SSEA-4 binding members

Abstract

The present invention relates to the expression of stage-specific embryonic antigen 4 (SSEA-4) in stem memory T cells (T SCM ), then SSEA‑4 can be used as a target to isolate, activate and expand this T cell subset in vivo and in vitro. It also involves combining SSEA‑4 to target T SCM Pharmaceutical antibody compositions and methods of use thereof. The antibodies disclosed herein recognize SSEA-4 glycolipids and induce T SCM Proliferation, the antibody can be used to sort this unique population from the blood for clinical expansion, for adoptive T cell transfer of T cell receptor (TCR)-transduced, chimeric antigen receptor (CAR)-T-transduced or cells for hematopoietic stem cell transplantation. Methods of use include, but are not limited to, in cancer treatment and diagnosis; as an agonist (IgG2) chimeric monoclonal antibody (mAb) to stimulate T in vivo in patients with cancer or chronic viral infection or after chemotherapy; SCM .

Description

[0001] background

[0002] The present invention relates to the expression of stage-specific embryonic antigen 4 (SSEA-4) in stem memory T cells (T SCM ), then SSEA-4 can be used as a target to isolate, activate and expand this T cell subset in vivo and in vitro. The present invention also relates to combining SSEA-4 to target T SCM The antibody disclosed herein recognizes SSEA-4 glycolipids and induces T SCM Proliferation, the antibody can be used to sort this unique population from the blood for clinical expansion, for adoptive T cell transfer of T cell receptor (TCR)-transduced, chimeric antigen receptor (CAR)-T-transduced or cells for hematopoietic stem cell transplantation. Methods of use include, but are not limited to, in cancer treatment and diagnosis; as an agonist (IgG2) chimeric monoclonal antibody (mAb) to stimulate T in vivo in cancer patients or patients with chronic viral infections or after chemotherapy; SCM .

[0003] SSEA is a globosyl glycolipid consisting of three species: SSEA-1, SSEA-3 and SSEA-4 (Suzuki et al. 2013). Sialyl galactosyl globoside (sialic acid Gb5Cer, SGG, MSGG) or SSEA-4 is a globosyl ganglioside synthesized from SSEA-3 by the enzyme ST3β-galactoside α-2,3-sialyltransferase 2 (ST3GAL2) (Saito et al. 2003). Due to the complexity of purification and the number of genes involved in their synthesis, the expression of these globosyl glycosides has been mainly defined by mAbs. The main limitation of this approach is that most of these mAbs have low specificity, making it difficult to interpret unique globosyl glycoside expressions. With this in mind, the expression of SSEA-4 was defined as follows: SSEA-4 is a component of the glycosynapse of the plasma membrane. During human preimplantation development, SSEA-4 is first observed on pluripotent cells of the inner cell mass and then lost upon differentiation (Tondeur et al. 2008). After birth, human germline stem cells in the testis and ovary (Harichandan, Sivasubramaniyan, and Buhring 2013) as well as mesenchymal stem cells (Gang et al. 2007) and cardiac stem cells (Sandstedt et al. 2014) express SSEA-4 (Gang et al. 2007). It was identified by immunizing animals with human embryonic carcinoma cells (human teratocarcinoma cells; tumors containing tissue derivatives of all three germ layers) (Shevinsky et al. 1982; Kannagi et al. 1983; Wright and Andrews 2009) and is widely used as a cell surface marker to define human embryonic stem cells and their malignant counterparts, embryonic carcinoma cells (Kannagi et al. 1983; Lou et al. 2014; Henderson et al. 2002). Among solid tumors, SSEA-4 overexpression has been found in glioblastoma (about 55% of grade I, about 55% of grade II, about 60% of grade III, and about 69% of grade IV astrocytomas) (Lou et al. 2014), renal cell carcinoma (Saito et al. 1997), breast cancer cells and breast cancer stem cells (Huang et al. 2013), basal cell lung cancer (Gottschling et al. 2013), epithelial ovarian cancer (Ye et al. 2010), and oral cancer (Noto et al. 2013). It is of great significance to identify ultra-specific glycan markers associated with and / or predictive of cancer and to develop antibodies against these markers for diagnosis and treatment of a broad spectrum of cancers.SSEA-4 is a glycan expressed on embryonic stem cells and downregulated in adult stem cells. However, the inventors unexpectedly showed that it is expressed in human and mouse T cells. SCM This is the first time that T SCM Unique mark on.

[0004] Memory T cells (including CD4 + and CD8 + Memory T cells include several subtypes: T SCM , central memory T cells (T CM ), transitional memory T cells (T TM )(Only in CD4 + memory T cells), effector memory T cells (T EM ) and terminal effector T cells (T TE )(Mateus et al. 2015; Takeshita et al. 2015). There is still much debate about which method should be used to induce the generation of T cells from naive lymphocytes, central memory lymphocytes, or tumor infiltrating lymphocytes (TILs). SCM There is currently controversy over the use of cells to generate more effective anti-tumor cells for human clinical trials (Klebanoff, Gattinoni, and Restifo 2012).

[0005] People SCM The cells are described as a long-lived population of memory T cells that have a similar phenotype to naive T cells (CD45RO - 、CCR7 + 、CD45RA + 、CD62L + 、CD27 + 、CD28 + and IL-7Rα + ), and also highly expressed CD95, IL-2Rβ (CD122) and CXCR3 (Gattinoni et al. 2011). SCM The cells are a clonally expanded primitive memory T subset that arises upon antigenic stimulation and exhibits a significantly enhanced proliferation and reconstitution capacity (Gattinoni et al. 2011).

[0006] The maintenance of long-term immunity is believed to be dependent on T SCM Cells, T SCM The cells account for a small proportion of the CD4 + and CD8 +T cells account for 2% to 4% of the total number of T cells (Gattinoni et al. 2011; Lugli, Gattinoni, et al. 2013). Zhang et al. (Zhang et al. 2005) first observed T cells in a mouse graft-versus-host disease (GVHD) model. SCM cells, they reported that post-mitotic CD44 低 CD62 高 CD8 + This T cell population is able to generate and maintain all allogeneic T cell subsets in GVHD responses. These alloreactive CD8 + T cells have been shown to have enhanced self-renewal and multipotency and to be able to differentiate into T CM , T EM and T TE cells (Chahroudi, Silvestri, and Lichterfeld 2015; Zhang et al. 2005). In humans, an example comes from the identification of yellow fever (YF)-specific naive CD8 + T cell populations that are stably maintained for more than 25 years and are capable of self-renewal ex vivo (Fuertes Marraco et al. 2015). SCM Cells can be identified by flow cytometry based on the simultaneous expression of several naive markers and the marker CD95 (Mahnke et al. 2013). SCM Reports on CD8 T cells are limited because the low frequency of these cells restricts detailed characterization. For example, <1% of total human T cells is defined as CD8 + CD45RA + CCR7 + CD127 + CD95 + Virus-specific T SCM Human CMV-specific T cells SCM T cells can be detected at a frequency similar to that observed in other subsets, which is approximately 1 / 10,000 T cells (Schmueck-Henneresse et al. 2015; Di Benedetto et al. 2015). SCM The cells are preferentially present in lymph nodes and less frequently in the spleen and bone marrow (Lugli, Dominguez, et al. 2013).

[0007] TSCM T cells may play an important role in specific anti-tumor responses and long-term immune surveillance against tumors (Darlak et al. 2014; Coulie et al. 2014; Martin 2014). SCM T cells are also emerging as important players in the maintenance of long-lived T cell memory and are therefore considered an attractive group for adoptive transfer-based cancer immunotherapy. However, the molecular signals that regulate their production remain poorly defined. Experiments in the context of adoptive immunotherapy have shown that T cells lacking two key transcription factors that control T cell differentiation, T-box transcription factor (T-bet) and eomesin (eomes), are unable to trigger antitumor responses and express T-like proteins. SCM Therefore, T SCM The antitumor potential of glioma cells seems to rely more on their further differentiation into effector memory cells rather than their intrinsic activity (Li et al. 2013).

[0008] Adoptive T cell therapy is an effective strategy for cancer immunotherapy, but the infused T cells often become exhausted and thus have a poor prognosis after transplantation into the patient. SCM Adoptive transfer of cells overcomes this disadvantage because T SCMThe cells are close to naive T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. Adoptive cell therapy using tumor-specific T cells derived from natural TCR or artificial CAR has entered the late stage of clinical trials. Immunotherapy of cancer using T cells expressing CAR is a relatively new method in adoptive cell therapy. CAR-T cells have shown significant success in some B-cell malignancies, however, the response rate for solid cancers has been less successful so far. The strategy is based on equipping T cell genes with a new synthetic receptor consisting of an antibody-like recognition extracellular domain and a T cell signaling intracellular domain. The direct recognition of intact antigens provided by the antibody-derived binding domain of the receptor enables T cells to bypass the limitations of antigen recognition mediated by the major histocompatibility complex (MHC), so that a given CAR can be used in any patient, regardless of their MHC haplotype. MHC independence gives CAR-T cells a fundamental anti-tumor advantage because some tumor cells downregulate MHC expression to escape TCR-mediated immune responses (Garrido et al. 1993). However, T cells engineered to express the CAR of interest are still able to recognize and eradicate tumor cells. In addition, by using CAR-T cells, the range of potential tumor targets can be expanded to include epitopes beyond the range of TCR-based recognition, such as the potential to include not only proteins but also carbohydrates (Mezzanzanoca et al. 1998) and glycolipids (Yvon et al. 2009) for tumor targeting.

[0009] The characteristics of T cells selected for expansion and adoptive transfer are critical in determining the persistence of the transferred cells. In the presence of infection or cancer, antigen-specific T cells can expand and differentiate into effector T cells that are specialized to rapidly clear pathogens as well as memory T cells that can persist long-term and prevent disease recurrence. Memory T cell classification is heterogeneous and contains multiple subsets with unique properties. The immune memory spectrum includes T SCM Cells, T SCM T cells express CD45RA, CCR7, and CD62L like naive T cells, and also express CD95. SCM Cells can differentiate into central memory T cells (T CM ) and effector memory T cells (T EM cells) and terminal effector T cells (T TE ), but they also have a significant self-renewal potential, as shown in serial transplantation experiments (Cieri et al. 2013). The contribution of different memory subsets to the maintenance of the overall memory assortment of antigen-specific T cells has not yet been fully elucidated, as T SCMThe low frequency of cells limits their detailed characterization (SchmueckHenneresse et al. 2015). It is necessary to fully define the generation, expansion, and activation of T SCM Cieri and colleagues describe the generation of large numbers of T cells by priming naive T cells with anti-CD3 / CD28 and low doses of IL-7 and IL-15. SCM This suggests that T cells can be generated, expanded, and genetically modified in vitro from naive precursors. SCM However, the expanded cells no longer expressed CD45RA but instead expressed CD45RO, so they were probably T CM In addition, in vitro generated T SCM The cells showed enhanced proliferation capacity after adoptive transfer into immunodeficient mice, a finding consistent with those of naturally occurring T SCM cells (Gattinoni and Restifo 2013; Cieri et al. 2013). SCM Cell subsets have profound implications for the design and development of effective vaccines and T cell-based therapies (Restifo and Gattinoni 2013; Gattinoni et al. 2011; Lugli, Dominguez, et al. 2013). SCM Cells can promote the clinical development of cell (CAR-T) immunotherapy (Han et al. 2013; Akinleye, Avvaru, et al. 2013; Breton et al. 2014; Akinleye, Chen, et al. 2013; Novero et al. 2014; Sureshet al. 2014). However, T cells in circulating lymphocytes SCM The low cell number limits their application (Gattinoni and Restifo 2013).

[0010] Altered glycosylation is a characteristic of cancer cells, and several glycan structures are well-known tumor markers (Meezan et al. 1969; Hakomori 2002). These abnormal changes may include an overall increase in N-linked glycan branching (Lau and Dennis 2008) and sialic acid content (van Beek, Smets, and Emmelot 1973), loss or overexpression of certain glycan epitopes (Sell 1990; Hakomori and Zhang 1997; Taylor-Papadimitriou and Epenetos 1994), the persistence of truncated neoglycans, or the appearance of new glycans (Huang et al. 2013). In fact, many tumors show increased expression of certain glycolipids, especially gangliosides, glycosphingolipids (GSLs), and sialic acids attached to glycan chains. Numerous studies have shown that aberrant glycosylation is responsible for the initial oncogenic transformation and plays a key role in inducing tumor invasion and metastasis (Hakomori 2002). Overexpression of multiple GSLs has been identified in various types of human malignancies: GD4 in melanoma (Nudelman et al. 1982), GD2 in neuroectodermal tumors (Cahan et al. 1982), fucosyl-GM1 in small cell lung cancer (Nilsson et al. 1986), Globo-H in breast and ovarian cancer (Chang et al. 2008), and stage-specific embryonic antigen (SSEA)-3 and SSEA-4 in breast cancer and breast cancer stem cells (Changet et al. 2008).

[0011] Successful cancer immunotherapy relies on the generation of mAbs with good specificity and potent killing. The complexity of the glycome and altered expression of glycosyltransferases associated with malignant transformation make cancer cell-associated carbohydrates excellent targets (Christiansen et al. 2014; Dalziel et al. 2014; Daniotti et al. 2013; Hakomori 2002). Glycolipids are particularly attractive due to their dense cell surface distribution, fluidity, and association with membrane microdomains, all of which facilitate their involvement in a wide range of cell signaling and adhesion processes (Fuster and Esko 2005; Hakomori 2002; Hakomori 2008). However, generating anti-glycolipid antibodies is a challenging task because they do not provide T cell help and mAbs are typically low-affinity IgMs. Summary of the invention

[0012] The FG2811.72 (also abbreviated as FG2811) mAb is a mouse IgG3 mAb produced by mice immunized with the glycol-engineered mouse fibroblast cell line SSEA-3 / -4-LMTK. Interestingly, the FG2811 mAb specifically recognizes SSEA-4. SSEA-4 is structurally similar to SSEA-3, except that it has an additional terminal sialic acid residue. The α-2,3-sialyltransferase encoded by the ST3GAL2 gene has been shown to be the primary enzyme that promotes sialylation of SSEA-3 to SSEA-4. The FG2811 mAb specifically binds to SSEA-4 and does not cross-react with SSEA-3. This is in contrast to the previously derived mAbs MC813 and MC613, which the inventors found also binds to SSEA-3 and Forssman, while MC613 binds to SSEA-3 and Globo-H. In contrast to MC813, FG2811 does not bind to erythrocytes, suggesting that these cells do not express SSEA-4 and that MC813 binding may be related to SSEA-3 / Forssman expression (Cooling and Hwang 2005). US2010 / 0047827 describes a mAb that binds to SSEA-4, but they also show that this mAb binds only to the terminal disaccharide Neu5Ac(α2-3)Gal, which may be expressed by a range of other erythrocyte glycosides, and also binds to SSEA-3 and Globo-H. US2016 / 0289340A1 does disclose some new anti-SSEA-4 binding mAbs that appear to be specific, but in their analysis MC813 was also SSEA-4 specific, in contrast to our results. Screening for binding to normal blood showed that FG2811, but not MC813, recognized a small population of lymphocytes. This was further characterized as T SCM Compared to the previous SSEA-4 mAb (MC813) that cross-reacts with SSEA-3 and Forssman antigens, the present disclosure describes a highly specific mAb for SSEA-4, FG2811, that can stimulate T SCM proliferation and maintenance.

[0013] In one aspect, the invention provides a specific binding member that specifically binds to SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc.

[0014] In another aspect, the present invention provides a method for identifying stem memory T cells (T SCM) by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on cells using a specific binding member of the invention.

[0015] In another aspect, the present invention provides purified stem memory T cells (T SCM ) by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on cells using a specific binding member of the invention.

[0016] In another aspect, the present invention provides a method for targeting stem memory T cells (T SCM In another aspect, the present invention provides a specific binding member capable of specifically binding to a stem memory T cell (T SCM In some aspects of the invention, the specific binding member is capable of inducing stem memory T cells (T SCM )’s proliferation.

[0017] In another aspect, the invention provides a specific binding member that binds SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc, wherein the isolated antibody or binding fragment or member thereof is super specific.

[0018] In another aspect, the present invention provides a specific binding member that binds to SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc, wherein the specific binding member is capable of stimulating stem memory T cells (T SCM )’s proliferation.

[0019] In another aspect, the present invention provides a specific binding member that binds to SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc, wherein the specific binding member is capable of activating stem memory T cells (T SCM ).

[0020] In some aspects of the invention, the stem memory T cells (T SCM ), the specific binding members of the present invention are able to stimulate stem memory T cells (T SCM) proliferation of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.

[0021] In some aspects of the invention, stem memory T cells (T SCM ) activation can be measured by the production of specific markers or by increased functional effects of the cells. In some aspects of the invention, the activation of stem memory T cells (T SCM ), the specific binding members of the present invention are able to activate stem memory T cells (T SCM ) at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.

[0022] In some aspects of the invention, a specific binding member may be capable of binding to a target protein in a volume of less than about 10 -8 The affinity of M (K d ) binds to the glycolipid Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(d1-4)Gal(β1-4)Glc. The specific binding member may be able to bind to the glycolipid Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(d1-4)Gal(β1-4)Glc at about 10 -9 The affinity of M (K d ) binds to the presented glycolipid. The specific binding member may be capable of binding to the presented glycolipid with less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The affinity of M (K d ) binds to the presented glycolipid.

[0023] Another aspect of the invention provides a specific binding member comprising heavy chain binding domains CDR1, CDR2 and CDR3, and light chain binding domains CDR1, CDR2 and CDR3. The invention may provide a specific binding member comprising one or more binding domains selected from Figure 2 The amino acid sequences of residues 27 to 38 (CDRH1), residues 56 to 65 (CDRH2), and residues 105 to 113 (CDRH3) of a and 2b.

[0024] The specific binding members of the invention may comprise essentially Figure 2 In one embodiment of the invention, the specific binding member of the invention comprises a binding domain, which comprises essentially Figure 2In this embodiment of the invention, the specific binding member may further comprise an amino acid sequence as shown in residues 105 to 113 of the amino acid sequence of a (CDRH3). Figure 2 One or two, preferably both, of the binding domains represented by residues 27 to 38 (CDRH1) and residues 56 to 65 (CDRH2) of the amino acid sequence shown in a.

[0025] In another aspect, the invention provides specific binding members comprising one or more binding domains selected from Figure 2 b Amino acid sequences of residues 27 to 38 (CDRL1), residues 56 to 65 (CDRL2), and residues 105 to 113 (CDRL3).

[0026] In one aspect of the invention, a binding domain may comprise essentially Figure 2 b) is an amino acid sequence as shown in residues 105 to 113 (CDRL3). In this embodiment of the invention, the specific binding member may further comprise substantially Figure 2 One or two, preferably both, of the binding domains represented by residues 27 to 38 (CDRL1) and residues 56 to 65 (CDRL2) of the amino acid sequence shown in b.

[0027] In some embodiments of the invention, the variable heavy chain and / or light chain may comprise HCDR1 to 3 and LCDR1 to 3 of antibody FG2811. In some embodiments of the invention, the variable heavy chain and / or light chain may comprise HCDR1 to 3 and LCDR1 to 3 of antibody FG2811, and the framework region of FG2811.

