 at 120 hours, right panel: Target cell re-challenge 3 at 192 hours. [Figures 11A-11E] Figure 11 shows the in vivo anti-tumor efficacy of T cells expressing anti-CD19 / CD22 bispecific CAR in a mouse cancer model. Figure 11A: Images showing cancer cell luciferase in control mice (untreated) and mice treated with CAR-T cells at 0.125x106 and 0.25x106 cells. Figure 11B: Diagram showing inhibition of tumor growth in mice treated with CAR-T cells by quantification of luciferase in tumor cells over the course of treatment. Figures 11C-11D: Diagram showing tumor cell luciferase quantification at days 14 and 33, respectively, after treatment. Figure 11E: Survival curves of control mice and mice treated with CAR-T cells. [Fig. 12A-12E] Figures showing CAR-T cell proliferation, phenotype, and persistence in vivo are included. Figure 12A: CAR-T cell numbers and phenotypes at 10, 19, and 33 days after treatment. Top panel: Group 2 mice treated with 0.125x106 CAR-T cells; bottom panel: Group 3 mice treated with 0.25x106 CAR-T cells. Figures 12B-12C: Day 33 / Day 19 ratios of T cell subtypes in Group 2 and Group 3 mice, respectively. Figures 12D-12E: Cell numbers in spleens at day 33 in Group 2 and Group 3 mice, respectively. [Figure 13A-13B] ELISA binding assay of anti-CD19 or CD22 scFv-Fc. Figure 13A: Binding of anti-CD22 scFv-Fc, anti-19 scFv-Fc, and anti-CD19 / CD22 scFv-Fc to CD19. Figure 13B: Binding of anti-CD22 scFv-Fc, anti-19 scFv-Fc, and anti-CD19 / CD22 scFv-Fc to CD22. [Figure 14A-14B] Shown are oxygen consumption of EPC-001-023 transduced T cells. Figure 14A: Oxygen consumption in EPC-001-023 CAR T cells at day 5. Figure 14B: Oxygen consumption in EPC-001-023 CAR T cells at day 11. [Figure 15] Western blot analysis of CD19 and CD22 proteins in CD19 and CD22 knockout cells and parental Raji cells is shown. [Figure 16A-16B]Quantitative FACS analysis of surface receptor expression is shown: Figure 16A: CD19 surface receptor counts on the indicated cell lines. Figure 16B: CD22 surface receptor counts on the indicated cell lines. [Figure 17] Shows the in vivo antitumor efficacy of EPC-001-23 CAR-T cells in an animal model engrafted with parental and knockout Raji cells. [Figures 18A-18C] Quantitative assessment of tumor bearing as determined by luciferase bioluminescence is shown in Figure 18A: Luciferase signal in parental Raji cells. Figure 18B: Luciferase signal in CD22 knockout Raji cells. Figure 18C: Luciferase signal in CD19 knockout Raji cells. [Figure 19A-19B] Phenotypic analysis of EPC-001-23 CAR-T cells performed on PBMC and spleen samples collected on day 36. Figure 19A: Phenotypic analysis of CD3+CAR+ cells in PBMC. Figure 19B: Phenotypic analysis of CD3+CAR+ cells in spleen. [Figures 20A-20C] Phenotypic analysis of EPC-001-23 CAR-T cells performed on PBMC and spleen samples collected on day 36. Figure 20A: CD3+CAR+ T cell counts in PBMC. Figure 20B: Quantitative PD1+, Tim3-CD3+CAR+ expression in PBMC. Figure 20C: Quantitative PD1+, Tim3-CD3+CAR+ expression in spleen. [Figures 21A-21C] Figures showing EPC-001-23 bispecific CAR-T cell proliferation and activation in vitro compared to tisagenlecleucel control. Figure 21A: T cell proliferation as indicated by CAR+ T cell counts, Figure 21B: T cell activation as indicated by granzyme B+ CAR-T cells at 72 hours. Figure 21C: CAR-T cytotoxicity. [Fig. 22A-22B]Figures 22A and 22B include figures showing in vivo cytotoxicity of EPC-001-23 bispecific CAR-T cells in mice implanted with Raji cells or CD22 KO Raji cells compared to tisagenlecleucel control. Figure 22A: Photographs showing tumor imaging in mice treated with the indicated CAR-T cells. Figure 22B: Diagram showing in vivo CAR-T cell proliferation and persistence. [Figure 23A-23B] Figures 23A and 23B include figures showing in vivo cytotoxicity of EPC-001-23 bispecific CAR-T cells in mice implanted with Raji cells or CD19 KO Raji cells compared to tisagenlecleucel control. Figure 23A: Photographs showing tumor imaging in mice treated with the indicated CAR-T cells. Figure 23B: Diagram showing in vivo CAR-T cell proliferation and persistence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The B-lymphocyte antigen CD19 is a member of the immunoglobulin superfamily that is expressed primarily on B-lineage cells and follicular dendritic cells. CD19 has been reported to function as an adaptor protein to recruit cytoplasmic signaling proteins and as a regulator to reduce the threshold of signaling pathways initiated by the B-cell receptor (through the CD19 / CD21 complex).

[0026] Cluster of differentiation 22 (CD22) is a member of the SIGLEC family of lectins. This molecule is expressed at high levels on the surface of mature B cells versus immature B cells. As an inhibitory receptor for B cell receptor (BCR) signaling, it plays a regulatory role in preventing overactivation of the immune system.

[0027] Both CD19 and CD22 have been established as promising targets for the treatment of certain diseases, such as leukemia. However, the efficacy of therapeutics targeting only CD19 or only CD22 may be affected, for example, by tumor antigen escape, resulting in reduced efficacy of the treatment.

[0028] Provided herein are bispecific chimeric antigen receptors (CARs) capable of binding to both CD19 and CD22, as well as engineered immune cells expressing such bispecific CARs. The anti-CD19 / CD22 bispecific CARs disclosed herein exhibited excellent binding activity to both surface-expressed CD19 and CD22, as well as excellent cytotoxic T lymphocyte-mediated cytotoxicity. Furthermore, the anti-CD19 / CD22 bispecific CARs exhibited excellent in vivo antitumor activity, as observed in mouse models. Furthermore, the bispecific CAR-T cells were shown to be effective in killing either CD19 / CD22 double-positive or cancer cells expressing only one of the two target antigens, indicating that the bispecific CAR-T cells maintain therapeutic efficacy in the context of either CD19 evasion or CD22 evasion.

[0029] Thus, immune cells expressing the bispecific CARs disclosed herein are capable of expressing CD19 + / CD22 + , CD19 - / CD22 + , or CD19 + / CD22 - It is expected to have a superior therapeutic effect in treating diseases involving diseased cells (e.g., cancer cells) and in addressing issues such as tumor antigen escape associated with monospecific CAR-T therapy or targeted therapy. The CD19 / CD22 bispecific tandem CAR has bivalent target engagement, thus locking CD19 and CD22 targets, preventing or delaying target escape, and potentially lowering the dose. In addition, each CD19 or CD22 binding domain can independently engage CD19 or CD22 expressing cells and mediate cancer cell death when either target is escaped.

[0030] Thus, the present disclosure provides an anti-CD19 / CD22 bispecific CAR, a nucleic acid encoding the same, a host cell, such as an immune cell (e.g., a T cell, a NK cell, or a macrophage) that expresses the bispecific CAR, and a method for the treatment of a CD19 / CD22 bispecific CAR. + and / or CD22 +Therapeutic uses of such immune cells in treating diseases associated with disease cells are featured.

[0031] I. Chimeric Antigen Receptor As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial immune cell receptor that can bind to an antigen expressed by an unwanted cell, such as a tumor-associated antigen (TAA) (e.g., CD19 or CD22). Generally, a CAR can include a fusion polypeptide that includes an extracellular antigen-binding domain (e.g., a single-chain variable fragment or scFv derived from an antibody specific for a target antigen), a costimulatory domain, and an intracellular signaling domain. In some cases, the fusion polypeptide can further include a hinge and a transmembrane domain located at the C-terminus of the extracellular antigen-binding domain. In some embodiments, the CAR disclosed herein is a T cell receptor. In other embodiments, the CAR disclosed herein can be a NK cell receptor.

[0032] A typical antibody molecule consists of a heavy chain variable region (V H ) and the light chain variable region (V L ), which are usually involved in antigen binding. H Area and V L The regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, known as "framework regions" ("FRs"). H and V Ltypically consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0033] In some embodiments, the antibody portions disclosed herein contain the same heavy and / or light chain complementarity determining regions (CDRs) or the same VDRs as a reference antibody. H and / or V L They may share a chain. H and / or V L Two antibodies that have CDRs mean that their CDRs are identical when determined by the same technique (e.g., the Kabat, Chothia, AbM, Contact, or IMGT techniques known in the art; see, e.g., bioinf.org.uk / abs / ). Such anti-CD19 antibodies have the same V and VD sequences as the exemplary antibodies described herein. H , the same V L , or both.

[0034] In some embodiments, the antibody moieties disclosed herein may share a certain level of sequence identity compared to a reference sequence. The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0035] In some embodiments, the antibody moieties disclosed herein may have one or more amino acid variations relative to a reference antibody. The amino acid residue variations disclosed in this disclosure (e.g., in framework regions and / or CDRs) may be conservative amino acid residue substitutions. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be prepared by methods for modifying polypeptide sequences known to those skilled in the art, such as those found in references that summarize such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley&Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V, (b) F, Y, W, (c) K, R, H, (d) A, G, (e) S, T, (f) Q, N, and (g) E, D.

[0036] Each of the anti-CD19 / CD22 bispecific CARs disclosed herein comprises an anti-CD19 portion and an anti-CD22 portion in the extracellular antigen-binding domain.

[0037] (a) Anti-CD19 binding part The anti-CD19 binding portion of any of the CARs disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein) comprises a heavy chain variable domain (V) of an anti-CD19 antibody connected by a peptide linker. H ) and the light chain variable domain (V L The scFv fragment may be in the form of an scFv, which is a fusion polypeptide comprising a V H and V LThe fragments can be in any orientation. In some cases, the scFv is N-terminal to C-terminal, V L A fragment, a peptide linker, and a V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment, a peptide linker, and a V L In some examples, the scFv may further comprise an N-terminal signal peptide to direct the CAR containing the scFv to the cell surface.

[0038] In some embodiments, the anti-CD19 binding portion may be derived from anti-CD19 antibody EPC-001-1 (see Table 1 below). The heavy and light chain complementarity determining regions provided in Table 1 are based on the Kabat definition. See also PCT / US2020 / 047035, filed August 19, 2020, the relevant disclosures of which are incorporated by reference for subject matter and for purposes of reference herein. [Table 1]

[0039] An anti-CD19 binding moiety (and an anti-CD22 binding moiety disclosed below) derived from a reference antibody refers to a binding moiety that has substantially similar structural and functional characteristics as the reference antibody. Structurally, a binding moiety may contain the same heavy and / or light chain complementarity determining regions or the same V H Chain and / or V L Alternatively, the binding moiety may have only a limited number of amino acid variations in one or more of the framework regions and / or one or more of the CDRs without significantly affecting the binding affinity and specificity relative to the reference antibody. See discussion below.

[0040] In some examples, the anti-CD19 binding moiety may contain the same heavy chain CDRs as those in antibody EPC-001-1, provided in Table 1 above. Alternatively, or in addition, the anti-CD19 binding moiety may have the same light chain CDRs as those in antibody EPC-001-1, also provided in Table 1 above. Such anti-CD19 binding moieties may have the same V and V CDRs as EPC-001-1. H and / or V L Alternatively, the anti-CD19 binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework regions of EPC-001-1. For example, the anti-CD19 binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions relative to the corresponding framework regions of EPC-001-1.

[0041] In some embodiments, the anti-CD19 portion may contain a certain level of variation in one or more CDRs relative to the CDRs of EPC-001-1. For example, the anti-CD19 portion may contain the V H Alternatively, or in addition, the anti-CD19 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of EPC-001-1. L The antibody may comprise light chain CDRs that, individually or collectively, have at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity to the CDRs. As used herein, "individually" means that one CDR of the antibody shares the sequence identity shown with the corresponding CDR of a reference antibody (e.g., EPC-001-1 or any of the anti-CD22 reference antibodies disclosed below). "Collectively" means that the three Vs of the combined antibody are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity. H CDR or V L The CDRs are aligned to the corresponding three V H CDR or V L For the CDRs, it is meant to share the indicated sequence identity.

