CD22-targeting fraction for the treatment of B-cell acute lymphoblastic leukemia (B-ALL)

Abstract

This invention provides a therapy for treating CD22-positive cancers such as B-cell acute lymphoblastic leukemia (B-ALL). Specifically, this invention provides an anti-CD22 monoclonal antibody for treating CD22-positive cancers, whose scFv, as part of a chimeric antigen receptor (CAR) T cell, can target the first Ig extracellular domain (i.e., the domain furthest from the membrane) of the CD22 antigen.

Description

Technical Field

[0001] This invention provides a therapy for treating CD22-positive cancers such as B-cell acute lymphoblastic leukemia (B-ALL). Specifically, this invention provides an anti-CD22 monoclonal antibody for treating CD22-positive cancers, wherein its F(AB')2 fragment, particularly the scFv fragment, can target the CD22 antigen. Background Technology

[0002] B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive cancer that can be diagnosed at any age throughout an individual's life and is the most common malignancy in children. Although the current 5-year disease-free survival rate is 80%, the prognosis for patients with refractory and relapsed (R / R) disease is very poor. The incidence of B-ALL is lower in adults, but even with high-dose chemotherapy regimens and allogeneic stem cell transplantation, unfavorable clinical outcomes are common.

[0003] Immunotherapy has brought unprecedented hope in cancer treatment. Adoptive cellular immunotherapy, based on genetically engineered human T cells containing chimeric antigen receptors (CARs) (which are redirected to tumor antigens on the cell surface), has shown great potential in relapsed / relapsed bacillary ALL due to the high efficiency and specificity of CD19-CAR T cells (response rate ~90% and complete remission rate ~50% after 12 months). However, CD19-CAR T cells are not always curative; ~60% of patients inevitably relapse, mainly due to poor persistence of CAR T cells or the presence of CD19-negative bacillary ALL clones. Therefore, unfortunately, there are few treatment options for patients with relapsed CD19-negative bacillary ALL after chemotherapy or CD19-directed immunotherapy. Consequently, new strategies are being explored to enhance the function and persistence of CAR T cells, including the development of novel target antigens, co-stimulatory domains, or effector cells.

[0004] Like CD19, CD22 is also expressed in a B-cell lineage-restricted manner. CD22-directed T cells are an attractive complement to current CD19-directed T cell strategies because i) they constitute CD19... – / dim B-ALL relapse alternatives, since CD22 is preserved in this relapse, and ii) they will target CD19 before the earliest VDJ rearrangement. - B lineage progenitor cells (pre-VDJ CD19) –B-lineage progenitors, these cells may escape CD19-CAR T-cell therapy. In fact, CD22 has been used as a target for B-ALL in clinical trials using anti-CD22 monoclonal antibodies (moAbs) linked to immunotoxins, and some CD22-CARs have also been reported. CD22-CAR T cells induce clinical remission in ~70% of R / R B-ALL patients who are either non-responsive or resistant to CD19-CAR T cells.

[0005] However, regarding whether B-ALL blast cells can escape CD22-CAR T cell-mediated lysis and subsequently relapse into CD22- / dim, these studies have reported conflicting results (Fry TJ, Shah NN, Orentas RJ, Stetler-Stevenson M, Yuan CM, Ramakrishna S et al. CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nature medicine. 2018; 24(1):20-8; and Pan J, Niu Q, Deng B, Liu S, Wu T, Gao Z et al. CD22CAR T-cell therapy in refractory or relapsed B acutely lymphoblastic leukemia. Leukemia. 2019). Interestingly, a comparative study from the Orentas lab (Blood. 2013 Feb 14; 121(7):1165-74. doi: 10.1182 / blood-2012-06-438002. Epub, 2012 Dec 14; and Adrienne H. Long, Waleed M. Haso & Rimas J. Orentas (2013) Lessons learned from a highly-active CD22 specific chimeric antigen receptor, OncoImmunology, 2:4, e23621) reported that, using targeted... proximal epitopes on cell surface The maximum therapeutic effect of CD22-CAR T cells can be obtained when anti-CD22 antibodies show high binding affinity for CD22.

[0006] In this paper, we have developed and identified a novel CD22-CAR that targets distal membrane CD22 epitope The report states that it can effectively eliminate primary B-ALL cells. Attached Figure Description

[0007] Figure 1 Design, detection, and expansion of CD22-CAR T cells. (A) CD22-CAR structure and intracellular mock-IC protocol. (B) Histogram showing the blocking of binding (PE signal) to the anti-CD22S-HCL-1 clone in CD22+B cell line Raji cells pre-incubated with hCD22.7 clone (left inset). The right inset illustrates the structure of CD22, showing the binding sites of hCD22.7 scFv (as well as m971 scFv, HA22scFv, and BL22 scFv). (C) Representative CAR detection in primary T cells. T cells transduced with CD22-CAR were detected as GFP+ and co-recognized by anti-human IgG F(Ab')2 and anti-His moAb (after incubation with rCD22-His). (D) Appropriate T cell activation was determined by CD25 and CD69 staining 48 hours after exposure to anti-CD3 / CD28 (PBMCs from n=3 healthy donors). (E) Robust expansion of untransduced T cells or activated T cells transduced with mock-IC or CD22-CAR (PBMCs from n=3 healthy donors). TM, transmembrane; mock-IC, intracellular mock; scFv, single-stranded variable fragment; rCD22-His, recombinant CD22-His; His, histidine; FSC, forward scattering; PBMC, peripheral blood mononuclear cells. The figure shows mean ± SEM (n=3 healthy donors).

[0008] Figure 2 CD22-CAR T cells specifically targeted and eliminated B-ALL cell lines in vitro.

[0009] (A) Experimental design for in vitro cytotoxicity assay. (B) Gating strategy for cytotoxicity analysis using FACS. Live target cells were identified as 7AAD-CD3-GFP-CD19+CD10+ / CD13+ (CD33+ for the MV4-11 cell line). CD22 was not used to avoid confounding the detection of target cells (due to potential antigen loss). Duplicate cells were removed from the analysis. (C) Percentage of live target cells after incubation with CD22-CAR T cells at a specified E:T ratio for 48 hours. Results were normalized relative to simulated-IC data (PBMCs from n=3 healthy donors). CD22 expression in different cell lines is shown in the preceding bar chart. (D) CD22-CAR specificity of CD19-KO SEM cells 529 and CD22-KO SEM cells 529 (PBMCs from n=3 healthy donors) generated using CRISPR-Cas9. CD22 expression in different cell lines is shown in the preceding bar chart. (E) Absolute count of live target cells as measured by FACS in a 48-hour cytotoxicity assay at an E:T ratio of 1:1 (PBMCs from n=3 healthy donors). (F) ELISA showing robust secretion of pro-inflammatory cytokines by CD22-CAR T cells 48 hours after exposure to B-ALL and AML cell lines (top inset) and to CD22-KO or CD19-KO SEM cell lines (bottom inset) (PBMCs from n=3 healthy donors). FSC, forward scatter; SSC, side scatter; FSC-H, forward scatter-high; FSC-A, forward scatter region; E:T, effector-to-target ratio; KO, knockout; wt, wild-type; MFI, median fluorescence intensity. Data are shown as mean ± SEM (n=3 healthy donors). *p<0.05, **p<0.01, ***p<0.001.

[0010] Figure 3CD22-CAR T cells effectively targeted and eliminated primary B-ALL cells in vitro, although CD22 expression levels affected their efficacy. (A) Median fluorescence intensity (MFI) of CD22 in different primary B-ALL samples (n=9). The mean CD22 MFI was used to classify primary B-ALL samples as CD22 high (ALL#1 to ALL#3) or CD22 low (ALL#4 to ALL#9). (B) Absolute counts of live target CD22 high and CD22 low cells as measured by FACS in cytotoxicity assays at 24 and 48 hours with an E:T ratio of 1:1. Statistical significance for each sample is shown. Two-way ANOVA (Sidak multiple comparison test) was used. (C) ELISA showed that CD22-CAR T cells similarly and robustly secreted pro-inflammatory cytokines 48 hours after exposure, regardless of CD22 expression levels. Data are shown as mean ± SEM (PBMCs from n=3 healthy donors). *P<0.05, **P<0.01.

[0011] Figure 4CAR T cells targeting the CD22 epitope at the distal membrane effectively eliminated PDX B-ALL blast cells of varying invasiveness in vivo. (A) Experimental design for in vivo experiments. (B) Gating strategy for analyzing FACS data from in vivo experiments. Leukemia cells were identified as hCD45+hHLA-ABC+hCD3-hCD19+hCD10+. CD22 was excluded from identification to avoid confounding the analysis (due to potential antigen loss). CAR T cells were identified based on CD3 and GFP expression. Duplicate cells were removed from the analysis. (C) Leukemia cell load (top inset) and T cell persistence (bottom inset) in peripheral blood were measured during a 26-week follow-up period (n=4–5 mice / group; n=3 PDX, high CD22 (ALL#1 and ALL#2) and low CD22 (ALL#10)). (D) Leukemia cell load in peripheral blood, ipsilateral and contralateral bone marrow, and spleen at sacrifice (pooled data from 14 mice from three PDXs). (E) Spleen weight (total animals, n=14) and macroscopic images (ALL#2 mice) of mice treated with simulated-IC and CD22-CAR T cells at sacrifice. (F) Peripheral blood erythrocyte counts (n=14) of mice treated with simulated-IC and CD22-CAR T cells at sacrifice. (G) CD22 MFI of persistent primary B-ALL in mice treated with simulated-IC and CD22-CAR T cells at sacrifice (ALL#2 mice, n=4 to 5). Representative FACS plots of contralateral bone marrow samples show CD22 levels of the target cell population in mice treated with simulated-IC and CD22-CAR T cells. D, Day; W, Week; it, Intratibial; iv, Intravenous; BM, Bone marrow; RBC, Erythrocytes; MFI, Median fluorescence intensity. Mean ± SEM is shown in the figures. *P<0.05, **P<0.01, ***P<0.001.

[0012] Figure 5 Immunogenicity prediction of hCD22.7-scFv. The immunogenicity potential of hCD22.7 scFv, m971 scFv, and HA22 CD22-scFv was compared by computer-aided predictive analysis. CD22-scFv was cleaved into a nonameric peptide, and HLA molecules showed a strong preference for the length of the nonameric peptide. When the half-maximal inhibitory concentration (IC50) < 500 nM, the peptide was considered to bind to MHC class I; when IC50 < 50 nM, the peptide was considered a strong binder (affinity value > 2, dashed line), and the amino acid sequence is shown in the figure. Only the most representative HLA supertypes were analyzed. The predicted epitope binding affinity was calculated as (1 / IC50 x 100). The predicted epitopes were distributed along the x-axis according to their distribution in the protein.

[0013] Figure 6 Generation, transduction, expansion, and detection of CD22 / CD19-dual CAR T cells. A) Protocol for the CAR constructs used. (S) and (L) represent the short-(G4S)4- and long-(G4S)7- sizes of the linker between scFv cells, respectively. B) T cell activation (left inset) determined by FACS based on CD25 and CD69 expression 48 hours after exposure to anti-CD3 / CD28 plus IL-7 and IL-15, and clusters of activated T cells under an optical microscope (right inset) (n=5). C) Transduction efficiency (left inset) and expansion (right inset) of activated T cells transduced with the specified CAR (n=5). Arrows indicate the time for harvesting CAR T cells for CAR surface detection on human T cells. D) Representative flow cytometry plots of CAR expression on human T cells, detected by GFP+ (top inset), anti-scFv (second row), CD19-Fc / anti-Fc (third row), and CD22-His tag (bottom inset). T cells transduced with CAR are shown in green. E)CD4 + and CD8 + Representative CAR transduction in T cells (n=5). Data are presented as mean ± SEM.