[0028] The present invention includes specific binding members comprising multiple binding domains or combinations thereof having the same or different sequences. Each binding domain can be carried by a human antibody skeleton. For example, one or more skeleton regions can replace the skeleton region of the entire human antibody or the skeleton region of its variable region.

[0029] An isolated specific binding member of the invention comprises essentially Figure 2 b The sequence shown by residues 1 to 123 (VL) of the amino acid sequence shown.

[0030] In some embodiments, having Figure 2 A specific binding member having a CDR sequence of Figure 2 b. Specific binding member combination of the CDR sequences of

[0031] In one embodiment, the specific binding member may comprise a light chain variable sequence and a heavy chain variable sequence, the light chain variable sequence comprising one or more (i.e. 1, 2 or 3) of LCDR1, LCDR2 and LCDR3, wherein:

[0032] LCDR1 contains SSVNY,

[0033] LCDR2 includes DTS, and

[0034] LCDR3 contains FQASGYPLT; and

[0035] The heavy chain variable sequence comprises one or more (i.e. 1, 2 or 3) of HCDR1, HCDR2 and HCDR3, wherein:

[0036] HCDR1 contains GFSLNSYG,

[0037] HCDR2 contains IWGDGST, and

[0038] HCDR3 comprises TKPGSGYAF.

[0039] In another aspect, the present invention provides a specific binding member comprising: Figure 2 a VH domain comprising residues 1 to 126 of the amino acid sequence of Figure 2 b. The VL domain of residues 1 to 123 of the amino acid sequence of

[0040] In certain embodiments, the specific binding member is a human antibody, a chimeric antibody or a humanized antibody. In some aspects of the invention, the specific binding member is a monoclonal antibody. In some aspects of the invention, the specific binding member is a polyclonal antibody.

[0041] The present invention also includes specific binding members as described above, but wherein the sequence of the binding domain is substantially as follows Figure 2 Thus, there is provided a specific binding member as described above, but wherein one or more binding domains are Figure 2 Those depicted in differ in 1 to 5, 1 to 4, 1 to 3, 2, or 1 amino acid substitution.

[0042] The present invention also includes Figure 2The epitope of an isolated antibody or binding fragment thereof or member thereof is the region in which the isolated antibody or binding fragment thereof or member thereof binds to its antigen. If two antibodies or binding fragments thereof or members each competitively inhibit (block) the binding of the other to the antigen, then they bind to the same or overlapping epitope. That is, as measured in a competitive binding assay, a 1×, 5×, 10×, 20× or 100× excess of one isolated antibody or binding fragment thereof or member thereof inhibits the binding of the other by at least 50%, but preferably at least 75%, 90% or even 99% compared to a control lacking a competing antibody (see, e.g., (Junghans et al. 1990), which is incorporated herein by reference).

[0043] In a preferred embodiment of the present invention, the competitive specific binding member and the Figure 2 a VH chain having an amino acid sequence of residues 1 to 126 of Figure 2 Antibodies with a VL chain having the amino acid sequence of residues 1 to 123 of b compete for binding to SSEA-4, only attached to the glycolipid Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc.

[0044] Preferably, the competing specific binding member is an antibody, such as a mAb, or any antibody fragment referred to throughout this document.

[0045] Once a single prototype mAb with the desired properties described herein is isolated, such as FG2811 mAb, it is simple to generate other mAbs with similar properties by using methods known in the prior art. For example, one can use, for example (Jespers, Roberts et al. 1994) (which is incorporated herein by reference) to guide the selection of mAbs with the same epitope and therefore similar properties as the prototype mAb. Using phage display, the heavy chain of the prototype antibody is first paired with a library of (preferably human) light chains to select mAbs that bind glycans, and then the new light chain is paired with a library of (preferably human) heavy chains to select mAbs that bind glycans with the same epitope as the prototype mAb (preferably human) mAbs.

[0046] The present invention includes a polypeptide that is capable of binding to SSEA-4 linked only to glycolipids and inducing ADCC and / or CDC and that binds to SSEA-4 in the VH and / or VL domains. Figure 2 A MAb having a VH or VL domain that is at least 90%, 95% or 99% identical to the VH or VL domain of the antibody. Reference to 90%, 95% or 99% identity may refer only to the framework regions of the VH and / or VL domain. In particular, the CDR regions may be identical, but the framework regions may differ by 1%, 5% or 10%. Preferably, such an antibody is identical to Figure 2 The sequences differ in a small number of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. In any embodiment of the invention, the specific binding pair can be an antibody or antibody fragment, Fab, (Fab′)2, scFv, Fv, dAb, Fd, or diabody. In some embodiments, the antibody is a polyclonal antibody. In other embodiments, the antibody is a monoclonal antibody. The antibodies of the present invention can be humanized antibodies, chimeric antibodies, or veneered antibodies, or can be non-human antibodies of any species. In one embodiment, the specific binding partner of the present invention is the mouse antibody FG2811, and the mouse antibody FG2811 comprises Figure 2 The heavy chain shown in a and Figure 2 b Light chain shown.

[0047] The specific binding members of the invention may carry a detectable or functional label.

[0048] In other aspects, the invention provides isolated nucleic acids encoding specific binding members of the invention and methods of making specific binding members of the invention, the methods comprising expressing the nucleic acid under conditions that result in expression of the binding member and recovering the binding member. The invention includes isolated nucleic acids encoding specific binding members that are capable of specifically binding to SSEA-4 and are at least 90%, at least 95%, or at least 99% identical to the sequences provided herein.

[0049] The specific binding members of the present invention can be used for treating or diagnosing a method of a human or animal body, such as a method for treating a tumor of a patient (preferably a human), the method comprising administering an effective amount of a specific binding member of the present invention to the patient. The present invention also provides a specific binding member of the present invention for use in medicine, preferably for treating a tumor, and the use of a specific binding member of the present invention in the preparation of a drug for diagnosing or treating a tumor. The tumor can be a gastric tumor, a colorectal tumor, a pancreatic tumor, a lung tumor, an ovarian tumor or a breast tumor.

[0050] Antigens that bind to specific binding members of the invention are disclosed herein. Preferably, SSEA-4 that can be bound (preferably specifically bound) by a specific binding member of the invention can be provided. SSEA-4 can be provided in an isolated form and can be used for screening to develop additional specific binding members for SSEA-4. For example, a compound library can be screened for library members that specifically bind to SSEA-4.

[0051] In other aspects, the present invention provides an isolated specific binding member (preferably the first aspect of the present invention) which is capable of binding to a polysaccharide containing SSEA (i.e., Neu5Ac(α2 3)Gal(β1 3)GalNAc(β1 3)Gal(α1 4)Gal(β1 4)Glc) for use in the diagnosis or prognosis of gastric tumors, colorectal tumors, pancreatic tumors, lung tumors, ovarian tumors and breast tumors.

[0052] In another aspect of the present invention, there is provided an in vitro induction of stem memory T cells (T SCM ) proliferation method, the method comprising causing stem memory T cells (T SCM ) is contacted with a specific binding member of the invention.

[0053] In another aspect of the present invention, there is provided a method for inducing stem memory T cells (T SCM ) in a cell culture medium for proliferation, the cell culture medium comprising a specific binding member of the invention.

[0054] In another aspect of the present invention, a method for inducing stem memory T cells (T SCM ) a method of proliferation comprising administering to a subject a specific binding member of the invention.

[0055] In another aspect of the invention, there is provided a binding member of the invention for use in therapy. In another aspect of the invention, there is provided a method of treating a patient, wherein the method comprises administering a specific binding member of the invention to a patient in need thereof.

[0056] In another aspect of the invention there is provided a specific binding member of the invention for use in a method of treating an autoimmune disease, HIV, adult T-cell leukemia or graft-versus-host disease.

[0057] In another aspect of the present invention, a method for treating or preventing cancer is provided, which method comprises administering a specific binding member of the present invention to a subject in need thereof.

[0058] In another aspect of the present invention, a method for treating or preventing a patient with a chronic viral infection is provided, comprising administering a specific binding member of the present invention to a subject in need thereof.

[0059] In another aspect of the present invention, a method for treating or preventing autoimmune diseases, HIV, adult T-cell leukemia or graft-versus-host disease is provided, which comprises administering a specific binding member of the present invention to a subject in need thereof.

[0060] In another aspect of the present invention, there is provided a method comprising stem memory T cells (T SCM) and a specific binding member of the invention, wherein the stem memory T cells (T SCM ) when compared to a corresponding cell culture of the same species.

[0061] In some aspects of the invention, a cell line comprising stem memory T cells (T cells) without a specific binding member of the invention is SCM ) compared to the corresponding cell cultures containing stem memory T cells (T SCM ) and a specific binding member of the invention, the proliferation rate of a cell culture is increased by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%.

[0062] In another aspect of the present invention, there is provided a method for purifying stem memory T cells (T cells) using a specific binding member of the present invention. SCM ), wherein the stem memory T cells (T) are expressed as compared to a corresponding cell population not purified using a specific binding member of the invention. SCM ) in the cell population is increased by at least about 10%.

[0063] In some aspects of the invention, the stem memory T cells (T cells) in the purified cell population are expressed when compared to a corresponding cell population that has not been purified using a specific binding member of the invention. SCM ) is increased by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%.

[0064] In some aspects of the invention, the specific binding members of the invention are isolated antibodies or binding fragments or members thereof.

[0065] The invention also provides a method for diagnosing cancer, the method comprising using a specific binding member of the invention to detect SSEA-containing glycans in a sample from an individual. In some diagnostic methods of the invention, the pattern of glycans detected by the binding member can be used to stratify treatment options for an individual.

[0066] These and other aspects of the invention will be described in further detail below.

[0067] As used herein, "specific binding members" are members of a pair of molecules that have binding specificity to each other. The members of the specific binding pair can be of natural origin or produced in whole or in part by synthesis. A member of the pair of molecules has an area on its surface, which can be a projection or a cavity, which specifically binds to the specific space and polarity organization of another member of the pair of molecules, and is therefore complementary to it. Therefore, the right members have the characteristic of specific binding to each other. Examples of the types of specific binding pairs include antigen-antibody, biotin-avidin, hormone-hormone receptor, 5 receptor-ligand, enzyme-substrate. The present invention generally relates to antigen-antibody type reactions, although it also relates to small molecules bound to antigens defined herein.

[0068] As used herein, "treatment" includes any regimen that can benefit a human or non-human animal, preferably a mammal. Treatment may be of an existing condition, or may be preventative (prophylactic treatment).

[0069] As used herein, a "tumor" is an abnormal growth of tissue. It can be local (benign) or invade nearby tissue (malignant) or distant tissue (metastatic). Tumors include neoplastic growths that cause cancer, including esophageal tumors, colorectal tumors, gastric tumors, breast tumors, ovarian tumors, and endometrial tumors, as well as cancerous tissues or cell lines, including but not limited to leukemia cells. As used herein, "tumor" also includes endometriosis within its scope.

[0070] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen binding site that specifically binds to an antigen, whether the molecule is natural or partially or wholly synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is an antibody binding domain or homologous to an antibody binding domain. These antibodies may be from natural sources, or they may be partially or wholly synthetically produced. Examples of antibodies of the present invention are: immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and isotype subclasses thereof; fragments comprising an antigen binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. Monoclonal antibodies may be referred to as "mAbs".

[0071] Monoclonal antibodies and other antibodies can be used and recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technology may involve introducing DNA encoding the immunoglobulin variable region or CDR of the antibody into the constant region or constant region plus framework region of a different immunoglobulin. For example, see EP-A-184187, GB2188638A or EP-A-239400. Hybridomas or other cells that produce antibodies may undergo genetic mutations or other changes, which may or may not change the binding specificity of the produced antibody.

[0072] Since antibodies can be modified in a variety of ways, the term "antibody" should be interpreted as covering any specific binding member or substance of a binding domain with desired specificity. Therefore, the term covers antibody fragments, derivatives, functional equivalents and homologues, humanized antibodies of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether the polypeptide is natural or synthesized in whole or in part. Therefore, a chimeric molecule comprising an immunoglobulin binding domain or an equivalent fused with another polypeptide is included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Humanized antibodies can be modified antibodies with the variable region of non-human (e.g., mouse) antibodies and the constant region of human antibodies. For example, a method for preparing humanized antibodies is described in U.S. Patent No. 5,225,539.

[0073] It has been shown that fragments of whole antibodies can perform the function of binding to antigen. Examples of binding fragments are: (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH domains (Ward et al. 1989); (v) isolated CDR regions; (vi) F(ab')2 fragments, i.e., bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al. 1988, Huston et al. 1993). al. 1988); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; (Holliger, Prospero, and Winter 1993)).

[0074] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region, the two domains being connected (e.g., by a peptide linker) but unable to associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide in the multimer with the second domain of another polypeptide in the multimer (WO94 / 13804).

[0075] Where bispecific antibodies are used, these may be conventional bispecific antibodies which may be made in a variety of ways (Holliger and Winter 1993) (e.g., by chemical preparation or by hybridization of hybridomas), or may be any of the bispecific antibody fragments described above. It may be preferred to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs may be constructed without the Fc region using only the variable domains, potentially reducing the effectiveness of anti-idiotypic responses. Other forms of bispecific antibodies include the single chain "Janusins" described in (Traunecker, Lanzavecchia, and Karjalainen 1991).

[0076] Bispecific diabodies, which are different from bispecific full antibodies, may also be useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities can be easily selected from libraries using phage display technology (WO 94 / 13804). If one arm of the diabody is to be kept constant, for example, specific for antigen X, a library can be created in which the other arm is varied and antibodies with appropriate specificity are selected.

[0077] "Binding domain" is a portion of a specific binding member that contains a region that specifically binds to part or all of an antigen and is complementary. In the case where the binding member is an antibody or an antigen-binding fragment thereof, the binding domain may be a CDR. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is referred to as an epitope. The antigen binding domain may be provided by one or more antibody variable domains. The antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0078] "Specificity" is generally used to refer to situations where one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner, and, for example, has less than about 30%, preferably 20%, 10% or 1% cross-reactivity with any other molecule. The term also applies, for example, to situations where an antigen binding domain is specific for a particular epitope carried by many antigens, in which case a specific binding member carrying the antigen binding domain will be able to bind to a variety of antigens carrying that epitope. Specific binding members of the invention may be able to bind to Le y Specifically binds in the sense that no binding to any other antigen (eg, any other glycan) can be detected when binding is tested according to the protocol outlined in "Glycomic Analysis" in the Examples herein.

[0079] According to the present invention, "isolated" refers to the state in which the specific binding members of the present invention or the nucleic acid encoding such binding members will preferably be. When this preparation is carried out by recombinant DNA technology implemented in vitro or in vivo, members and nucleic acids will generally not contain or substantially contain substances naturally associated with them, such as other polypeptides or nucleic acids found in their natural environment or the environment (such as cell culture medium) in which they are prepared. Specific binding members and nucleic acids can be formulated together with diluents or adjuvants, but are still separated for practical purposes. For example, if used to coat microtiter plates for immunoassays, members will generally be mixed with gelatin or other carriers, or when used for diagnosis or treatment, members are mixed with pharmaceutically acceptable carriers or diluents. Specific binding members can be naturally glycosylated or glycosylated by heterologous eukaryotic cell systems, or they can be (for example, if produced by expression in prokaryotic cells) non-glycosylated.

[0080] "Substantially as shown..." means that the amino acid sequence of the present invention will be identical or highly homologous to the amino acid sequence referred to. "Highly homologous" contemplates that 1 to 5, 1 to 4, 1 to 3, 2 or 1 amino acid substitutions may be made in the sequence.

[0081] The present invention also includes within its scope Figure 2 A polypeptide having an amino acid sequence as shown in Figure 2The polynucleotides of the nucleic acid sequences shown, and sequences having substantial identity thereto (e.g., having at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 99% identity thereto). The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the first sequence for optimal alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is an alignment between two sequences that achieves the highest percent identity. The percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (i.e., % identity = number of identical positions / total number of positions × 100).

[0082] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul in 1990 (Karlin and Altschul 1990), which was improved by Karlin and Altschul in 1993 (Karlin and Altschul 1993). The algorithm was incorporated into the NBLAST and XBLAST programs of Altschul et al. in 1990 (Altschul et al. 1990). BLAST nucleic acid searches can be performed with the NBLAST program (score = 100, wordlength = 12) to obtain nucleic acid sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described by Altschul et al. in 1997 (Altschul et al. 1997). Alternatively, PSI-Blast can be used to perform iterative searches to detect long-distance relationships (Id.) between molecules. When using BLAST, GappedBLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller in 1989 (Myers and Miller 1989). The ALIGN program (version 2.0) as a part of the GCG sequence alignment software package incorporates this algorithm. Other algorithms known in the art for sequence analysis include ADVANCE and ADAM as described by Torellis and Robotti (Torelli and Robotti 1994) in 1994, and FASTA as described by Pearson and Lipman (Pearson and Lipman 1988) in 1988. In FASTA, ktup is a control option for setting the sensitivity and speed of the search.

[0083] The isolated specific binding member of the invention is capable of binding to a SSEA-4 carbohydrate, which may be SSEA-4 ceramide or may be on a protein moiety. Figure 2 Residues 105 to 116 of (CDRH3) and Figure 2The binding domain having the amino acid sequence shown by residues 105 to 113 can be held in a structure that allows these regions to bind to the SSEA-4 carbohydrate.

[0084] The structures used to carry the binding domains of the present invention generally have antibody heavy or light chain sequences or substantial portions thereof, wherein the binding domains are located in positions corresponding to the CDR3 regions of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and positions of immunoglobulin variable domains can be determined by reference to http: / / www.imgt.org / . Figure 1 a and Figure 1 b as the CDR3 in the human heavy chain variable domain or a substantial portion thereof, and may carry essentially the same Figure 1 The amino acid sequence listed as residues 105 to 113 of c is used as CDR3 in the human light chain variable domain or a substantial portion thereof.

[0085] The variable domain can be derived from any germline or rearranged human variable domain, or can be a synthetic variable domain based on a consensus sequence of a known human variable domain. Recombinant techniques can be used to introduce the CDR3 source sequence of the present invention into a library of variable domains lacking a CDR3 region. For example, Marks et al. (Marks et al. 1992) described a method for producing an antibody variable domain library in 1992, wherein a common primer at or near the 5′ end of the variable domain region is used in combination with a common primer in the third framework region of a human VH gene to provide a VH variable domain library lacking CDR3. Mark et al. (Marks et al. 1992) also described how to combine the library with the CDR3 of a specific antibody in 1992. Using similar techniques, the sequence of the CDR3 source of the present invention can be shuffled with a library of VH or VL domains lacking CDR3, and the complete VH or VL domains of the shuffle are combined with the same VL or VH domains to provide a specific binding member of the present invention. The library can then be displayed in a suitable host system (e.g., the phage display system of Wo92 / 01047) so that suitable specific binding members can be selected. 4 More than 10 individual members (e.g. 6 Up to 10 8 or 10 10 members).

[0086] A similar promiscuous or combinatorial technique was also disclosed by Stemmer in 1994 (Stemmer 1994), who described the technique in relation to β-lactamase genes, but noted that the method could be used to generate antibodies. Another alternative is to generate new VH or VL regions carrying the CDR3-derived sequences of the invention using, for example, random mutagenesis of the FG2811 VH or VL genes to generate mutations throughout the variable domain. This technique was described by Gram et al. in 1992 (Gram et al. 1992), which used error-prone PCR.