[0042] In some cases, the anti-CD19 portion may include up to 10 amino acid variations (e.g., up to 9, 8, 7.6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively relative to those of the CDRs of EPC-001-1. In some cases, the anti-CD19 portion may include a heavy chain CDR3 that is the same as the heavy chain CDR3 of EPC-001-1 and may include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0043] In some examples, the anti-CD19 moieties disclosed herein can comprise the amino acid sequence of SEQ ID NO: 9. Alternatively, the anti-CD19 moieties can comprise an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to SEQ ID NO: 9. In other examples, the anti-CD19 moieties disclosed herein can comprise the same V H and V L The V H and V L It has the reverse orientation of the fragments.

[0044] Any of the anti-CD19 moieties disclosed herein (e.g., SEQ ID NO: 9 or the reverse V H and V L or its counterpart with the same orientation) can be used to construct the anti-CD19 / CD22 bispecific CAR disclosed herein.

[0045] (b) Anti-CD22 binding part The anti-CD22 binding portion of any of the CARs disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein) comprises a heavy chain variable domain (V) of an anti-CD22 antibody connected by a peptide linker. H ) and the light chain variable domain (V L The scFv fragment may be in the form of an scFv, which is a fusion polypeptide comprising a V H and V LThe fragments can be in any orientation. In some cases, the scFv is N-terminal to C-terminal, V L A fragment, a peptide linker, and a V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment, a peptide linker, and a V L In some examples, the scFv may further comprise an N-terminal signal peptide to direct the CAR containing the scFv to the cell surface.

[0046] In some embodiments, the anti-CD22 binding moiety may be derived from anti-CD22 antibody EPC-001-2, EPC-001-3, or EPC-001-4 (see Table 2 below). The heavy and light chain complementarity determining regions provided in Table 1 are based on the Kabat definition. See also PCT / US2020 / 047479, filed August 21, 2020, the relevant disclosure of which is incorporated by reference for subject matter and for purposes of reference herein. [Table 2] JPEG2024504162000004.jpg64170

[0047] In some examples, an anti-CD22 binding moiety may contain the same heavy chain CDRs as those in antibody EPC-001-2, provided in Table 2 above. Alternatively, or in addition, an anti-CD22 binding moiety may have the same light chain CDRs as those in antibody EPC-001-2, also provided in Table 2 above. Such an anti-CD22 binding moiety may have the same V and V CDRs as EPC-001-2. H and / or V LAlternatively, the anti-CD22 binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework regions of EPC-001-2. For example, the anti-CD22 binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions relative to the corresponding framework regions of EPC-001-2.

[0048] In some embodiments, the anti-CD22 portion may contain a certain level of variation in one or more CDRs relative to the CDRs of EPC-001-2. For example, the anti-CD22 portion may contain the V H Alternatively, or in addition, the anti-CD22 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of EPC-001-2. L It may comprise light chain CDRs that, individually or collectively, are at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identical to the CDRs.

[0049] In some cases, the anti-CD22 portion may include up to 10 amino acid variations (e.g., up to 9, 8, 7.6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively relative to those of the CDRs of EPC-001-2. In some cases, the anti-CD22 portion may include a heavy chain CDR3 that is the same as the heavy chain CDR3 of EPC-001-2 and may include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0050] In some examples, the anti-CD22 moieties disclosed herein can comprise the amino acid sequence of SEQ ID NO: 18. Alternatively, the anti-CD22 moieties can comprise an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to SEQ ID NO: 18. In other examples, the anti-CD22 moieties disclosed herein can comprise the same V H and V L The V sequence may be the same as SEQ ID NO: 18. H and V L It has the reverse orientation of the fragments.

[0051] In some examples, the anti-CD22 binding moiety may contain the same heavy chain CDRs as those in antibody EPC-001-3, provided in Table 2 above. Alternatively, or in addition, the anti-CD22 binding moiety may have the same light chain CDRs as those in antibody EPC-001-3, also provided in Table 2 above. Such anti-CD22 binding moieties may have the same V and V CDRs as EPC-001-3. H and / or V L Alternatively, the anti-CD22 binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework regions of EPC-001-3. For example, the anti-CD22 binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions relative to the corresponding framework regions of EPC-001-3.

[0052] In some embodiments, the anti-CD22 portion may contain a certain level of variation in one or more CDRs relative to the CDRs of EPC-001-3. For example, the anti-CD22 portion may contain the V H Alternatively, or in addition, the anti-CD22 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of EPC-001-3. LIt may comprise light chain CDRs that, individually or collectively, are at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identical to the CDRs.

[0053] In some cases, the anti-CD22 portion may include up to 10 amino acid variations (e.g., up to 9, 8, 7.6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively relative to those of the CDRs of EPC-001-3. In some cases, the anti-CD22 portion may include a heavy chain CDR3 that is the same as that of EPC-001-3 and may include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0054] In some examples, the anti-CD22 moieties disclosed herein can comprise the amino acid sequence of SEQ ID NO: 27. Alternatively, the anti-CD22 moieties can comprise an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to SEQ ID NO: 27. In other examples, the anti-CD22 moieties disclosed herein can comprise the same V H and V L The V sequence may be the same as SEQ ID NO:27. H and V L It has the reverse orientation of the fragments.

[0055] In some examples, an anti-CD22 binding moiety may contain the same heavy chain CDRs as those in antibody EPC-001-4, provided in Table 2 above. Alternatively, or in addition, an anti-CD22 binding moiety may have the same light chain CDRs as those in antibody EPC-001-4, also provided in Table 2 above. Such an anti-CD22 binding moiety may have the same V and V CDRs as EPC-001-4. H and / or V LAlternatively, the anti-CD22 binding moiety may comprise an amino acid variation in one or more of the framework regions relative to the corresponding framework regions of EPC-001-4. For example, the anti-CD22 binding moiety may collectively comprise up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in one or more framework regions relative to the corresponding framework regions of EPC-001-4.

[0056] In some embodiments, the anti-CD22 portion may contain a certain level of variation in one or more CDRs relative to the CDRs of EPC-001-4. For example, the anti-CD22 portion may contain the V H Alternatively, or in addition, the anti-CD22 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the V CDRs of EPC-001-4. L It may comprise light chain CDRs that, individually or collectively, are at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identical to the CDRs.

[0057] In some cases, the anti-CD22 portion may include up to 10 amino acid variations (e.g., up to 9, 8, 7.6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy and light chain CDRs collectively relative to those of the CDRs of EPC-001-4. In some cases, the anti-CD22 portion may include a heavy chain CDR3 that is the same as that of EPC-001-4 and may include one or more amino acid variations in one or more of the other heavy and light chain CDRs.

[0058] In some examples, the anti-CD22 moieties disclosed herein can comprise the amino acid sequence of SEQ ID NO: 36. Alternatively, the anti-CD22 moieties can comprise an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or more) identical to SEQ ID NO: 36. In other examples, the anti-CD22 moieties disclosed herein can comprise the same V H and V L The V sequence may be the same as SEQ ID NO:36. H and V L It has the reverse orientation of the fragments.

[0059] Any of the anti-CD22 moieties disclosed herein can be used to construct the anti-CD19 / CD22 bispecific CARs disclosed herein. In some examples, the anti-CD22 moiety has the amino acid sequence of SEQ ID NO: 18, or the reverse V H and V L In some examples, the anti-CD22 moiety may comprise the amino acid sequence of SEQ ID NO: 27, or its counterpart with a reversed V H and V L In some examples, the anti-CD22 moiety may comprise the amino acid sequence of SEQ ID NO: 36, or its counterpart with a reversed V H and V L It may include its counterpart with orientation.

[0060] (c) Other components of the chimeric antigen receptor construct In addition to the extracellular antigen binding domains disclosed herein, any of the CARs, including the anti-CD19 / CD22 bispecific CARs, may further comprise one or more intracellular signaling domains (e.g., costimulatory and cytoplasmic signaling domains), and optionally, a hinge domain, a transmembrane domain, an N-terminal signal peptide, or a combination thereof. In some cases, the CAR may be co-expressed with a suicide gene (e.g., a truncated EGFR gene) in the host immune cells. For example, the CAR coding sequence and the suicide gene may be configured in a bicistronic expression cassette, and the CAR coding sequence and the suicide gene may be linked via a self-cleaving peptide (e.g., P2A or T2A) coding sequence. Examples are provided in Table 3 below. [Table 3]

[0061] Signaling domains Any of the CAR constructs disclosed herein, including anti-CD19 / CD22 bispecific CARs, typically contain one or more intracellular signaling domains, including a costimulatory domain and a cytoplasmic signaling domain. A "costimulatory signaling domain" refers to at least a fragment of a costimulatory signaling protein that mediates intracellular signaling to induce an immune response, such as an effector function (secondary signal). The cytoplasmic signaling domain can be any signaling domain involved in eliciting cell signaling (primary signaling) that leads to the proliferation and / or activation of immune cells. The cytoplasmic signaling domains described herein are not costimulatory signaling domains known in the art that relay costimulatory or secondary signals to fully activate immune cells.

[0062] In some embodiments, the costimulatory signaling domain and the cytoplasmic signaling domain are for use in a CAR construct disclosed herein that is introduced into a T cell. In some embodiments, the costimulatory signaling domain and the cytoplasmic signaling domain are for use in a CAR construct disclosed herein that is introduced into a NK cell.

[0063] In some cases, the costimulatory signaling domain can be derived from a costimulatory protein involved in T cell response, such as a member of the B7 / CD28 family, a member of the TNF superfamily, a member of the SLAM family, or any other costimulatory molecule. Examples include, but are not limited to, 4-1BB, CD28, OX40, ICOS, CD40, CD40L, CD27, GITR, HVEM, TIM1, LFA1 (CD11a), or CD2. In a particular example, the costimulatory signaling domain is a 4-1BB signaling domain (e.g., SEQ ID NO: 44 in Table 3 above).

[0064] The cytoplasmic signaling domain may include an immunoreceptor tyrosine-based activation motif (ITAM) domain or may be ITAM-free. "ITAM" as used herein generally refers to a conserved protein motif present in the tail of signaling molecules expressed in many immune cells. Exemplary cytoplasmic signaling domains include the signaling domain of CD3zeta, e.g., SEQ ID NO:45.

[0065] In some cases, the costimulatory signaling domain can be derived from a costimulatory protein involved in NK cell response. Examples include, but are not limited to, DAP10, DAP12, 2B4, NKG2D, FcRIγ, NKp30, NKp44, or NKp46. Exemplary cytoplasmic signaling domains for use in NK cell CARs include, but are not limited to, CD3ζ, e.g., SEQ ID NO:45.

[0066] Hinge and transmembrane domains In some cases, the CAR constructs disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs) may contain a transmembrane domain, which may be a hydrophobic alpha-helix that spans the membrane. A "transmembrane domain" may be a peptide fragment that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain may provide stability to the CAR that contains it. An exemplary transmembrane domain may be the CD8 transmembrane domain, or the CD28 transmembrane domain. In one example, the transmembrane domain may include SEQ ID NO: 43, as shown in Table 3 above.

[0067] Alternatively, or in addition, the CAR constructs disclosed herein may also include a hinge domain, which may be located between the extracellular antigen binding domain and the transmembrane domain, or between the transmembrane domain and the intracellular signaling domain. The hinge domain may function to provide flexibility to the CAR or a domain thereof, or to prevent steric hindrance of the CAR or a domain thereof. The hinge domain may comprise 5-20 amino acid residues. In some embodiments, the hinge domain may be a CD8 hinge domain or an IgG hinge. Other hinge domains may be used. In one example, the hinge domain may comprise SEQ ID NO: 42, as shown in Table 3 above.

[0068] (d) Anti-CD19 / CD22 bispecific CAR In some aspects, provided herein is an anti-CD19 / CD22 bispecific CAR comprising an anti-CD19 portion (e.g., an anti-CD19 scFv such as those disclosed herein), an anti-CD22 portion (e.g., an anti-CD22 scFv such as those disclosed), one or more intracellular signaling domains, such as a costimulatory signaling domain and a cytoplasmic signaling domain, and optionally a hinge domain and a transmembrane domain as disclosed herein. In some cases, the anti-CD19 / CD22 bispecific CAR can be a single polypeptide comprising both the anti-CD19 portion and the anti-CD22 portion. In other cases, the anti-CD19 / CD22 bispecific CAR can be a multi-chain (e.g., two-chain) molecule. The anti-CD19 portion and the anti-CD22 portion can be located on separate polypeptides.