[0014] Figure 7 Robust anti-leukemic efficacy and specificity of CD22 / CD19-dual CAR T cells in vitro. A) Absolute number (A) and percentage (B) of live target cells (SEM or NALM6) after 48 hours of incubation with specified CAR T cells and an E:T ratio. Results in A are based on Mock-CAR data normalized (PBMCs of independent healthy donors, n=3). C) Pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α produced by CAR T cells after 48 hours of exposure to SEM or NALM6 target cells at a 1:1 E:T ratio (PBMCs of independent healthy donors, n=3). D) Different CD22 / CD19 combination phenotypes in CRISPR / Cas9-edited SEM cells. E) Percentage (E) and absolute number (F) of live target cells after 48 hours of incubation with specified CAR T cells and an E:T ratio. Results in E are based on Mock-CAR data normalized (PBMCs of independent healthy donors, n=5). G) IL-2, IFN-γ, and TNF-α production by CAR T cells 48 hours after exposure to the specified SEM phenotype (n=5). Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Tukey post-hoc test.

[0015] Figure 8Using both NALM6 and SEM B-ALL cell lines, CD22 / CD19(S)-CAR T cells were as effective as CD19-CAR T cells in vivo. A) Experimental design for in vivo experiments. 1×10 5 One NALM6 / SEM cell expressing Luc was transplanted into the bone marrow of NSG mice (n=6 / group). Four days later, 4×10⁴ cells were injected intravenously. 6 Mock-CAR T cells, CD19-CAR cells, or CD22 / CD19(S)-CAR T cells were used. Leukemia cell burden was monitored weekly by bioluminescence (BLI) using an in vivo imaging system (IVIS). Mice were sacrificed when BLI confirmed complete leukemia in mice treated with Mock-CAR, and leukemia cell burden and CAR T cell persistence were analyzed by FACS. B) IVIS imaging of NALM6 leukemia cell burden by BLI at specified time points. C) Total radiation quantification of NALM6 (p / sec / cm2 / sr) at specified time points. D, E) NALM6 leukemia cell burden in peripheral blood (PB) (D) and contralateral bone marrow (BM) (E) at sacrifice of mice treated with Mock-CAR, CD19-CAR, and CD22 / CD19(S)-CAR, respectively. F) Representative FACS plots show the persistence of NALM6 cells (blue) and human T cells (red) in the bone marrow of mice treated with Mock-CAR, CD19-CAR, and CD22 / CD19(S)-CAR, respectively, at the time of sacrifice. GK) Same as BF for SEM target cells. Each point represents one mouse. Data are shown as mean ± SEM, *p<0.05, ***p<0.001, ****p<0.0001; one-way ANOVA and Tukey post-hoc test.

[0016] Figure 9 CD22 / CD19(S)-CAR T cells eliminated primary and PDX B-ALL cells. A) CD22 and CD19 FACS expression in primary B-ALL blast cells (n=3). B) Absolute number of live primary B-ALL cells after 24 hours of incubation with CAR T cells at a 2:1 E:T ratio. C) Pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α produced by CAR T cells after 24 hours of exposure to primary B-ALL blast cells at a 2:1 E:T ratio (PBMCs from 3 independent healthy donors). DF) Same as AB for PDX B-ALL samples. Data are shown as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001; two-way ANOVA and Tukey post-hoc test.

[0017] Figure 10.CD22 / CD19(S)-CAR effectively delayed relapse in long-term follow-up of B-ALL PDX. A) Describe the experimental design. 0.5 × 10⁻⁶ CARs were intravenously transplanted into NSG mice (n = 6–8 / group). 6 10 B-ALL PDX#1 cells and 1×10 6 5 × 10⁶ B-ALL PDX#2 cells. After B-ALL was implanted into the bone marrow, mice were randomized and intravenously injected with 5 × 10⁶ cells on day 17 or day 31. 6 Mock-CAR, CD19-CAR, or CD22 / CD19(S)-CAR T cells were administered. Leukemia cell burden and human T cell persistence in peripheral blood were monitored every two weeks using FACS. Bone marrow aspirates were analyzed using FACS at sacrifice (week 4) and at the endpoint analysis (week 13) of Mock-CAR treated mice. B) Leukemia cell burden in peripheral blood (top left inset) and bone marrow (bottom left inset) at specified time points after CAR T cell infusion. The right inset shows bone marrow FACS analysis before CAR T cell infusion (3 days prior) and at sacrifice (week 4) of Mock-CAR treated mice. C) Follow-up of CD19-CAR-treated mice compared to CD22 / CD19(S)-CAR-treated mice at specified time points (n = 7-8 animals / group). D) Leukemia cell load at sacrifice / endpoint (week 13 post-CAR T cell infusion) in CD19-CAR-treated mice compared to CD22 / CD19(S)-CAR-treated mice (n = 7 mice / group). Relapse was defined as a bone marrow blast percentage >1% (dashed horizontal line). The bottom inset shows the FACS analysis, which illustrates the persistence of B-ALL cells (blue) and human T cells (red) in each independent CD19-CAR and CD22 / CD19(S)-CAR-treated mouse. Each point represents one mouse. Data are shown as mean ± SEM, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA and Tukey post-hoc test.

[0018] Figure 11 The structure of CD22 with 7 domains is shown. Binding with an anti-CD22 antibody is illustrated.

[0019] Figure 12The binding domain of Inotuzumab (distal) and the binding domain of hCD22.7 (domain 1, distal). The detected epitope of Inotuzumab (blue) overlaps with the exposed region adjacent to the epitope recognized by hCD22.7 (red). The epitope of Inotuzumab is slightly extended than that of hCD22.7 and is mainly composed of acidic / basic residues. Currently, only the structural information of the three first domains (1-300) of CD22 is shown in this figure.

[0020] Figure 13 Structural models of the CD22-itocilizumab scFv complex and the CD22-hCD22.7 scFv complex. For hCD22.7, the structure was predicted based on the identified CD22 binding epitope (red) and the heavy and light chain sequences of hCD22.7 scFv (blue and pink, respectively). For itocilizumab, the structure was predicted based on the identified CD22 binding epitope (blue) and the heavy and light chain sequences of itocilizumab scFv (yellow and green, respectively). The modeling of itocilizumab was based on structure 5czx (with high sequence identity to both heavy and light chains, heavy chain: 90%; light chain: 92%) obtained from the PDB database.

[0021] Figure 14 A) Cytotoxicity assay. 1×10 5 The percentage of live target NALM6 cells (CD1a+) and Jurkat cells (CD1a-, as control) after incubation with CD22-CAR T cells at specified numbers (1 / 1, 1 / 2, 1 / 4, and 1 / 8 E:T ratio) for 24 to 48 hours. Results were normalized relative to simulated data (data on PBMCs were from n=3 healthy donors, as well as from two independent experiments). B) ELISA showed the pro-inflammatory cytokines secreted by different CD22-CAR T cells 24 to 48 hours after exposure to NALM6 cells. Data are shown as mean ± SEM (n=3 healthy donors).

[0022] Figure 15 2.5 × 10⁻⁶ mmol / L was injected intratibially (IT) into NSG mice (n = 4 groups). 6 NALM 6 cells expressing Luc / GFP ( Figure 16-18 ) or SEM cells ( Figure 20-22 Three days later, administer a single intravenous injection (intravenous) of 5×10. 6One MOCK or different CD22 CART. Tumor burden was monitored every 4 days according to BLI (using IVIS imaging). When the MOCK-treated animals became completely leukemic, they were euthanized, and leukemia cell burden and CART persistence were analyzed using FACS.

[0023] Figure 16 A) IVIS imaging of tumor burden in NALM6-Luc cells monitored by BLI at specified time points. B) Mean radiometric quantification at specified time points. Left side, linear scaling. Right side, logarithmic scaling.

[0024] Figure 17 Leukemia cell load (NALM6-Luc cells) in peripheral blood (PB), bone marrow (BM), and spleen at the time of sacrifice.

[0025] Figure 18 Persistence of T cells (NALM6-Luc cells) in peripheral blood, bone marrow and spleen at the time of sacrifice.

[0026] Figure 19 A) Use 1×10 at the specified time 5 Cytotoxicity assays were performed using SEM WT cells (left) and SEM CD22 KO cells (CD22 KO) as target cells (T). The percentage of viable target cells after incubation with effector cells (E, CD22-CAR T) at specified cell numbers (1 / 1, 1 / 2, 1 / 4, and 1 / 8 E:T ratios) for 24 and 48 hours were measured. Results were normalized relative to simulated data (from n=3 healthy donors and one experimental PBMC). B) ELISA showed pro-inflammatory cytokines secreted by different CD22-CAR T cells 24 to 48 hours after exposure to SEM cells. Data are shown as mean ± SEM (n=3 healthy donors).

[0027] Figure 20 A) IVIS imaging of tumor burden in SEM-luc cells monitored by BLI at specified time points. (B) Mean radiometric quantification at specified time points. Left side, linear scale. Right side, logarithmic scale.

[0028] Figure 21 Leukemia cell load (SEM-Luc cells) in peripheral blood, bone marrow, and spleen at the time of sacrifice.

[0029] Figure 22 The persistent euthanasia load (SEM-Luc cells) of T cells in peripheral blood, bone marrow, and spleen at the time of sacrifice.

[0030] Figure 23. The protein sequences of g5 / 44 scFv and CD22.7 scFv have been aligned for comparison. A) Comparison of the heavy chains; B) Comparison of the light chains for each of these scFv. For each amino acid, the degree of homology is expressed as a score from 1 to 10 (10 is *). As shown in the figure, the CDRs of these g5 / 44 and CD22.7 scFv are indicated by boxes. G5 / 44 and m5 / 44 are the human and mouse versions of the same antibody (itotuzumab).

[0031] Figure 24. Humanization process according to the sequence-based (and structure-assisted) CDR implantation pathway. Figure 24 shows a comparison of mouse and humanized versions of the heavy and light chains of the new anti-CD22 (clone hCD22.7). Figure 25 A) shows the humanized heavy chain, Figure 25 B) shows the humanized light chain.

[0032] Figure 25 The humanization process follows the "de-mouse-derived" approach. Figure 25 The identification of regions in the mouse sequence that are immunogenic and individual changes in the sequence (i.e., point mutations) to eliminate this tendency are shown. Detailed Implementation

[0033] definition

[0034] The act of “administering” or “administration of” a drug to a patient (and its grammatical equivalents) refers to direct administration (which can be administered by a healthcare professional to a patient), self-administration, and / or indirect administration (which can be the act of prescribing a drug). For example, when a doctor instructs a patient to self-administer a drug or provides a prescription for a drug, this constitutes administering the drug to the patient.

[0035] The term “affibody” refers to a protein derived from the Z domain of protein A and modified to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

[0036] The term "antibody" refers to a molecule that contains at least one immunoglobulin domain that binds to or reacts immunologically with a specific target. The term includes the entire antibody and any antigen-binding portion thereof, or a single chain thereof, and combinations thereof; for example, the term "antibody" specifically includes bivalent antibodies and bivalent bispecific antibodies.

[0037] A typical type of antibody consists of at least two heavy chains (“HC”) and two light chains (“LC”) linked together by disulfide bonds.

[0038] Each "heavy chain" consists of a "heavy chain variable structure domain" (abbreviated as "VH" here) and a "heavy chain constant structure domain" (abbreviated as "CH" here). The heavy chain constant structure domain typically includes three constant structure domains, namely CH1, CH2, and CH3.

[0039] Each “light chain” comprises a “light chain variable structure domain” (abbreviated here as “VL”) and a “light chain constant structure domain” (“CL”). The light chain constant structure domain (CL) can be κ-type or λ-type. The VH and VL structure domains can be further subdivided into hypervariable regions (called complementarity-determining regions (“CDR”)) interspersed within more conservative regions (called “frame regions” (“FW”)).

[0040] Each VH and VL consists of three CDRs and four FWs, arranged in the following order from the amino terminus to the carboxyl terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. This disclosure specifically proposes the VH and VL sequences and the corresponding subsequences of CDR1, CDR2, and CDR3.

[0041] The precise amino acid sequence boundaries of a given CDR can be determined using several well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Maryland, Bethesda (“Kabat” numbering scheme), and Al-Lazikani et al. (1997), JMB273, 927-948 (“Chothia” numbering scheme).