[0087] Another method that can be used is to directly mutagenize the CDR region of the VH or VL gene. Barbas et al. (Barbas et al. 1994) and Schier et al. (Schierl et al. 1996) disclosed this technology in 1994. The substantial portion of the immunoglobulin variable domain will generally include at least three CDR regions and their intervening framework regions. The portion may also include at least about 50% of one or both of the first framework region and the fourth framework region, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The additional residues at the N-terminal or C-terminal end of the substantial portion of the variable domain may be residues that are not usually associated with naturally occurring variable domain regions. For example, the construction of the specific binding members of the present invention by recombinant DNA technology may result in the introduction of N-terminal or C-terminal residues encoded by a linker, which is introduced to facilitate cloning or other manipulation steps, including the introduction of a linker to connect the variable domain of the present invention to other protein sequences including immunoglobulin heavy chains, other variable domains (e.g., when producing diabodies) or protein tags, as discussed in more detail below.

[0088] The present invention provides specific binding members comprising a pair of binding domains based essentially on Figure 2 The amino acid sequences of the VL and VH regions shown, i.e., Figure 2 Amino acids 1 to 127 (VH) and Figure 2 A single binding domain based on any of these sequences forms further aspects of the invention. Figure 2 In the case of a binding domain of the listed VH region amino acid sequence, such a binding domain can be used as a targeting agent, because it is known that immunoglobulin VH domains can bind to target antigens in a specific manner. In the case of any single-chain specific binding domain, these domains can be used to screen for complementary domains that can form a dual domain specific binding member with in vivo properties as good or equal to the FG2811 antibody disclosed herein.

[0089] This can be achieved by phage display screening methods using a so-called hierarchical dual combinatorial approach as disclosed in WO92 / 01047, in which a single colony containing either H or L chain clones is used to infect a complete clone library encoding the other chain (L or H), and the resulting double-chain specific binding members are selected according to phage display technology (e.g. as described in this reference). This technology was also disclosed by Marks et al. 1992.

[0090] The specific binding members of the present invention may also comprise an antibody constant region or a portion thereof. Figure 2 The specific binding member of the VL region shown in a can be linked to the antibody light chain constant domain at its C-terminus. Figure 2 The specific binding members of the VH regions shown may be linked at their C-termini to all or part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE and IgM) and any isotype subclass (particularly IgGl, IgG2 and IgG4).

[0091] The specific binding members of the invention can be used in methods of diagnosing and treating tumors in human or animal subjects.

[0092] When used in diagnostics, the specific binding members of the invention may be detectably labeled (e.g., radiolabeled, such as 131 I or 99 The invention also provides a method for labeling a specific binding member of the present invention with a Tc-labeled antibody, which can be attached to a specific binding member of the present invention using conventional chemical methods known in the field of antibody imaging. Labels also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties, such as biotin, which can be detected by binding to a specific homologous detectable moiety (e.g., a labeled avidin).

[0093] In addition, the specific binding members of the present invention can be used alone or in combination with other treatments simultaneously or in sequence, depending on the disease treated. Therefore, the present invention further provides a product containing the specific binding members of the present invention and an activating agent as a combined preparation for treating tumors simultaneously, individually or in sequence. Activating agent can include chemotherapeutic agents or cytotoxic agents, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin (oxaliplatin) and tamoxifen (tamoxifen), which can synergize with the binding members of the present invention. Other activating agents can include analgesics of suitable doses, such as nonsteroidal anti-inflammatory drugs (such as aspirin, paracetamol, ibuprofen or ketoprofen) or opiates (such as morphine), or antiemetics.

[0094] Although not wishing to be bound by theory, the ability of the binding members of the invention to synergize with active agents to enhance tumor killing may not be due to an immune effector mechanism, but may be a direct result of binding of the binding members to SSEA-4 glycans bound to the cell surface. Cancer immunotherapy involving antibodies directed against immune checkpoint molecules has shown effectiveness against a variety of malignancies and in combination with different immuno-oncology treatment modalities.

[0095] The specific binding members of the present invention will generally be applied in the form of a pharmaceutical composition, which may include at least one component in addition to the specific binding members. In addition to the active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabilizer or other substances well known to those skilled in the art. This substance should be nontoxic and should not interfere with the effect of the active ingredient. The exact nature of the carrier or other substances will depend on the route of administration, which may be oral or injected (e.g., intravenous). It is expected that injection will be the main route of therapeutic administration of the composition, but catheters or other surgical tubes are also used to deliver. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration. Powder formulations can be reconstituted into liquid formulations for use.

[0096] For intravenous injection or injection at the affected part, the active ingredient will be in the form of a pyrogen-free, pH-appropriate, isotonic and stable enterally acceptable aqueous solution. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles (e.g., sodium chloride injection, Ringer's injection, sodium lactate Ringer's injection). Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0097] The pharmaceutical composition for oral administration can be in tablet, capsule, powder or liquid form. Tablet can comprise solid carrier, for example gelatin or adjuvant. Liquid pharmaceutical composition generally comprises liquid carrier, for example water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Can comprise physiological saline solution, dextrose or other sugar solution, or glycerol such as ethylene glycol, propylene glycol or polyethylene glycol. When preparation is liquid, it can be physiological saline solution, for example containing non-phosphate buffer of pH 6.8-7.6, or lyophilized powder.

[0098] The composition can also be administered by microspheres, liposomes, other microparticle delivery systems or slow-release formulations placed in certain tissues (including blood). Suitable examples of slow-release carriers include semi-permeable polymer matrices in the form of shared articles (e.g., suppositories or microcapsules). Implantable or microcapsule slow-release matrices include polylactide (U.S. Patent No. 3,773,919; EP-A-0058481) copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al. 1983), poly (2-hydroxyethyl-methacrylate). The liposomes containing the polypeptide are prepared by known methods: DE 3,218,121A; (Eppstein 1985); (Hwang, Luk, and Beaumie 1980); EP-A-0052522; EP-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (about 200 angstroms to 800 angstroms) unilamellar types, wherein the lipid content is greater than about 30 mol.% cholesterol, the selected ratio being adjusted to obtain an optimal polypeptide leakage rate. The composition can be administered topically to a tumor site or other desired site, or can be delivered in a manner targeted to a tumor or other cells.

[0099] The composition is preferably administered to an individual in a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount applied, the rate of administration and the time course will depend on the nature and severity of the disease being treated. Treatment prescriptions, such as dosage decisions, are the responsibility of general practitioners and other physicians, and generally require consideration of the disease being treated, the condition of the individual patient, the delivery site, the method of administration and other factors known to the physician. The composition of the present invention is particularly related to the treatment of existing tumors (particularly cancer), and the treatment of preventing such conditions from recurring in the initial treatment or after surgery. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980 (Remington 1980).

[0100] The optimal dosage can be determined by the physician based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered, and the route of administration. In general, serum concentrations of polypeptides and antibodies that saturate the receptors are desirable. Concentrations in excess of about 0.1 nM are usually sufficient. For example, 100 mg / m 2 The antibody dose provided a serum concentration of approximately 20 nM that lasted for approximately eight days.

[0101] As a rough guide, a total of 10 mg / m2 of antibody may be given per week. 2 Up to 300 mg / m 2 . Equivalent doses of the antibody fragment should be used at more frequent intervals to maintain serum levels above the concentration that saturates the SSEA4 carbohydrate energy. The dosage of the composition will depend on the characteristics of the binding member, for example, its binding activity and plasma half-life in vivo, the concentration of the polypeptide in the formulation, the route of administration, the site and rate of administration, the clinical tolerance of the patient involved, the pathological condition suffered by the patient, etc., which is well within the skill of the physician. For example, a dose of 300 μg of antibody per administration per patient is preferred, although the dose range may be about 10 μg to 6 mg per dose. Different doses are used during a series of sequential vaccinations; the practitioner may perform an initial vaccination and then boost with a relatively smaller dose of the antibody.

[0102] The present invention also relates to an optimized immunization regimen for enhancing a protective immune response against cancer. The present invention provides an immunization regimen for enhancing a protective immune response against cancer.

[0103] Binding members of the present invention can be generated in whole or in part by chemical synthesis. The binding members can be easily prepared according to established standard liquids or preferred solid phase peptide synthesis methods, and the general description of the method can be widely obtained (for example, see JM Stewart and JD Young, 1984 (Stewart and Young 1984), M. Boαanzsky and A. Bodanzsky, 1984 (Bodanzsky and Bodanzsky 1984)); Or, binding members can be prepared in solution by liquid phase method or by any combination of solid phase method, liquid phase method and solution chemistry, for example, first complete each peptide part, then if necessary and suitable, after removing any protecting group present, introduce residue x by the reaction of each carbonic acid or sulfonic acid or its reactive derivative.

[0104] Another convenient way of producing a binding member according to the invention is by expressing a nucleic acid encoding the binding member using the nucleic acid in an expression system.

[0105] The present invention also provides isolated nucleic acids encoding specific binding members of the present invention. Nucleic acids include DNA and RNA. In a preferred aspect, the present invention provides nucleic acids encoding specific binding members of the present invention as defined above. Examples of such nucleic acids are shown in Figure 2 The skilled artisan will be able to determine substitutions, deletions and / or additions to such nucleic acids that will still provide a specific binding member of the invention.

[0106] The present invention also provides a construct in the form of a plasmid, a vector, a transcription cassette or an expression cassette containing at least one of the above-mentioned nucleic acids. The present invention also provides a recombinant host cell comprising one or more of the above-mentioned constructs. As described above, the nucleic acid encoding the specific binding members of the present invention constitutes an aspect of the present invention; the method for producing the specific binding members also constitutes an aspect of the present invention, and the method includes expressing by the encoding nucleic acid. By culturing a recombinant host cell containing the nucleic acid under appropriate conditions, expression can be conveniently achieved. After production by expression, any suitable technology can be used to separate and / or purify the specific binding members, and then use as appropriate.

[0107] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines that can be used in the art to express heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, and many other cells. Common preferred bacterial hosts are Escherichia coli (E. coli). The expression of antibodies and antibody fragments in prokaryotic cells (e.g., E. coli) is well established in the art. For a review, see Plückthun, 1991 (Pluckthun 1991). Those skilled in the art may also choose to use expression in eukaryotic cell culture to generate specific binding members, and recent reviews are shown, for example, in Reff, 1993 (Reff 1993); Trill et al., 1995 (Trill, Shatzman, and Ganguly et al. 1995).

[0108] Suitable vectors containing appropriate regulatory sequences can be selected or constructed, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. As appropriate, the vector can be a plasmid, a virus, such as a phage or a phagemid. For more details, see, for example, Sambrook et al., 1989 (Sambrook 1989). Many known techniques and protocols for nucleic acid manipulation in, for example, preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis are described in detail in Ausubel et al., 1992 (Ausubel 1992).

[0109] Therefore, another aspect of the present invention provides a host cell containing nucleic acids disclosed herein. In another aspect, a method is provided comprising introducing such nucleic acids into host cells. Any available technique can be used for introduction. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia virus, or for insect cells, baculovirus). For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophage. Expression of the nucleic acid can be initiated or allowed after introduction, for example, by culturing host cells under expression conditions of the gene.

[0110] The nucleic acid of the invention can be integrated into the genome (eg chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome according to standard techniques.

[0111] The present invention also provides a method, which comprises using the construct as described above in an expression system to express the specific binding member or polypeptide as described above.

[0112] The preferred features of each aspect of the invention apply mutatis mutandis to each other aspect.The prior art documents mentioned herein are incorporated herein to the maximum extent permitted by law.

[0113] In certain aspects, the present disclosure provides a pharmaceutical composition comprising a mAb or binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0114] Immunomodulatory mAb is intended to block the key inhibitory pathways (checkpoint blockers) that inhibit effector T cells or engage co-stimulatory immune receptors (immunostimulation) in an exciting manner. In this patent, we show that 2811mAb can stimulate T cell proliferation in vitro and in vivo. It has been shown that isotype-dependent FcγRIIB engagement is necessary for immune-stimulating mAb activity. These agents stimulate signal transduction through their target receptors (usually members of the tumor necrosis factor receptor (TNFR) superfamily), and the interaction of mAb Fc with FcγRIIB promotes receptor aggregation and subsequent downstream signal transduction. As cancer therapeutic agents, they are intended to enhance tumor immunity by engaging co-stimulatory receptors such as CD40, 4-1BB or OX40 on APC or effector T cells, or promote apoptosis by stimulating death receptors such as DR4, DR5 or Fas (CD95) on cancer cells. In contrast to directly targeted agents, the agonistic activity of these mAbs depends on their ability to engage inhibitory FcγRIIB, and mAbs with a high ratio of binding to activating receptors rather than inhibitory receptors (A:I) (e.g., mouse IgG2a, human IgG1) are essentially inactive in preclinical models, whereas those with a low A:I ratio (e.g., mouse IgG1 and hIgG2) are highly agonistic. Signaling through FcγRIIB is not necessary to confer activity; rather, it provides a cross-linking scaffold for the mAb to promote TNFR aggregation and activation (Beers, Glennie, and White 2016). In this regard, FG2811 mIgG1 is used to stimulate T cells in vivo. SCM , while plate-bound 2811 hIgG1 or mIgG3 can be used in vitro. An alternative approach is to use the hIgG2 isotype. This human isotype, which has limited binding affinity for FcγRIIB, has an intrinsic ability to drive receptor aggregation through its unique hinge disulfide bond configuration (White 2015; Liu 2019; Yu 2020). When synthesized, hIgG2 is converted into a range of isotypes through disulfide rearrangements of its hinge and CH1 domains, with more compact and rigid forms showing potent FcγRIIB-independent receptor aggregation in vitro and in vivo. Thus, we demonstrated that 2811 hIgG2-induced T SCM Stimulate

[0115] In some aspects, the present invention provides for isolating stem memory T cells (T cells) ex vivo by binding of an isolated specific binding member of the present invention to an SSEA-4 antigen. SCM In some aspects, the invention provides a method for ex vivo proliferation of stem memory T cells (T cells) by binding of an isolated specific binding member of the invention to an SSEA-4 antigen. SCMIn some aspects, the invention provides methods for isolating and proliferating stem memory T cells (T cells) in vitro by binding of the isolated specific binding member of the invention to the SSEA-4 antigen. SCM ) method.

[0116] In certain aspects, the invention provides methods for isolating and / or expanding stem memory T cells (T cells) ex vivo using any mouse or human isotype of mAb 2811. SCM In certain aspects, the present invention provides methods for isolating and / or proliferating stem memory T cells (T cells) in vitro using isolated antibodies or binding fragments thereof or members thereof. SCM ), wherein the antibody or binding fragment or member thereof comprises a binding domain from mAb 2811 and any framework region from any mouse or human antibody isotype.

[0117] In some aspects, the present invention provides for isolating stem memory T cells (T cells) in vivo by binding of an isolated specific binding member of the present invention to an SSEA-4 antigen. SCM In some aspects, the invention provides a method for in vivo proliferation of stem memory T cells (T) by binding of an isolated specific binding member of the invention to an SSEA-4 antigen. SCM In some aspects, the invention provides methods for isolating and proliferating stem memory T cells (T cells) in vivo by binding of the isolated specific binding members of the invention to the SSEA-4 antigen. SDM ) method.

[0118] In some aspects, the present invention provides an isolated specific binding member of the present invention for use in isolating stem memory T cells (T cells) in vivo by binding to the SSEA-4 antigen. SCM In some aspects, the invention provides an isolated specific binding member of the invention for use in in vivo proliferation of stem memory T cells (T cells) by binding to the SSEA-4 antigen. SCM In some aspects, the invention provides an isolated specific binding member of the invention for use in isolating and proliferating stem memory T cells (T cells) in vivo by binding to the SSEA-4 antigen. SCM ) method.

[0119] In certain aspects, the invention provides methods for isolating and / or expanding stem memory T cells (T cells) in vivo using any mouse or human isotype of mAb 2811. SCM In certain aspects, the present invention provides methods for isolating and / or proliferating stem memory T cells (T cells) in vivo using isolated antibodies or binding fragments thereof or members thereof. SCM), wherein the antibody or binding fragment or member thereof comprises a binding domain from mAb 2811 and any framework region from any mouse or human antibody isotype.

[0120] In some aspects of the invention, the proliferation of stem memory T cells (T SCM ) refers to increasing cell proliferation and / or promoting cell division.

[0121] In some aspects of the present invention, by using binding members targeting of the present invention or labeled cells, then apply cell sorting or cell separation method to identify or purify cells.In some aspects of the present invention, binding members of the present invention can be used together with cell sorting or cell separation method, such as fluorescence activated cell sorting (FACS), flow cytometry, immunomagnetic cell separation, immune density cell separation, immune guided laser capture microdissection.In a preferred embodiment, binding members of the present invention can be used together with fluorescence activated cell sorting (FACS).In a preferred embodiment, binding members of the present invention can be used together with flow cytometry method.

[0122] In certain aspects, the invention provides a method for treating cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a separated specific binding member of the invention. In some methods of the invention, the administered binding members stimulate the proliferation of the separated specific binding members of the invention in the subject.

[0123] In some embodiments, provided methods treat a cancer selected from the group consisting of brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, stomach cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, bone cancer, skin cancer, cervical cancer, ovarian cancer, and prostate cancer.

[0124] In certain aspects, the invention provides a pharmaceutical composition comprising the isolated specific binding members of the present invention, the pharmaceutical composition is used to treat a method for treating a cancer of an object in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to the object. In some methods of the present invention, the binding members administered stimulate the proliferation of the isolated specific binding members of the present invention in the object.

[0125] In certain embodiments, the pharmaceutical composition used according to the methods of the present invention is selected from the group consisting of cancers of the brain, lung, breast, oral, esophagus, stomach, liver, bile duct, pancreas, colon, kidney, bone, skin, cervix, ovary, and prostate.

[0126] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] As used herein, symbols, graphics, and textual nomenclature used to describe glycans and related structures are well established and understood in the art, including, for example, "Symbols Nomenclatures for Glycan Representation" by Ajit Varki et al. (Varki et al. 2009).

[0128] legend

[0129] Figure 1 : Schematic diagram of the generation of SSEA-3 and SSEA-4 glycans from lactosylceramide (LC). The LMTK mouse fibroblast cell line was transduced with A4GALT, B3GALNT1, and B3GALT5 genes to produce α-1,4-galactosyltransferase, β-1,3-N-acetylgalactosaminyltransferase, and β-1,3-galactosyltransferase, respectively, which in turn add glycans to LC to generate SSEA-3 and SSEA-4 glycans.

[0130] Figure 2 : Amino acid and nucleotide sequences of FG2811 IgG3 heavy chain and kappa light chain variable regions and mIgG1, hIgG1 and hIgG2 constant regions.

[0131] (A) Nucleotide and amino acid sequences of the mature FG2811 heavy chain variable region, showing framework regions (FR) 1 to 3 and complementarity determining regions (CDR) 1 to 3.

[0132] (B) Nucleotide and amino acid sequence of the mature FG2811 kappa chain variable region, showing framework regions (FRs) 1 to 3 and complementarity determining regions (CDRs) 1 to 3.