[0069] In some embodiments, an anti-CD19 / CD22 bispecific CAR disclosed herein may comprise an anti-CD19 binding portion (e.g., scFv) derived from EPC-001-1 and an anti-CD22 binding portion derived from EPC-001-2. In other embodiments, an anti-CD19 / CD22 bispecific CAR disclosed herein may comprise an anti-CD19 binding portion (e.g., scFv) derived from EPC-001-1 and an anti-CD22 binding portion derived from EPC-001-3. In yet other embodiments, an anti-CD19 / CD22 bispecific CAR disclosed herein may comprise an anti-CD19 binding portion (e.g., scFv) derived from EPC-001-1 and an anti-CD22 binding portion derived from any one of EPC-001-2-4.

[0070] Various combinations of anti-CD19 and anti-CD22 antibodies were investigated to generate anti-CD19 / CD22 bispecific cell engagement. Many of such bispecific scFvs showed low expression levels or low binding activity to CD19 and / or CD22. Unexpectedly, it was found that anti-CD19 / CD22 bispecific scFvs generated from anti-CD19 parent clone EPC-001-1 and three of the anti-CD22 parent clones, EPC-001-2, EPC-001-3, and EPC-001-4, showed desirable levels of bispecific scFv expression and maintained strong and specific target cell engagement compared to monoclonal anti-CD19 or anti-CD22 scFvs.

[0071] The anti-CD19 binding moiety (e.g., scFv) derived from EPC-001-1 can be any of the anti-CD19 moieties related to EPC-001-1 disclosed above. In some cases, it can contain the same heavy and / or light chain CDRs as EPC-001-1. In certain examples, the scFv can contain the same V and V CDRs as EPC-001-1. H and / or the same V L In some cases, the scFv may comprise, from the N-terminus to the C-terminus, L A fragment (e.g., SEQ ID NO: 8), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment (e.g., SEQ ID NO: 4), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V L In one particular example, the anti-CD19 portion may comprise SEQ ID NO:9.

[0072] The anti-CD22 binding moiety (e.g., scFv) derived from EPC-001-2 can be any of the anti-CD22 moieties related to EPC-001-2 disclosed above. In some cases, the scFv contains the same heavy and / or light chain CDRs as EPC-001-2. In certain examples, the scFv contains the same V and V CDRs as EPC-001-2. H and / or the same V LIn some cases, the scFv may comprise, from the N-terminus to the C-terminus, L A fragment (e.g., SEQ ID NO: 17), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment (e.g., SEQ ID NO: 14), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V L In one particular example, the anti-CD19 portion may comprise SEQ ID NO:18.

[0073] The anti-CD22 binding moiety (e.g., scFv) derived from EPC-001-3 can be any of the anti-CD22 moieties related to EPC-001-3 disclosed above. In some cases, the scFv contains the same heavy and / or light chain CDRs as EPC-001-3. In certain examples, the scFv contains the same V and V CDRs as EPC-001-3. H and / or the same V L In some cases, the scFv may comprise, from the N-terminus to the C-terminus, L A fragment (e.g., SEQ ID NO: 26), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment (e.g., SEQ ID NO: 22), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V L In one particular example, the anti-CD19 portion may comprise SEQ ID NO:27.

[0074] The anti-CD22 binding portion (e.g., scFv) derived from EPC-001-4 can be any of the anti-CD22 portions related to EPC-001-3 disclosed above. In some cases, the scFv contains the same heavy and / or light chain CDRs as EPC-001-4. In certain examples, the scFv contains the same V and V CDRs as EPC-001-4. H and / or the same V LIn some cases, the scFv may comprise, from the N-terminus to the C-terminus, L A fragment (e.g., SEQ ID NO: 35), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V H Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, H A fragment (e.g., SEQ ID NO: 31), a peptide linker (e.g., any one of SEQ ID NOs: 38 to 41), and V L In one particular example, the anti-CD19 portion may comprise SEQ ID NO:36.

[0075] In some embodiments, an anti-CD19 / CD22 bispecific CAR may comprise a fusion polypeptide comprising both the anti-CD19 and anti-CD22 moieties disclosed herein, which may be connected via a flexible peptide linker, e.g., any one of SEQ ID NOs: 38-41. In some cases, the linker may be a short G / S-rich linker (e.g., having up to 5 amino acid residues), e.g., GGGGS (SEQ ID NO: 38). The anti-CD19 and anti-CD22 moieties may be in any orientation shown in FIG. 2A. For example, the fusion polypeptide may comprise, from N-terminus to C-terminus, an anti-CD19 moiety (e.g., anti-CD19 scFv), a peptide linker, and an anti-CD22 moiety (e.g., anti-CD22 scFv). Alternatively, the fusion polypeptide may comprise, from N-terminus to C-terminus, an anti-CD22 moiety (e.g., anti-CD22 scFv), a peptide linker, and an anti-CD19 moiety (e.g., anti-CD19 scFv).

[0076] Any of the fusion polypeptides comprising anti-CD19 and anti-CD22 moieties may further comprise a costimulatory signaling domain and a cytoplasmic signaling domain such as those disclosed herein. Optionally, the fusion polypeptide may further comprise a hinge domain and a transmembrane domain, also disclosed herein. A schematic diagram of an exemplary design of a bispecific CAR is provided in FIG. 3. In some examples, the bispecific CAR may be included in a polycistronic expression cassette with a suicide gene (e.g., a truncated EGFR) via a self-cleaving peptide linker, as shown in FIG. 3.

[0077] Exemplary anti-CD19 / CD22 bispecific scFvs and CARs are provided in Table 4 below. [Table 4] JPEG2024504162000007.jpg255164JPEG2024504162000008.jpg255166JPEG2024504162000009.jpg255169JPEG2024504162 000010.jpg255166JPEG2024504162000011.jpg244170JPEG2024504162000012.jpg255170JPEG2024504162000013.jpg70170

[0078] Also within the scope of the present disclosure are bispecific anti-CD19 / CD22 antibodies comprising an anti-CD19 binding portion derived from a parent anti-CD19 antibody provided in Table 1 and an anti-CD22 binding portion derived from a parent anti-CD22 antibody provided in Table 2 herein. Such bispecific antibodies can be in any suitable form known in the art. For example, a bispecific antibody can comprise an anti-CD19 scFv and an anti-CD22 scFv in tandem repeats (e.g., a bispecific antigen-binding portion in any of the bispecific CARs disclosed herein). In some cases, such bispecific antibodies can further comprise an Fc fragment to form a scFv-Fc fusion polypeptide.

[0079] (e) Anti-CD19 and anti-CD22 CAR Also within the scope are anti-CD19 and anti-CD22 CARs that comprise any of the anti-CD19 and anti-CD22 binding moieties disclosed herein.

[0080] In some aspects, provided herein is an anti-CD19 CAR, a nucleic acid encoding the same, and a host cell expressing the same. The anti-CD19 CAR may comprise (a) an extracellular binding domain, which may be any of the anti-CD19 binding moieties, for example, an anti-CD19 scFv derived from EPC-001-1, (b) a costimulatory signaling domain, such as those disclosed herein, and (c) a cytoplasmic signaling domain, such as those disclosed herein. The anti-CD19 CAR may further comprise a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen binding domain. In one example, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 62.

[0081] In some aspects, provided herein are anti-CD22 CARs, nucleic acids encoding same, and host cells expressing same. In some examples, the anti-CD22 CARs may comprise (a) an extracellular binding domain that may be any of the anti-CD22 binding moieties, such as anti-CD19 scFv from EPC-001-2, EPC-001-3, or EPC-001-4, (b) a costimulatory signaling domain, such as those disclosed herein, and (c) a cytoplasmic signaling domain, such as those disclosed herein. The anti-CD22 CARs may further comprise a hinge domain and a transmembrane domain located at the C-terminus of the extracellular antigen binding domain. In one example, the anti-CD22 CAR comprises the amino acid sequence of SEQ ID NO: 61.

[0082] II. CAR-expressing immune cells In some aspects, provided herein are engineered immune cells, such as T cells, NK cells, or macrophages, having surface expression of any of the anti-CD19, anti-CD22, or anti-CD19 / CD22 bispecific CAR constructs disclosed herein. In some cases, the engineered immune cells are T cells that express any of the anti-CD19 / CD22 bispecific CARs provided in Table 4 above (e.g., SEQ ID NO: 63).

[0083] Any of the CAR-expressing immune cells disclosed herein can be engineered with additional mechanisms to reprogram the CAR-expressing cells to enhance the bioactivity and / or persistence of the CAR-expressing cells, thereby enhancing the overall therapeutic effect. For example, the CAR-expressing immune cells can be further engineered to knock-in one or more immune modulatory genes, one or more immune checkpoint inhibitor genes, or a combination thereof. Alternatively, the CAR-expressing immune cells disclosed herein can be further engineered to knock-down or knock-out one or more inhibitory genes.

[0084] (a) Preparation of CAR-expressing immune cells The genetically engineered immune cells disclosed herein can be prepared by introducing an expression cassette encoding any of the CAR constructs disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein, such as those provided in Table 4) into suitable immune cells and harvesting the resulting engineered immune cells expressing the CAR on the cell surface.

[0085] The population of immune cells as starting parent cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as spleen, lymph nodes, thymus, stem cells, or tumor tissue. A suitable source for obtaining the desired type of host cells will be apparent to one of skill in the art. In some embodiments, the population of immune cells is derived from PBMCs. The desired type of host cells (e.g., T cells, NK cells, macrophages, or a combination thereof) can be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules. As a non-limiting example, anti-CD3 and anti-CD28 antibodies can be used for the expansion of T cells. In some embodiments, a specific type of cell (e.g., T cells, NK cells, or macrophages) can be enriched from the immune cell population. Such enriched cell subpopulations can be expanded and / or activated in vitro before being genetically engineered for the introduction of a CAR-encoding expression cassette.

[0086] To construct immune cells expressing any of the CAR polypeptides described herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein, such as those provided in Table 4), an expression vector for stable or transient expression of the CAR polypeptide can be made and introduced into immune host cells via conventional methods. For example, a nucleic acid encoding a CAR polypeptide can be cloned into a suitable expression vector, such as a viral vector, operably linked to a suitable promoter. Non-limiting examples of useful vectors of the present disclosure include viral vectors, such as retroviral vectors, including gamma retroviral vectors, adeno-associated viral vectors (AAV vectors), and lentiviral vectors. The nucleic acid and vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and bind with a ligase. Alternatively, a synthetic nucleic acid linker can be ligated to the ends of the nucleic acid encoding the CAR polypeptide. The synthetic linker can contain a nucleic acid sequence that corresponds to a particular restriction site in the vector. The choice of expression vector / plasmid / viral vector depends on the type of host cell for expression of the CAR polypeptide, but must be suitable for integration and replication in eukaryotic cells. Any such nucleic acid encoding a CAR and expression vectors containing the same are also within the scope of this disclosure.

[0087] For the expression of the CAR polypeptide described herein, various promoters may be used, including but not limited to, cytomegalovirus (CMV) intermediate early promoter, viral LTR such as rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, or herpes simplex tk virus promoter. Additional promoters for the expression of the CAR polypeptide include any constitutively active promoter in immune cells. Alternatively, any regulatable promoter may be used so that its expression can be regulated in immune cells. In some embodiments, the promoter may be a pEF1α promoter.

[0088] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as a neomycin gene or a kanamycin gene, for selecting stable or transient transfectants in the host cell; an enhancer / promoter sequence derived from the immediate early gene of human CMV for high level transcription; a transcription termination and RNA processing signal derived from SV40 for mRNA stability; an SV40 polyomavirus origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a "suicide switch" or "suicide gene" in case of inducing a cause for the death of cells carrying the vector (e.g., an inducible caspase such as HSV thymidine kinase or iCasp9), and a reporter gene for assessing expression of the CAR polypeptide.

[0089] In a particular embodiment, such a vector may also include a suicide gene. As used herein, the term "suicide gene" refers to a gene that kills cells that express the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, e.g., a drug, to a cell in which the gene is expressed, causing the cell to die when the cell comes into contact with or is exposed to the agent. Suicide genes are known in the art (see, e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8. In particular examples, the suicide gene can encode a truncated EGFR, such as the truncated EGFR provided in Tables 3 and 4 above.