[0042] Therefore, those skilled in the art will understand that sequences FW1, FW2, FW3, and FW4 are also disclosed. For a specific VH, FW1 is the subsequence between the N-terminus of VH and the N-terminus of H-CDR1, FW2 is the subsequence between the C-terminus of H-CDR1 and the N-terminus of H-CDR2, FW3 is the subsequence between the C-terminus of H-CDR2 and the N-terminus of H-CDR3, and FW4 is the subsequence between the C-terminus of H-CDR3 and the C-terminus of VH. Similarly, for a specific VL, FW1 is the subsequence between the N-terminus of VL and the N-terminus of L-CDR1, FW2 is the subsequence between the C-terminus of L-CDR1 and the N-terminus of L-CDR2, FW3 is the subsequence between the C-terminus of L-CDR2 and the N-terminus of L-CDR3, and FW4 is the subsequence between the C-terminus of L-CDR3 and the C-terminus of VL.

[0043] The variable domains of the heavy and light chains contain regions that interact with the binding target; these target-interacting regions are also referred to herein as “antigen-binding sites” or “antigen-binding sites”. The constant domains of an antibody can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (C1q) of the classical complement system. Exemplary antibodies disclosed herein include typical antibodies, but also include bivalent fragments and their variants (such as F(Ab')2).

[0044] As used herein, the term “antibody” includes complete polyclonal antibodies, complete monoclonal antibodies, bivalent antibody fragments (such as F(Ab')2), multispecific antibodies (such as bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecules that contain antigen-binding sites.

[0045] Antibodies can be any of the five major types (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subtypes (such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ based on the characteristics of their heavy chain constant domains. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional conformations. Antibodies can be naked or conjugated with other molecules (such as therapeutic or diagnostic agents) to form immunoconjugates.

[0046] The term "antigen-binding fragment" or "Fab" refers to an antibody fragment containing one constant domain and one variable domain, respectively, of the heavy and light chains. Fab fragments can be obtained by digesting an intact monoclonal antibody with papain.

[0047] The term “cancer” refers to a group of diseases that can be defined as any abnormal growth of benign or malignant tissue that does not have a physiological function, is caused by uncontrolled and usually rapid cell proliferation, and has the potential to invade or spread to other parts of the body.

[0048] "CD22-positive" cancer (including "CD22-positive" carcinomatosis) is a disease in which cells containing CD22 are present on their cell surface. The term "CD22-positive" also refers to cancer that produces sufficient levels of CD22 on its cell surface, thereby enabling the CAR-containing cells of the present invention to have a therapeutic effect mediated by the binding of CAR to CD22. In some embodiments, CD22-positive cancer is B-cell acute lymphoblastic leukemia (B-ALL), particularly CD19. - B-ALL relapse.

[0049] The term “chimeric antigen receptor” or “CAR” refers to a synthetic receptor that targets T cells to selected antigens and reprograms the function, metabolism, and persistence of T cells (see Rivière & Sadelain, 2017. Mol Ther. 25(5):1117-1124). Similarly, the term “CART” refers to T cells containing a CAR.

[0050] As used herein, “combination therapy,” “in combination with,” or “in conjunction with” means any form of simultaneous, concurrent, synchronous, sequential, or intermittent treatment using at least two different modalities of treatment (i.e., compounds, components, targets, or therapeutic agents). Therefore, these terms refer to the administration of one modality of treatment before, during, or after the administration of another modality of treatment to the subject. Combinations can be administered in any order. They can be administered together with therapeutically effective modalities (e.g., simultaneously in the same or separate composition, formulation, or unit dose form) or separately (e.g., on the same or different days, in any order according to appropriate dosing regimens for individual compositions, formulations, or unit dose forms). Typically, each modality will be administered according to the dosage and / or timing schedule determined for that modality. Optionally, three or more modalities may be used in combination therapy. Furthermore, the combination therapies described herein can be used in conjunction with other types of treatment. For example, other anticancer treatments may be selected from groups consisting of chemotherapy, surgery, radiation therapy (radiation), and / or hormone therapy, as well as other treatments relevant to the subject’s current standard of care.

[0051] "Complete response," "complete remission," or "CR" indicates the disappearance of all target lesions as defined in the RECIST v1.1 guidelines. This does not always mean that the cancer has been cured.

[0052] The term "co-stimulatory signaling domain" refers to the signal transduction portion that provides signals to T cells (in addition to the primary signal provided by the CD3ζ chain of the TCR / CD3 complex) mediating T cell responses, including but not limited to activation, proliferation, differentiation, and cytokine secretion. Co-stimulatory domains may include, but are not limited to, all or some of the following: CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, 1COS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83. In some embodiments, the co-stimulatory signaling domain is an intracellular signaling domain that interacts with other intracellular mediators to mediate cellular responses, including activation, proliferation, differentiation, and cytokine secretion.

[0053] As used herein, the term "effective amount" for a pharmaceutical agent (e.g., a therapeutic agent such as CAR-T) refers to an amount sufficient to produce a beneficial or anticipated outcome (e.g., clinical outcome), and therefore, "effective amount" depends on the context in which it is applied. For example, in the context of administering a therapeutic agent for the treatment of T-ALL, an effective amount may reduce the number of cancer cells; reduce the size or burden of the tumor; inhibit (i.e., to some extent slow down and, in some embodiments, stop) the penetration of cancer cells into surrounding organs; inhibit (i.e., to some extent slow down and, in some embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate one or more cancer-related symptoms to some extent; and / or result in a favorable response, such as increased progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), reduced disease progression (PD), shortened time to progression (TTP), or any combination thereof. The term "effective amount" may be used interchangeably with "effective dose," "therapeutic effective amount," or "therapeutic effective dose."

[0054] The terms “individual,” “patient,” or “object” are used interchangeably in this application to refer to a human being and are not intended to be limiting in any way. An “individual,” “patient,” or “object” can be of any age, sex, and physical condition. The term “patient in need” generally refers to a patient with CD22-positive cancer.

[0055] "Infusion" or "infusing" refers to the introduction of a solution containing a therapeutic agent into the body via a vein for therapeutic purposes. Infusion is typically achieved using an intravenous infusion bag.

[0056] As used herein, “intracellular signaling domain” refers to all or part of one or more domains of a molecule (here, a chimeric receptor molecule) that provides for lymphocyte activation. The intracellular domains of such molecules mediate signaling through interactions with cell mediators, leading to proliferation, differentiation, activation, and other effector functions. Examples of intracellular signaling domains used in the CAR of this invention include intracellular sequences of the CD3ζ chain, and / or co-receptors that coordinate to initiate signal transduction upon CAR binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional properties. T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation and provide T cell receptor-like signals (primary cytoplasmic signaling sequences) and those sequences that provide secondary or co-stimulatory signals in an antigen-independent manner (secondary cytoplasmic signaling sequences). Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs referred to as receptor tyrosine-based activation motifs or ITAMs. Examples of primary cytoplasmic signaling sequences containing ITAM include those from CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0057] A “partial response” or “PR” is defined as a reduction of at least 30% in the total diameter of the target lesion relative to the baseline total diameter, in response to treatment, as defined in the RECIST v1.1 guidelines.

[0058] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable diluent” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Acceptable carriers, excipients, or stabilizers that are non-toxic to the receptor at the doses and concentrations employed, without limiting the scope of the invention, include: additional buffers; preservatives; co-solvents; antioxidants (including ascorbic acid and methionine); chelating agents (such as EDTA); metal complexes (e.g., Zn-protein complexes); biodegradable polymers (such as polyesters); salt-forming counterions (such as sodium, polyols); amino acids (such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine); organic sugars or sugar alcohols (such as lactitol, stachyose, mannose, and sorbitol). Xylose, ribose, ribitol, myonisitose, myonisitol, galactose, galactitol, glycerol, cyclic polyols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents (e.g., urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, [α]-monothioglycerol, and sodium thiosulfate); low molecular weight proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins); and hydrophilic polymers (e.g., polyvinylpyrrolidone). Other pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in Remington's Pharmaceutical Sciences, 16th edition, Osol, A, ed., (1980), may also be included in the pharmaceutical compositions described herein, provided that they do not adversely affect the intended properties of the pharmaceutical composition.

[0059] "Progressive disease" or "disease that has progressed" refers to the presence of one or more new lesions or tumors and / or existing non-target lesions as defined in the RECIST v1.1 guidelines, indicating clear progression. Progressive disease or disease that has progressed may also refer to tumor growth exceeding 20% ​​since the start of treatment, whether due to an increase in mass or an increase in tumor spread.

[0060] Progression-free survival (PFS) refers to the time from enrollment to disease progression or death. PFS is typically measured using the Kaplan-Meier method and the Responsive Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria. Generally, progression-free survival means that a patient remains alive without the cancer progressing.

[0061] The term “RECIST” refers to the response assessment criteria for solid tumors. RECIST guidelines, criteria, or standards describe standard methods for measuring solid tumors and definitions of objective assessment of changes in tumor size for use in clinical trials of cancer in adults and children. RECIST v1.1 refers to version 1.1 of the revised RECIST guidelines, published in the European Journal of Cancer 45 (2009) 228-247.

[0062] The term "favorable response" generally refers to a beneficial state produced in a subject. In the context of cancer treatment, this term refers to the provision of a therapeutic effect on the subject. Positive therapeutic effects in cancer can be measured in a variety of ways (see Weber, 2009. J Nucl Med. 50 Suppl 1: 1S-10S). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess the therapeutic efficacy of anticancer therapies. Regarding tumor growth inhibition, according to NCI criteria, a T / C ≤ 42% is the minimum level of antitumor activity. A T / C < 10% is considered a high level of antitumor activity, where T / C (%) = median tumor volume in the treated patient / median tumor volume in the control patient × 100. A favorable response can be assessed, for example, by increasing progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases, stable disease (SD), reduced progressive disease (PD), shortened time to progression (TTP), or any combination thereof.

[0063] The term "sequence identity" refers to the percentage value obtained when comparing two sequences using a pairwise sequence alignment tool. In the case of this application, sequence identity was obtained using the global alignment tool "EMBOSS Needle" with default settings (Rice et al., 2000. Trends Genet. 16(6):276-7; Li et al., 2015. Nucleic Acids Res. 43(W1):W580-4). The global alignment tool is available at https: / / www.ebi.ac.uk / Tools / psa / .

[0064] The term "single-chain antigen-binding fragment" or "scFab" refers to a fusion protein containing a variable domain and a constant domain of an antibody light chain linked to a variable domain and a constant domain of an antibody heavy chain, wherein the heavy and light chains are linked together by a short peptide.

[0065] The term "single-chain variable fragment" or "scFv" refers to a fusion protein containing a heavy-chain variable domain and a light-chain variable domain of an antibody, which are interconnected by a peptide linker. The term also includes disulfide-stabilized Fv (dsFv). A method for stabilizing scFv using disulfide bonds is disclosed in Reiter et al., 1996. Nat Biotechnol. 14(10):1239-45.

[0066] "Stable disease" refers to disease that has not progressed or relapsed as defined in the RECIST v1.1 guidelines. In stable disease, there is neither sufficient tumor shrinkage to meet the criteria for partial response nor sufficient tumor enlargement to meet the criteria for progressive disease.

[0067] "Time to progression of tumor" (TTP) refers to the time from enrollment to disease progression. TTP is typically measured using the RECIST v1.1 standard.

[0068] As used in this application, the terms "treatment" and "therapy" refer to a set of hygienic, pharmacological, surgical, and / or physical means used for the purpose of curing and / or alleviating a disease and / or symptoms, with the goal of remedying a health problem. The terms "treatment" and "therapy" include both preventative and therapeutic methods, as both aim to maintain and / or restore the health of an individual or animal. Regardless of the source of the symptoms, disease, and defect, within the scope of this application, the application of appropriate medicine to alleviate and / or cure a health problem should be interpreted as treatment or therapy.

[0069] describe

[0070] CD19-CAR T-cell therapy has shown undisputed potential in relapsed / relapsed B-ALL. Unfortunately, 40%–60% of patients eventually relapse due to poor persistence of CAR T-cell therapy or the presence of CD19-negative B-ALL clones. Regarding CD19… - Treatment options for B-ALL relapse are very limited, therefore new strategies (including the development of novel target antigens) should be explored to enhance the function and persistence of CAR T cells. CD22 is a pan-B cell antigen and an attractive alternative to CD19 for CAR T cell therapy because it will target CD19-negative relapsed patients who typically retain CD22 expression.