[0133] (C) Comparison of nucleotide and amino acid sequences of mIgG1 with germline

[0134] (D) Comparison of nucleotide and amino acid sequences of hIgG2 with germline

[0135] (E) Comparison of nucleotide and amino acid sequences of hIgG1 with germline

[0136] Figure 3 : Binding pattern of 2811 mouse IgG (IgG1 and IgG3) isotypes to chimeric IgG (IgG1 and IgG2) isotypes to SSEA-3 / -4-LMTK cells.

[0137] FG2811mG3, FG2811mG1, CH2811hG1, CH2811hG2, MC813 (anti-SSEA-4 mAb; mouse IgG1), MC631 (anti-SSEA-3 mAb; rat IgM), FG88.7 (anti-Lewis a / c / x mAb; mouse IgG3), anti-mouse secondary and tertiary antibodies only, anti-human secondary and tertiary antibodies only, and medium only to SSEA-3 / -4-LMTK cells. The results are expressed as geometric mean (Gm) values.

[0138] Figure 4 : Evaluation of the specificity of FG2811mG3 for SSEA-4.

[0139] (A) Evaluation of FG2811mG3 mAb binding to lipid antigens by HPTLC. Thin layer chromatography analysis of 1) wild-type LMTK and 2) SSEA-3 / -4-LMTK plasma membrane lipid extracts was performed using 5 μg / ml of i) FG2811mG3 mAb, ii) MC631 mAb, and iii) MC813 mAb;

[0140] (B) Binding of FG2811mG3 mAb to SSEA-3 / -4-LMTK cell surface antigens. Binding of 5 μg / ml of i) secondary antibody only, ii) MC813, iii) FG2811mG3, and iv) MC631 to SSEA-3 / -4-LMTK cells was evaluated by direct immunofluorescence staining and flow cytometry analysis. Results are expressed as Gm values;

[0141] (C) Reactivity of FG2811mG3 mAb with HSA-coupled glycan antigens (SSEA-3, SSEA-4, Globo-H, and Forssman). Binding of 5 μg / ml of i) FG2811mG3, ii) MC813, iii) MC631, iv) M1 / 87, and v) KM93 mAbs to HSA-coupled glycans was assessed by ELISA. MC631 (anti-SSEA-3 and SSEA-4), MC813 (anti-SSEA-4), M1 / 87 (anti-Forssman), and KM93 (anti-sialyl-Lewis x ) was included as a positive control mAb. Antibody activity was measured by absorbance at 450 nm. Error bars represent the mean ± standard deviation of quadruplicate wells (***p<0.0001; *p<0.05 compared with control, ANOVA followed by Bonferroni multiple comparison test, GraphPad Prism6);

[0142] (D) Binding of FG2811mG3 mAb to the Consortium for Functional Glycomics glycan array (CFG, Core H, version 5.1). Sp represents the length of the spacer between glycans on the slide.

[0143] Figure 5 : Evaluation of the affinity of FG2811mG3 for antigen.

[0144] (A) The binding kinetics of FG2811mG3 mAb to SSEA-3 / -4-LMTK plasma membrane lipid antigen was detected using SPR (Biacore X);

[0145] (B) SSEA-3 / -4-LMTK plasma membrane lipid ELISA. A range of FG2811mG3 mAb concentrations were incubated in microwells coated with SSEA-3 / -4-LMTK plasma membrane lipids. 50 Value (6.8×10 -10 M) obtained by nonlinear regression of log-transformed data (GraphPadPrism6);

[0146] (C) SSEA-3 / -4-LMTK cell surface binding. SSEA-3 / -4-LMTK cells were incubated with a range of FG2811mG3 mAb concentrations and analyzed for cell binding by flow cytometry. Background-subtracted data were fitted to a site-specific binding model (GraphPad Prism6) to generate K d Values. Representative binding curves from three independent experiments are shown.

[0147] Figure 6: Binding of FG2811mG3 antibody to a panel of human cancer cell lines.

[0148] (A) Antibody binding to brain cancer cell lines (U251, KNS42, DAOY, SF188, U87, and UW2283). The binding of 5 μg / ml of antibody FG2811mG3, MC813, mouse IgG3κ isotype control, and secondary antibody only (no primary antibody) to brain cancer cell lines was evaluated by flow cytometry, and the results are expressed as Gm values;

[0149] (B) Antibody binding to ovarian (SKOV3, IGROV1, and OVCAR-5), breast (T47D, MCF7, DU4475, and HCC1187), and colorectal (Colo205 and HCT15). Binding of 5 μg / ml of antibody FG2811mG3, anti-HLA-A, B, C (W6 / 32), and secondary antibody only (no primary antibody) to a panel of cancer cell lines was assessed by flow cytometry and the results are presented as Gm values.

[0150] Figure 7 : Cytotoxic activity of FG2811mG3 antibody.

[0151] (A) ADCC killing of cancer cells by FG2811mG3 mAb. Dose-dependent ADCC activity of FG2811mG3 mAb on SKOV3 and T47D cells. 51 Cr-labeled cancer target cells were incubated with increasing concentrations of FG2811mG3 mAb (0.003 μg / ml to 10 μg / ml) and human PBMC (target cells: PBMC; 100:1). 51 Cr, and the total released by 10% Triton-X 51 The percentage of Cr is expressed. Anti-CD55 mAb (791T / 36) was used as a negative control mAb. The significance compared with the PBMC control was determined by ANOVA followed by Bonferroni multiple comparison test, GraphPad Prism 6. (***, P < 0.001 compared with the control);

[0152] (B) CDC killing of cancer cells by FG2811mG3 mAb. Dose-dependent CDC activity of FG2811mG3 mAb on SKOV3 and T47D cells. 51 Cr-labeled cancer target cells were incubated with increasing concentrations of FG2811mG3 mAb (0.003 μg / ml to 10 μg / ml) and human serum. 51 Cr, and the total released by 10% Triton-X 51 The percentage of Cr is expressed. Anti-CD55 mAb (791T / 36) was used as a negative control mAb. The significance compared with the PBMC control was determined by ANOVA followed by Bonferroni multiple comparison test, GraphPad Prism 6. (*, P < 0.005; ***, P < 0.001 compared with the control);

[0153] (C) FG2811mG3 induces direct cell death in cancer cells at 37°C. Propidium iodide (PI) uptake after mAb exposure was assessed by flow cytometry analysis. SSEA-3 / -4-LMTK cells were incubated with 30 μg / ml FG2811mG3 mAb at 37°C. Hydrogen peroxide (H2O2) and culture medium alone were included as positive and negative controls, respectively;

[0154] (D) Phase contrast imaging of FG2811mG3 treated cancer cells. Images (magnification x10) show SSEA-3 / -4-LMTK, SKOV3 and LMTK cells after incubation with 30 μg / ml of FG2811mG3 mAb and medium alone for 72 hours.

[0155] Figure 8: Normal red blood cell binding.

[0156] (A) Binding of FG2811mG3 mAb to healthy donor erythrocytes was assessed by flow cytometry. Binding of FG2811mG3 mAb and 791T / 36 positive control mAb (anti-CD55 mAb) to erythrocytes was compared by flow cytometry. Both mAbs were used at 10 μg / ml. Isotype control mAb alone and culture medium were used as negative controls. Results are representative of 5 donors;

[0157] (B) Hemagglutination assay. Hemagglutination of erythrocytes caused by different concentrations (0.625 μg / ml to 10 μg / ml) of FG2811mG3 mAb was compared with 791T / 36 and anti-blood group positive control antibodies. PBS was used as negative control. Results are representative of 5 donors.

[0158] Figure 9: Binding of FG2811mG1 to human blood cells.

[0159] (A) Binding of FG2811mG1 to whole blood PBMCs from healthy donors. Binding of healthy donor whole blood to FG2811mG1, MC813, mouse IgG1 isotype control antibody (isotype ctrl), OKT3 (anti-CD3), 198 (anti-CEACAM6), and anti-mouse IgG Fc-specific FITC secondary antibody alone (no primary antibody) was evaluated by indirect immunofluorescence staining and flow cytometry analysis. All mAbs were used at 5 μg / ml. The results shown are representative of whole blood from 7 different healthy donors. The results are shown in dot plots and histograms;

[0160] (B) PBMC phenotyping. Consecutive panels depict the CD3 + Flow cytometry gating strategy for FG2811mG3+PBMC. Draw gates to identify CD3 + FG2811mG3 + Cells were analyzed; CD3 + FG2811mG3 + The expression of CD45RA and CD45RO in cells was further examined. + 、CD45RA + RO + and CD45RO + Expression of CD62L, CD95 and CCR-7 markers in cells.

[0161] Figure 10: Transcriptional analysis of CH2811hG1-enriched naive T cells and CD122 / CD95-enriched naive T cells from four healthy donors (BD3, BD13, BD61, BD96) using bulk RNAseq.

[0162] (A) Venn diagram showing the common genes between the two groups of differentially expressed (DE) genes obtained by comparing naive CD8 T cells (GSE83808) with CH2811hG1-enriched naive T cells and CD122 / CD95-enriched naive T cells, respectively. The stemness characteristics of the 2227 common genes identified were analyzed using StemChecker (Pinto et al. 2015). The table shows the statistically significant enrichment of genes associated with stem cell subsets and the significantly enriched stemness-related transcription factor targets;

[0163] (B) Heatmap and hierarchical clustering (Euclidean distance) of transcriptome profiles enriched in CH2811hG1 and CD122 / CD95 based on 257 overlapping genes from ESCs and 113 overlapping genes from HSCs, respectively (both from the table in A). There is no clear segregation of the two enriched populations, suggesting that their stemness characteristics share commonalities;

[0164] (C)(i) Heat map based on CD8 + T SCM and T N DE genes between (>2-fold, p<0.001) from Gattinoni et al. 2011, and based on (ii) transcription factor subsets shown to be involved in effector differentiation, effector function, exhaustion, and homing adhesion genes from Pilipow et al. 2018. Donors 1 to 6 are from GSE114765, and CD8 / CD4 naive and memory datasets are from GSE23321.

[0165] Fig.11 : PBMC proliferation induced by CH2811hG1 antibody

[0166] (A) PBMCs were isolated from whole blood of two healthy donors (BD3 and BD18) and labeled with CSFE dye. CSFE-labeled T cells from healthy donors were stimulated with plate-bound i) PHA, ii) CH2811hG1 mAb, and iii) culture medium, and cells were collected on day 11 to examine CD4 and CD8 T cell proliferation. The percentage of proliferation of specific T cell populations was assessed by CSFE dye dilution analysis. Results are representative of 2 donors;

[0167] (B) Summary of CD4 and CD8 PBMC proliferation.

[0168] FIG. 12 : CH2811hG1 antibody-induced T cell proliferation.

[0169] (A) T cell purity and CSFE labeling check. Pure T cells were isolated from whole blood of four healthy donors (BD61, BD2, BD3, BD26) and labeled with CSFE dye. T cell purity was checked by staining T cells with anti-CD3 antibody and CSFE labeling was checked in the FITC channel;

[0170] (B) CH2811hG1 plate-bound antibody induced T cell proliferation at 5 μg / ml. CSFE-labeled T cells from healthy donors were stimulated with plate-bound i) CH2811hG1, ii) anti-CD3 antibody, and iii) culture medium, and cells were collected on days 7, 11, and 14 to examine CD4 and CD8 T cell proliferation. The percentage of specific T cell population proliferation was assessed by CSFE dye dilution analysis. Results are representative of 4 donors;

[0171] (C) Summary of i) total T cell proliferation, ii) CD4 T cell proliferation, and iii) CD8 T cell proliferation from 4 healthy donors (BD61, BD2, BD3, BD26) assessed by CSFE dye dilution analysis;

[0172] (D) Percentage of CD4 T cells that divided a certain number of times after 11 days of CH2811hG1 stimulation in vitro. T cells were loaded with CSFE dye and stimulated with i) anti-CD3, ii) CH2811hG1 or iii) culture medium on day 0. On day 11, CSFE profiles were analyzed by flow cytometry. The number of cell divisions is indicated by boxes, and the percentage of cells that divided a certain number of times is indicated above the boxes.

[0173] Figure 13: Evaluation of TCR repertoire clonotypes of CH2811hG1 stimulated T cells.

[0174] T cell repertoires were examined in extracted RNA from CSFE high and low CH2811hG1-stimulated T cells from 2 donors on day 19 (BD3) and day 14 (BD26), respectively. TCR repertoire diversity is shown in the tree diagram, where each rounded rectangle represents a unique entry: VJ-uCDR3, and the size of the dots indicates the relative frequency;

[0175] (A) Diversity plots of CH2811 stimulated T cells for CFSE high (B) and CSFE low (C) TRA chains, and CFSE high (D) and CSFE low (E) TRB chains. The higher the diversity of the sample, the closer the solid line is to the dashed line. This line converges to a curve that describes the overall diversity of the sample, with the "perfect" diversity being the black dashed line (each unique clonotype receives the same number of reads, i.e., no clonal expansion or dominant clones).

[0176] Fig.14 : Kinetics of individual cytokine / chemokine responses.

[0177] Pure T cells isolated from 4 healthy donors (BD61, BD2, BD3, BD26) were stimulated with CH2811hG1 (5 μg / ml) on day 0. Unstimulated cells (culture medium) were included as negative controls. Supernatants were collected on days 7, 11 and 14 and the concentrations (pg / ml) of IFNγ, TNFα, IL-8, IL-10, IL-2, IL-5, IL-17A, IL-7 and IL-21 were evaluated. Each point represents a different donor. The cytokine / chemokine results between CH2811hG1-stimulated T cells and unstimulated cells were compared and analyzed by applying an unpaired Student's t-test, and the P values ​​were calculated accordingly (***, P < 0.0001, **, P < 0.01, *, P < 0.05; GraphPad Prism 6).

[0178] Fig.15 : T cells stimulated by CH2811hG1 remained viable for more than 2 months in vitro.

[0179] (A) T cells were stimulated with plate-bound CH2811hG1 (5 μg / ml) or anti-CD3 antibody (0.005 μg / ml) or medium on day 0. On day 35, under an optical microscope, all cells stimulated with i) anti-CD3 antibody and ii) medium died, except for cells stimulated with iii) CH2811hG1 (magnification ×20);

[0180] (B) Phenotypic analysis of live CH2811hG1 stimulated T cells on day 35. Consecutive panels depict the phenotype of FG2811mG3 + Flow cytometry gating strategy for cells. Draw gates to i) FG2811mG3 + and ii) FG2811mG3- cells were analyzed; their CD3 and CD122 expression was examined. + Expression of CD45RA, CD45RO, CD62L, and CD95 markers in cells (results represent 1 donor);

[0181] (C) CH2811hG1-stimulated T cells remained viable and maintained proliferation capacity at day 35 in vitro. On day 33, surviving CH2811hG1-stimulated T cells were restimulated with plate-bound CH2811hG1 (5 μg / ml) or a combination of plate-bound anti-CD3 (0.005 μg / ml) and anti-CD28 (5 μg / ml) antibodies. Under light microscopy, i) anti-CD3 / CD28 restimulated T cells underwent massive T cell proliferation and formed T cell blasts at day 39; ii) On day 70, CH2811hG1-stimulated cells remained viable and showed significant T cell expansion (magnification ×10 and ×20);

[0182] (D) IL-7 and IL-21 may be key self-maintaining cytokines for the long-term survival of T cells stimulated by CH2811hG1 in vitro. i) Representative cytokine / chemokine expression levels (pg / ml) in T cells restimulated by CH2811hG1 and ii) anti-CD3 / CD28. T cells were stimulated with CH2811 on day 0, and then restimulated with CH2811hG1 on days 33 and 64 or with anti-CD3 / CD28 antibodies on day 33. Supernatants were collected and the concentrations (pg / ml) of IFNγ, IL-10, IL-17A, IL-2, IL-21, IL-5, IL-7, IL-8 and TNF-α were assessed at days 7, 11, 14, 39, 54 and 70. Triangles and arrows depict the restimulation days of 2811 and CD3 / CD28 antibodies, respectively.

[0183] Fig.16 Expression of SSEA-4 on mouse immune cells.

[0184] HHDII / DP4 mice were euthanized and spleen, mesenteric lymph nodes, and inguinal lymph nodes were harvested. i) Splenocytes, ii) mesenteric lymph node cells, and iii) inguinal lymph node cells were stained with FITC-labeled CH2811hG1 antibody and evaluated using flow cytometric analysis.

[0185] Fig.17 : FG2811mG1 induces phenotypic T in C57 / B6 mice SCM cell.

[0186] (A) On day 16, the total cell number of splenocytes from groups A and B was counted using trypan blue exclusion analysis;

[0187] (B) On day 16, splenocytes from individual mice in groups A and B were stained with CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies and evaluated using flow cytometric analysis;

[0188] (C) On day 16, splenocytes from groups A and B were cultured with (A+2811 and B+2811) or without (A-2811 and B-2811) plate-bound FG2811mG1 (5 μg / ml) and harvested on day 24. Splenocytes from day 24 were stained with CD3, CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies and evaluated using flow cytometric analysis;

[0189] (D) On days 24, 27, and 30, A+2811 splenocytes were harvested and stained with anti-CD3, CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 and evaluated using flow cytometric analysis.

[0190] Figure 18: T cells from HHDII and HHDII / DP4 mice SCM Direct ex vivo phenotyping of cells

[0191] HHDII and HHDII / DP4 naive mice were sacrificed, and splenocytes were harvested and stained with CD3, CD44, CD62L, SCA-1, and CH2811hG2-PeCy7 antibodies and evaluated using flow cytometric analysis.

[0192] (A) Representative flow cytometric images of directly ex vivo stained splenocytes from HHDII mice.

[0193] (B) Summary of phenotyping results of splenocytes isolated from HHDII mice.

[0194] (C) Representative flow cytometry images of directly ex vivo stained splenocytes from HHDII / DP4 mice.

[0195] (D) Summary of phenotyping results of splenocytes isolated from HHDII / DP4 mice.

[0196] (E) Summary of phenotyping results of CD4 and CD8 T cells isolated from HHDII / DP4 mice.

[0197] FIG. 19 : Mouse splenocytes proliferate in response to plate-bound FG2811mG1 and FG2811hG1

[0198] HHDII unsensitized mice were sacrificed, spleen cells were harvested, pan T cells were enriched and CFSE-labeled. CFSE-labeled T cells were then plated in wells containing plate-bound 2811 mouse IgG1 (5ug / ml) or human IgG1 (5ug / ml) or anti-CD3 (1ug / ml) and incubated at 37°C. On days 7, 12, and 14, cells were sampled and stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry.

[0199] (A) Representative flow cytometry images of T cells proliferating in response to FG2811mG1 and FG2811hG1 at day 12.

[0200] (B) Total percentage of cells proliferating on day 12 in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3.

[0201] (C) Total percentage of CD8 T cells proliferating on day 12 in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3.

[0202] (D) Total percentage of CD4 T cells proliferating on day 12 in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3.

[0203] Figure 20: Anti-CD3 and CD28 induce ex vivo proliferation of cells with stem cell-like properties from HHDII-naive mice, driving expansion of effector memory cells

[0204] HHDII unsensitized mice were sacrificed, spleen cells were harvested, pan T cells were enriched and CFSE-labeled. CFSE-labeled T cells were then plated in wells containing anti-CD3 and anti-CD28 (1 ug / ml each) and incubated at 37°C. On days 7, 12 and 14, cells were sampled and stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry.