[0090] The nucleic acids disclosed herein may contain two coding sequences, one for any of the CAR constructs disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein, such as those provided in Table 4), and the other for a suicide gene product. The two coding sequences may be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically separate) polypeptides. To this end, the nucleic acids described herein may contain a third nucleotide sequence located between the first and second coding sequences. This third nucleotide sequence may, for example, encode a ribosomal skipping site. A ribosomal skipping site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of an additional open reading frame from one messenger RNA. This third nucleotide sequence can encode a self-cleaving peptide, such as, for example, a P2A, T2A, or F2A peptide (see, e.g., Kim et al., PLoS One. 2011;6(4):e18556). See also FIG. 3.

[0091] Any of the vectors containing a nucleic acid sequence encoding an ACTR polypeptide as described herein are also within the scope of this disclosure.

[0092] Such vectors, or sequences encoding a CAR polypeptide contained therein, can be delivered into a host cell, such as a host immune cell (e.g., a T cell, a NK cell, or a macrophage), by any suitable method. Methods of delivering vectors to immune cells are well known in the art and can include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction, such as lentiviral transduction).

[0093] After introducing a vector encoding any of the CAR polypeptides provided herein (e.g., any of the anti-CD19 / CD22 bispecific CARs disclosed herein, such as those provided in Table 4) into a host cell, the cell can be cultured under conditions that allow expression of the CAR polypeptide. If expression of the CAR polypeptide is regulated by a regulatable promoter, the host cell can be cultured under conditions in which the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter, and the immune cells are cultured in the presence of an inducing molecule or under conditions in which the inducing molecule is produced. Determining whether a CAR polypeptide is expressed will be apparent to one of skill in the art and can be assessed by any known method, for example, detection of mRNA encoding the CAR polypeptide by quantitative reverse transcriptase PCR (qRT-PCR), or detection of the CAR polypeptide protein by methods including western blotting, fluorescence microscopy, and flow cytometry. Alternatively, expression of a functional CAR can be determined by binding activity and / or CTL activity against cells expressing a target antigen, e.g., CD19 and / or CD22.

[0094] Methods for preparing host cells expressing any of the CAR polypeptides described herein may also include activating the host cells ex vivo. Activating a host cell means stimulating the host cell to an activated state in which it may perform an effector function. The method of activating the host cell will depend on the type of host cell used to express the CAR polypeptide. For example, T cells may be activated ex vivo in the presence of one or more molecules, including but not limited to anti-CD3 antibodies, anti-CD28 antibodies, IL-2, and / or phytohemoagglutinin. In other examples, NK cells may be activated ex vivo in the presence of one or more molecules, such as 4-1BB ligand, anti-4-1BB antibodies, IL-15, anti-IL-15 receptor antibodies, IL-2, IL12, IL-21, and / or K562 cells. In some embodiments, host cells expressing any of the CAR polypeptides described herein (CAR-expressing cells) are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be apparent to one of skill in the art and may include assessing the expression of one or more cell surface markers associated with cellular activation, cytokine expression or secretion, and cell morphology.

[0095] Methods for preparing host cells expressing any of the CAR polypeptides described herein can include growing the host cells ex vivo. Growing the host cells can include any method that results in an increase in the number of cells expressing the CAR polypeptide, for example, allowing the host cells to grow or stimulating the host cells to grow. Methods for stimulating the growth of host cells will depend on the type of host cell used to express the CAR polypeptide and will be apparent to one of skill in the art. In some embodiments, the host cells expressing any of the CAR polypeptides described herein are grown ex vivo prior to administration to a subject.

[0096] In some embodiments, the host cells expressing the CAR polypeptide are expanded and activated ex vivo before administering the cells to a subject. Activation and expansion of the host cells can be used to allow for integration of the viral vector into the genome and expression of the gene encoding the CAR polypeptide described herein. When mRNA electroporation is used, activation and / or amplification may not be required, although electroporation may be more effective if performed on activated cells.

[0097] In some cases, the CAR polypeptide is transiently expressed (e.g., for 3-5 days) in a suitable host cell. Transient expression can be advantageous when there is potential toxicity and needs to be useful in the early stages of clinical trials for possible side effects.

[0098] (b) Pharmaceutical Composition Any of the engineered immune cells expressing a CAR disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs, such as those provided in Table 4 above) can be mixed with a pharma- ceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0099] The phrase "pharmaceutical acceptable" when used in connection with compositions of the present disclosure refers to molecular entities and other components of such compositions that are physiologically acceptable and typically do not produce adverse reactions when administered to a mammal (e.g., human). Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the US Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, particularly humans. "Acceptable" means that the carrier is compatible with the active components of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition is administered. Any of the pharmaceutical compositions used in the present methods may contain pharmaceutical acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.

[0100] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0101] For examples of additional useful agents, see also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0102] IV. Therapeutic uses Any of the engineered immune cells (e.g., T cells, NK cells, or macrophages) expressing a CAR disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs such as those provided in Table 4 above) may be used for therapeutic purposes, e.g., to eliminate unwanted cells that express CD19 and / or CD22. In some examples, the engineered immune cells are CAR-T cells expressing any of the anti-CD19 / CD22 bispecific CARs such as those provided in Table 4 above.

[0103] To practice the methods described herein, an effective amount of immune cells (NK cells, T lymphocytes, or macrophages) expressing any of the CARs described herein (e.g., any of the anti-CD19 / CD22 bispecific CARs such as those provided in Table 4 above), or a pharmaceutical composition thereof, can be administered to a subject in need of treatment via a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of the respective agent (e.g., NK cells, T lymphocytes, or macrophages expressing a CAR) that confers a therapeutic effect on the subject upon administration. Determining whether an amount of the cells or compositions described herein has achieved a therapeutic effect will be apparent to one of skill in the art. Effective amounts will vary according to the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, gender, and weight, duration of treatment, nature of combination therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner, as will be recognized by those of skill in the art. In some embodiments, an effective amount alleviates, relieves, improves, enhances, reduces the symptoms of, or delays the progression of, any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient.

[0104] As used herein, the term "therapeutically effective" as applied to a dose or amount refers to that amount of a compound or pharmaceutical composition sufficient to produce a desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would have been effective when administered individually. Within the context of the present disclosure, the term "therapeutically effective" refers to that amount of a compound or pharmaceutical composition sufficient to delay the onset, halt the progression, reduce, or alleviate at least one symptom of the disorder treated by the method of the present disclosure.

[0105] In some embodiments, the methods of the present disclosure can be used to eliminate or inhibit disease cells that express CD19 and / or CD22. Thus, any of the immune cells disclosed herein can be CD19 + and / or CD22 + CD19 in cancer cells + and / or CD22 + The methods disclosed herein can be used to treat diseases associated with disease cells. + and / or CD22 + It may be used to treat cancers involving cancer cells, such as hematopoietic cancers. In certain embodiments, the cancer may be a solid tumor.

[0106] In some embodiments, an effective amount of any of the engineered immune cells expressing a CAR disclosed herein (e.g., any of the anti-CD19 / CD22 bispecific CARs such as those provided in Table 4 above) may be provided to a subject in need of treatment via a suitable route, e.g., intravenous infusion. + and / or CD22 + CD19 in cancer cells + and / or CD22 + The patient may be a human patient having a disease associated with disease cells. In some cases, the human patient may be a human patient having a disease associated with disease cells. + and / or CD22 + The human patient has a cancer comprising the cancer cells. In some cases, the human patient may have a hematopoietic cancer. In other cases, the human patient may have a solid tumor.

[0107] In some instances, the human patient is + and / or CD22 +The patient may have a B cell malignancy involving diseased B cells. Examples include, but are not limited to, non-Hodgkin's lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma.

[0108] In some examples, the human patient may have a T-cell malignancy. Examples include, but are not limited to, T lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (e.g., cutaneous T-cell lymphoma, adult T-cell leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), or peripheral T-cell lymphoma not otherwise specified (PTCL, NOS)).

[0109] In some embodiments, immune cells (e.g., NK and / or T cells) for use in the treatments disclosed herein can be autologous to the subject, i.e., immune cells can be obtained from a subject in need of treatment, genetically engineered for expression of a CAR polypeptide, and then administered to the same subject. In one particular embodiment, prior to reintroduction into the subject, autologous immune cells (e.g., T lymphocytes, NK cells, or macrophages) are activated and / or expanded ex vivo. Administration of autologous cells to a subject can result in reduced host cell rejection compared to administration of non-autologous cells.

[0110] Alternatively, the genetically engineered immune cells (e.g., T cells, NK cells, or macrophages) can be allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of a CAR polypeptide, and administered to a second subject of the same species but different from the first subject. For example, the allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor. In certain embodiments, the T lymphocytes are allogeneic T lymphocytes in which expression of endogenous T cell receptors has been inhibited or eliminated. In one particular embodiment, the allogeneic T lymphocytes are activated and / or expanded ex vivo prior to being introduced into the subject. The T lymphocytes can be activated by any method known in the art, for example, in the presence of anti-CD3 / CD28, IL-2, and / or phytohemoagglutinin.

[0111] NK cells can be activated by any method known in the art, for example, in the presence of one or more agents selected from the group consisting of CD137 ligand protein, CD137 antibody, IL-15 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, and K562 cell line. For example, see U.S. Patent Nos. 7,435,596 and 8,026,097 for a description of useful methods for expanding NK cells. For example, NK cells used in the methods of the present disclosure lack or poorly express major histocompatibility complex I and / or II molecules and can be preferentially expanded by exposure to cells genetically modified to express membrane-bound IL-15 and 4-1BB ligand (CDI37L). Such cell lines include K562 [ATCC, CCL243, Lozzio et al., Blood 45(3):321-334(1975); Klein et al., Int. J. Cancer 18:421-431(1976)], and the Wilms tumor cell line HFWT (Fehniger et al., Int Rev Immunol 20(3-4):503-534(2001); Harada H, et al., Exp Hematol 32(7):614-621(2004)), endometrial tumor cell line HHUA, melanoma cell line HMV-II, hepatoblastoma cell line HuH-6, small cell lung carcinoma cell lines Lu-130 and Lu-134-A, neuroblastoma cell lines NB19 and N1369, testicular-derived embryonal carcinoma cell line NEC14, cervical carcinoma cell line TCO-2, and bone marrow metastatic neuroblastoma cell line TNB1 [Harada, et al., Jpn. J. Cancer Res 93:313-319(2002)], but need not be limited thereto. Preferably, the cell line used lacks or expresses poorly both MHCI and II molecules, such as K562 and HFWT cell lines. A solid support may be used instead of a cell line. Such a support should preferably have at least one molecule attached to its surface that is capable of binding to NK cells and inducing a primary activation event and / or a proliferative response, or capable of binding a molecule having such an affect, thereby functioning as a scaffold.The support may have CD137 ligand protein, CD137 antibody, IL-15 protein, or IL-15 receptor antibody bound to its surface. Preferably, the support has IL-15 receptor antibody and CD137 antibody bound to its surface.

[0112] According to the present disclosure, a patient is 5 ~10 9 Patients may be treated by injecting immune cells, such as T lymphocytes or NK cells, expressing a CAR polypeptide, such as an anti-CD19 / CD22 bispecific CAR (e.g., SEQ ID NO: 23) listed in Table 4 above, in a therapeutically effective amount ranging from 10 CAR+ cells. Infusions may be repeated as frequently and as many times as the patient can tolerate until a desired response is achieved. Appropriate infusion doses and schedules will vary from patient to patient, but can be determined by the treating physician for a particular patient. In some instances, infusions may be repeated in a therapeutically effective amount ranging from about 10 CAR+ cells. 6 Inject an initial dose of cells / Kg, 10 8 The total number of cells per kg can be increased to more than 100.

[0113] The particular dosage regimen used in the methods described herein, i.e., dose, timing, and repetition, will depend on the particular subject and the subject's medical history. The appropriate dosage of CAR-expressing immune cells used will depend on the type of cancer being treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to immune cell therapy, and the discretion of the attending physician.