[0071] In this series, some CD22-CARs have been reported to induce clinical remission in 70% of R / R B-ALL patients (who are either non-responsive to or resistant to CD19-CAR T cells). However, conflicting results have been reported in Phase I clinical studies based on different CD22 scFvs (m971 versus YK-CD22BB-002) regarding CD22 loss and immune escape after contact with CD22-CAR T cells. Previous meta-analyses of several anti-CD22moAbs have suggested that epitope selection is crucial for the antileukemic activity of CD22-CARs, and that m971 scFv, targeting the proximal Ig extracellular domains (5 to 7) of the CD22 membrane, appears to be more effective than BL22 scFv and HA22 scFv, which target proximal Ig domain 3.

[0072] In this invention, we report the preclinical development of a novel CD22-targeting motif, preferably an anti-CD22 antibody, more preferably an anti-CD22 antibody fragment selected from a list consisting of scFv, Fab, and scFab. This CD22-targeting motif can help broaden the limited existing CD22-CAR library. To this end, three CD22-reactive hybridomas were generated using conventional moAb generation techniques, producing three highly specific IgG1 isotype anti-CD22 moAbs. Finally, an hCD22.7 clone was selected to construct the CAR. To our knowledge, this hCD22.7 scFv is the first to be used in CD22-CAR development, recognizing the epitope of Ig extracellular domain 1 from the distal end of the CD22 membrane, as shown in SEQ ID NO 15. Using clinically relevant primary B-ALL cells, these distal membrane epitope CD22-CAR T cells exhibited high performance in vitro and in vivo. In fact, they were highly effective in controlling several B-ALL PDXs with varying degrees of invasiveness, while also exhibiting long-term (up to 26 weeks) T cell persistence. Importantly, the experimental design of this application used a rigorous "mock" control, in which all structural motifs, cytolysis motifs, and co-stimulatory motifs were expressed in effector T cells but lacked the extracellular anti-CD22 scFv region. This intracellular mock-IC validated the high specificity and sensitivity of the CD22-CAR in this application.

[0073] As illustrated in the embodiments provided herein, CD22 expression levels were used to classify nine primary B-ALL samples into CD22-positive groups. 高 and CD22 低This application demonstrates that all primary B-ALL cells were effectively recognized and eliminated by CD22-CAR in vitro. Furthermore, CD22-CAR T cells were capable of controlling several B-ALL PDXs with different invasiveness over a longer period (up to 26 weeks) in vivo, which is related to the long-term persistence of T cells, and the very few surviving / resistant B-ALL cells did not show any signs of CD22 antigen loss.

[0074] Therefore, we have produced a CD22-CAR from the outset that is different from existing CD22-CARs, and it appears to recognize the first Ig extracellular domain of the CD22 antigen (i.e., the domain furthest from the membrane).

[0075] In the context of this invention, "the first Ig extracellular domain of the CD22 antigen (i.e., the domain furthest from the membrane)" is understood to be the CD22 domain having the following amino acid sequence:

[0076] [DSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSE] (SEQ ID NO 14).

[0077] In the context of this invention, "epitope from the first Ig extracellular domain (i.e., the domain furthest from the membrane) of the CD22 antigen" is understood to be an epitope region having the following amino acid sequence: [ESTKDGKVP] (SEQ ID NO 15).

[0078] This CD22-CAR has demonstrated high efficacy both in vitro and in vivo when used in clinically relevant B-ALL patient samples. Specifically, the CAR of the present invention, derived from the distal end of the target membrane of hCD22.7, exhibits high efficacy when used with different PDXs. Completely under control within the body disease This is related to the persistence of CD22-CAR.

[0079] Therefore, a first aspect of the present invention provides a CD22 targeting portion, wherein the CD22 targeting portion has binding affinity for the first Ig extracellular domain of the CD22 antigen (i.e., the domain furthest from the membrane), specifically, it has binding affinity for SEQ ID NO 14, and more specifically, it has binding affinity for SEQ ID NO 15.

[0080] In a preferred embodiment of the invention, a first aspect provides a chimeric antigen receptor (CAR) comprising an extracellular domain (containing a CD22 targeting portion), a transmembrane domain, and an intracellular signaling domain, wherein the CD22 targeting portion has binding affinity for the first Ig extracellular domain (the domain furthest from the membrane) of the CD22 antigen, specifically for SEQ ID NO 14, and more specifically for SEQ ID NO 15. Binding molecules that specifically bind to the first Ig extracellular domain (the domain furthest from the membrane) of the CD22 antigen, particularly those specifically binding to SEQ ID NO 15, are considered particularly useful or suitable for the diagnosis and treatment of the aforementioned conditions, particularly B-cell acute lymphoblastic leukemia (B-ALL), and more particularly CD19-negative relapsed B-ALL.

[0081] Methods for phage display and assembly to generate the aforementioned antibodies are known in the art (described, for example, Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; Fuchs et al., (1991) Bio / Technology 9:1370-1372; Hay et al., (1992) Human Antibody Hybridoma 3:81-85; Huse et al., (1989) Science 246:1275-1281; Griffths et al., (1993) EMBO J 12:725-734; Hawkins et al., (1992) J Mol Biol 226:889-896; Clackson et al., (1991) Nature 352:624-628; Gram et al., (1992) PNAS 89:3576-3580; Garrad et al., (1991) Bio / Technology 9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res 19:4133-4137; and Barbas et al., (1991) PNAS 88:7978-7982, all of which are incorporated herein by reference).

[0082] In some embodiments, the CD22 targeting portion is an antibody, scFv, Fab, or scFab comprising a VL domain and a VH domain, wherein the VL domain comprises LCDR1, LCDR2, and LCDR3 peptides, the VH domain comprises HCDR1, HCDR2, and HCDR3 peptides, and LCDR1 is composed of [QSLLDSDGKTY] (SEQ ID NO: 1), LCDR2 is composed of [LVS] (SEQ ID NO: 2), LCDR3 is composed of [WQGTHFPWT] (SEQ ID NO: 3), HCDR1 is composed of [GDSITSGY] (SEQ ID NO: 4), HCDR2 is composed of [ISYSGST] (SEQ ID NO: 5), and HCDR3 is composed of [ARYPSPDAMNY] (SEQ ID NO: 6).

[0083] In some embodiments, the CD22 targeting portion is an antibody, F(AB')2, scFv, Fab, or scFab comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8. In some other embodiments, the CD22 targeting portion is an antibody, F(AB')2, scFv, Fab, or scFab comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 23 and the VH domain is composed of SEQ ID NO: 22. In some other embodiments, the CD22 targeting portion is an antibody, F(AB')2, scFv, Fab, or scFab comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 21 and the VH domain is composed of SEQ ID NO: 20.

[0084] In some embodiments, the CD22 targeting portion is an scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8. In some other embodiments, the CD22 targeting portion is an scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 23 and the VH domain is composed of SEQ ID NO: 22. In some other embodiments, the CD22 targeting portion is an scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 21 and the VH domain is composed of SEQ ID NO: 20.

[0085] VL domain (SEQ ID NO: 7)

[0086] [DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGS GSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRA]

[0087] VH domain (SEQ ID NO: 8)

[0088] [EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYMQLKSVTTEDTATYYCARYPSPDAMNYWGQGTSVTVSS]

[0089] In some implementations, the CD22 targeting portion is an scFv containing SEQ ID NO: 9 or consisting of SEQ ID NO: 9.

[0090] scFv (SEQ ID NO: 9) from clone hCD22.7

[0091] [EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYMQLKSVTTEDTATYYCARYPSPDAMNYWGQGTSVTVSSGGGGSGGGGSGGGGSGGG GSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIKRA]

[0092] In a preferred embodiment of the first aspect of the invention, the transmembrane domain of the chimeric antigen receptor (CAR) can be derived from a natural or synthetic source. When the source is natural, the domain can originate from any membrane-binding or transmembrane protein. The transmembrane region may include at least the transmembrane region of the α, β, or ζ chain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0093] The transmembrane domain can be synthetic or a variant of a naturally occurring transmembrane domain. In some embodiments, the synthetic transmembrane domain or its variants primarily contain hydrophobic residues such as leucine and valine.

[0094] In some embodiments, the transmembrane domains include transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or variants thereof, wherein the variants have 95% sequence identity.

[0095] In some embodiments, the transmembrane domains include transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or variants thereof, wherein the variants have 98% sequence identity.

[0096] In some implementations, the transmembrane domains include transmembrane domains of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0097] In some implementations, the transmembrane domain includes the transmembrane domain of CD8 or a variant thereof, wherein the variant has 95% sequence identity.

[0098] In some implementations, the transmembrane domain includes the transmembrane domain of CD8 or a variant thereof, wherein the variant has 98% sequence identity.

[0099] In some implementations, the transmembrane domain includes the transmembrane domain of CD8.

[0100] In some embodiments, the transmembrane domain comprises SEQ ID NO: 10 or a sequence having 95% sequence identity with SEQ ID NO: 10.

[0101] In some embodiments, the transmembrane domain comprises SEQ ID NO: 10 or a sequence having 98% sequence identity with SEQ ID NO: 10.

[0102] In some embodiments, the transmembrane domain includes SEQ ID NO: 10. In some embodiments, the transmembrane domain consists of SEQ ID NO: 10.

[0103] Transmembrane domain from CD8 (SEQ ID NO: 10):

[0104] [TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC]

[0105] In another preferred embodiment, the intracellular signaling domain of the chimeric antigen receptor (CAR) is prepared for activation of at least one function of the CAR-expressing cell upon binding to a ligand expressed on tumor cells. In some embodiments, the intracellular signaling domain comprises one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is a portion and / or variant of an intracellular signaling domain prepared for activation of at least one function of the CAR-containing cell.

[0106] In some implementations, the intracellular signaling domain comprises intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b or variants thereof, wherein the variants have 95% sequence identity.

[0107] In some implementations, the intracellular signaling domain comprises intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66b or variants thereof, wherein the variants have 98% sequence identity.

[0108] In some implementations, the intracellular signaling domains include intracellular domains of CD3ζ, FcRγ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66b.

[0109] In some implementations, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant has 95% sequence identity.

[0110] In some implementations, the intracellular signaling domain comprises the intracellular domain of CD3ζ or a variant thereof, wherein the variant has 98% sequence identity.

[0111] In some implementations, the intracellular signaling domain includes the intracellular domain of CD3ζ.

[0112] In some implementations, the intracellular signaling domain contains SEQ ID NO: 11 or a sequence having 95% sequence identity with SEQ ID NO: 11.

[0113] In some implementations, the intracellular signaling domain contains SEQ ID NO: 11 or a sequence having 98% sequence identity with SEQ ID NO: 11.

[0114] In some implementations, the intracellular signaling domain contains SEQ ID NO: 11 or a sequence that has 99% sequence identity with SEQ ID NO: 11.

[0115] In some embodiments, the intracellular signaling domain includes SEQ ID NO: 11. In some embodiments, the intracellular signaling domain consists of SEQ ID NO: 11.

[0116] Intracellular signaling domain from CD3ζ (SEQ ID NO: 11)

[0117] [RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

[0118] In some embodiments, the CAR may further include a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain includes intracellular domains of CD27, CD28, CD137 (also known as human 4-1BB), CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or variants thereof, wherein the variants have 95% sequence identity.

[0119] In some implementations, the co-stimulatory signaling domain comprises intracellular domains of CD27, CD28, CD137 (also known as human 4-1BB), CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, CD276, or variants thereof, wherein the variants have 98% sequence identity.

[0120] In some implementations, the co-stimulatory signaling domain includes intracellular domains of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276.

[0121] In some implementations, the co-stimulatory signaling domain comprises an intracellular domain of CD137 or a variant thereof, wherein the variant has 95% sequence identity.

[0122] In some implementations, the co-stimulatory signaling domain comprises an intracellular domain of CD137 or a variant thereof, wherein the variant has 98% sequence identity.

[0123] In some implementations, the co-stimulatory signaling domain includes the intracellular domain of CD137.

[0124] In some implementations, the co-stimulatory signaling domain contains SEQ ID NO: 12 or a sequence having 95% sequence identity with SEQ ID NO: 12.