[0205] (A) On days 11, 15, and 20, cells were harvested and stained with CH2811hG2-PeCy7 and / or anti-CD3. (i) CD3 + Cell 2811 + Cell percentage, (ii) CD3 + Low percentage of CSFE in cells, (iii) 2811 + The number of cells (×10 4 / mL)(iv)CD3 + The number of T cells (×10 5 / mL, (n=2 wells).

[0206] (B) Representative flow cytometry images of splenocytes stained 11 days after CD3 / CD28 stimulation, cells were stained with anti-CD3, CD44, CD62L and CH2811hG2-PeCy7 and evaluated using flow cytometric analysis.

[0207] (C) Eleven days after CD3 / CD28 stimulation, 2811 + Total number of effector memory T cells, central memory T cells, effector T cells and naive T cells (n=2 wells).

[0208] (D) Eleven days after CD3 / CD28 stimulation, 2811 + Percentages of effector memory T cells, central memory T cells, effector T cells, and naive T cells (n=2 wells).

[0209] Figure 21: Human 2811hG2 and mouse 2811mG1 induce ex vivo proliferation of cells with stem cell-like properties from HHDII / DP4-naive mice, driving expansion of effector memory cells

[0210] HHDII / DP4 unsensitized mice were sacrificed, spleen cells were harvested, pan T cells were enriched and CFSE-labeled. CFSE-labeled T cells were then plated in wells containing soluble human IgG2 (5ug / mL) or mouse IgG1 2811 Ab (5ug / mL) or anti-CD3 (1μg / ml) and CD28 stimulation in the presence or absence of AKTi, and the cells were incubated at 37°C. On days 11 and 15, cells were sampled and stained with anti-CD3, CD44, CD62L, SCA1 and CH2811hG2-PeCy7 and evaluated using flow cytometric analysis.

[0211] (A) Representative flow cytometry images of T cells proliferating in response to FG2811mG1 and 2811hG2 (n=2 wells) on day 11.

[0212] (B) Eleven days after stimulation with CD3 / CD28, FG2811mG1, or 2811hG2, the expression of 2811 + Total number of effector memory T cells, central memory T cells, effector T cells and naive T cells (n=2 wells).

[0213] (C) Eleven days after stimulation with CD3 / CD28, FG2811mG1, or 2811hG2, the expression of 2811 + Percentages of effector memory T cells, central memory T cells, effector T cells, and naive T cells (n=2 wells).

[0214] Figure 22: Anti-CD3 and CD28 induce 2811 isolated from healthy donors + In vitro proliferation of cells

[0215] PBMCs were isolated from buffy coats, pan-T cell enrichment was performed, and approximately 2 × 10 cells / well of a 24-well plate were incubated in the presence of anti-CD3 / CD28 with or without additional cytokines (IL-7 or IL-21). 6 On days 15 and 20, cells were sampled and stained with CD45RA, CD62L, CD122, CD95, CD3, CCR7, and 2811hGPe-Cy7.

[0216] (A) Representative flow cytometry plots phenotyping T cells proliferating in response to anti-CD3 / CD28 stimulation on day 20 (n=2 wells).

[0217] (B) 15 and 20 days after CD3 / CD28 stimulation, (i) 2811 + CD3 + Percentage of T cells, (ii) 2811 + The total number of cells (×10 4 / mL, n = 2 wells).

[0218] Fig.23 : T cells stimulated with anti-CD3 / CD28 SCM The frequency of cells increased

[0219] PBMCs were isolated from buffy coats and enriched for pan-T cells. Approximately 2 × 10 cells / well of a 24-well plate were incubated in the presence of anti-CD3 / CD28. 6 On days 15 and 20, cells were collected and analyzed according to CD3 + CD45RA + CCR7 + CD95 + CD122 低 Expression of T SCM CD3 T cell population T SCM Percentage of cells (i), 2811 + T SCM The percentage of cells (ii), T SCM 2811 + 、CD3 + Percentage of cells (iii).

[0220] Figure 24: Soluble FG2811mG1 can stimulate mouse T cells through Fc cross-linking

[0221] Splenocytes were isolated from HHDII and HHDII / FDP4 mice, and pan T cells were enriched from splenocytes harvested from HHDII mice. HHDII pan T cells and HHDII / DP4 splenocytes were CFSE labeled. CFSE-labeled T cells were cultured alone or mixed with HHDII / DP4 splenocytes at a 1:1 ratio in the presence of soluble FG2811mG1, and controls included medium only (negative) and LPS (positive control).

[0222] (A)(i) Representative flow cytometry plots of T cells cultured in the presence of FG2811mG1, LPS, or medium alone on day 15. Proliferating (CSFE low) and non-proliferating (CFSE 高 )cell.

[0223] (ii) Representative flow cytometry plots of T cells cultured with splenocytes in the presence of FG2811mG1, LPS, or medium alone on day 15. Proliferating (CSFE low) and non-proliferating (CFSE 高 )cell.

[0224] (B) Summary of the proliferative responses of CD4 and CD8 T cells when cultured with or without splenocytes in the presence of FG2811mG1, LPS, or medium alone. DETAILED DESCRIPTION

[0225] method

[0226] Plasma membrane glycolipid extraction

[0227] SSEA-3 / -4-LMTK cell aggregates (5×10 7 Cells) were resuspended in 500 μl of mannitol / HEPES buffer (50 mM mannitol, 5 mM HEPES, pH 7.2, all Sigma) and passed through 3 needles (23G, 25G, 27G), 30 times each. 5 μl of 1M CaCl2 was added to the cells and passed through 3 needles, 30 times each, as described above. The sheared cells were incubated on ice for 20 minutes and then spun at 3,000g for 15 minutes at room temperature. The supernatant was collected and spun at 48,000g for 30 minutes at 4°C and the supernatant was discarded. The pellet was resuspended in 1 ml of methanol and then in 1 ml of chloroform and rolled and incubated at room temperature for 30 minutes. The sample was then spun at 1,200g for 10 minutes to remove precipitated proteins. The supernatant containing plasma membrane glycolipids was collected and stored at -20°C.

[0228] Liposome preparation

[0229] SSEA-3 / -4-LMTK plasma membrane (pm) glycolipid extract (5×10 7 Cell equivalents) were mixed with lipids [cholesterol, dicetyl phosphate (DCP), phosphatidylcholine (PC), and α-GalCer] at a total concentration of 10 mgs in a round-bottom flask at different ratios (Table 2). The lipid mixture was then dried using a rotary evaporator at 60°C until the solvent evaporated, leaving a uniform lipid film on the wall of the flask. The flask was allowed to cool to room temperature, and then 100 μl of sterile PBS was added. The opening of the flask was covered with a parafilm and then immersed in an ultrasonic bath for 10 minutes to generate liposomes. (All work with chloroform and methanol was performed in a fume hood).

[0230] Immunization regimen

[0231] BALB / c mice were 6 to 8 weeks old (Charles River, UK). Normal mouse serum (NMS) was collected by tail bleeding before immunization as a negative control and stored at -20°C. SSEA-3 / -4-LMTK cells (1×10 per mouse per immunization) were used at two-week intervals using a 1 ml insulin syringe (BD Bioscience, Spain). 6 Mice were immunized intraperitoneally (ip) with SSEA-3 / -4-LMTK cells (1×10 cells per mouse per immunization). Antisera were collected 7 days after the second immunization and every 7 days thereafter by tail bleeding and screened for IgG and IgM antibody responses. Once a high titer IgG response was obtained, SSEA-3 / -4-LMTK cells (1×10 cells per mouse per immunization) were used to screen for IgG and IgM antibody responses. 5 Animals were boosted intravenously (iv) with 100 cells per 100 mL dose and sacrificed 5 days later.

[0232] mAb generation

[0233] Isolation of splenocytes - euthanize mice and remove spleens. After washing with 5 ml serum-free medium (RPMI 1640) using a 25-gauge needle, gently stir the spleen with sterile tweezers to harvest splenocytes. 5 ml splenocytes are collected in a sterile 25 ml universal tube while discarding excess fat and connective tissue. The total liquid volume containing splenocytes is increased to 25 ml with serum-free medium (RPMI 1640) and centrifuged at 100 g for 10 minutes. Remove the supernatant, leaving 1 ml of culture medium and splenocytes, then resuspend the splenocytes in 5 ml serum-free medium (RPMI1640) and count them using a hemocytometer by trypan blue staining to assess viability.

[0234] Fusion of spleen cells with NS0 myeloma cells: Washed spleen cells were mixed with healthy NS0 myeloma cells at a ratio of 1:10 (NS0: spleen cells; 1×10 7 ∶1×10 8The ratio of 100 cells) was mixed in a 25 ml universal tube and centrifuged at 317 g for 5 minutes. The supernatant was aspirated and the combined cell pellet was gently and gradually resuspended in 800 μl polyethylene glycol (PEG) within 1 minute. The cell mixture was gently stirred for 1 minute, and then 1 ml of serum-free medium (RPMI1640) was added within 1 minute while continuing to stir. 20 ml of serum-free medium (RPMI1640) was added within 1 minute while continuing to stir. The cell mixture was then centrifuged at 317 g for 5 minutes, the supernatant was removed and the cell mixture was resuspended in 15 ml of hybridoma culture medium [500 ml of hybridoma serum-free medium (Gibco): 10 ml of HT (hypoxanthine thymidine) supplement (50×Hybri-Max; Sigma): 31 μl (31 μg) methotrexate (1 mg / ml; Sigma): 25 ml of hybridoma cloning factor (Opti-CloneII; MP): 50 ml of filtered NS0 spent culture medium]. The cell suspension was evenly distributed on a 96-well flat-bottom plate and incubated at 37°C in a cell culture incubator (5% CO2).

[0235] CH2811hG1 generation

[0236] Trizol (Invitrogen, Paisley, UK) was used according to the manufacturer's protocol with 5 × 10 6 Total RNA was prepared from 100 FG2811 hybridoma cells. First-strand cDNA was prepared from 3 μg of total RNA using a first-strand cDNA synthesis kit and AMV reverse transcriptase according to the manufacturer's protocol (Roche Diagnostic). PCR and sequencing of the heavy and light chain variable regions were performed by Syd Labs, Inc (Natick, MA 01760, USA), and variable region family usage was analyzed using the IMGT database (Lefranc et al. 2018). The FG2811 variable region was then cloned into the hIgG1 / kappa dual expression vector pDCOrig-hIgG1 (Metheringham et al. 2009), and the sequence was confirmed by sequencing.

[0237] Monoclonal Antibody Characterization

[0238] mAb isotyping Spent hybridoma serum-free medium (Invitrogen Scotland, UK) was collected and 150 μl was diluted 1 / 10 in PBS 1% (w / v) BSA and then pipetted into a development tube of a mouse mAb isotyping test kit (AbD Serotec, Kidlington, UK) and incubated at room temperature for 30 seconds. The test tube was vortexed briefly to ensure complete resuspension of the colored microparticle solution. An isotyping strip was placed in the test tube with the solid red end of the strip at the bottom of the test tube for 5 to 10 minutes. The results were interpreted by checking that the blue band appeared in one of the class or subclass windows and above the letters in the K or λ window of the strip (indicating the heavy and light chain composition of the mAb).

[0239] Mouse mAb Purification 2 liters of spent hybridoma serum-free medium (Invitrogen Scotland, UK) were collected and 0.2% sodium azide (Sigma) was added. The spent medium was then filtered through Whatman paper and then filtered using a 0.2 μmsteritop filter (Sigma). According to the manufacturer's recommendations, HiTrap protein G HP antibody purification columns (GE Healthcare) were used for purification. The mAb binding buffer consisted of PBS-Tris pH 7.0, and mAbs were eluted using Tris-Glycine pH 12.0. Fractions containing IgG mAbs were combined, the pH was neutralized using 10 M HCl, and dialyzed overnight through PBS, then aliquoted and stored at -80 °C.

[0240] Transient mAb Production Using ExpiFectamine TM 293 Transfection Kit (Gibco, Life Technologies) transient transfection Expi293 TM HEK293 cells in suspension (100 ml, 2 × 10 6 cells / ml), and the conditioned medium was harvested on day 7 after transfection.

[0241] Tumor cell lines

[0242] Cell lines were maintained by regular replacement of complete medium and splitting to maintain logarithmic growth. All cell lines were regularly checked for mycoplasma contamination and verified using short tandem repeat (STR) analysis (Table 1).

[0243]

[0244]

[0245] Table 1: Cancer cell lines GBM: Glioblastoma multiforme

[0246] Antibodies that bind to cancer cell lines and mouse fibroblasts.

[0247] 1×10 5 Cells were incubated with 50 μl of primary antibody (various concentrations) at 4 ° C for 1 hour. The cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 minutes. The supernatant was discarded and FITC-conjugated anti-mouse / anti-human IgG / IgM Fc specific antibodies or biotin-conjugated anti-mouse / anti-human IgG / IgMFc specific antibodies (Sigma) diluted 1 / 100 in RPMI 10% FCS were used as secondary antibodies. The cells were incubated at 4 ° C in the dark for 1 hour. The cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 minutes. Biotinylated secondary antibodies were detected using 50M1 streptavidin-FITC (Sigma) or Strep-PeCy7 (eBioscience) diluted 1 / 100 in RPMI 10% FCS. The cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 minutes. Cells were fixed with 0.4% formaldehyde and analyzed on a Beckman Coulter Fc-500 flow cytometer (Beckman Coulter, High Wycombe, UK) or a MACSQ flow cytometer (Miltenyi Biotech, Bisley, UK).

[0248] Antibodies bind to whole blood

[0249] 50 μl of healthy donor whole blood was incubated with 50 μl of primary antibody at 4 ° C for 1 hour. The blood was washed with 150 μl of RPMI 10% NBCS and spun at 100g for 5 minutes. The supernatant was discarded and 50 μl of FITC-conjugated anti-mouse / anti-human IgG Fc specific antibody or biotin-conjugated anti-mouse / anti-human IgG Fc specific antibody (Sigma; 1 / 100 in RPMI 10% NBCS) was used as a secondary antibody. The cells were incubated at 4 ° C in the dark for 1 hour, then washed with 150 μl of RPMI 10% NBCS and spun at 100g for 5 minutes. 50 μl of streptavidin-FITC (Sigma; 1 / 100 in RPMI 10% NBCS) or streptavidin-PE-Cy7 (eBioscience; 1 / 100 in RPMI 10% NBCS) was used to detect the biotinylated secondary antibody. Cells were incubated at 4°C in the dark for 1 hour, then washed with 200 μl of RPMI 10% NBCS and spun at 100 g for 5 minutes. After discarding the supernatant, 50 μl / well Cal-Lyse (Invitrogen, Paisley, UK) was used, followed by 500 μl / well distilled water to lyse the red blood cells. The blood was then spun at 100 g for 5 minutes, the supernatant was discarded and the cells were resuspended in 500 μl PBS. Samples were analyzed on a FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

[0250] TLC analysis of glycolipid binding

[0251] LMTK and SSEA-3 / -4-LMTK plasma membrane lipid samples were blotted onto silica gel plates and developed twice in chloroform (Sigma) / methanol (Sigma) / distilled water (60:30:5, volume ratio), then twice in hexane (Sigma): ether (Sigma): acetic acid (Sigma) (80:20:1.5, volume ratio). The dried plates were sprayed with 0.1% polyisobutyl methacrylate (Sigma) in acetone. After air drying, the plates were blocked with PBS 2% (w / v) BSA for 1 hour at room temperature and incubated with primary antibodies diluted in PBS 2% (w / v) BSA at 4°C overnight. The plates were then washed 3 times with PBS and incubated with a biotin-conjugated anti-mouse IgG Fc-specific secondary antibody (Sigma) diluted 1 / 1000 in PBS 2% (w / v) BSA for 1 hour at room temperature. The plates were then washed again in PBS and then incubated with IRDye 800CW Streptavidin (LICOR Biosciences, Cambridge, UK) diluted 1 / 1000 in PBS 2% (w / v) BSA for 1 hour at room temperature in the dark. The plates were then washed 3 more times with PBS and air-dried in the dark. Lipid bands were visualized using a LICOR Odyssey scanner.

[0252] Glycan (coupled with HSA) ELISA

[0253] ELISA plates (Becton Dickinson, Oxford, UK) were coated overnight at 4°C with 100 ng / well of SSEA-3, SSEA-4, Forssman, Globo-H and sialyl-Lewis x (SLex) polysaccharide-HSA conjugates resuspended in PBS (Elicityl, Crolles, France), blocked with 200 μl / well of PBS 5% (w / v) BSA for 1 hour at room temperature, and then incubated with 50 μl / well of primary antibody (5 μg / ml). Primary antibodies were detected using biotinylated anti-mouse IgG or anti-rat IgM secondary antibodies (Sigma) diluted 1 / 5000 in PBS 1% (w / v) BSA. After incubation with streptavidin horseradish peroxidase (HRPO) conjugate (Invitrogen) diluted 1 / 5000 in PBS 1% (w / v) BSA and development with 3,3',5,5'-tetramethylbenzidine (TMB; Sigma), the plates were read at 450 nm using a Tecan Infinite F50.

[0254] Erythrocyte binding assay

[0255] Healthy donor erythrocytes were washed three times in PBS and resuspended in PBS with 10 times the packed cell volume. Then 50 μl of washed erythrocytes were incubated with 50 μl of primary antibody at 37 ° C for 1 hour. The cells were washed with 150 μl of PBS and spun at 100 g for 5 minutes. The supernatant was discarded and the cells were resuspended in 50 μl of FITC-conjugated anti-mouse IgG Fc specific secondary antibody (Sigma) diluted 1 / 100 in PBS 1% (w / v) BSA. The cells were incubated at 37 ° C in the dark for 1 hour, then washed with 150 μl of PBS and spun at 100 g for 5 minutes. The supernatant was discarded and the cells were resuspended in 500 μl of PBS. The samples were analyzed by FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

[0256] Red blood cell hemagglutination assay

[0257] 4 ml of normal donor whole blood was collected into a heparin tube (Becton Dickinson). The whole blood was transferred to a 15 ml sterile conical tube and washed with sterile PBS. The washed blood was centrifuged at 100 g for 5 minutes. The supernatant was aspirated. The washing step was repeated twice. After the last wash, the blood cell pellet was diluted with sterile PBS to prepare a final working concentration of 0.5% red blood cells. 50 μl of 0.5% red blood cells were added to each well of a U-bottom 96-well plate. On top of the red blood cells, the primary antibody was added at 50 μl / well and incubated at room temperature for 1 hour or until the red blood cells agglutinated.

[0258] Monoclonal antibody affinity analysis

[0259] The kinetic parameters of FG2811mG3 mAb binding to liposomes containing SSEA-4 were determined by surface plasmon resonance (SPR, Biacore 3000, GE Healthcare). L1 sensor chips (GE-healthcare) were pretreated with 40mM octyl D-glucoside, then coated with liposomes containing SSEA-4 (6000RU) and subjected to short pulses of NaOH (10mM) to remove loosely bound liposomes. The reference flow cell was treated in the same manner except that liposomes without SSEA-4 were used. Since the injection of HSA (0.1mg / ml) induced a slight increase in RU (50RU to 60RU), the coverage of both flow cells was nearly complete. After the signal of the two flow cells stabilized, FG2811mG3 mAb with increasing concentrations (0.3nmol / L to 200nmol / L) was injected and then regenerated (10mM glycine, pH1.5) after circulation. Binding curves were fit to a 1 :1 (Langmuir) binding model using BIAevaluation 4.1.