[0114] In some embodiments, engineered immune cells expressing any of the CAR constructs disclosed herein (e.g., anti-CD19 CAR, anti-CD22 CAR, or anti-CD19 / CD22 bispecific CAR) may be utilized in combination with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, etc. Such therapy may be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure. When co-administered with additional therapeutic agents, the suitable therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0115] V. KITS FOR THERAPEUTIC USE The present disclosure also provides kits for the use of engineered immune cells (e.g., T lymphocytes, NK cells, or macrophages) expressing an anti-CD19 CAR, an anti-CD22 CAR, or an anti-CD19 / CD22 bispecific CAR described herein. Such kits can include one or more containers containing the engineered immune cells, which can be formulated into a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0116] In some embodiments, the kits described herein include engineered immune cells that can be expanded in vitro. The immune cells can express any of the CARs disclosed herein, for example, any of the anti-CD19 / CD22 bispecific CARs, such as those provided in Table 4 above.

[0117] In some embodiments, the kit may additionally include instructions for use in any of the methods described herein. The included instructions may include instructions for administration of the engineered immune cells disclosed herein to achieve an intended activity in a subject, e.g., eliminating target disease cells, such as cancer cells expressing CD19, CD22, or both. The kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment.

[0118] The instructions for use of the engineered immune cells described herein generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. The instructions provided with the kits of the present disclosure are typically instructions on a label or insert. The label or insert indicates that the engineered immune cells are used to treat, delay the onset of, and / or alleviate a disease or disorder associated with CD19 and / or CD22 positive disease cells in a subject.

[0119] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, bottles, flexible packaging, etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices, or injection devices, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container may have a sterile access port.

[0120] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0121] general technology The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985>>, Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984>>, Animal Cell Culture (R.I. Freshney, ed. (1986>>, Immobilized Cells and Enzymes (lRL Press, (1986>>, and are described in detail in the literature such as B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0122] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0123] Example 1.CD19 + and / or CD22 + Generation and characterization of cell lines This example describes the generation of cell lines expressing either or both of the CD19 and CD22 surface antigens. (a) Generation of CD19 / CD22-positive recombinant cell lines K562 cells (ATCC) were transfected with 10ug of pCMV6-Entry vector carrying a nucleotide sequence encoding full-length human CD19 or CD22 fused to a flag or Myc tag at the C-terminus. A polyclonal, drug-resistant pool of CD19- or CD22-expressing cells was obtained by a G418 drug selection process. In parallel, parental cell lines transfected with an empty pCMV6-Entry vector were generated for use as negative controls. CD19- or CD22-expressing cells were sorted by FACS to obtain pools of CD19- or CD22-expressing cells. The pools were expanded under G418 drug selection. Single cell sorting was then performed, followed by further drug selection to generate clonal cell lines. Clonal lines were screened for CD19 or CD22 expression by FACS.

[0124] To generate CD19 / CD22 double positive cell lines, 10ug of CD22 plasmid was transfected into 5M CD19 expressing cell line and selected under G418. Single cell sorting was performed for both CD19 and CD22 high, followed by G418 selection and clonal FACS screening to obtain high CD19 / CD22 / K562 double positive cell lines.

[0125] (b) Quantification of recombinant and endogenous CD19 and CD22 receptor numbers in target cell lines. To further characterize the recombinant and endogenous CD19 and CD22 expression levels in the target cell lines (as shown herein), quantitative FACS assays were performed using Bangs Laboratories Inc QUANTUM Alexa Fluor® 647 (fluorescent dey) MESF microsphere beads for standard calibration according to the manufacturer's protocol. The parental K562 cell line showed undetectable CD19 and CD22 expression. In single or double positive recombinant cell lines, the expression level of CD19 was about 5-fold higher than that of CD22. Raji cells showed more CD19 and CD22 expression than Nalm6 cells. In both Raji and Nalm6 cells, the expression level of CD19 was about 4-9-fold higher than that of CD22. The CD19 and CD22 receptor copy numbers are summarized in Table 5. See also Figure 1. [Table 5]

[0126] (c) Generation of CD19, CD22, and CD19 / CD22GFP positive cell lines. K562, CD19 K562, CD22 K562, CD19 / CD22 K562, Raji, and Nalm6 cell lines were further engineered to introduce a GFP expression cassette using Incucyte CytoLight green lentiviral transduction. Cells were sorted for GFP positivity and selected under G418 drug selection to establish stable cell lines. GFP-positive cell lines were utilized for imaging-based cytotoxicity assays on a Cytation® 5 instrument (cell imaging multimode reader).

[0127] Example 2. Characterization of CD19-CD22 bispecific scFv Exemplary anti-CD19 / CD22 bispecific antibodies were characterized as follows.

[0128] (a) Preparation of CD19-CD22 bispecific scFv CD19 / CD22 bispecific scFvs were cloned into pET22b bacterial periplasmic vector in CD19-CD22 and CD22-CD19 orientations and expressed in Rosetta II strain. Figure 2A shows an exemplary design of CD19 / CD22 bispecific scFvs. To purify the antibody fragments expressed in E. coli cells, 3 μl of Ni Sepharose® Excel resin (GE) was mixed with 1 mL of filtered supernatant and packed into a 10 mL or 20 mL BioRad Econo-Pac® column. Prior to loading, the resin of the column was equilibrated with at least 20 column volumes (CV) of Buffer A (1×PBS, pH 7.4, with additional NaCl added to 500 mM). The filter-sterilized supernatant was purified by gravity flow, either by controlling the flow rate at 1 mL / min or by pouring twice over the same packed resin bed. The column was then washed with the following buffers: 10 CV Buffer A, 20 CV Buffer B (1x PBS, pH 7.4, with additional NaCl to 500 mM and Imidazole at 30 mM). Detox® buffer was used to remove endotoxin if necessary.

[0129] To purify antibody fragments from 250 mL expression cultures, the antibody binding column was washed sequentially with 20 CV Buffer C (1x PBS pH 7.4 with extra NaCl to 500 mM, 1% Tx114), 20 CV Buffer D (1x PBS pH 7.4 with extra NaCl to 500 mM, 1% Tx100 + 0.2% TNBP) and 40 CV Buffer E (1x PBS pH 7.4 with extra NaCl to 500 mM).

[0130] The protein was eluted with Elution Buffer F (1x PBS pH 7.4 with additional NaCl to 500mM and Imidazole 500mM) in a total of 6 fractions (0.5CV pre-elution, 5x 1CV elution). Fractions were run through a Bradford assay (100ul of diluted Bradford solution + 10ul sample). Fractions with a bright blue colour were pooled and their protein concentration was measured by A280 elongation index. An SDS-PAGE gel assay was performed to analyse the purity of the purified antibody.

[0131] (b) Binding activity of anti-CD19, anti-CD22, and anti-CD19 / CD22 bispecific scFv antibodies to cell surface antigens by FACS analysis. To determine the specific target cell binding activity after conversion into different bispecific scFv formats, CD19, CD22 monospecific scFvs and CD19 / CD22 bispecific scFvs were tested in a FACS binding assay using K562, CD19 K562, CD22 K562, and CD19 / CD22 K562 recombinant cell lines. Briefly, each bispecific scFv was diluted to 200 nM and incubated with 100,000 K562, CD19 K562, CD22 K562, and CD19 / CD22 K562 cell lines in a 96-well plate with shaking at 4° C. for 1 hour. Cells were spun down at 1300 rpm for 5 minutes at 4° C. to remove unbound antibody. Cells were then washed once with 200 μL PBS per well. The samples were mixed with Alexa Fluor® 647 (fluorochrome) conjugated anti-His antibody (secondary antibody, 100 μL, 1:1000 dilution) and incubated at 4° C. for 30 minutes in the dark with shaking. The samples were then spun down at 1300 rpm for 5 minutes at 4° C. and washed twice with 200 uL of 1×PBS per well. The resulting samples were reconstituted in 200 uL of 1×PBS and read on an Attune™ NxT flow cytometer. Analysis was performed by counting only Alexa Fluor® 647-(fluorochrome) positive cells and then plotted with GraphPad Prism® 8.1 software.

[0132] The binding activity of anti-CD19, anti-CD22, and anti-CD19 / CD22 bispecific scFvs to cell surface antigens is provided in Figure 3. Of the bispecific scFvs tested, four bispecific scFvs retained similar binding activity to the parental monospecific scFvs for both CD19 and CD22 target cell lines. Two bispecific scFvs showed similar binding to the CD19 target cell line and reduced binding to the CD22 target cell line compared to the parental monospecific scFv.

[0133] Four bispecific scFvs with full binding activity were then tested for binding activity to Raji cells expressing endogenous CD19 and CD22 by FACS assay, and their EC 50 The EC values ​​were determined. Briefly, each purified bispecific scFv protein was titrated from 200 nM in 3-fold serial dilutions in complete medium. The diluted samples were incubated with 100,000 Raji cell line cells in 96-well plates at 4° C. for 1 hour with shaking. Washing, detection, and analysis were performed as described above. The EC values ​​of these exemplary anti-CD19 / CD22 bispecific scFv antibodies were 50 The values ​​are provided in Table 6 below. [Table 6]

[0134] Example 3: Construction of anti-CD19 / CD22 bispecific chimeric antigen receptor This example describes the construction of an exemplary anti-CD19 / CD22 bispecific chimeric antigen receptor (CAR) and the introduction of such a construct into a host cell for expression via viral transduction.

[0135] (a) Exemplary CAR Format and Structure The bispecific CD19 / CD22 scFv was converted to CD19-CD22 or CD22-CD19 orientation. The scFv fragment was linked in tandem with a modified IgG4 hinge, CD28 transmembrane domain, 4-1BB costimulatory domain, and CD3z intracellular signaling domain (see sequence listing below). In some examples, the bispecific CAR construct was further linked to a truncated EGFR fragment. An exemplary bispecific CAR construct is shown in FIG. 2B. The sequences were cloned into a pEF1a-based lentiveral vector. Table 7 below provides exemplary CAR constructs. [Table 7]

[0136] (b) Lentivirus production and characterization Bispecific CAR-encoding lentiviral vectors were co-transfected with LV-MAX packaging mix into Expi293™ (HEK293 cells) using polyethyleneimine (PEI) transfection reagent according to the manufacturer's protocol. Transfected cells were grown for 72 hours at 37°C shaking with 8% CO2 level. Supernatants were centrifuged at 3200 rpm for 10 minutes at room temperature and harvested by vacuum filtration using a 0.45um PES membrane. Viruses were concentrated by ultracentrifugation (Beckman Coulter) at 18000 rpm for 2 hours at 4°C. Pellets were then resuspended in lentiviral stabilizer, immediately aliquoted, and stored at -80°C.

[0137] Viral titers were measured using a p24 ELISA kit (Qiagen) according to the manufacturer's protocol and calculated based on the standard curve set for each assay. Based on the results of the P24 ELISA, functional titers TU / mL were measured by transducing different amounts of virus into a fixed number of HEK293 cells. The percentage of CAR+ expressing cells was checked by flow cytometry at different time points after transfection starting from 24 hours.

[0138] Example 4. Characterization of immune cells expressing anti-CD19 / CD22 bispecific CARs Density gradient centrifugation: PBMCs were isolated from fresh healthy donors in an LRS chamber using Lymphoprep™ (density gradient medium) and the SepMate™ 50 PBMC isolation kit from Stemcell Technology. CD3 T cells were then isolated from the PBMCs using the EasySep™ (density gradient medium) human T cell isolation kit according to the Stemcell technology protocol. + Pan T cells were isolated. Pan T cells were activated with human T activator CD3 / CD28 Dynabeads® beads at a 1:1 bead to cell ratio for 24 hours and then transduced with lentivirus in the presence of Dynabeads® (anti-CD3 / CD28 antibody conjugated beads) and 1 mg / mL protamine sulfate. Spinoculation was performed at 300g for 2 hours at 25°C. Cells and virus were incubated at 37°C for 24 hours. The next day, cells were removed from beads and virus. Cells were grown in 5% human serum containing recombinant human IL15 and IL7 (Peprotech) in X-VIVO™ 15 (Lonza) medium for 2 weeks. Medium was changed and fresh cytokines were added every 2-3 days.