[0125] In some implementations, the co-stimulatory signaling domain contains SEQ ID NO: 12 or a sequence having 98% sequence identity with SEQ ID NO: 12.

[0126] In some implementations, the co-stimulatory signaling domain contains SEQ ID NO: 12 or a sequence that has 99% sequence identity with SEQ ID NO: 12.

[0127] In some embodiments, the co-stimulation signaling domain includes SEQ ID NO: 12. In some embodiments, the co-stimulation signaling domain consists of SEQ ID NO: 12.

[0128] Co-stimulatory signaling domain from CD137 (SEQ ID NO: 12)

[0129] [KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL]

[0130] In some implementations, the CAR includes:

[0131] (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain contains polypeptides LCDR1, LCDR2 and LCDR3, and the VH domain contains polypeptides HCDR1, HCDR2 and HCDR3, wherein LCDR1 is composed of SEQ ID NO: 1, LCDR2 is composed of SEQ ID NO: 2, LCDR3 is composed of SEQ ID NO: 3, HCDR1 is composed of SEQ ID NO: 4, HCDR2 is composed of SEQ ID NO: 5 and HCDR3 is composed of SEQ ID NO: 6;

[0132] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity with SEQ ID NO: 10;

[0133] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 95% sequence identity with SEQ ID NO: 11; and

[0134] (iv) A co-stimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity with SEQ ID NO: 12.

[0135] In some implementations, the CAR includes:

[0136] (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain contains polypeptides LCDR1, LCDR2 and LCDR3, and the VH domain contains polypeptides HCDR1, HCDR2 and HCDR3, wherein LCDR1 is composed of SEQ ID NO: 1, LCDR2 is composed of SEQ ID NO: 2, LCDR3 is composed of SEQ ID NO: 3, HCDR1 is composed of SEQ ID NO: 4, HCDR2 is composed of SEQ ID NO: 5 and HCDR3 is composed of SEQ ID NO: 6;

[0137] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity with SEQ ID NO: 10;

[0138] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 98% sequence identity with SEQ ID NO: 11; and

[0139] (iv) A co-stimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity with SEQ ID NO: 12.

[0140] In some implementations, the CAR includes:

[0141] (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain contains polypeptides LCDR1, LCDR2 and LCDR3, and the VH domain contains polypeptides HCDR1, HCDR2 and HCDR3, wherein LCDR1 is composed of SEQ ID NO: 1, LCDR2 is composed of SEQ ID NO: 2, LCDR3 is composed of SEQ ID NO: 3, HCDR1 is composed of SEQ ID NO: 4, HCDR2 is composed of SEQ ID NO: 5 and HCDR3 is composed of SEQ ID NO: 6;

[0142] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity with SEQ ID NO: 10;

[0143] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 99% sequence identity with SEQ ID NO: 11; and

[0144] (iv) A co-stimulatory signal domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity with SEQ ID NO: 12.

[0145] In some implementations, the CAR includes:

[0146] (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain contains polypeptides LCDR1, LCDR2 and LCDR3, and the VH domain contains polypeptides HCDR1, HCDR2 and HCDR3, wherein LCDR1 is composed of SEQ ID NO: 1, LCDR2 is composed of SEQ ID NO: 2, LCDR3 is composed of SEQ ID NO: 3, HCDR1 is composed of SEQ ID NO: 4, HCDR2 is composed of SEQ ID NO: 5 and HCDR3 is composed of SEQ ID NO: 6;

[0147] (ii) A transmembrane domain comprising SEQ ID NO: 10;

[0148] (iii) Intracellular signaling domains, which include SEQ ID NO: 11; and

[0149] (iv) A co-stimulatory signaling domain containing SEQ ID NO: 12.

[0150] In some implementations, the CAR includes:

[0151] (i) an scFv comprising a VL domain and a VH domain, wherein the VL domain contains the polypeptides LCDR1, LCDR2 and LCDR3, the VH domain contains the polypeptides HCDR1, HCDR2 and HCDR3, and LCDR1 is composed of SEQ ID NO: 1, LCDR2 is composed of SEQ ID NO: 2, LCDR3 is composed of SEQ ID NO: 3, HCDR1 is composed of SEQ ID NO: 4, HCDR2 is composed of SEQ ID NO: 5 and HCDR3 is composed of SEQ ID NO: 6;

[0152] (ii) A transmembrane domain, which consists of SEQ ID NO: 10;

[0153] (iii) Intracellular signaling domain, which consists of SEQ ID NO: 11; and

[0154] (iv) Co-stimulatory signaling domain, which consists of SEQ ID NO: 12.

[0155] In some implementations, the CAR includes:

[0156] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8;

[0157] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95% sequence identity with SEQ ID NO: 10;

[0158] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 95% sequence identity with SEQ ID NO: 11; and

[0159] (iv) A co-stimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 95% sequence identity with SEQ ID NO: 12.

[0160] In some implementations, the CAR includes:

[0161] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 23 and the VH domain is composed of SEQ ID NO: 22;

[0162] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10;

[0163] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11; and

[0164] (iv) A co-stimulatory signal domain comprising SEQ ID NO: 12 or a sequence having 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 12.

[0165] In some implementations, the CAR includes:

[0166] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 21 and the VH domain is composed of SEQ ID NO: 20;

[0167] (ii) A transmembrane domain comprising or consisting of the following: SEQ ID NO: 10 or a sequence having 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10;

[0168] (iii) An intracellular signaling domain comprising or consisting of: SEQ ID NO: 11 or a sequence having 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 11; and

[0169] (iv) A co-stimulatory signal domain comprising or consisting of the following: SEQ ID NO: 12 or a sequence having 95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 12.

[0170] In some implementations, the CAR includes:

[0171] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8;

[0172] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 98% sequence identity with SEQ ID NO: 10;

[0173] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 98% sequence identity with SEQ ID NO: 11; and

[0174] (iv) A co-stimulatory signaling domain comprising SEQ ID NO: 12 or a sequence having 98% sequence identity with SEQ ID NO: 12.

[0175] In some implementations, the CAR includes:

[0176] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8;

[0177] (ii) A transmembrane domain comprising SEQ ID NO: 10 or a sequence having 99% sequence identity with SEQ ID NO: 10;

[0178] (iii) An intracellular signaling domain comprising SEQ ID NO: 11 or a sequence having 99% sequence identity with SEQ ID NO: 11; and

[0179] (iv) A co-stimulatory signal domain comprising SEQ ID NO: 12 or a sequence having 99% sequence identity with SEQ ID NO: 12.

[0180] In some implementations, the CAR includes:

[0181] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8;

[0182] (ii) A transmembrane domain comprising SEQ ID NO: 10;

[0183] (iii) Intracellular signaling domains, which include SEQ ID NO: 11; and

[0184] (iv) A co-stimulatory signaling domain containing SEQ ID NO: 12.

[0185] In some implementations, the CAR includes:

[0186] (i) scFv comprising a VL domain and a VH domain, wherein the VL domain is composed of SEQ ID NO: 7 and the VH domain is composed of SEQ ID NO: 8;

[0187] (ii) A transmembrane domain, which consists of SEQ ID NO: 10;

[0188] (iii) Intracellular signaling domain, which consists of SEQ ID NO: 11; and

[0189] (iv) Co-stimulatory signaling domain, which consists of SEQ ID NO: 12.

[0190] In some embodiments, the CAR includes or consists of: SEQ ID NO: 13 or a sequence having 95% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR includes or consists of: SEQ ID NO: 13 or a sequence having 98% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR includes or consists of: SEQ ID NO: 13 or a sequence having 99% sequence identity with SEQ ID NO: 13. In some embodiments, the CAR includes or consists of SEQ ID NO: 13.

[0191] The full-length sequence of CAR (SEQ ID NO: 13)

[0192] [EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYMQLKSVTTEDTATYYCARYPSPDAMNYWGQGTSVTVSSG GGGSGGGGSGGGGSGGGGSDVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFP WTFGGGTKLEIKRATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR]

[0193] In some other embodiments, any of the CARs described above may further include a CD19-targeting portion or any other targeting portion, such as a CD20-targeting portion. Specifically, the CAR is preferably a bispecific CD22 / CD19-BiCAR.

[0194] In a second aspect, the invention provides a nucleic acid encoding any of the CARs of the invention (including any of the CARs disclosed above). The nucleic acid sequence encoding the chimeric receptor is linked to modular components that can be excised and replaced with other components to customize the chimeric receptor.

[0195] In some embodiments, the nucleic acid is suitable for transducing or transforming cells. In some embodiments, the nucleic acid is suitable for transducing or transforming T cells for adoptive immunotherapy.

[0196] In some implementations, the nucleic acid is a codon optimized for expression in mammalian cells. Codon optimization methods are known in the art (e.g., see Parret et al., 2016, Curr Opin StructBiol. 39: 155-162).

[0197] The nucleic acids of the present invention can be contained in γ-retroviral or lentiviral vectors that can be used to transduce or transform T cells (see Rivière & Sadelain, 2017. Mol Ther. 25(5):1117-1124). The nucleic acids can also be inserted into cells using DNA transposons, RNA transfection, or genome editing technologies (such as TALEN, ZFN, and CRISPR / Cas9) (see Rivière & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0198] A third aspect of the invention provides a cell comprising the nucleic acid of the invention and / or the CAR of the invention. In some embodiments, the cell is a T cell (referred to as CAR).

[0199] In some implementations, the cell is an immature T cell, a memory stem T cell, or a central memory T cell. These cells are currently considered more suitable for adaptive immunotherapy (see Rivière & Sadelain, 2017. Mol Ther. 25(5):1117-1124).

[0200] In some implementations, the cell is an autologous T cell. The term "autologous cell" refers to a cell obtained from the same patient to be treated using any of the methods of the present invention.

[0201] In some implementations, the cell is an allo-tolerant T cell. The term "allo-tolerant cell" refers to a cell that has been engineered to reduce the risk of graft-versus-host disease (GVHD). In some implementations, this is achieved through genome editing-mediated deletion of the TCR and / or β2-microglobulin. 15,19 Allogeneic tolerant cells are known in the field (see the section on allogeneic T cells in Rivière & Sadelain, 2017. Mol Ther. 25(5): 1117-1124).

[0202] In some other embodiments, the cell may further comprise a CAR containing a CD19-targeting portion. Preferably, the CAR is a bispecific CD22 / CD19-dual CAR.

[0203] A fourth aspect of the invention provides a pharmaceutical composition comprising a CD22-targeting portion as defined in the invention and / or a plurality of cells of the invention, as well as a pharmaceutically acceptable carrier or diluent.

[0204] The pharmaceutical compositions described herein may also contain other substances. These substances include, but are not limited to, cryoprotectants, surfactants, antioxidants, and stabilizers. As used herein, the term "cryoprotectant" includes agents that provide stability to CART against cryo-induced stress. Non-limiting examples of cryoprotectants include sugars (such as sucrose, glucose, trehalose, mannitol, mannose, and lactose); polymers (such as dextran, hydroxyethyl starch, and polyethylene glycol); surfactants (such as polysorbates (e.g., PS-20 or PS-80)); and amino acids (such as glycine, arginine, leucine, and serine). Cryoprotectants that exhibit low toxicity in biological systems are typically used.

[0205] In some implementations, the cells are prepared as follows: first, the cells are harvested from their culture medium, then washed and concentrated to a therapeutically effective amount in a suitable medium and container system (“pharmaceutically acceptable” carrier). Suitable infusion media can be any isotonic formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextran or lactated Ringer's solution in water may also be used. Human serum albumin, fetal bovine serum, or other human serum components may be added to the infusion medium.

[0206] In one aspect, the present invention provides cells according to the invention for use as medicines or pharmaceutical compositions according to the invention.

[0207] A fifth aspect of the invention provides a method for treating CD22-positive cancer, wherein preferably the CD22-positive cancer is B-cell acute lymphoblastic leukemia (B-ALL), especially CD19-negative B-ALL relapse, the method comprising administering the cells of the invention or the pharmaceutical composition of the invention to a patient in need.