[0260] Glycomic analysis of FG2811mG3 (Functional Glycomics Consortium)

[0261] To determine the fine specificity of the FG2811mG3 antibody, the antibody was FITC labeled and sent to the Functional Glycomics Consortium, where it was screened against ≥600 natural and synthetic glycans (Core H Group, Version 5.1). Synthetic glycans and mammalian glycans with amino linkers were printed onto glass microscope slides activated with N-hydroxysuccinimide (NHS) to form amide bonds. The printed slides were incubated with 5 μg / ml of the antibody at room temperature for 1 hour, and then binding was detected with Alexa488-conjugated goat anti-mouse IgG. The slides were then dried, scanned, and the screening data was compared with the Functional Glycomics Consortium database.

[0262] CSFE T cell proliferation assay

[0263] PBMC isolation

[0264] Whole blood (buffy coat) was obtained from the National Blood Service (Sheffield) or collected from healthy donors in a syringe containing lithium heparin (1000 units / ml; Sigma H0878). Whole blood was diluted 1:1 with RPMI 1640 and layered on lymphocyte separation medium (Histopaque-1077; Sigma) and then centrifuged at 800 g with brake off for 25 minutes. After centrifugation, plasma was collected from the top layer and PBMCs were collected from the buffy coat. PBMCs were washed twice with RPMI 1640 and spun at 317 g for 5 minutes. The number of PBMCs was counted and the cells were ready for naive T cell isolation.

[0265] Pure T cell isolation

[0266] Each 1×10 7 PBMCs were resuspended in 40 μl of cold MACS buffer [PBS 1% (v / v) FCS 1% (v / v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665; 0.5 M pH 8 EDTA: Invitrogen). 7 Add 10 μl of pan-T cell biotin antibody (Miltenyi) to each 1×10 cells and incubate at 4°C in the dark for 5 minutes. 7 Add 30 μl of cold MACS buffer to each 1×10 cells. 720 μl of pan-T cell microbeads (Miltenyi) were added to each cell and incubated at 4°C in the dark for 10 minutes. The cells were added to an LS column (Miltenyi) and the flow-through containing CD3 purified T cells was collected. The cells retained in the column are non-T cells.

[0267] CSFE loading

[0268] Wash the purified T cells with RPMI1640 and count the number of cells. Spin the cells at 317 g for 5 minutes and remove the supernatant. 7 The purified T cells were resuspended in 1 ml of PBS 10% FCS. CSFE was dissolved in 18 μl DMSO (Invitrogen) and then dissolved in 1.8 ml of PBS 10% FCS. Then, 1×10 7 110 μl of diluted CSFE was added to each T cell and incubated at room temperature in the dark for 5 min. The CSFE-loaded cells were washed with PBS 10% FCS and then incubated at 1×10 6 Cells (2 × 10 cells / ml in 2 mL) were resuspended in complete medium (RPM1640 2% (v / v) Hepes 1% (v / v) L-glutamine 1% (v / v) penicillin-streptomycin) 10% donor plasma. 6 ) were added to each well of a 24-well plate pre-coated with CH2811hG1 antibody (5μg / ml), FG2811mG1 (5μg / ml), CH2811hG2 antibody (5μg / ml) or containing anti-CD3 antibody (OKT-3; 0.005μg / ml), anti-CD3eAb (1ug / ml, eBioscience, 16-0031-85) and anti-CD28Ab (1ug / ml eBioscience16-0281-85) or culture medium alone. On days 7, 11 and 14, cells were harvested and stained with relevant antibodies against CD3 (eBioscience, 17-0031), SCA-1 (Miltenyi, 130-102-343), CD62L (Miltenyi, 130-102-543), CD44 (Miltenyi, 130-116-495), anti-CD4-APC-780 (eBioscience 47-0049), anti-CD8-VioGreen (Miltenyi, 130-102-805, Tim3-PE (eBioscience, 130-118-563) or with CH2811hG2-PeCy7 (in-house, diluted 1:50), and then analyzed using a MACSQ flow cytometer (MACSQUANT analyzer 10).

[0269] Luminex [Milliplex Map Kit Human High Sensitivity T-cell Magnetic Bead Panel (96-well plate assay)]

[0270] According to the manufacturer's recommendation, the determination of 9-well format is carried out on the filter plate. In total, 200 μl of wash buffer is added to each hole of the 96-well filter plate (Millipore). The plate is sealed and mixed at room temperature for 10 minutes on a plate shaker. Wash buffer is removed by flipping the plate and patting on a paper towel. Then 25 μl of each standard, control and sample (culture supernatant of CSFE proliferation assay) are added to each hole, 25 μl of serum matrix is ​​added to each standard and control well, and 25 μl of assay buffer is added to each sample well. Use immediately after vortexing the working bead mixture. Next, 25 μl of mixed beads are added to each hole. The plate is then sealed, wrapped with aluminum foil, and incubated for 16 to 18 hours at 4 ° C with stirring on a plate shaker (500 rpm to 800 rpm). After incubation, the plate is placed on a handheld magnet for 60 seconds, and then the liquid is removed from the plate by inverting the plate and patting on a paper towel. The plate is washed twice with 200 μl of wash buffer each time. After the second wash, the bottom of the plate was dried by patting on a paper towel, and 25 μl of detection antibody was added to each well. The plate was then sealed, wrapped with aluminum foil, and incubated for 1 hour at room temperature on a plate shaker. Next, 25 μl of streptavidin-phycoerythrin was added to each well containing 25 μl of detection antibody. The plate was shaken for another 30 minutes at room temperature. After incubation, the plate was placed on a handheld magnet for 60 seconds, and then the liquid was removed from the plate by inverting the plate and patting on a paper towel. The plate was washed twice with 200 μl of wash buffer each time. Then 150 μl of sheath fluid (Luminex) was added to each well. The beads were resuspended on a plate shaker for 5 minutes and then read on a Bio-Plex 3D instrument (Bio-Rad, Hercules, CA). The instrument was set to collect at least 50 beads for each analyte. The raw data was measured as mean fluorescence intensity (MFI).

[0271] Naive T cell isolation

[0272] Whole blood was collected from a normal donor in a syringe containing lithium heparin (1000 units / ml; Sigma H0878). Whole blood was diluted 1:1 with RPMI 1640 and layered on lymphocyte separation medium (Histopaque-1077; Sigma), then centrifuged at 800g with brake off for 25 minutes. After centrifugation, plasma was collected from the top layer and PBMCs were collected from the buffy coat. PBMCs were washed twice with RPMI 1640 and spun at 317g for 5 minutes. The number of PBMCs was counted and the cells were ready for naive T cell isolation. Each 1×10 7 1×10 PBMC were resuspended in 40 μl of cold MACS buffer [PBS 1% (v / v) FCS 1% (v / v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665; 0.5 M pH 8 EDTA: Invitrogen). 7 Add 10 μl of naive pan-T cell biotin antibody (Miltenyi) to each 1×10 cells and incubate at 4°C in the dark for 5 minutes. 7 Add 30 μl of cold MACS buffer to each 1×10 cells. 7 Add 20 μl of naive pan T cell beads (Miltenyi) to each cell and incubate at 4°C in the dark for 10 minutes. Add the cells to an LS column (Miltenyi) and collect the flow-through containing CD3 purified T cells. The cells retained in the column are non-T cells. Stain naive T cells with CH2811hG1 or CD95 / CD122 antibody combination for 30 minutes. Wash the cells with MACS buffer and perform cell sorting. CH2811hG1 + and CD95 / CD122 + Cells were sorted into RNAProtect (QIAGEN) and stored at -80°C.

[0273] Sample extraction and quality control

[0274] 8 T cell samples were provided in RNA protection reagent. The entire sample volume was extracted using Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The quantity and integrity of the extracted RNA samples were evaluated using NanoDrop 8000 spectrophotometer V2.0 (Thermo Scientific, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) respectively with eukaryotic RNA Pico bioanalyzer chips. The samples showed low levels of degradation, with RNA integrity values ​​(RIN) of 7.4 to 10 and an average yield of 110 ng.

[0275] eDNA synthesis

[0276] Use sequencing v4 The Low Input RNA Kit (Clontech, Mountain View, CA, USA) was used to generate full-length cDNA molecules from 1 ng of total RNA from each sample. The quality of cDNA was determined using The cDNA was measured using an Agilent 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and quality checked using an Agilent 2200 Tapestation and a high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples showed good cDNA quality with molecular sizes ranging from 400 bp to 10,000 bp.

[0277] Library generation and RNA sequencing

[0278] Sequencing libraries were prepared using the Illumina Nextera XT sample preparation kit (Illumina Inc., Cambridge, UK) with 150 pg cDNA input per sample. A final PCR amplification was performed for 11 cycles. The final library was quantified and characterized using an Agilent 2200 Tapestation (Agilent Technologies, Waldbronn, Germany) equipped with a high-sensitivity D1000 screentape and a 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Equimolar amounts of each sample library were pooled together for sequencing using lumina Sequencing was performed using the 500 Mid-output kit to generate 75 bp paired-end reads.

[0279] Differential expression analysis

[0280] After quality check using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc), 75 bp paired-end reads were aligned to the Homo sapiens reference genome hg19 using STAR (version 2.6.1d). Default parameters were used for profiling and reads were counted using GeneCounts. Differential expression analysis (DE) was performed on 2811-enriched and similar CD95 / CD122-enriched T cells using a dataset of CD4 and CD8 naive T cells from GSE114765 (Pilipow et al. JCI insight 2018) using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple test correction to estimate FDR folds (FDR < 0.05). Common genes between the DE sets (two for CD8 and two for CD4) T cells were identified using Venny 2.1 and used as input in the StemChecker database to identify “stemness” signatures (Pinto et al. 2015).

[0281] Transcriptional analysis using RNAseq

[0282] Eight T cell samples (four of which were CH2811 + , 4 were CD122 / CD95+). The entire sample volume was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The quantity and integrity of the extracted RNA samples were assessed using a NanoDrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) in combination with a eukaryotic RNA Pico Bioanalyzer chip, respectively. The samples showed low levels of degradation, with RNA integrity numbers (RIN) ranging from 7.4 to 10 and an average yield of 110 ng. Sequencing was performed using a v4 Full-length cDNA molecules were generated from 1 ng of total RNA from each sample using the Low Input RNA Kit (Clontech, Mountain View, CA, USA). The quality of cDNA was determined using 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) was used to measure, and the quality was checked using Agilent 2200 Tapestation and high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples showed good cDNA quality, with molecular sizes ranging from 400 bp to 10,000 bp. Sequencing libraries were prepared using the Illumina Nextera XT sample preparation kit (Illumina Inc., Cambridge, UK), with 150 pg cDNA input for each sample. A final PCR amplification of 11 cycles was performed. Using The final library was quantified and qualified using an Agilent 2200 Tapestation (Agilent Technologies, Waldbronn, Germany) equipped with a high-sensitivity D1000 screentape and a 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). Equimolar amounts of each sample library were pooled together for sequencing using lumina Sequencing was performed using the 500 Mid-output kit to generate 75 bp paired-end reads. After quality checking using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc), the 75 bp paired-end reads were aligned to the Homo sapiens reference genome (Ensembl assembly GRCh37 (hg19)) using STAR (version 2.6.1d). Default parameters were used for mapping, and reads were counted using GeneCounts.

[0283] Using a dataset of CD4 and CD8 naive T cells from GSE83808 (Hosokawa et al. 2017), differential expression analysis (DE) was performed for CH2811-enriched and similar CD95 / CD122-enriched transformed profiles using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple test correction to estimate FDR folds (FDR < 0.05). Common genes between the DE groups (two for CD8 and two for CD4) T cells were identified using Venny2.1 and used as input in the StemChecker database to identify "stemness" features and overlap with gene sets associated with hematopoietic stem cells (HSC) and embryonic stem cells (ESC) (Pinto et al. 2015). The distribution of significantly enriched genes was shown by heat map analysis (https: / / software.broadinstitute.org / morpheus / ). In parallel, using http: / / bioinformatics.sdstate.edu / idep / , the profiles enriched for CH2811 and CD95 / CD122 were compared to published profiles for naive and memory CD4 and CD8 T cells (GSE23321) and activated naive CD8 T cells (GSE114765). SCM and visualization of the distribution of effector T differentiation-related genes.

[0284] Mouse studies

[0285] C57BL / 6J mice (Charles River), HHDII / HLA-DP4 (DP*0401) mice (EM: 02221, European Mouse Mutant Archive), and HHDII mice (Pasteur Institute) aged 8 to 12 weeks were used. All work was carried out under a project license approved by the Ministry of the Interior. Six mice were randomly divided into two groups (Group A and Group B), and the researchers were not blinded. On day 0, endotoxin-free FG2811mG1 mAb was immunized into Group A mice (250μg / mouse) by the intraperitoneal route (ip). Group B mice were used as non-immunized control groups. Spleens were harvested for analysis on day 16, and then spleen cells from the same group were combined and restimulated in the presence or absence of plate-bound FG2811mG1 antibodies (5μug / ml). Splenocytes were harvested from culture on days 24, 27, and 30 for analysis using anti-CD3, CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies.

[0286] Mouse tissue staining

[0287] HHDIIDP4 unsensitized mice were used. All work was performed under a project license approved by the Ministry of the Interior. Spleen, mesenteric lymph nodes, inguinal lymph nodes, bone marrow, and blood samples from unsensitized mice were collected for analysis. Tissues were incubated with CH2811hG2-PeCy7 (in-house, 1:50 dilution), anti-CD3 (eBioscience, 17-0031), SCA-1 (Miltenyi, 130-102-343), CD62L (Miltenyi, 130-102-543), CD44 (Miltenyi, 130-116-495), anti-CD4-APC-780 (eBioscience, 47-0049), anti-CD8-VioGreen (Miltenyi, 130-102-805), and Tim3-PE (eBioscience, 130-118-563).

[0288] Example

[0289] The present invention will now be further described with reference to the following examples and figures.

[0290] Example 1. Generation of FG2811.72

[0291] Generation and characterization of FG2811 mAb

[0292] BALB / c mice were immunized intraperitoneally (ip) with a cell line expressing SSEA-3 / -4 (SSEA-3 / -4-LMTK) and boosted intravenously (iv) within 3 months. This cell line was generated by transducing wild-type LMTK mouse fibroblasts with the genes for α-1-4 galactosyltransferase (A4GALT), β-1-3-N-acetylgalactosaminyltransferase (B3GALNT1), and β-1-3-galactosyltransferase (B3GALT5) (Cid et al. 2013). This cell line has endogenous sialyltransferases that add sialic acid to the termini of SSEA-3 glycans, thereby generating SSEA-4 glycans ( Figure 1 ).

[0293] To generate anti-SSEA-4 specific mAb, spleen cells from immunized mice were fused with myeloma NS0 cells. After multiple rounds of screening and limiting dilution cloning, the anti-SSEA-4 monoclonal antibody FG2811mG3 was obtained.

[0294] SSEA-3 and SSEA-4 are known to be globulin series glycolipids. To confirm that FG2811mG3 mAb recognizes cell surface glycolipid antigens on SSEA-3 / -4-LMTK cells, SSEA-3 / -4-LMTK plasma membrane lipid extracts were subjected to high performance thin layer chromatography (HPTLC) analysis and immunostaining using FG2811mG3 mAb ( Figure 4 A). MC631 (commercial anti-SSEA-3 mAb) and MC813 (commercial anti-SSEA-4 mAb) mAbs were included as comparisons, and wild-type LMTK cells were used as a negative control cell line. Both FG2811mG3 mAb and MC631mAb stained glycolipids expressed on SSEA-3 / -4-LMTK cells, but not on wild-type LMTK cells. FG2811mG3 mAb showed very specific glycolipid staining, while MC631 stained three different glycolipid antigens, indicating that MC631 mAb cross-reacts with two additional glycolipid antigens expressed on SSEA-3 / -4-LMTK cells. MC813 failed to stain glycolipids from SSEA-3 / -4-LMTK and LMTK cells, likely due to its lower affinity for the SSEA-4 antigen. Subsequent cell surface antigen binding showed that MC631 (rat IgM; Gm: 55.93) had the strongest binding to SSEA-3 / -4-LMTK cell surface antigen, followed by FG2811mG3 mAb (mouse IgG3; Gm: 20.77) and MC813 mAb (mouse IgG1; Gm: 8.77) ( Figure 4 B). Secondary antibody only (Gm: 0.34) was used as negative control. ELISA assays were then performed to screen FG2811mG3 mAb against HSA-conjugated SSEA-3, SSEA-4, Globo-H, Forssman, and sialyl Lewis x glycans ( Figure 4C). ELISA results showed that FG2811mG3mAb had SSEA-4 glycan specificity (1.2OD units) and M1 / 87mAb had Forssman glycan specificity (1.1OD units). In contrast, both MC631 and MC813 cross-reacted with other glycans. MC631mAb recognized SSEA-3 (1.0OD units), SSEA-4 (1.0OD units), and GloboH (0.5OD units) glycans. MC813mAb bound to SSEA-3 (0.8OD units), SSEA-4 (1.2OD units), and Forssman (0.7OD units). To determine the fine specificity of FG2811mG3, the Functional Glycoscience Consortium (CFG) screened against ≥600 natural and synthetic glycans, and FG2811mG3 only bound to SSEA-4 glycans, confirming its specificity for SSEA-4 ( Figure 4 D).

[0295] The binding kinetics of the FG2811mG3 mAb to the SSEA-3 / -4-LMTK plasma membrane lipid antigen were examined using surface plasmon resonance (SPR; Biacore X). Fitting of the binding curve revealed that the apparent functional affinity of the 2811 mAb was strong (K d ~2×10 -9 M) and fast association rate (~10 5 1 / Ms) and a slow dissociation rate (~10 -4 1 / s)( Figure 5 A). This is consistent with the EC of SSEA-3 / -4-LMTK plasma membrane lipid ELISA 50 Value (EC 50 =6.8×10 -10 M)( Figure 5 B) and cell function affinity (K d =5×10 -9 M) consistent ( Figure 5 C).

[0296] Example 2. FG2811 antibody sequence

[0297] DNA sequencing revealed that FG2811mG3 mAb belongs to the IGHV2-3*01 heavy chain and IGKV4-63*01 light gene families ( Figure 2 A and B). Mutational assessment revealed 9 nucleotide differences between the FG2811 heavy chain and the germline sequence, resulting in 5 changes in amino acid residues. Similarly, there were 7 nucleotide differences between the FG2811 kappa chain and the germline sequence, resulting in 5 changes in amino acid residues. The nature and pattern of mutations suggest somatic hypermutation and affinity maturation.

[0298] The FG2811 heavy and light chain variable regions were cloned into mouse IgG1, human IgG2, and IgG1 expression vectors ( Figure 2 C to E). They were transfected into HEK293 cells and the antibodies were purified on protein G. mIgG3 mAb, mIgG1 mAb, hIgG1 mAb, and hIgG2 mAb bound to the SSEA-3 / -4LMTK cell line ( Figure 3 ).