[0139] The expression of CAR on the cell surface was assessed by surface staining using anti-EGFR antibody or CD22-Fc directly conjugated with Mix-n-stain Alexa Fluor® 647 or CF 640R antibody labeling kit (Sigma). Briefly, 100,000 lentiviral transduced T cells were incubated with 25 nM anti-EGFR-Alexa Fluor® 647 or recombinant human CD22 / Fc-CF 640R for 1 hour in the dark with shaking at 4°C. The cells were spun down at 1,300 rpm for 5 minutes, the supernatant was removed, and washed with 200 uL 1xPBS. The resulting samples were reconstituted in 200 uL 1xPBS. The surface expression percentage was quantified by reading the fluorescently stained cells on an Attune™ NxT flow cytometer. CAR surface expression was imaged at 20x magnification on a Cytation® 5 instrument using a Cy5 cube and a DAPI cube (BioTek). 1:1000 diluted Hoechst34580 was used to stain the nuclei of the cells.

[0140] The CAR expression levels of the various CAR constructs on human T cells, detected by conjugated anti-EGFR or CD22-Fc recombinant protein in FACS assays, range from 35-85%. Table 8 below summarizes the percentage of CAR+ cells in PMBCs transduced with the listed CAR constructs. [Table 8]

[0141] The various CARs showed uniform surface expression patterns on T cells imaged by Cytation® 5 using both Alexa Fluor® 647-labeled anti-EGFR antibody and Alexa Fluor® 647-labeled CD22-Fc fusion protein. See Figures 4A and 4B using EPC-001-19 as an example. Similar patterns were observed with the various CARs.

[0142] Example 5: Cytotoxic T lymphocyte (CTL) activity of T cells expressing anti-CD19 / CD22 bispecific CAR Human PBMC and Pan T cell isolation, viral transduction, and T cell expansion were described above. To screen for different CAR activities, a real-time image-based CTL activity assay was performed using GFP-engineered target cells. Briefly, 20,000 CAR-transduced T cells were incubated with 20,000 K562-GFP, CD19 / K562-GFP, CD22 / K562-GFP, CD19 / CD22 / K562-GFP, Raji-GFP, and Nalm-6-GFP cells, respectively, at a 1:1 effector to target cell ratio in RPMI medium containing 10% FBS. No cytokines were added. The assay was performed for 96 hours, and GFP in target cells was imaged and quantified by a Cytation® 5 scanner. IFNγ was detected with a Human IFNγ DuoSet® ELISA kit (R&D System) after the CTL assay. Briefly, after the CTL assay was terminated at 96 hours, the supernatants were collected. Recombinant IFNγ was serially diluted and included in the assay to generate a standard curve. Supernatant IFNγ and recombinant IFNγ were assayed according to the manufacturer's protocol provided. Data was analyzed using GraphPad Prism® 8.0 software.

[0143] Real-time CTL activity of CAR-expressing cells against various target cells (including K562, CD22 K562, CD19 K562, CD19 / Cd22 K562, Raji cells, and Nalm6 cells) was monitored, and the end points of CTL activity of various CAR constructs (see Examples above) were calculated. As shown in Figures 5A-5D, the level of IFNγ secretion correlated with CTL activity, as observed with the various CARs tested. Multiple donors were screened with the various CARs and showed similar results.

[0144] Example 6: CTL assay of EPC-001-16 at different effector to target cell ratios To further evaluate the CTL activity of CAR-T cells, multiple donors were transduced with lentivirus carrying EPC-001-16 (used as a representative anti-CD19 / CD22 bispecific CAR), and the resulting CAR-T cells were expanded as described above. For cells derived from donor 1, transduced or non-transduced T cells were co-cultured with K562, CD19 K562, CD22 K562, and CD19 / CD22 K562 GFP cells at effector-to-target cell ratios of 5:1, 2.5:1, and 1:1 for 96 hours. Dose-dependent target-specific CTL activity was observed as shown in Figure 6A. For cells derived from donor 2 and donor 3, the effector-to-target cell ratios tested were 2.5:1 and 1:1. Similar results were observed as shown in Figure 6B. The CTL activity of CAR-T cells against CD22 / K562-GFP cells was lower than that against CD19 / K562-GFP cells. This may be due to the low copy number of CD22 in CD22 K562 cells, which is 5-10 times lower than the copy number of CD19 in CD19 / K562 and CD19 / CD22 K562 cells. Differences in engineered GFP expression in individual target cell lines may also play a role. The results of IFNγ secretion are shown in Figure 6C.

[0145] Example 7: Mechanistic study of CAR-T cells expressing anti-CD19 / CD22 bispecific CAR by CTL assay To study the mechanism of individual scFv-based CAR activity, CD19 and CD22 scFv monospecific CARs and corresponding bispecific CARs were constructed (see Table 9 below). [Table 9]

[0146] PBMCs and Pan T cells were isolated from two donors and transduced with lentivirus carrying EPC-001-16, EPC-001-1-17, or EPC-001-18. The resulting transduced cells were expanded as described and then co-cultured with K562, CD19 K562, CD22 K562, or CD19 / CD22 K562 GFP cells at effector to target cell ratios of 2.5:1 and 1:1 for 60 hours, and GFP was quantified by imaging every 2 hours on a Cytation® 5. The EPC-001-16 CD19 / CD22 bispecific CAR showed similar CTL activity compared to the CD19 and CD22 monospecific CARs against all tested target cell lines. FIG. 7 showed the CTL cell killing results of various anti-CD19 / CD22 bispecific CARs or anti-CD19, anti-CD22 monospecific CARs against K562 cells, CD22 K562 cells, CD19 K562 cells, and CD19 / CD22 K562 cells at different E:T ratios.

[0147] Example 8: CTL activity of exemplary bispecific CARs with various linkers To further test whether different linkers between two different scFvs affect CAR activity, we used the same anti-CD19 and anti-CD22 scFv fragments as EPC-001-16 and different linkers, i.e., (G 4 S)1, (G 4 S) 2 , and (G 4 S) 3 A CAR construct was constructed having the following structure: See Table 10 below. CTL activity was compared to EPC-001-16. [Table 10]

[0148] PBMCs and Pan T cells were isolated, transduced with EPC-001-19, EPC-001-1-20, EPC-001-21, and EPC-001-22 lentiviruses, and expanded as described. Transduced T cells were co-cultured with K562, CD19 / K562, CD22 / K562, and CD19 / CD22 / K562 GFP cells at an effector to target cell ratio of 1:1 for 60 hours. CTL activity was imaged by target cell GFP levels on a Cytation® 5 every 2 hours. The percentage of CTL activity was analyzed using GraphPad Prism® 8.0. As shown in Figure 8, EPC-001-19 showed better CTL activity compared to EPC-001-20, EPC-001-21, and EPC-001-22.

[0149] Example 9: Cytokine analysis of exemplary bispecific CARs by FACS following CTL assay To further examine the cytokine profile of CAR-T cells expressing the anti-CD19 / CD22 bispecific CAR disclosed herein upon engagement with target cells, Pan T cells were transduced with EPC-001-19 (as a representative bispecific CAR) and expanded. Transduced T cells were co-cultured with K562, CD19 / K562, CD22 / K562, CD19 / CD22 / K562, Raji, or Nalm-6GFP cells at an effector-to-target cell ratio of 5:1 for 96 hours. IFNγ and granzymes were detected by intracellular staining by FACS. Briefly, 4 hours before taking samples, cells were Golgi blocked using a cell activation cocktail containing Bredfeldin A according to the manufacturer's recommendations (Biolegend). Cells were spun down at 1,300 rpm for 5 minutes at room temperature. The cells were washed once with 1×PBS and then stained with Zombie Aqua™ fixed cell viability dye (Biolegend) diluted 1:1000 in 1×PBS with 5 min incubation at room temperature. The cells were then washed twice with 1×PBS. The cells were then stained with anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD22, CD22-Fc for 30 min at room temperature in the dark. The cells were then fixed using eBioscience Foxp3 / Transcription Factor Fixation / Permeabilization kit (Thermo Fisher scientific) for 20 min at room temperature in the dark. The cells were then washed twice with 1×PBS and resuspended in 1×PBS and stored overnight at 4C. The next day, the cells were permeabilized using permeabilization buffer (Thermo Fisher Scientific) for 15 min at room temperature in the dark. The cells were then stained for IFNγ and granzyme intracellular proteins for 30 min at room temperature in the dark. Cells were washed twice with 1× permeabilization buffer, then resuspended in 1× PBS and read on an Attune™ NxT flow cytometer.

[0150] At 96 hours, CD8+ T cells, ranging from 40-80% of the CD8+ population, show specific secretion of both IFNγ and granzyme upon incubation with target cells. A good correlation was observed with IFNγ and granzyme positive CD8 T cells, as shown in Table 11 below. Without being bound by theory, the differences observed between different cell lines may be due to variations in target expression levels in the different cell lines. [Table 11]

[0151] Example 10: CAR-T cell proliferation upon target cell engagement To further test the proliferation of CAR-T cells upon target cell engagement, a 7-day proliferation assay was performed using EPC-001-19 as an example. EPC-001-19 was transduced into Pan T cells and expanded. The transduced T cells were labeled with Cell Trace Far Red at a final concentration of 1 uM. 20,000 target T cells were co-cultured with 20,000 K562, CD19 / K562, CD22 / K562, CD19 / CD22 / K562, and Raji cells at an E:T ratio of 1:1, respectively. The assay was set up in RPMI medium with 10% FBS, and fresh medium was added to the cells every 2 days. No cytokines were added to the medium during the assay. CAR-T proliferation was analyzed with an Attune™ NxT flow cytometer. CAR-T cells show target cell-specific proliferation upon engagement over 7 days, which correlates with the target expression level on the cells. Figure 9 showed CAR-T cell proliferation upon engagement with target cells at an E:T ratio of 1:1.

[0152] Example 11: Persistence of CAR-T cells upon multiple rounds of target cell challenge To further investigate the persistence of CAR-T cells, multiple rounds of target cell challenge experiments were performed using EPC-001-19 as an example. EPC-001-19 was transduced into Pan T cells and expanded. 20,000 transduced T cells were challenged with 20,000 K562, CD19 / K562, CD22 / K562 or CD19 / CD22 / K562 GFP cells for 48 hours, followed by re-challenging the transduced T cells with 20,000 fresh target cells for another 72 hours, and then re-challenging the transduced T cells with 20,000 fresh target cells for another 72 hours, for a total of three challenges. Taget cells GFP CTLs were imaged every 2 hours on a Cytation®5 and quantification was analyzed using GraphPad Prism® 8.0 software. During each re-challenge, 50ul of supernatant was collected for cytokine measurement.

[0153] CAR-T cells expressing EPC-001-19 showed sustained CTL activity in three rounds of target challenge and rechallenge experiments over 8 days. Similar levels of IFNγ secretion were observed at all time points measured. Figure 10A shows the percentage of cell killing in response to one, two, or three rounds of target cell challenge. Figure 10B shows the corresponding IFNγ secretion of CAR-T cells.

[0154] Example 12: Bispecific CAR-transduced Pan T and naive T cell phenotypes CAR-T cell phenotype correlates with in vivo T cell persistence. EPC-001-19 was transduced into human Pan T and naive T cells, and T cell phenotype was analyzed using a FACS assay with a panel of antibodies detecting T cell differentiation markers. Briefly, transduced T cells were stained using anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD62L, anti-CCR7, anti-EGFR as described above. Analysis was performed by Attune™ NxT software. CD4 and CD8 positive Tcm and Tem cells were gated, and the results showed a higher Tcm population and less Tem in transduced naive T cells than in Pan T cells.

[0155] Example 13: In vivo antitumor efficacy using the disseminated Raji-luciferase model The in vivo antitumor efficacy of EPC-001-23 CAR-T was evaluated in a disseminated Raji cell model in NCG mice. EPC-001-23CAR was generated by deleting EGFRt from EPC-001-19. EPC-001-23 was transduced into naive Pan T cells and expanded in vitro for 4 days. 1e6 Raji-luciferase cells were inoculated into NCG mice. On day 3, mice were administered PBS control (group 1), 0.125e6 (group 2) and 0.25e6 (group 3) EPC-001-23 CAR-T cells. Mice were imaged and weighed every 2-3 days. Blood was collected from mice on days 10, 19, and 33. Spleens were also collected on day 33. CAR-T cell phenotype was analyzed by FACS assay immediately after blood and tissue collection.

[0156] As shown in Figures 11A-11B, dose-dependent tumor growth inhibition was observed on days 6-14. Group 3 animals showed more significant tumor growth inhibition than the control group. On day 31, cancer cells were eradicated from mice treated with CAR-T cells at both doses (Group 2 and Group 3 mice). Figures 11C-11D: Tumor cell luciferase quantification on days 14 and 33 after treatment, respectively.