[0208] In some implementations, a therapeutically effective amount of cells is administered to the patient. In some implementations, at least 10... 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 Or 10 10The number of cells will depend on the intended end use of the composition and the cell types contained in the composition. For example, if cells specific to a particular antigen are required, the cell population will contain more than 70%, typically more than 80%, 85%, and 90 to 95% of such cells. For the purposes described herein, the cell volume is typically one liter or less, and may be 500 ml or less, even 250 ml or less, or 100 ml or less. Clinically relevant numbers of cells may be allocated to multiple infusions, accumulating to equal or greater than 10. 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 Or 10 10 Each cell.

[0209] In some embodiments, the cells or drug composition are injected intravenously or intraperitoneally into the bone marrow, lymph nodes, and / or cerebrospinal fluid.

[0210] In some implementations, the method includes combination therapy. In some implementations, the method further includes administration of CD19-CAR T cells.

[0211] In some embodiments, the cell or pharmaceutical composition described herein is administered in combination with chemotherapeutic agents and / or immunosuppressants. In some embodiments, the patient is first treated with a chemotherapeutic agent that inhibits or destroys other immune cells, followed by the cell or pharmaceutical composition described herein. In some cases, chemotherapy can be completely avoided.

[0212] In some embodiments, the patient to be treated by the method of the present invention is in complete or near-complete remission after treatment with another therapy. In some embodiments, the patient to be treated by the method of the present invention has previously been treated with another therapy that produces a partial response, a complete response, disease stabilization, reduction in progressive disease, shortening of tumor progression time, or any combination thereof.

[0213] The following embodiments are merely illustrative of the invention and do not limit the scope of the invention.

[0214] Example

[0215] Example 1

[0216] Materials and methods

[0217] Generation of CD22 single-stranded variable fragments (ScFv)

[0218] MoAb, which reacts with human CD22, is obtained by using NS-1 myeloma cells with a previously treated 30 × 10⁻⁶ cells. 6 The hybridomas were generated by fusion of spleen cells from Balb / c mice immunized three times with 300.19 cells transfected with CD22. The supernatant from wells containing the hybridomas was screened by flow cytometry for moAbs that reacted with 300.19 cells transfected with CD22. Three reactive hybridomas were selected for further identification, and subcloning (clones hCD22.7, hCD22.316, and hCD22.401) was performed using a limiting dilution method. All three antibodies were IgG1κ isotypes, as confirmed using a mouse moAb isotype kit (Boehringer Mannheim, Germany). Antibody specificity was further validated using the CD22-transfected COS cell line (pUNO1-hCD22; InvivoGen, Toulouse, France), cell lines Raji, Daudi, PRMI 82229, U266, and Jurkat, and peripheral blood (PB) mononuclear cells (PBMCs). The CD22 epitope binding to the hCD22.7 clone was determined by cross-blocking with an antibody against the N-terminal domain of CD22 (S-HCL-1). CD22-specific scFvs from the hCD22.7 clone were obtained using a mouse IgG library primer set (Progen, Heidelberg, Germany).

[0219] CAR design and vectors, lentivirus generation and T cell transduction

[0220] CD22-specific scFv was cloned into a pCCL lentiviral backbone containing a human CD8 transmembrane (TM) domain, human 4-1BB and CD3ζ intracellular domains (second-generation CAR), and a T2A-GFP cassette. The same lentiviral vector containing a CD8 TM-4-1BB-CD3ζ domain linked to a His-tag was used as a mock-IC control. HEK 293T cells were co-transfected with the pCCL vector and packaging plasmids VSV-G and psPAX2 using polyethyleneimine (PEI, Polysciences Inc., Warrington, PA) to generate VSV-G pseudotyped CAR-expressing viral particles. Supernatants were collected at 48 and 72 hours post-transfection and concentrated by ultracentrifugation.

[0221] PBMCs were isolated from the buffy coat of erythrocyte sedimentation rate (ESR) of healthy volunteers using Ficoll-Hypaque gradient centrifugation (GE Healthcare, Chicago, Illinois). The ESR buffy coat was obtained from the Barcelona Blood and Tissue Bank with Institutional Review Board approval (HCB / 2018 / 0030). T cells were activated by coating plates with anti-CD3 antibody (OKT3) and anti-CD28 antibody (CD28.2) (BD Biosciences, Franklin Lake, New Jersey) for 2 days, and transduced with CAR-expressing lentivirus at an infection fold of 10 in the presence of interleukin-7 (IL7) and IL15 (10 ng / mL; Miltenyi Biotec, Bergischgradbach, Germany). T cells expanded for up to 10 days in RPMI-1640 medium (Gibco / Invitrogen, Waltham, MA), which contained 10% heat-inactivated fetal bovine serum (FBS) (Sigma, St. Louis, Missouri), penicillin-streptomycin (Gibco / Invitrogen), and IL-7 and IL-15 (10 ng / mL). Surface expression of CD22-CAR was tracked using fluorescence-activated cell sorting (FACS).

[0222] cell lines

[0223] SEM, NALM6, MV4-11, and REH cell lines were purchased from the DSMZ cell line library (Brunschweig, Germany). CD19 knockout (KO) SEM cells and CD22-KO SEM cells were generated via CRISPR-mediated genome editing. Briefly, 200,000 cells (Neon transfector, ThermoFisher Scientific, Waltham, MA) were electroporated using a Cas9 protein / tracrRNA / crRNA complex (IDT, Coralville, IA). Two primers were designed for each gene: CD19-exon 2.1, CAGGCCTGGGAATCCACATG and CD19-exon 14.1, AGACATGGATAATCCCGAT; and CD22-exon 3.2, TCATGACAGTGGTCAGCTG and CD22-exon 9, CAGGTGTAGTGGGAGACGGG. After electroporation, cells were allowed to recover, and then CD19-negative or CD22-negative cells were sorted by FACS (purity >99%).

[0224] In vitro cytotoxicity assay and determination of cytokine release

[0225] Target cells (cell lines or primary B-ALL cells; 100,000 target cells per well in a 96-well plate) were incubated with CD22-CAR T cells or simulated ICT cells at different effector-to-target ratios (E:T) for specified time periods. Cell lines were cultured in RPMI-1640, 10% FBS, and penicillin-streptomycin. Primary cells were cultured in StemSpan medium. TM SFEM medium (StemCell Technologies, Vancouver, Canada), 20% FBS, penicillin-streptomycin, insulin-transferrin-selenium (ITS) (Gibco / Invitrogen), hSCF (100 ng / mL), hFLT3L (100 ng / mL), hIL3 (10 ng / mL), and hIL7 (10 ng / mL) (all from Miltenyi Biotec, Bergisch-Gladbach, Germany). CAR T cell-mediated cytotoxicity was determined by analyzing residual viable target cells (7-aminoactinomycin D negative; 7-AAD negative) at each time point and E:T ratio. For absolute cell counts, BD TruCount was used. TM Absolute counting tubes (BD Biosciences). Quantification of pro-inflammatory cytokines IL2, TNFα, and IFNγ in supernatants harvested 24 hours (cell line) and 48 hours (primary cells) after T cell exposure at a 1:1 E:T ratio using BD OptEIA. TM The human ELISA kit (BDBiosciences) was used to determine this.

[0226] Flow cytometry

[0227] Cell surface expression of CD22-CAR was determined by GFP expression and binding to AffiniPure F(Ab')2 fragment goat anti-mouse IgG (H+L)-APC (Jackson ImmunoResearch, Westgrove, PA) and anti-His-APC (J095G46; BioLegend, San Diego, CA) (pre-incubated with human recombinant CD22-His (rCD22-His) (ThermoFisherScientific)). Activation of transduced T cells was determined by surface staining with CD3-PE (UCHT1; BD Biosciences), CD25-APC (M-A251; BD Biosciences), and CD69-VioBlue (REA824; Miltenyi Biotec) 48 hours after plating on PBMCs. The following human antibodies were used to identify T cells and target cells in in vitro assays: CD3-PE (UCHT1), CD22-APC (HIB22), CD19-BV421 (HIB19), and CD10-PECy7 (HI10a) or CD13-PECy7 (WM15) (for SEM cell lines) or CD33-APC (WM53) (for MV4-11 cell lines) (all from BD Biosciences). Dead cells were discarded by 7AAD staining. Cells collected from mouse PB, bone marrow (BM), and spleen were stained with HLA-ABC-PE (G46-2.6), CD45-BV510 (HI30), CD3-PerCP (SK7), CD22-APC, CD19-BV421, and CD10-PECy7 (all from BD Biosciences). Cells were incubated for 30 minutes and then stained with BD FACS. TM Lysis buffer (BDBiosciences) was used for lysis and fixation. A fluorescence minus one control (FMO) was used to set the gating. A FACSDiva-equipped... TM FACSCanto software TM -II flow cytometer analysis (BDBiosciences).

[0228] In vivo xenograft models of B-ALL and CAR T cells

[0229] 12-week-old non-obese diabetes (NOD) Cg-Prkdc sc id Il2rgtm1Wjl / SzJ(NSG) mice (The Jackson Laboratory, Bar Harbor, Maine) were housed under pathogen-free conditions. All in vivo procedures were approved by the local ethics committee (HRH-17-0029-P1). A total of 0.5 × 10⁻⁶ mice were used. 6 Up to 1.5 × 10 6 One PDX B-ALL cell was transplanted into the bone marrow of NSG mice subjected to sublethal radiation (2 Gy), and 4 × 10⁶ cells were intravenously infused two weeks later. 6 One CD22-CAR T cell (mean transduction rate ~51.3%) or simulated IC T cell (mean transduction rate ~54.2%) was used. Engraftment of B-ALL in peripheral blood was monitored every week. Bone marrow aspirate and spleen were analyzed six weeks after transplantation and at the time of sacrifice. Red blood cell (RBC) counts were determined using a 2800VET V-Sight hematology analyzer (A. Menarini Diagnostics, Badalona, ​​Spain).

[0230] Statistical analysis

[0231] All data are expressed as mean ± SEM. Unless otherwise noted, two-tailed unpaired Student's t-tests were used to assess differences between groups. All analyses were performed using Prism software version 8.0 (GraphPad Software, San Diego, CA).

[0232] result

[0233] CD22.7-CAR T cells effectively eliminated B-ALL cells in vitro.

[0234] The novel anti-CD22 scFv (cloned hCD22.7) of this application was cloned into a second-generation CAR construct, which consists of anti-CD22 scFv, a CD8 transmembrane spacer, and intracellular signaling domains from 4-1BB and CD3ζ, and is linked to the GFP frame via a T2A sequence. Figure 1 a).

[0235] To determine the CD22 domain recognized by hCD22.7 moAb, we performed a cross-blocking assay using the anti-CD22 clone H-SCL-1 (known to bind to the distal Ig extracellular domain 1 of CD22) (Engel P, Wagner N, Miller AS, Tedder TF. Identification of the ligand-binding domains of CD22, a member of the immunoglobulin superfamily that uniquely binds a sialic acid-dependent ligand. The Journal of Experimental Medicine. 1995; 181(4):1581-6). In cells pre-incubated with antibody hCD22.7, binding to the labeled H-SCL-1 clone was blocked, indicating that both moAbs overlap in binding to the distal Ig domain of CD22. Figure 1 b). Healthy human T cells were successfully transduced using both CD22-CAR lentiviral vector and mock-IC lentiviral vector, with transduction efficiencies ranging from 20% to 50%. CD22-CAR expression in T cells was determined by co-detection with scFv and GFP, and by using anti-His moAb after incubation with human rCD22-His. Figure 1 c). Importantly, the activated (CD69) + CD25 + CD22-CAR T cells ( Figure 1 d) It continued to expand >100-fold within a 15-day period, similar to simulated ICT cells. Figure 1 e) indicates that CAR expression does not inhibit the proliferation of T cells.

[0236] Subsequently, in vitro cytotoxicity assays were performed on CD22-CAR T cells from three different healthy donors using an increased E:T ratio against B-ALL cell lines NALM6, SEM, and REH (with AML cell line MV4-11 as a negative control). Figure 2 a) Target cells that survived exposure to CD22-CAR T cells were identified as 7AAD. – CD3 – CD19 + CD10 + (or CD13 in the case of SEM cell lines) + In the case of the MV4-11 cell line, it is 7AAD. – CD3 – CD33+ ()( Figure 2 b). Compared to mimic-IC T cells, CD22-CAR T cells specifically eliminate CD22 in a manner dependent on a relatively low E:T ratio. + B-ALL cells ( Figure 2 c). The specificity of CD22-CAR T cells was further demonstrated using CRISPR / Cas9-mediated CD19-KO SEM cells and CD22-KO SEM cells. Figure 2 d).