[0299] Example 3. Binding of 2811 to a panel of human cancer cell lines

[0300] SSEA-4 has been reported to be overexpressed on glioblastoma cancer cell lines, as demonstrated by MC813 mAb. Therefore, SSEA-4 expression was assessed on a panel of brain cancer cell lines using 5 μg / ml of FG2811mG3 mAb and MC813 mAb by flow cytometry analysis ( Fig. 6A ). Mouse IgG3 κ isotype control and medium only (no primary antibody) were used as negative controls. Cancer cell lines U251 and U87 are adult GBM cells, SF188 and KNS42 are pediatric GBM cells, and UW2283 and DAOY are medulloblastoma cancer cells. FG2811mG3 bound weakly to DAOY (Gm: 50) and UW2283 (Gm: 36), but failed to bind to other cancer cell lines. In contrast, MC813 bound weakly to U251 (Gm: 27) and U87 (Gm: 38), and strongly to DAOY (Gm: 169) and UW2283 (Gm: 152); it failed to bind to KNS42 and SF188. Due to the low specificity of the MC813 antibody, this result suggests that SSEA-4 expression may be found only in the DAOY and U251 cell lines, and the expression level is low. The binding of FG2811mG3 antibody to a panel of cancer cell lines consisting of ovarian, breast, and colorectal cells was further evaluated by FACS ( Figure 6B FG2811mG3 strongly bound to SKOV3 (Gm: 203), moderately bound to T47D (Gm: 95) and MCF7 (Gm: 76), weakly bound to IGROV1 (Gm: 41) and OVCAR-5 (Gm: 87), and failed to bind to DU4475 (Gm: 26), HCC1187 (Gm: 22), Colo205 (Gm: 13) and HCT15 (Gm: 22).

[0301] Example 4. Cytotoxicity of 2811 mAb

[0302] The ability of FG2811mG3 mAb to induce tumor cell death via ADCC was investigated. Figure 7A). Human PBMCs were used as a source of effector cells, while SKOV3 and T47D cells served as target cells. After incubation at 37°C for 18 hours, the number of cells killed by FG2811mG3 mAb was measured. 50 :10 -10 M) were sensitive to FG2811mG3 mAb killing in a concentration-dependent manner, showing a maximum of 66% cell lysis. Although FG2811mG3 mAb bound to T47D, the mAb failed to induce T47D cell killing by ADCC, indicating that the killing effect was dependent on SSEA-4 expression levels. It is well known that CDC is an important mechanism involved in the elimination of tumor cells in vivo. The ability of CDC induced by FG2811mG3 mAb to kill SKOV3 and T47D cells was determined in the presence or absence of human serum as a complement source ( Figure 7 B). FG2811mG3 mAb showed SKOV3 (EC 50 :10 -9 The maximum cell lysis rate of M) cells was 48%. Similarly, FG2811mG3 failed to induce T47D cell killing by CDC. To investigate whether FG2811mG3 mAb can induce direct killing of tumor cells, PI uptake assay was performed on SSEA-3 / -4-LMTK and SKOV3 cells using 30 μg / ml of FG2811mG3 mAb ( Figure 7 C). Hydrogen peroxide and medium alone were included as positive and negative controls, respectively. FG2811mG3 mAb induced 74.7% PI uptake on SSEA-3 / -4-LMTK and weaker induction on SKOV-3 cells; 28.6% PI uptake was induced. To confirm that the PI assay truly reflects cell death in growing cells, cell viability of SSEA-3 / -4-LMTK and SKOV3 cells treated with 30 μg / ml of FG2811mG3 mAb was assessed under light microscopy ( Figure 7 D). LMTK wild-type cells and cells treated with medium (RPMI) alone were used as negative controls. Following addition of FG2811mG3 mAb, SSEA-3 / -4-LMTK cells were observed to aggregate within seconds. However, this phenomenon did not occur when SKOV3 and LMTK cells were incubated with FG2811mG3 mAb. FG2811mG3-treated SSEA-3 / -4-LMTK and SKOV3 cells showed evidence of growth inhibition 72 hours after mAb addition. FG2811mG3 mAb had no effect on LMTK cells. Cells incubated with medium alone showed no growth inhibition and reached 100% confluence within the 72-hour incubation period, with some cell death.

[0303] Example 5. 2811 staining of erythrocytes

[0304] SSEA-3 and SSEA-4 are reported to be expressed in most human erythrocytes. Therefore, the binding of FG2811mG3 mAb to erythrocytes from five donors was evaluated by flow cytometry over a range of concentrations (10 μg / ml). Fig. 8A ). Anti-CD55 mAb (791T / 36; 10 μg / ml; Gm: 202) was included as a positive control, and IgG isotype control and PBS were used as negative controls. FG2811mG3 (Gm: 10) did not bind to erythrocytes from all 5 donors. The erythrocyte agglutination assay further confirmed that FG2811mG3 mAb (0.625 μg / ml to 10 μg / ml) did not agglutinate erythrocytes from 5 donors. In contrast, 791T / 36 mAb and antiserum antibodies agglutinated erythrocytes from all donors. PBS was used as a negative control ( Figure 8B ).

[0305] Example 6. 2811 and stem memory T cells (T SCM )Combined

[0306] T SCM The discovery of T cells and the fact that SSEA-4 is a stem cell marker led us to hypothesize that 2811 mAb could recognize T SCM Cells. Whole blood samples were collected from seven healthy donors (BD3, BD13, BD18, BD27, BD38, BD96, BD31) and stained with FG2811mG1 mAb ( Fig.9A ). MC813 mAb was included as a comparison; mouse IgG1 isotype control antibody and secondary antibody only (no primary antibody) were used as negative controls; 198 antibody (anti-CEACAM6) and OKT3 antibody (anti-CD3) were used as positive control antibodies against granulocytes and PBMCs, respectively. FG2811mG1 mAb stained a small population of peripheral blood mononuclear cells (PBMCs), ranging from 0.8% to 2.3% in 7 healthy donors. MC813 mAb, which recognizes SSEA-3, SSEA-4, and Forssman antigens, did not stain any blood cells in the 7 donors. 198 mAb stained granulocytes, and OKT3 mAb stained CD3 + T cell staining. Secondary antibody and medium alone did not show cell staining. Next, to investigate these 2811 + Are PBMCs T SCMPBMCs were collected from two healthy donors and co-stained with FG2811, CD3, CD122, CD45RA, CD45RO, CD62L, and CD95 antibodies and analyzed by multiparameter flow cytometry ( Fig. 9B , Table 2). First, CD3 + Total T cells were then identified, 2811 + Of the two donors, 2811 + The frequency of cells ranged from 0.32% to 0.41%. + 2811 + The expression of CD45RA and CD45RO markers was analyzed in the CD3 + 2811 + CD45RA + (37.5% to 38.6%), CD45RO + (38% to 47.8%) and CD45RA + RO + Finally, the CD45RA + 、CD45RO + and CD45RA + RO + Expression of CD62L, CD95 and CCR-7 in the population. + (88.7% to 90%), CD45RA + RO + (79.5% to 89.6%) and CD45RO + (64.5% to 67.1%) Most of them are CD62L + ; 27.6% to 59.7% CD45RA + , 29.5% to 85.7% CD45RA + RO + and 81.2% to 86.1% CD45RO + CD95 + ; 56.1% to 78.9% CD45RA + , 51.4% to 78.1% CD45RA + RO + and 53.7% to 61.2% CD45RO + CCR-7 + These results suggest that 2811 / CD45RA + T cells SCM Cells, while 2811 / CD45RA + RO +The cells may be activated T SCM ,2811 / CD45RO + The cells may be activated T SCM or T CM cell.

[0307]

[0308] Table 2. PBMC phenotype. PBMC were isolated from two healthy donors (BD13 and BD38) and stained with a panel of antibodies (CD3, FG2811, CD45RA, CD45RO, CD62L, CD95 and CCR7). The phenotype of PBMC was determined using flow cytometry and the results are expressed as the percentage of positive cells.

[0309] In the hierarchical model of human T cell differentiation, after antigen sensitization, naive T cells (T N ) gradually differentiate into stem memory T cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), which eventually differentiate into terminally differentiated effector T cells (T TE / TEMRA ). These T cell subsets are distinguished by the combined expression of different markers (Table 3) (Gattinoni et al. 2017).

[0310]

[0311] Table 3: Hierarchical model of human T cell differentiation.

[0312] Example 7. RNA sequencing of 2811 positive T cells

[0313] By transcriptome analysis, we investigated the putative T SCM Cells (Gattinoni et al. 2017) and 2811 + The degree of correlation between T cells. CD95 and CD122 (IL-2Rβ) markers distinguish T N Cells and T SCM cells; the CD45RO marker distinguishes other memory T cell subtypes from T SCM Therefore, a pan-naive human T cell isolation kit ( Naive T cells were isolated from four healthy donors using the human T cell isolation kit (Miltenyi), which contains a cocktail of biotinylated antibodies for depletion of memory T cells and non-T cells. Purified naive T cells (CD45RA +) were subsequently stained with CH2811hG1 or CD95 / CD122 combination to separate 2811 + and the presumed T SCM RNA sequencing of CH2811hG1-enriched and CD95 / CD122-enriched T cells and differential gene expression (DE) analysis using a dataset from CD8 naive T cells showed that of the 5036 genes significantly upregulated or downregulated in SSEA-4-positive (CH2811) cells, 2227 (44%) were in common with DE genes upregulated or downregulated in CD95 / CD122-positive T cells, indicating a large number of overlapping genes between the two populations ( Fig. 10A Of the common genes, 257 overlapped significantly with the embryonic stem cell gene set, 103 overlapped with hematopoietic stem cells, and 78 overlapped with embryonal carcinomas ( Fig. 10A ), which means that SSEA-4 is indeed associated with a T cell subset with stem cell-like behavior. The distribution profiles of the first two overlapping gene sets in our dataset are shown using heat map analysis. In addition, the T cell subsets in our CH2811hG1-enriched and CD95 / CD122-enriched T cell profiles are also shown. SCM and effector differentiation gene subsets, and comparison with CD8 / CD4 naive and memory T cells and activated CD8 naive T cells (“donors”) ( Fig. 10B Hierarchical clustering showed a clear separation of the CH2811hG1 and CD95 / CD122 samples, suggesting that they are more similar to each other than true naive / memory T cells or activated naive T cells and may represent different T cell subsets with stem cell-like behavior ( Fig. 10C ).

[0314] Example 8. T cell proliferation and expansion

[0315] According to the co-stimulatory paradigm, TN cells require the engagement of T cell receptor (TCR) signal 1 and co-stimulatory signal 2 for full activation, leading to proliferation and differentiation. However, a subclass of CD28-specific antibodies known as CD28 superagonists, which differ from traditional CD28 antibodies, are able to fully activate T cells without additional stimulation of the TCR. We investigated whether plate-bound CH2811hG1 mAb could induce proliferation of CD4 and CD8 T cells. Initially, PBMCs were isolated from two healthy donors (BD3 and BD18) and, after CSFE labeling, antibody stimulation with 5 μg / ml of plate-bound CH2811hG1mAb revealed that CD4 (13% to 20%) and CD8 (2% to 31%) T cells proliferated on day 11 ( Fig.11 A to B). PBMCs stimulated with PHA and medium alone were positive and negative controls.

[0316] To exclude that this was due to Fc activation of antigen-presenting cells, purified T cells (96% purity; Fig. 12A ), CSFE labeling was performed and then stimulated with 5 μg / ml of plate-bound CH2811hG1 mAb. On day 14, 8% to 18% of CD4 + T cells and 3% to 7% CD8 + T cell proliferation, indicating that proliferation is not mediated by Fc interactions ( Fig. 12B To C). Cells stimulated with anti-CD3 mAb (0.005 μg / ml) and medium alone were used as positive and negative controls, respectively. The percentage of cells undergoing cell division was different, as most SSEA-4 positive cells underwent at least 4 cell divisions during the 14-day period (96%), whereas only 55% of CD3 stimulated cells underwent 4 cell divisions ( Fig.12D )

[0317] Example 10. Evaluation of TCR library clonotypes.

[0318] The clonality of CH2811IgG1 stimulated T cells was evaluated from 2 donors (BD3 and BD26), the TCR repertoire was determined, and fully automated multiplex PCR was performed to generate TCRα (TRA) and TCRβ (TRB) chain libraries for next generation sequencing (NGS) analysis of unique CDR3 (uCDR3). Dendrogram analysis (Figure 13) revealed that there were some relatively dominant clonal types in the CSFE low populations of the two donors. The non-proliferating population diversity of the TRA and TRB chains was 18.9 and 12.8, respectively. As expected, the diversity of the 2811 stimulated population was 3 and 3.3 for the TRA and TRB chains, respectively. The less diversity indicates that these cells represent cells that have been stimulated by antigens.

[0319] Example 11. Dynamics of individual cytokine / chemokine responses

[0320] T SCM The cells have been shown to have high proliferative capacity, self-renewal and multipotency, and they can further differentiate into other T cell subsets. We hypothesized that in the absence of any cytokine supplementation, FG2811 + T SCM The cells can proliferate and self-renew in vitro. We first aimed to identify FG2811 + T SCM Cytokines released by cells after stimulation with CH2811hG1 antibody can then be designed to expand and maintain putative FG2811 + T SCMMethods for cell stemness. T cells were purified from 4 healthy donors and stimulated with plate-bound CH2811hG1 mAb, and supernatants were collected on days 7, 11, and 14 and evaluated for cytokine or chemokine release. Unstimulated cells (medium only) were used as negative controls. Multiplex cytokine assays (Luminex technology) were used to measure secretion of nine cytokines / chemokines (IFNγ, IL-10, IL-17A, IL-2, IL-21, IL-5, IL-7, IL-8, and TNFα). Fig.14 ). The chemokine IL-8 was strongly upregulated after CH2811hG1 mAb stimulation, whereas more modest levels of TNFα, IL-10, and IL-5 were detectable from day 7 to day 14.

[0321] As shown by trypan blue exclusion, unstimulated and anti-CD3 stimulated T cells survived no longer than 14 days in culture, and only CH2811hG1 stimulated T cells survived more than 14 days ( Fig.15 A). On day 35, live CH2811hG1 stimulated T cells were collected and characterized by staining the cells with a panel of antibodies (FG2811, CD3, CD122, CD45RA, CD45RO, CD62L and CD95) and analyzed using multiparameter flow cytometry ( Fig.15 B) Of the 32.47% of live cells, 3% were FG2811 + , the remaining 97% is FG2811 - .FG2811 + cell[ Fig.15 B(i)] is 99% CD3 + and CD122+, of which 60% were CD45RA / RO double positive (CD45RA / RO + ), 30% are CD45RA + .CD45RA / RO + and CD45RA + All cells were CD62L + and CD95 + , indicating that they are T SCM Cells. CD45RA / RO + The group may be activated T SCM cells, while CD45RA + Maybe a more childish T SCM Cell. FG2811 - group[ Fig.15 B(ii)] is 99% CD3 + , but only 34% for CD122 + .FG2811 - CD3+ The population was 62% CD45RO + and 17%CD45RA + CD62L in 49% of FG2811 - CD3 + CD45RO + CD95 was expressed on 79% of FG2811 cells. - CD3 + CD45RO + CD45RO / CD62L / CD95 triple positive cells (CD45RO / CD62L / CD95 + ) can be activated T SCM or T CM cells, while CD45RO / CD95 + The cells can be T EM or T EMRA .CD45RA + The cell population contains more CD62L + cells (~76%) but less CD95 + Cells (~28%). CD45RA / CD62L / CD95 + The cells can be T SCM cells. This result suggests that CH2811hG1 stimulation enables the long-term maintenance of T cells in culture with "stem cell-like" and memory characteristics, which may differentiate into other T cell types. The proliferative potential of these viable cells was assessed by restimulating them with anti-CD3 / CD28 antibodies on day 33 or with CH2811hG1 mAb on days 33 and 64. Under light microscopy, on day 39, cells restimulated with anti-CD3 / CD28 antibodies formed T cell blasts [ Fig.15 C(i)], where most CD3 / CD28 restimulated T cells died, leaving only a few viable cells. In contrast, cells restimulated twice with CH2811 antibody were still alive at day 70 and showed a significant expansion in number [ Fig.15 C(ii)]. Supernatants from both cultures were collected on days 39, 54, and 70 and screened for cytokines and chemokines ( Fig.15 D). In CH2811hG1 restimulated cultures, IL-7 and IL-21 levels gradually increased, although other cytokine / chemokine levels gradually decreased from day 14 to undetectable levels on day 70. Fig.15 D(i)]. This result suggests that IL-7 and IL-21 can be self-sustaining in culture FG2811 + T SCM IL-7 plays an important role in T cells. SCMIL-21 plays a key role in inhibiting the differentiation of effector T cells (Lugli, Dominguez, et al. 2013). In cultures restimulated with anti-CD3 / 28 antibodies, all cytokines and chemokines increased, indicating activation of variants of different T cell subsets. Fig.15 D(ii)]. For example, Th1 cells are characterized by the secretion of IL-2, IFNγ, and TNFα, Th2 cells secrete IL-5, Th17 cells secrete IL-17A and IL-21, and regulatory T cells (Tregs) secrete IL-10 (Raphausen et al. 2015).

[0322] Example 12. Identification of FG2811 in mice + T SCM cell.

[0323] Next, we used the CH2811hG1 antibody to investigate the expression of SSEA-4 on mouse splenocytes, mesenteric lymph node cells, and inguinal lymph node cells ( Fig.16 ). These results show that the CH2811hG1 antibody stained 0.5% of spleen cells, 0.37% of mesenteric lymph node cells, and 0.52% of inguinal lymph node cells.

[0324] Example 13. FG2811 (mouse IgG1) induces phenotypic T in C57B / 6J mice SCM cell.

[0325] To determine the T cell agonist effect of FG2811mG1 in vivo, a group of three mice (Group A) were immunized ip with 250 μg of FG2811 on day 0 (Group A). ​​Three non-immunized mice were included as a control group (Group B). On day 16, both groups of mice were euthanized and spleens were harvested. The total cell count of splenocytes in Group A mice was higher than that in Group B mice, at 7 × 10 7 Up to 1×10 8 cells and 3.9×10 7 to 6.2×10 7 Cells ( Fig.17 A). Splenocytes from individual mice in each group were stained with CH2811hG1, anti-CD4, CD8, CD19, SCA-1, CD44, CD62L, CD11b, and F4 / 80 antibodies and analyzed by multiparameter flow cytometry ( Fig.17 B) CH2811hG1 mAb was used to identify SSEA-4 +Anti-CD4 and CD8 on splenocytes, T cells, CD44 and CD62L on T and B cell subsets, SCA-1 on stem-like cells (for identification of hematopoietic stem cells and mouse T SCM Cell markers and other markers) and CD11b and F4 / 80 of macrophages. + (0.97% to 1.2%), CD62L + (5.51% to 10.83%) and CD62 + CD44 + The frequencies of CD4 T cells in the 2 groups were 8.74% to 15.03%, which were lower than those in group B (1.62% to 1.74%, 17.39% to 19.2%, and 18.4% to 27.34%, respectively). + 、CD8 + 、CD19 + 、CD11b + 、F4 / 80 + and CD11b + F4 / 80 + There were few differences in the percentage of cells, except that mouse A3 contained lower CD8 + T cell population (Table 4). This result may indicate that FG2811mG1 antibody immunization induces 2811 + Cell proliferation and differentiation, leading to naive-like cells in vivo (2811 + , SCA1+ and CD62L + )reduce.