[0157] Due to cancer cell overgrowth, the control mouse was euthanized on day 14. As shown in Figure 11E, one mouse from the low-dose and high-dose CAR-T treatment groups, respectively, was euthanized on days 31 and 33 due to GVHD. There was no significant change in mouse weight over the course of treatment.

[0158] Using the EPC-001-23 construct as an example, CAR-T cell proliferation and persistence was demonstrated over the course of treatment, as shown in Figures 12A-12C. CAR-T cells differentiated and expanded from Tscm to Tcm and Tem over the course of treatment. In addition, both CD4 and CD8 Tcm and Tem of EPC-001-23 CAR-T cells were found to home to the spleen. 12D-12E show the cell numbers in the spleens of mice from groups 2 and 3 on day 33, respectively.

[0159] Example 14: Characterization of anti-CD22-CD19 bispecific antibodies This example describes the characterization of an exemplary anti-CD19 / CD22 bispecific antibody in a scFv-Fc fusion format.

[0160] (a) Preparation of CD19-CD22 bispecific scFv Anti-CD19 scFv-Fc, anti-CD22 scFv-Fc, and anti-CD22 scFv-CD19 scFv-Fc antibodies were transiently expressed in Expi293F™ cells in a freestyle system (Invitrogen) according to standard protocols. Cells were grown for 5 days before harvesting. Supernatants were collected by centrifugation and filtered through a 0.2 μm polyethersulfone (PES) membrane. Fusion proteins were purified by MabSelect™ Prism A Protein A resin (GE Health). Proteins were eluted with 100 mM glycine pH 2.5 + 150 mM NaCl and quickly neutralized with 20 mM citrate pH 5.0 + 300 mM NaCl. Antibodies were then further purified by a Superdex® 200 16 / 600 column. Monomeric peak fractions were pooled and concentrated. The final purified protein had less than 10 EU / mg endotoxin and was kept in 20 mM histidine pH 6.0 + 150 mM NaCl.

[0161] (b) Binding activity of anti-CD19, anti-CD22, and anti-CD19 / CD22 bispecific antibodies An ELISA assay was developed to determine the EC50 of anti-CD19-Fc, anti-CD22-Fc, and anti-CD22-CD19-Fc fusion proteins. Briefly, 384-well plates were immobilized with HIS-tagged human CD19 or CD22 recombinant proteins at a maximum concentration of 2 μg / mL in 1×PBS in a total volume of 25 μL per well. Plates were incubated overnight at 4° C., followed by blocking with 80 μL of Superblock per well for 1 hour. Purified anti-CD19, CD22, or CD22-CD19 scFvFc fusion proteins were serially diluted 2-fold starting at 25 nM, and 25 μL was added to human CD19 or CD22 immobilized wells and incubated for 1 hour with shaking. CD19 or CD22 binding was detected by adding 25 μL of anti-hFcHRP diluted 1:5000 in 1×PBST. Between each step, the plate was washed 3 times with 1XPBST in a plate washer. The plate was then developed with 20 μL of TMB substrate for 5 minutes and stopped by adding 20 μL of 2N sulfuric acid. The plate was read at OD450nm (BIOTEK plate reader) and the data was plotted using GraphPad Prism® 8.1 software. The ELISA binding results are shown in Figures 13A-13B. EC of anti-CD19 or CD22 scFv-Fc to CD19 and CD22 50 The values ​​are shown in Table 12. [Table 12]

[0162] (c) Anti-CD19 / CD22 scFv bispecific antibody binding to endogenous cell lines by FACS analysis. To determine the specific target cell binding activity of antibodies in scFv and bispecific scFv-Fc fusion formats, anti-CD19 or anti-CD22 monospecific scFv and anti-CD19 / CD22 bispecific scFv-Fc fusion antibodies were tested in a FACS binding assay using CD19 and CD22 expressing cell lines Raji and Nalm-6, and the negative cell line U87MG. Briefly, each scFv-Fc fusion was diluted to 25 nM and incubated with 100,000 Raji, Nalm6, and U87MG cells in a 96-well plate for 1 hour at 4° C. with shaking. The cells were spun down at 1300 rpm for 5 minutes at 4° C. to remove unbound antibodies. The cells were then washed once with 200 μL PBS per well. The samples were mixed with Alexa Fluor® 647-conjugated anti-hFc antibody (secondary antibody, 100 μL, 1:1000 dilution) and incubated at 4° C. for 30 minutes in the dark with shaking. The samples were then spun down at 1300 rpm for 5 minutes at 4° C. and washed twice with 200 μL of 1×PBS per well. The resulting samples were reconstituted in 200 μL of 1×PBS and read on an Attune™ NxT flow cytometer. Analysis was performed by counting only Alexa Fluor® 647-positive cells and then plotting using GraphPad Prism® 8.1 software.

[0163] As observed by FACS analysis, all anti-CD19, anti-CD22, and anti-CD19 / CD22 antibodies in Fc fusion format showed specific binding activity to CD19- and CD22-expressing Raji and Nalm6 cell lines, but not to U87MG and U251MG cells, which do not express CD19 or CD22.

[0164] (d) Binding kinetics of anti-CD19 / CD22 bispecific antibodies by surface plasmon resonance (SPR). Kinetic analysis of anti-CD19, anti-CD22, and anti-CD22 / CD19 scFv-Fc fusions was evaluated by SPR technology using a Biacore™ T200. Assays were performed using the Biacore™ T200 control software version 2.0. For each cycle, 1 μg / mL of Fc fusion protein was captured on flow cell 2 in 1×HBSP buffer on a protein A sensor chip for 60 seconds at a flow rate of 10 ul / min. Two times consecutive human CD19 or CD22-HIS tagged proteins were injected into both reference flow cell 1 and Fc fusion protein capture flow cell 2 for 150 seconds at a flow rate of 30 μl / min, followed by a 300 second wash. The flow cells were then regenerated with glycine pH 2 for 60 seconds at a flow rate of 30 ul / min. Eight concentration points from 100 to 0 nM were assayed for each Fc fusion in a 96-well plate. The kinetics of anti-CD19, CD22, CD22-CD19 binding to CD19 or CD22 proteins were analyzed using Biacore™ T200 evaluation software version 3.0. Specific binding response units were derived from subtraction of binding to reference flow cell-1 from Fc fusion protein capture flow cell-2. Table 13 below shows the binding kinetics of anti-CD19, CD22, and CD22-CD19 scFv-Fc fusion proteins to CD19 or CD22. [Table 13]

[0165] Example 15: Evaluation of CAR-T metabolism from CAR-T production. To predict CAR-T persistence in vivo, a CAR-T metabolic assay was developed to evaluate mitochondrial oxygen consumption. EPC-001-23 was transduced into activated human naive T cells and allowed to expand for up to 11 days. On days 5 and 11, CAR-T cells and non-transduced T cells were tested in the assay using an Agilent Seahorse® instrument.

[0166] 250,000 CAR-T cells were seeded on poly-D-lysine treated plates according to the manufacturer's protocol. First, 1.5 μM oligomycin was added to inhibit mitochondrial ATP synthase in port A. Then, 1 μM carbonyl cyanide-4 (trifluoromethyoxy) phenylhydrazone (FCCP) was added to port B. Finally, 0.5 μM rotenone and antimycin were added together in port C to inhibit complexes I and III of the electron transport chain to block mitochondrial respiration. Between each drug addition, samples were mixed and read three times. Plates were read on an Agilent Seahorse® XFe96 instrument using the XF Mito Stress program. Analysis was performed using the Agilent Seahorse® analyzer software. Figures 15A-15B show that CAR candidate EPC-001-023 showed 2-3 times more oxygen consumption on day 5, which continued until day 11.

[0167] Example 16: CD19 or CD22 Raji-Luc KO cell lines generated by CRISPR technology This example evaluates the mechanisms underlying the ability of EPC-001-023 CAR-T cells to overcome the avoidance of CD19- or CD22-expressing targets.

[0168] (a) Generation of CRISPR cell lines To identify the mechanism of EPC-001-023 CAR-T-mediated effects, CD19 or CD22 knockout cell lines were developed using CRISPR technology and animal models. Briefly, CRISPR sgRNA sequences were designed using the Broad Institute's CRISPick database. The top five selective sgRNA sequences targeting CD19 and CD22 were designed (Table 14). [Table 14]

[0169] sgRNAs were synthesized by Integrated DNA Technologies and cloned into the lentiCRISPRv2 vector using an enzymatic digestion method. Plasmid sequences were confirmed. Lentiviruses of these constructs were generated using Expi293™ cells transfected with polyethyleneimine (PEI). Viruses were concentrated using ultracentrifugation and then directly transduced into Raji luciferase cells followed by spinoculation for 2 hours at room temperature. After 2 days, cells were stained with commercially available anti-human CD19 Alexa Fluor® 647 or anti-human CD22 Alexa Fluor® 647 followed by single cell sorting for CD19 or CD22 knockout expression. Cells were maintained in culture for 1 month under drug selection pressure. Cells were characterized by flow cytometry and western blot for CD19 and CD22 expression.

[0170] (b) Confirmation of CD19 or CD22 knockout cell lines by FACS assay CD19 and CD22 knockout single clones were screened by FACS. Briefly, 100,000 cells of each clone were seeded in a 96-well plate with shaking at 4°C for 1 hour. 25 nM anti-human CD19-FITC or anti-human CD22-Alexa Fluor® 647 was incubated with the cells in a final volume of 100 μL at 4°C for 1 hour. The cells were spun down at 1300 rpm for 5 minutes at 4°C to remove unbound antibodies. The cells were then washed twice with 200 μL PBS per well. The resulting samples were reconstituted in 200 μL 1×PBS and read on an Attune™ NNxTT flow cytometer. Analysis was performed by counting only Alexa Fluor® 647- or FITC-positive cells, and then plotted using GraphPad Prism® 8.1 software.

[0171] As determined by FACS analysis, CD22 knockout cells showed a complete loss of CD22 expression, but full expression of CD19, compared to parental Raji cells. Similarly, CD19 knockout cells showed a complete loss of CD19 expression, but full expression of CD22, compared to parental Raji cells.

[0172] (c) Confirmation of CD19 or CD22 knockout cell lines by Western blot assay CD19 or CD22 knockout cell lines were further confirmed by Western blot. Briefly, for each cell line, 1e6 cells were lysed with 100 μL of 1× cell lysis buffer (Cell Signaling Cat. No. 9803) containing PMSF and protease inhibitor cocktail (Cell Signaling Cat. No. 5871). Samples were incubated on ice for 20 minutes and then spun down at 13,000 rpm for 20 minutes at 4C. Supernatants were transferred to new tubes. 25 μL of total cell lysate in SDS loading buffer and β-mercaptoethanol was loaded onto a 12-well SDS-PAGE gel and run at a constant 200 voltage for 22 minutes. Proteins were transferred onto PVDF membranes using iBlot™2 according to the manufacturer's instructions. Membrane blots were blocked with 5% milk powder in 1×PBST (0.05% polysorbate 20 in 1×PBS) for 1 hour at room temperature. Then washed 3 times with 1xPBST, each wash for 10 minutes at room temperature. Primary was added to the blot at 1:1000 dilution and incubated overnight at 4°C. The next day, blot was washed 3 times with 1xPBST (each 10 minutes at room temperature). Secondary antibody was added at 1:1000 dilution anti-rabbit HRP and incubated for 1 hour at room temperature. Blot was then washed 3 times with 1xPBST (each 10 minutes at room temperature), developed using ECL™ reagent (Cell Signaling Cat #6883), and subsequently read on ProteinSimple® Fluorchem E-gel imager. As shown in Figure 15, no CD19 or CD22 protein was detected in the CD19 and CD22 knockout cell lines, while the parental Raji cell line showed abundant CD19 and CD22 expression.