[0237] Importantly, in the 48-hour absolute number assay at a 1:1 E:T ratio, B-ALL cells showed almost no survival after exposure to CD22-CART cells. Figure 2 e). CD22-CAR T cells produced high levels of pro-inflammatory cytokines IL2, TNFα, and IFNγ after co-culturing with target cells, confirming their cytotoxicity. Figure 2 f).

[0238] CD22 expression levels affect the efficiency of CD22.7-CAR T cells in killing primary B-ALL cells.

[0239] Shorter remission periods following CD22-CAR T-cell therapy have been reported, and this is associated with reduced CD22 expression, while high CD22 expression is beneficial to CD22-CAR T-cell activity. Furthermore, whether CD22– / dim B-ALL relapse is common after CD22-CAR T-cell therapy remains controversial. Based on this information, we next tested the efficacy of the CD22-CAR T-cells of this invention in vitro in nine primary B-ALL samples with varying CD22 expression levels. Primary B-ALL was classified as CD22-positive using a median CD22 fluorescence intensity (MFI) level that was above or below the mean (MFI = 6752). 高 Or CD22 低 ( Figure 3 a).

[0240] Cytotoxicity was measured 24 and 48 hours after exposure to CD22-CAR T cells or mimic-IC T cells. Overall, CD22-CAR T cells significantly eliminated primary B-ALL blast cells (…). Figure 3 b). However, although CD22-CAR T cells persistently eliminate CD22 throughout the entire CAR T cell exposure window. 高 B-ALL blastocytes (n=3) Figure 3 b, the small image on the left), but CD22-CAR T cell-mediated CD22 低The cytotoxicity of B-ALL blast cells (n=6) was evident but not strong, and only occurred during the initial or most recent 24 hours of exposure to CD22-CAR T cells, except for ALL#7. Figure 3 b, the small image on the right). However, in CD22 高 and CD22 低 In all B-ALL blastocytes, the pro-inflammatory cytokines IL2, TNFα, and IFNγ were robustly produced to a similar degree. Figure 3 c) This demonstrates that the CD22.7-CAR T cells of the present invention effectively recognize and kill primary B-ALL cells.

[0241] CAR T cells guided by distal CD22 epitopes effectively eliminated [the virus] in xenotransplantation from clinically relevant patients. B-ALL blastocytes in the body

[0242] We will use CD22 next. 高 (ALL#1 and ALL#2) and CD22 低 The activity of our CD22-CAR in vivo was evaluated using PDX B-ALL samples of (ALL#10). Figure 4 ). 0.5×10 6 Up to 1.5 × 10 6 Original CD22 + B-ALL blastocytes were transplanted into the bone marrow of NSG mice, and then 4×10⁻⁶ cells were infused two weeks later. 6 CD22-CAR T cells / mimetic-IC T cells. Leukemia cell engraftment and T cell persistence in peripheral blood were monitored every two weeks using FACS, and any animal was sacrificed when it developed obvious signs of disease, with bone marrow and spleen analyzed. Figure 4 (ab). The kinetics of leukemic remodeling (invasiveness) detectable between weeks 4 and 20 post-transplantation are unique for each PDX, CD22. 高 and CD22 低 PDX implantation in mice treated with simulated I-C T cells gradually tended to increase over time. Figure 4 c). However, importantly, CD22-CAR T cells completely eliminate (ALL#1 and ALL#10) or largely control (ALL#2) the growth of B-ALL cells. Figure 4 c, upper inset). T cell persistence adapted to the regeneration kinetics of each PDX was detected in all three PDXs, even at 26 weeks post-CAR T cell infusion (range, week 6 (n=15) to week 26 (n=5): 2.38% ± 0.65 to 0.34% ± 0.34 in the simulated-IC group; 2.79% ± 1.13 to 0.14% ± 0.09 in the CAR T cell group). Figure 4c, bottom small image). Quantifying tumor burden via FACS ( Figure 4 d) Lack of splenomegaly ( Figure 4 e) and signs of hematopoietic metastasis ( Figure 4 f) Further analysis was conducted to determine disease control in the ipsilateral bone marrow (injected tibia) and contralateral bone marrow (both femur and uninjected tibia), peripheral blood, and spleen at the time of sacrifice. Importantly, the persistent leukemia cells at sacrifice maintained full CD22 expression, ruling out immune escape due to antigen loss. Figure 4 g).

[0243] Finally, any modified murine or human scFv expressed on autologous T cells, if presented on the major histocompatibility complex (MHCI) class I, can be immunogenic because the peptides derived from it can be recognized as “non-self” antigens by the adaptive immune system. Therefore, we used the computer-based tool NetMHC 4.0 to perform a side-by-side comparison of the immunogenicity of our hCD22.7-scFv with that of the m971 and HA22 clones, a tool that predicts the affinity of the peptide for binding to the most representative HLA supertype. Figure 5 As shown, for hCD22.7, m971, and HA22, we found very similar total numbers of nonameric binding peptides (29, 23, and 26) and strong nonameric binding peptides (8, 9, and 6). In summary, the presented data indicate that this novel 4-1BB-based CD22-CAR (targeting distal membrane epitopes) exhibits high performance in controlling the growth of primary B-ALLPDX leukemia cells with varying degrees of invasiveness.

[0244] Example 2. Plotting the epitopes of Inotuzumab and the novel anti-CD22 scFv (clone hCD22.7) against CD22.

[0245] The CD22 sequence was translated into peptides of 7, 10, and 13 amino acids, with overlapping portions of 6, 9, and 12 amino acids, respectively. The resulting conformational CD22 peptide microarrays contained 2556 different peptides (printed in duplicate) and were framed with additional control peptides. The CD22 peptide microarrays were incubated with human IgG4 antibody itutuzumab at concentrations of 10 μg / ml and 65 μg / ml in incubation buffer, followed by staining with secondary and control antibodies. The microarrays were read out using a LICOR Odyssey imaging system at a 7 / 7 (red / green) scan intensity.

[0246] use The analyzer quantifies the spot intensity, generates an intensity map, and highlights interactions within the peptide map. For example... Figure 11 and Figure 12As shown, the detected epitope of itutuzumab (blue) overlaps with the exposed region of the adjacent hCD22.7 recognition epitope (red). The epitope of itutuzumab is slightly extended than that of the hCD22.7 recognition epitope and is mainly composed of acidic / basic residues. Furthermore, in Figure 13 The diagram shows structural models of the CD22-itocilizumab scFv complex and the CD22-hCD22.7 scFv complex. For hCD22.7, its structure was predicted based on identified binding epitopes of the heavy and light chains of CD22 (shown in red) and hCD22.7 scFv (shown in blue and pink, respectively). For itocilizumab, its structure was predicted based on identified binding epitopes of the heavy and light chains of CD22 (shown in blue) and itocilizumab scFv (shown in yellow and green, respectively).

[0247] Different bioinformatics analyses have shown that the predicted binding energies (expressed in kcal / mol) indicate that hCD22.7 has a more favorable binding energy than itutuzumab. Furthermore, despite a smaller contact surface area for hCD22.7 compared to itutuzumab, its energy is more advantageous.

[0248] Example 3. Experiments using the B-cell leukemia cell line NALM6

[0249] Materials and methods

[0250] -CAR vector, lentivirus generation and T cell transduction

[0251] Next, CD22 scFv was cloned into the pCCL lentiviral vector and located upstream of the CAR cassette (CD8 hinge and transmembrane domain, 4-1BB and CD3ζ inner domains, and T2A-GFP): CD22.7, g5 / 44 (patent WO2017216561), and M971 (TJFry et al.; Nat Med 2018 Vol. 24, No. 1, pp. 20-28). VSV-G pseudotyped CAR-expressing viral particles were generated by co-transfecting HEK 293T cells with the pCCL vector and packaging plasmids VSV-G, pMDL / pRRE, and pRSV-Rev using polyethyleneimine (PEI, Polysciences, Warrington, PA, USA). Supernatants were collected at 48 and 72 hours post-transfection and concentrated by ultracentrifugation. PBMCs were isolated from the erythrocyte sedimentation rate (ESR) amber layer of healthy volunteers by Ficoll-Hypaque gradient centrifugation (GE Healthcare, Chicago, Illinois, USA). T cells were activated by coating plates with anti-CD3 (OKT3) and anti-CD28 (CD28.2) antibodies (BD Biosciences, Franklin Lake, New Jersey, USA) for 2 days, and transduced with CAR-expressing lentivirus at an infection fold of 10 in the presence of interleukin-7 (IL-7) and IL-15 (10 ng / mL; Miltenyi Biotec, Bergischgradbach, Germany). T cells were expanded for up to 10 days in RPMI-1640 medium (Gibco / Invitrogen, Waltham, MA) containing 10% heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, Missouri, USA), penicillin / streptomycin (Gibco / Invitrogen), and IL-7 and IL-15 (10 ng / mL). GFP-positive cells were identified by fluorescence-activated cell sorting (FACS) to track CD22-CAR expression. For typical experiments, transduction efficiency could vary from 30% to 70%. The same percentage of infected (GFP-positive) cells was used in all in vitro and in vivo experiments, and equilibrated with untransduced cells (UT) if necessary. The minimum percentage of transduced cells was 30%. Cell surface expression of CD22-CAR was determined by the expression of green fluorescent protein (GFP) and its binding to recombinant human CD22-His (rCD22-His, ThermoFisher Scientific) and anti-His-APC (J095G46; BioLegend, San Diego, California, USA).Activation of transduced T cells was determined by surface staining with CD3-PE (UCHT1; BD Biosciences), CD25-APC (M-A251; BD Biosciences), and CD69-VioBlue (REA824; Miltenyi Biotec) 48 hours after PBMC plating.

[0252] - In vitro cytotoxicity assays, determination of cytokine release, and in vivo xenotransplantation models

[0253] NALM6 cells were purchased from DSMZ (Germany) and expanded according to DSMZ's recommendations. Target cells were incubated with different CAR22 or MOCK T cells at different effector-to-target ratios (E:T) for specified periods (24 hours and 48 hours). CAR-T-mediated cytotoxicity was determined by analyzing the residual surviving (7-AAD-) target cells at each time point and E:T ratio. Target cells (1 × 10⁻⁶) were incubated with different CAR22 or MOCK T cells at different effector-to-target ratios (E:T) for specified periods. 5 This assay was used for all cytotoxicity assays. In in vitro assays, the following human antibodies were used by flow cytometry to identify T cells and target cells: CD3-PE (UCHT1), CD22-APC (HIB22), CD19-BV421 (HIB19), and CD10-PECy7 (HI10a). The production of pro-inflammatory cytokines IL-2, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) was assessed by ELISA (Human ELISA SET, BD Biosciences) using in vitro supernatants harvested 24 to 48 hours after exposure to T cells.

[0254] The following experimental design was used for in vivo experiments (xenograft model): 1 × 10⁻⁶ xenografts were implanted into the tibia of NSG mice (8 to 12 weeks old, Jackson Laboratory, n = 4 groups) for intratibial transplantation (IT). 5 100 Luc-GFP (NALM6) cells were injected intravenously (IV) 3 days later. 6 CD22-CART from healthy PBMCs. Tumor burden was monitored at specified time points using bioluminescence (BLI) via the Xenogen In Vivo Imaging System (IVIS) 50 imaging system (Perkin Elmer). Animals treated with MOCK were euthanized when they became completely leukemic, and leukemic cell burden and CART persistence were analyzed by FACS. For human chimeras, bone marrow (BM), peripheral blood (PB), and spleen were analyzed by FACS at the time of sacrifice. For lymphocytes, cells were stained with anti-HLA ABC-PE, CD3-APC, CD10-PECy7, CD45-AmCyan, and CD19-BV421 (see [link to documentation]). Figure 15 ).