[0173] (d) Quantification of CD19 and CD22 receptor numbers in knockout (KO) and knockdown (KD) cell lines. To further characterize the CD19 and CD22 expression levels in the knockout cell lines, quantitative FACS assays were performed using QUANTUM Alexa Fluor® 647MESF microsphere beads (Bangs Laboratories Inc) for standard calibration according to the manufacturer's protocol. The parental Raji cell line showed high levels of CD19 and CD22 expression. In the parental Raji cell line, the expression level of CD19 was about 5-fold higher than that of CD22. The CD19 and CD22 knockout cell lines showed undetectable CD19 or CD22 expression. The CD19 or CD22 knockdown cell lines showed very undetectable CD19 or very low CD22 copy numbers. The CD19 and CD22 receptor copy numbers are summarized in Table 15. See also Figures 16A-16B. [Table 15]

[0174] (e) EPC-001-023 mono- and bispecific scFv-Fc binding to CD19 or CD22 knockout cell lines Binding of mono- and bispecific antibody fragments (in scFv-Fc fusion format) corresponding to the antigen-binding portion in EPC-001-23 CAR-T cells to Raji parental, CD19 knockout, and CD22 knockout cells was tested by FACS as described above. The results show that all scFvs bound to the cell lines tested, whereas no binding of CD19 scFv and CD22 scFv to Raji CD19 or CD22 knockout cell lines was observed.

[0175] Example 17: EPC-001-23 in vivo mechanistic study in mouse models engrafted with CD19 or CD22 knockout Raji cells This example dissects the mechanisms that underscore the in vivo functionality of EPC-001-23 using bispecific and monospecific targeting.

[0176] The NCG mouse model was used to study the effect of EPC-001-23 CAR-T on seeded parental Raji cells, and CD19 KO or CD22 KO Raji cells. EPC-001-23 CARs were generated as described above. All Raji cells expressed luciferase for the purposes of imaging and quantification of tumor bearing.

[0177] On day 3, mice were dosed with PBS control (group 1), 0.25e6 EPC-001-23 CAR-T cells, and anti-CD19 control CAR-T cells (tisagenlecleucel) and engrafted with 0.3e6 parental Raji. 1e6 EPC-001-23 was dosed with CD19 knockout Raji and CD22 knockout Raji cell lines. Mice were imaged and weighed every 3-4 days. On day 36, blood was collected and spleens were removed for analysis of CAR-T cell phenotype by FACS. Control mice were euthanized on day 14 due to cancer cell overgrowth.

[0178] As shown in Figure 17, tumor growth inhibition was observed in all CAR-T cell treatment groups. Parental Raji cells were more robustly eliminated from CAR-T cell-treated mice by EPC-001-23 CAR-T cells through bispecific targeting of CD19 and CD22. Antitumor activity was also observed with CD19 or CD22 knockout Raji cells through monospecific engagement of EPC-001-23 CAR cells to target cells. Quantitative assessment of tumor bearing is shown in Figures 18A-18C. Phenotypic analysis performed on blood and spleen demonstrated the proliferation and persistence of EPC-001-23 CAR-T cells over the course of treatment, with CAR-T cells differentiating into Tcm and Tem in PBMCs, expanding, and homing to the spleen (Figures 19A-19B). Furthermore, both CD4+ and CD8+ CAR-T cells were present in similar proportions in PBMCs and spleens on day 36 and were detected as PD1+, Tim3-CAR+ T cells without depletion (Figures 20A-20C).

[0179] In summary, the results provided in this example (using EPC-001-23 as an example) show that anti-CD19 / CD22 bispecific CAR-T cells can target not only cancer cells expressing both CD19 and CD22, but also cancer cells expressing only one of the two target antigens. Such a feature is desirable in addressing potential evasion of monospecific CAR-T cell therapy.

[0180] Example 18. Effect of EPC-001-23 on CD19 KO or CD22 KO Raji cells in vitro and in vivo (a) In vitro cytotoxicity EPC-001-23 and tisagenlecleucel (anti-CD19 CAR-T cells) were produced as described above. CAR-T cells were incubated with parental Raji, CD19 KO, CD22 KO, CD19 knockdown (CD19KD), or CD22 KD cells at an E:T ratio of 5:1 for 72 hours. CAR-T cell proliferation and activation were assessed by counting CAR+T cells and Granzyme B+CAR+T cells, respectively, using a FACS assay. EPC-001-23 showed more robust CAR-T cell proliferation (Figure 21A) and activation (Figure 21B) at 72 hours compared to tisagenlecleucel (used as a control).

[0181] Target cell killing activity was also examined in the target cell re-challenge assay disclosed above. Briefly, CAR-Ts cells were produced and expanded in vitro. 100,000 CAR+T cells were incubated with 5000 target cells at a ratio of 20:1 on day 4 after transduction in a 96-well plate in 10% FBS / RPMI at stimulation 1. The samples were spun down at 1300 rpm for 5 minutes at room temperature and then incubated at 37C with 5% CO2 for 72 hours. After 3 days of incubation, the plate was spun down at 1,300 rpm for 5 minutes. 50uL of the supernatant was carefully removed and discarded. For re-challenge 2, 10,000 fresh target cells in 50ul of 10% FBS / RPMI were added to the plate containing CAR-T cells. Plates are spun down at 1,300 rpm for 5 min, followed by incubation at 37C with 5% CO2 for 72 h. Rechallenge is repeated three times as described. After the final 72 h incubation, target cells are counted by staining with 5uL of anti-human CD19 AF647 (Biolegend, Cat. No. SJ25C1) and 5uL of anti-human CD22 AF647 (Biolegend, Cat. No. HIB22) as described above. Stained cells are resuspended in 200uL of 1xPBS and read on an Attune™ 3-laser flow cytometer (Thermo Fisher scientific). Analysis was performed with Attune™ software and GraphPad Prism® 8.

[0182] As seen in Figure 21C, EPC-001-23 and tisagenlecleucel exhibited similar target cell killing activity in the presence of low levels of viable target cells, including parental Raji cells and CD22 KO Raji cells, whereas EPC-001-23 exhibited much stronger target cell killing activity against CD19 KO Raji cells compared to tisagenlecleucel.

[0183] (b) In vivo cytotoxicity The in vivo cytotoxicity of EPC-001-23 bispecific CAR-T cells and tisagenlecleucel (as a positive control) was also examined in NCG mouse models implanted with luciferase-expressing Raji cells and CD19 KO Raji cells. CAR-T cells were generated as described above. On day 3, PBS control (group 1), 0.2e6 EPC-001-23 CAR-T cells, and tisagenlecleucel CAR-T cells were dosed in mice implanted with 0.3e6 parental Raji. Mice were imaged and weighed every 3-4 days. As shown in Figures 22A and 22B, bispecific EPC-001-23 CAR-T cells showed stronger and more sustained antitumor activity compared to tisagenlecleucel in mice implanted with parental Raji cells. Figure 22B. Similarly, bispecific EPC-001-23 CAR-T cells demonstrated stronger and more durable antitumor activity compared to tisagenlecleucel in mice implanted with CD19 KO parental Raji cells, Figures 23A and 23B.

[0184] The results from this example support the cytotoxicity of bispecific CAR-T cells against cells expressing both target antigens and cells expressing only one target antigen. Thus, bispecific CAR-T cells are expected to maintain therapeutic efficacy in the setting of target escape, which can be an issue associated with monospecific CAR-T therapy.

[0185] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0186] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present invention to adapt it to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0187] equivalent While several embodiments of the invention have been described and illustrated herein, those of ordinary skill in the art will readily envision various other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application in which the teachings of the invention are used. Those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0188] All definitions and those used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0189] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0190] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0191] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in combination with open-ended language such as "comprising", may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0192] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be construed as inclusive, i.e., including at least one, but also more than one, of a number or list of elements, and optionally additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0193] As used herein in the specification and claims of this specification, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements of the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements of the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") may refer in one embodiment to at least one A, optionally including more than one, (and optionally including elements other than B), where B is absent, in another embodiment to at least one B, optionally including more than one, (and optionally including elements other than A), in yet another embodiment to at least one A, optionally including more than one, and at least one B, optionally including more than one, (and optionally including other elements), etc.

[0194] It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

1. A bispecific chimeric antigen receptor (CAR) specific for CD19 and CD22, comprising a first antigen-binding portion specific for CD19, a second antigen-binding portion specific for CD22, a costimulatory signaling domain, and a cytoplasmic signaling domain; the first antigen-binding portion comprises the same heavy chain complementarity determining regions (CDRs) and the same light chain CDRs as a reference antibody EPC-001-1 that binds to CD19; The second antigen-binding portion is a bispecific CAR that comprises the same heavy chain CDRs and the same light chain CDRs as reference antibodies EPC-001-2, EPC-001-3, or EPC-001-4, each of which binds to CD22.

2. The first antigen-binding portion comprises the same heavy chain variable region (V Ｈ ) and the same light chain variable region (V Ｌ ) and / or The second antigen-binding portion comprises the same heavy chain variable region (V Ｈ ) and the same light chain variable region (V Ｌ 2. The bispecific CAR of claim 1 , comprising:

3. 3. The bispecific CAR of claim 1 or 2, wherein the first antigen binding moiety, the second antigen binding moiety, or both, are single chain variable fragments (scFv).

4. the first antigen-binding portion is an scFv comprising the amino acid sequence of SEQ ID NO:9; and / or 4. The bispecific CAR of claim 3, wherein the second antigen binding portion is an scFv comprising the amino acid sequence of SEQ ID NO: 18, 27, or 36.

5. the costimulatory signaling domain is derived from a costimulatory molecule selected from CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L; and / or The bispecific CAR of any one of claims 1 to 4, wherein the cytoplasmic signaling domain is derived from CD3ζ.

6. The bispecific CAR comprises: (a) a fusion polypeptide comprising, from N-terminus to C-terminus, (i) the first antigen-binding portion, (ii) the second antigen-binding portion, (iii) the costimulatory signaling domain, and (iv) the cytoplasmic signaling domain; or (b) a fusion polypeptide comprising, from N-terminus to C-terminus, (i) the second antigen binding portion, (ii) the first antigen binding portion, (iii) the costimulatory signaling domain, and (iv) the cytoplasmic signaling domain.

7. The bispecific CAR of claim 6, further comprising a hinge domain and a transmembrane domain located between (ii) and (iii).

8. 8. The bispecific CAR of claim 6 or 7, further comprising a peptide linker connecting the first antigen binding moiety and the second antigen binding moiety.

9. 9. The bispecific CAR of claim 8, wherein the peptide linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 38), GGGGSGGGGS (SEQ ID NO: 39), GGGGSGGGGSGGGGGS (SEQ ID NO: 40), or GSTSGSGKPGSGEGSTKG (SEQ ID NO: 41).

10. The bispecific CAR of claim 9, comprising any one of the amino acid sequences of SEQ ID NOs: 48 to 53.

11. The bispecific CAR of claim 10, comprising any one of the amino acid sequences of SEQ ID NOs: 55 to 60 and 63 to 67.

12. A nucleic acid or set of nucleic acids that collectively encodes the bispecific CAR of any one of claims 1 to 11.

13. The nucleic acid or set of nucleic acids according to claim 12, comprising a nucleotide sequence encoding the bispecific CAR according to any one of claims 6 to 11.

14. 14. The nucleic acid or set of nucleic acids according to claim 13, further comprising a nucleotide sequence encoding a truncated epidermal growth factor receptor (EGFR) domain comprising the extracellular and transmembrane domains of EGFR, and a nucleotide sequence encoding a self-cleaving peptide located between said nucleotide sequence encoding said bispecific CAR and said nucleotide sequence encoding said truncated EGFR domain.

15. 15. The nucleic acid or set of nucleic acids according to claim 14, wherein the truncated EGFR domain comprises the amino acid sequence of SEQ ID NO:

68.

16. The nucleic acid or set of nucleic acids according to any one of claims 12 to 15, wherein the nucleic acid is an expression vector, which expression vector is optionally a viral vector.

17. A genetically engineered immune cell expressing the bispecific CAR of any one of claims 1 to 11.

18. 18. The genetically engineered immune cell of claim 17, comprising a nucleic acid according to any one of claims 12 to 17, encoding said bispecific CAR.

19. 19. The genetically engineered immune cell of claim 17 or 18, which is a T cell, a NK cell, or a macrophage, optionally wherein the immune cell is a T cell.

20. 20. A pharmaceutical composition comprising a genetically engineered immune cell for use in treating cancer in a subject, said genetically engineered immune cell being the genetically engineered immune cell of any one of claims 17 to 19, and said subject being a human cancer patient having CD19 + and / or CD22 + cancer cells.

21. 21. The pharmaceutical composition of claim 20, wherein the human cancer patient has a hematopoietic malignancy, optionally a T-cell malignancy or a B-cell malignancy.

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