[0255] result

[0256] Figure 14 This refers to cytotoxicity assays. In Figure 14 In A), the figure shows 1×10 5 The percentage of live target NALM6 cells (CD19+) and Jurkat cells (as controls) after incubation with CD22-CART cells at specified numbers (E:T ratios of 1 / 1, 1 / 2, 1 / 4, and 1 / 8) for 24 to 48 hours. Results were normalized relative to simulated data (data on PBMCs were from n=3 healthy donors and from two independent experiments). Furthermore, in Figure 14 In section B), the results of an ELISA assay are shown, revealing pro-inflammatory cytokines secreted by different CD22-CAR T cells 24 to 48 hours after exposure to NALM6 cells. Data are presented as mean ± SEM (n = 3 healthy donors). Furthermore, Figures 16 to 18 The results obtained from in vivo experiments using a xenograft model are shown.

[0257] like Figure 14 As shown in A), at 1×10 5 After incubating NALM6 cells with CD22-CAR T cells at 1 / 1 and 1 / 2 E:T ratios for 24 to 48 hours, the percentage of live target NALM6 (CD19+) cells decreased, similar to CD22.7 (from 37% to 12% live cells at 24 and 48 hours at a 1 / 1 ratio) and CD22-M971 (the gold standard) (from 55% to 27% live cells at 24 and 48 hours at a 1 / 1 ratio). However, the percentage of live target NALM6 (CD19+) cells obtained using CD22-G5 / 44 was significantly higher than that obtained using either CD22.7 or CD22-M971. As a control, the viability of Jurkat cells was not significantly affected by either of the CAR-T cells used. Figure 14 A). For example Figure 14 As shown in Figure B, compared to CD22-M971 or CD22-G5 / 44, the presence of CD22.7 CAR-T cells resulted in the release of higher levels of pro-inflammatory cytokines. Figure 16 As shown, the tumor burden at the specified time points monitored by BLI was significantly higher in the case of CD22-G5 / 44 compared to the tumor burden obtained using either CD22.7 or CD22-M971. Finally, as Figure 17As shown, the leukemia cell load in peripheral blood (PB), bone marrow (BM), and spleen at the time of sacrifice was significantly higher in the case of CD22-G5 / 44 compared to the leukemia cell load obtained using either CD22.7 or CD22-M971.

[0258] These results clearly demonstrate that CD22.7 appears to be significantly more effective than CD22-G5 / 44 both in vitro (cytotoxic) and in in vivo models (xenograft models). Consequently, the results also show that higher levels of pro-inflammatory cytokines are released in the presence of CD22.7.

[0259] Example 4. Experiments using SEM (CD22 positive) of acute lymphoblastic leukemia cell line.

[0260] Materials and methods

[0261] -CAR vector, lentivirus generation and T cell transduction

[0262] The method for generating the pCCL vector and lentiviral particles and transducing T cells is the same as described in Example 3.

[0263] - In vitro cytotoxicity assays, determination of cytokine release, and in vivo xenotransplantation models

[0264] Cell SEMs were purchased from DSMZ (Germany) and expanded according to DSMZ's recommendations. Target cells were incubated with different CAR22 or MOCK T cells at different effector-to-target ratios (E:T) for specified periods (24 hours and 48 hours). CAR22-mediated cytotoxicity was determined by analyzing residual surviving (7-AAD-) target cells at each time point and E:T ratio. Target cells (1 × 10⁻⁶) were incubated with different CAR22 or MOCK T cells at different effector-to-target ratios (E:T) for specified periods. 5 This is used in all cytotoxicity assays. In in vitro assays, the following human antibodies are used by flow cytometry to identify T cells and target cells: CD3-PE (UCHT1), CD22-APC (HIB22), CD19-BV421 (HIB19), and CD13-PECy7 (WM15) (see [link to in vitro assay]). Figure 19 A). The production of pro-inflammatory cytokines IL-2, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) was assessed using in vitro supernatant harvested 24 to 48 hours after exposure to T cells via ELISA (Human ELISA SET, BD Biosciences). (See also: [link to ELISA]) Figure 19 B).

[0265] The following experimental design was used for in vivo experiments (xenograft model): 1 × 10⁻⁶ xenografts were implanted into the tibia of NSG mice (8 to 12 weeks old, Jackson Laboratory, n = 4 groups) for intratibial transplantation (IT).5 5 × 10⁶ Luc-mCherry (SEM) cells were injected intravenously 3 days later. 6 CD22-CART from healthy PBMCs. Tumor burden was monitored at specified time points using bioluminescence (BLI) via the Xenogen In Vivo Imaging System (IVIS) 50 imaging system (PerkinElmer). Animals treated with MOCK were euthanized when they became completely leukemic, and leukemic cell burden and CART persistence were analyzed by FACS. For human chimeras, bone marrow (BM), peripheral blood (PB), and spleen were analyzed by FACS at the time of sacrifice. For lymphocytes, cells were stained with anti-HLA ABC-PE, CD3-PerCP, CD45-AmCyan, and CD19-BV421 (see [link to FACS]). Figure 20 ).

[0266] result

[0267] Figure 19 In vitro results are shown, using 1×10 5 SEM WT cells (left) and SEMCD22 KO cells (CD22KO) as target cells (T) were used for cytotoxicity assays at specified time points (A). Figure 19 In section B), the results of an ELISA assay are shown, revealing pro-inflammatory cytokines secreted by different CD22-CAR T cells 24 to 48 hours after exposure to SEM WT cells or SEM CD22 KO cells. Figures 20 to 2 3 shows the in vivo results.

[0268] like Figure 19 As shown in A), similar to the presence of CD22.7 and CD22-M971 (gold standard), the percentage of live target SEM WT cells (but not CD22 KO cells) decreased after 24 to 48 hours. The percentage of live target SEM WT cells obtained using CD22-G5 / 44 was significantly higher than that obtained using either CD22.7 or CD22-M971, especially at ratios of 1:4 and 1:8 (see SEM WT cell results (left)). Thus, as Figure 19 As shown in Figure B, CD22.7 or CD22-M971 strongly and similarly induced the release of pro-inflammatory cytokines. Figure 20 As shown, the tumor burden detected by BLI at specified time points in the case of CD22-G5 / 44 was significantly higher than that obtained using either CD22.7 or CD22-M971. Furthermore, as... Figure 21As shown, the leukemia cell load at sacrifice in the case of CD22-G5 / 44 was significantly higher in peripheral blood (PB), bone marrow (BM), and spleen compared to the leukemia cell load obtained using either CD22.7 or CD22-M971.

[0269] Example 5. Humanization of a novel anti-CD22 (cloned hCD22.7) scFv

[0270] Two different approaches were used for humanization.

[0271] 1. Sequence-based (and structure-aided) CDR porting; and

[0272] 2. Remove mouse-derived ingredients.

[0273] Strictly speaking, sequence-based (and structure-assisted) CDR transplantation is the only "humanization" approach, which involves transplanting mouse CDRs onto the human scFv framework. "De-mouse de-derived" is not itself humanization of scFv. This method relies on identifying immunogenic regions in mouse sequences and individually altering those sequences (i.e., point mutations) to eliminate this predisposition.

[0274] The sequence-based (and structure-assisted) CDR transplantation method yields the following humanized heavy and light chains.

[0275] Heavy chain (SEQ ID NO 20):

[0276] QVQLQESGPGLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEWIGYISYSGSTYYNPSLKSRITISRDTSKNQYSLKLSSVTTEDTATYYCARYPSPDAMNYWGQGTSVTVSS

[0277] Light chain (SEQ ID NO 21):

[0278] DVVMTQTPLTLPVTLGQPASISCKSSQSLLDSDGKTYLNWLQQRPGQSPKRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPWTFGGGTKLEIKRA

[0279] Figure 24 shows a comparison between mouse and human versions of the new anti-CD22 (clone hCD22.7) heavy and light chains.

[0280] The “de-mouse-derived” approach yields the following humanized heavy and light chains.

[0281] Heavy chain (SEQ ID NO 22)

[0282] QVQLQESGPSLVKPGQTLSLTCSVTGDSITSGYWNWIRQSPGNKLEYMGYISYSGSTYYNPTLKGRISITRDNSSSQYYLQLKSVTSEDTATFYCARYPSPDAMNYWGQGTSVTVSS

[0283] Light chain (SEQ ID NO 23)

[0284] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKLLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEADDLGVYFCWQGTHFPWTFGGGTKLEIKRA

[0285] Figure 25 The identification of regions in the mouse sequence that are prone to immunogenicity and individual sequence changes (i.e., point mutations) to eliminate this tendency are shown.

Claims

1. A CD22 targeting moiety, wherein the CD22 targeting moiety has binding affinity for a region of the first Ig extracellular domain of a CD22 antigen as defined by SEQ ID NO 15, and wherein the CD22 targeting moiety comprises a VL domain and a VH domain, wherein the VL domain comprises a LCDR1, a LCDR2 and a LCDR3 polypeptide, the VH domain comprises a HCDR1, a HCDR2 and a HCDR3 polypeptide, and wherein the LCDR1 consists of [QSLLDSDGKTY], the LCDR2 consists of [LVS], the LCDR3 consists of [WQGTHFPWT], the HCDR1 consists of [GDSITSGY], the HCDR2 consists of [ISYSGST], and the HCDR3 consists of [ARYPSPDAMNY].

2. The CD22 targeting moiety of claim 1, wherein the CD22 targeting moiety is an antibody, F(ab’)2, Fab, scFab or scFv, and wherein the VL domain consists of SEQ ID NO: 21 and the VH domain consists of SEQ ID NO:

20.

3. The CD22 targeting moiety of claim 1, wherein the CD22 targeting moiety is an antibody, F(Ab’)2, Fab, scFab or scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO:

8.

4. The CD22 targeting moiety of claim 3, wherein the CD22 targeting moiety is a scFv comprising a VL domain and a VH domain, wherein the VL domain consists of SEQ ID NO: 7 and the VH domain consists of SEQ ID NO:

8.

5. The CD22 targeting moiety of claim 2, wherein the CD22 targeting moiety is a scFv consisting of SEQ ID NO:

9.

6. A chimeric antigen receptor (CAR) comprising: a. an extracellular domain comprising a CD22 targeting moiety, wherein the CD22 targeting moiety is as defined in any one of claims 1 to 5; b. a transmembrane domain; and c. an intracellular signaling domain.

7. The chimeric antigen receptor of claim 6, wherein the transmembrane domain comprises a transmembrane domain of CD28, CD3, CD45, CD4, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154.

8. The chimeric antigen receptor of claim 7, wherein the transmembrane domain comprises a transmembrane domain of CD8.

9. The chimeric antigen receptor of any one of claims 6-8, wherein the intracellular signaling domain comprises an intracellular domain of CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66b.

10. The chimeric antigen receptor of claim 9, wherein the intracellular signaling domain comprises an intracellular domain of CD3 zeta.

11. The chimeric antigen receptor of any one of claims 6-8, wherein the chimeric antigen receptor further comprises a costimulatory signaling domain.

12. The chimeric antigen receptor of claim 11, wherein the costimulatory signaling domain comprises an intracellular domain of CD27, CD28, CD137, CD134, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, or CD276.

13. The chimeric antigen receptor of claim 11, wherein the costimulatory signaling domain comprises an intracellular domain of CD137.

14. The chimeric antigen receptor of any one of claims 6-8, wherein the CD22 targeting moiety is an scFV.

15. The chimeric antigen receptor of claim 14, wherein the scFV comprises a VL domain consisting of SEQ ID NO: 7 and a VH domain consisting of SEQ ID NO:

8.

16. The chimeric antigen receptor of claim 14, wherein the scFV comprises a VL domain consisting of SEQ ID NO: 21 and a VH domain consisting of SEQ ID NO:

20.

17. A nucleic acid encoding the chimeric antigen receptor of any one of claims 6-16.

18. A T cell comprising the nucleic acid of claim 17.

19. Use of the cell of claim 18 or the CD22 targeting moiety of any one of claims 1-5 in the manufacture of a medicament for treating a CD22-positive cancer, wherein the CD22-positive cancer is a B-cell type acute lymphoblastic leukemia.

20. The use of claim 19, wherein the CD22-positive cancer is CD19 - B-ALL relapse.

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