[0326] Markers 2811 Immunization (Group A) Control group (Group B) 2811 0.97-1.2％ 1.62-1.74％ SCA-1 37.23-55.06％ 42.22-61.73％ CD62L 5.51-10.83％ 17.39-19.2％ CD62L, CD44 8.74-15.03％ 18.4-27.34％

[0327] Table 4: Summary of the frequencies of different immune cell subsets in splenocytes of groups A and B on day 16.

[0328] Splenocytes from each group were pooled and then cultured in the presence (A+2811 and B+2811) or absence (A-2811 and B-2811) of plate-bound FG2811mG1 mAb at 5 μg / ml. Subsequently, on days 24, 27, and 30, these cells were harvested and stained with FG2811, CD3, CD4, CD8, CD44, CD62L, SCA1, CD11b, F4 / 80, and CD19 antibodies and analyzed by multiparameter flow cytometry ( Fig. 17CTo D). On day 24, group A splenocytes restimulated with FG2811mG1 mAb (A+2811) or without restimulated with FG2811mG1 mAb (A-2811) formed small and large cell populations, as shown by forward and side scatter (FSC / SSC) spectra. In contrast, group B splenocytes restimulated with FG2811mG1 mAb (B+2811) or without restimulated with FG2811mG1 mAb (B-2811) did not generate large populations ( Fig. 17C Large colonies continued to persist in A+2811 and A-2811 splenocyte cultures until day 30 ( Fig.17D ). The large cell population is mainly composed of CD3 中高 (CD3 mo-hi )CD4 高 (CD4 hi ) and CD8 高 (CD8 hi )T cells, while the small cell population consists of CD3 低中 (CD3 lo-mo )CD4 低 (CD4 1o ) and CD8 低 (CD8 1o )T cells.

[0329] In the hierarchical model of mouse T cell differentiation, after antigen sensitization, naive T cells (TN) gradually differentiate into stem memory T cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ). These T cell subsets were distinguished by the combined expression of different markers (Table 5).

[0330]

[0331] Table 5: Phenotypic markers of murine T cell populations

[0332] Phenotypic analysis showed that CD3 mo-hi The group is mainly composed of - CD62L + (T N and / or T SCM ; 28.8% to 32.49%) and CD44 + CD62L + (T CM ; 37.92% to 41.08%) phenotype T cells, followed by CD44 + CD62L - (T EF / T EM; ~27.8%) phenotype and a small portion with CD44 - CD62L - In contrast, CD3 lo-mo CD44 + CD62L - (T EF / T EM ; 66.09% to 70.83%) phenotype, followed by CD44 - CD62L - Phenotype (26.77% to 32.17%). N and / or T SCM Cells and T CM The percentages of cells ranged from 0.08% to 0.24% and from 1.5% to 2.29%, respectively. In addition to T cells, the large cell population also contained CD19 hi 、CD62L + and CD62L + SCA-1 + CD19 was detected only in the small cell population. lo Interestingly, CD11b + F4 / 80 + The percentage of macrophage populations was significantly reduced. FG2811mG1 antibody stimulated splenocytes from unimmunized mouse splenocyte B+2811 cultures in vitro without the formation of these large cell populations even on day 30, indicating that the generation of this cell population is an in vivo FG2811 antibody immune effect.

[0333] Example 14. T in HHDII and HHDII transgenic mice SCM Cell identification

[0334] To determine HHDII( Fig.18A and B) and HHDII / DP4 mice ( Fig. 18C To E) T SCM To determine the frequency of SSEA-4 cells, splenocytes were harvested from unprimed mice and stained with CH2811hG2-PeCy7, anti-CD3, CD4, CD8, CD44, CD62L, and SCA-1, and then analyzed by multiparameter flow cytometry. + Splenocytes, anti-CD4 and CD8 of T cells, CD44 and CD62L of T cell subsets, SCA-1 of stem-like cells (for identification of hematopoietic stem cells and mouse T SCMIn HHDII mice, 10.88% of the cells were 2811 + CD3 + cells, which translates to 1.85 × 10 5 cells / ml, and 24.61% of CD3 + Group is T SCM cell( Fig.18B ). 2811 in HHDII mice + The frequency of the HHDII population (10.88%) was higher than that previously observed in C57 / B6 mice (2.42% to 3.60%). + Further phenotypic analysis of the population ( Fig.18B ) showed 33.38% CD44 + CD62L - , 47.98% are CD44 + CD62L + We also identified 2811 cells expressing the stem cell marker SCA-1. + The percentage of cells, this marker compared with other markers (CD44 - CD62L + ) can be used in combination to define T in these mice SCM Cell. Most 2811 + SCA-1 + The cells also expressed CD44, indicating that they were antigen-stimulated.

[0335] In HHDII / DP4 mice, 12.01% of cells were 2811 + CD3 + cells per ml, which translates to 0.91 × 10 5 cells, in addition, 6.98% of CD3 + The group is also 2811 + of( Fig. 18C and D) 2811 in HHDII / DP4 mice + The frequency of 2811 in HHDII / DP4 mice (12.01%) was similar to that in HHDII mice and also higher than that previously observed in C57 / B6 mice (2.42% to 3.60%). + Further phenotypic analysis of the population ( Fig.18D ) showed 12.09% were CD44 + CD62L - , 77.15% were CD44 + CD62L + We also identified 2811 cells expressing the stem cell marker SCA-1. +The percentage of cells. Most 2811 + SCA-1 + The cells also expressed CD44 (75.51% CD44 + CD62L + , 30.03% CD44 + CD62L - ).

[0336] A more detailed phenotypic analysis of the T cell population from HHDII / DP4 mice was performed, which looked at the expression of 2811 in both CD4 and CD8 T cell subsets, but also at the expression of the exhaustion marker Tim3. The percentage of CD4+ T cells in HHDII / DP4 mice was 14.30%, however, CD8 + The percentage of T cells was very low, only 0.50% CD8+ cells ( Fig.18E ), of which 9.47% were CD4+ T cells 2811 + , and 10.14% of CD8 T cells were 2811 + The exhaustion marker Tim3 is expressed in CD4 + 2811 + (0.42%) cells and CD8+2811 + The expression of TNF-α on WT cells was low (0.34%), which was consistent with their stem cell characteristics.

[0337] Example 15. Plate-bound human (IgG1) and mouse (IgG1) 2811 induces ex vivo proliferation of mouse splenocytes

[0338] We investigated whether plate-bound CH2811hG1 and FG2811mG1 mAbs could induce proliferation of CD4 and CD8 T cells. Splenocytes were harvested from HHDII-naive mice and enriched for pan T cells (CD3 + ) and labeled with CFSE, followed by antibody stimulation using plate-bound CH2811hG1 mAb or FG2811mG1, anti-CD3 as a positive control, and culture medium as a negative control ( Fig.19A The proliferation responses of CD3, CD4, and CD8 T cell populations were measured on days 7, 12, and 14 ( Fig.19BTo D). The results show that CD3, CD4 and CD8 T cells proliferated in response to stimulation with plate-bound CH2811hG1 and FG2811mG1 mAbs, which was equal to or slightly higher than the medium control. On day 7, for CD3 T cells, 2.72% proliferated in response to FG2811mG1, 2.24% proliferated in response to CH2811hG1, for CD8 T cells, 2.47% proliferated in response to FG2811mG1, 1.46% proliferated in response to CH2811hG1, and for CD4 T cells, 1.32% proliferated in response to FG2811mG1, 0.96% proliferated in response to CH2811hG1. On day 12, proliferative responses to plate-bound CH2811hG1 and FG2811mG1 increased, with 6.46% proliferating in response to FG2811mG1 and 6.27% proliferating in response to CH2811hG1 for CD3 T cells, 6.21% proliferating in response to FG2811mG1 and 3.33% proliferating in response to CH2811hG1 for CD8 T cells, and 5.79% proliferating in response to FG2811mG1 and 2.82% proliferating in response to CH2811hG1 for CD4 T cells. On day 14, the proliferative response to plate-bound CH2811hG1 and FG2811mG1 increased further, with 10.07% proliferating in response to FG2811mG1 and 8.7% proliferating in response to CH2811hG1 for CD3 T cells, 7.87% proliferating in response to FG2811mG1 and 6.61% proliferating in response to CH2811hG1 for CD8 T cells, and 7.29% proliferating in response to FG2811mG1 and 5.15% proliferating in response to CH2811hG1 for CD4 T cells. These results indicate that murine splenocytes proliferate ex vivo in response to plate-bound CH2811hG1 and FG2811mG1 mAbs.

[0339] Example 16. Anti-CD3 and CD28 induced HHDII mice 2811 + Ex vivo expansion of cells

[0340] We investigated whether anti-CD3 and anti-CD28 could induce 2811 isolated from HHDII mice. + Cell proliferation. Splenocytes were harvested from HHDII-naive mice and enriched for pan-T cells (CD3 + ) and labeled with CFSE, then stimulated with anti-CD3 and anti-CD28 (1 μg / mL). CH2811hG2-PeCy7 mAb was used to determine the expression of 2811 on days 11, 15, and 20. + The proliferation response of the group Fig. 20A ). After stimulation with anti-CD3 and anti-CD28, 2811+ CD3 + The percentage of T cells increased, and by day 11, 61.2% of T cells were 2811 + By the 15th day, it further increased to 69.84%, but by the 20th day, 2811 + The percentage of T cells decreased to 57.58%. + The decrease in cell percentage was also associated with a decrease in cell viability, and T cells and 2811 + Decreased total T cell count ( Fig. 20A iii and iv). In the case of direct in vitro culture, only the culture medium control was 2811 + The percentage of cells was 10%, which increased to 20 to 30% on the 11th and 15th days, but also decreased on the 20th day.

[0341] 2811 cells amplified by stimulation with anti-CD3 and anti-CD28 + Cells were phenotyped. On day 11, cells were stained with CH2811hG2-PeCy7, anti-CD3, CD44, and CD62L ( Fig. 20B ). The identified T cell subsets are CD44 + CD62L - Definite effector memory T cells (T EM ), by CD44 + CD62L + Definitive central memory T cells (T CM ), by CD44 - CD62L - Definite effector T cells (T EFF ) and CD44 - CD62L + Clearly naive T cells (TN). Phenotyping results on day 11 ( Fig. 20C ) showed that stimulation with anti-CD3 and CD28 increased the + T EM (average 67.35×10 3 ), T CM (average 61.15×10 3 ), T EFF (average 141×10 3 ) and T N (average 16.45×10 3 2811 were stimulated with anti-CD3 and anti-CD28 + The phenotype of the cells is pushed toward a more effector T cell phenotype ( Fig.20D ).2811 + , T EFFThe percentage of cells was 47.7% (average), while T CM and T EM The percentage of cells has decreased below that of unstimulated cells (medium only).

[0342] These results indicate that anti-CD3 and anti-CD28 induce 2811 from HHDII mice + Ex vivo expansion of cells. Stimulation with anti-CD3 and anti-CD28 resulted in 2811 11 to 15 days after stimulation + The number and percentage of cells increased. 2811 + The total number of T cells increased (T CM TN, T EM , T EFF ), but stimulation does push T cells toward more of an effector T cell phenotype.

[0343] Example 17. Human (IgG2) and mouse (IgG1) 2811 induces in vitro proliferation of spleen cells from HHDII / DP4 mice

[0344] We investigated whether plate-bound CH2811hG2 and FG2811mG1 mAbs could induce proliferation of CD4 and CD8 T cells. Splenocytes were harvested from HHDII-naive mice and enriched for pan T cells (CD3 + ) and labeled with CFSE, followed by antibody stimulation using plate-bound CH2811hG2, FG2811mG1, anti-CD3 / CD28 (+ / -AKTi) as a positive control, and medium as a negative control. The proliferation response of the CD3 T cell population was measured on days 11, 15, and 20 ( Fig.21A ). The results show that 2811 + CD3 T cells proliferated in response to stimulation with plate-bound CH2811hG2 and FG2811mG1 mAbs. On day 11, 8.73% of 2811 + CD3 + T cells proliferated in response to FG2811mG1, 50.47% proliferated in response to CH2811hG2, and on day 15, 20.48% of 2811 + CD3 + T cells proliferated in response to FG2811mG1, 40.55% proliferated in response to CH2811hG2, and by day 20, this percentage decreased slightly, with 21.41% of 2811 + CD3 + T cells proliferated in response to FG2811mG1, and 35.13% proliferated in response to CH2811hG2. + The percentage of cells, 2811+ CD3 + and 2811 + The total number of cells was 2811. + In the presence or absence of AKTi, stimulation with anti-CD3 / CD28 also induced 2811 + Cell proliferation, of which 80% of 2811 at each time point after CD3 / CD28 stimulation + CD3 + T cells, this percentage dropped to 60% with the addition of AKTi, which is slightly toxic to the cells. These results indicate that CH2811hG2 induced 2811 T cells at all time points after stimulation. + Cell proliferation was also observed in FG2811mG1, but to a lesser extent.

[0345] The 2811 cells that had been expanded after stimulation with anti-CD3 / CD28 (+ / - AKTi), CH2811hG2, and FG2811mG1 mAbs were then + Cells were phenotyped. On day 11, cells were stained with CH2811hG2-PeCy7, anti-CD3, CD44, and CD62L ( Fig. 20B ). The identified T cell subsets are CD44 + CD62L - Definite effector memory T cells (T EM ), by CD44 + CD62L + Definitive central memory T cells (T CM ), by CD44 - CD62L - Definite effector T cells (T EFF ) and CD44 - CD62L + Clearly naive T cells (TN). Phenotyping results on day 11 ( Fig.21B ) showed that 2811 was stimulated with CH2811hG2 or FG2811mG1 mAb + T EM The total number increased to 62.8×10 3 cells (after CH2811hG2 stimulation) and increased to 5.24×10 after FG2811mG1 stimulation. 3 After CH2811hG2 stimulation, 2811 + T CM The total number increases to 6.95×10 3 cells, which increased to 1.8×10 after FG2811mG1 stimulation. 3 2811+ T EFF The total number increased to 29.05×10 after CH2811hG2 stimulation. 3 cells, which increased to 7.02×10 after FG2811mG1 stimulation. 3 2811 + The total number of TN cells increased only slightly after CH2811hG2 stimulation, increasing to 0.61×10 3 Cells (medium only 0.07×10 3 We also examined the expression of anti-CD3 / CD28, FG2811mG1, and CH2811hG2 in 2811 cells. + The percentage of T cells ( Fig. 21C ), 2811 + T EFF The percentage of cells was 34.82% after FG2811mG1 stimulation, 57.59% after CH2811hG2 stimulation, and 47.36% after anti-CD3 / CD28 stimulation. These results suggest that T cells from HHDII / DP4 mice are less skewed toward the effector phenotype compared with the results obtained from HHDII mice ( Fig.20D Compared with HHDII mice, T CM and T EM The percentages were also higher in the subpopulations.

[0346] These results suggest that splenocytes from HHDII / DP4 mice proliferate ex vivo in response to plate-bound CH2811hG2 and FG2811mG1 mAbs, which results in the induction of + In addition to the increase in the number of effector memory T cells, central memory T cells, effector T cells and naive T cells, 2811 + CD3 + The total number of T cells also increased. The proliferative ex vivo response to CH2811hG2 was greater than that to FG2811mG1, thus resulting in a greater number of 2811 + cell.

[0347] Example 18. Anti-CD3 and CD28 induced 2811 in healthy donors + Ex vivo expansion of cells

[0348] T SCM T cells isolated from healthy donors have been shown to have a high proliferative capacity, self-renewal and multipotency, and they can further differentiate into other T cell subsets. We investigated whether anti-CD3 / CD28 stimulation or the addition of different cytokines could induce T cell proliferation in T cells isolated from healthy donors. SCMCells were propagated in vitro. PBMCs were isolated from 4 healthy donors (buffy coats), pan-T cell enrichment was performed, and T cells were cultured in the presence of anti-CD3 / CD28, IL-7, IL-15 or IL-21. Phenotypic analysis was performed using anti-CD3, CD45RA, CD45RO, CD62L, CD95, CD122 and CCR7 on days 15 and 20, and the expression of different markers used to identify T cell populations is listed in Table 6.

[0349]

[0350] Table 6: Phenotypic markers of human T cell populations

[0351] CD3 + T cells were phenotyped and the CD3 + T cells were expanded after stimulation with anti-CD3 / CD28 or after the addition of IL-7, IL-15, and IL-21 (added in a range of combinations). Staining was performed on days 15 and 20 ( Fig.22A ). Phenotyping results on day 15 ( Fig. 22B ) showed that stimulation with CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21 increased + CD3 + The percentage of cells is also consistent with 2811 + and CD3 + On day 15, after anti-CD3 / CD28 stimulation, 2811 + CD3 + The percentage of T cells was 19.64% and 23.94%, which was higher than that of T cells cultured in the presence of cytokines alone (without CD3 / CD28 stimulation). Adding IL-7 / IL-21 or IL-7 / IL-15 / IL-21 combined with anti-CD3 / CD28 stimulation did slightly increase the 2811 + CD3 + When cells were cultured in the presence of CD3 / CD28 and IL-7 / IL-21, 2811 + CD3 + The percentage of T cells increased to 23.8% and 27.4%, and after the addition of IL-15, the percentage increased to 31.53%. + CD3 + The increase in the percentage of T cells was also associated with an increase in the total number of cells, with 80×10 4 2811 + CD3 + On day 20, when cultured with CD3 / CD8, IL-7 / IL-21 / IL-15, 2811+ CD3 + The percentage dropped from 31.53% on the 15th day to 16.45% and 17.56%. + CD3 + The decrease in T cell percentage was also related to 2811 + This is associated with a decrease in the total number of T cells.

[0352] Further and more detailed phenotyping of T cells from 2 donors was performed to identify T cells cultured in the presence of CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21. SCM Cells. Human T SCM The frequency of T cells was low in four healthy donors. SCM The percentage ranges from 0.64% to 3.48%. We studied T SCM Can it be expanded in the presence of CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21? Fig.23 ). In the presence of anti-CD3 / CD28, in the presence of IL-7 / IL-21 (3.51% and 6.32% T SCM ) or with IL-7 / IL-15 / IL-21 (3.62%T SCM ) When culturing T cells, it was observed that T SCM Cell expansion is the greatest. For one donor, T SCM Cells expanded 5-fold and 8-fold for the second donor. On day 20, in the presence of anti-CD3 / CD28, in the presence of IL-7 / IL-21 (14.84% and 11.33% T SCM ) or when T cells were cultured with IL-7 / IL-15 / IL-21 (13.67%), T SCM The percentage of cells further increased, and for one donor, T SCM The cells expanded 3-fold for the first donor and 9-fold for the second donor (compared to the culture control on day 20). We next determined what percentage of T SCM The cells were also 2811 positive ( Fig.23 ii). On day 0, 2811 + T SCM The cell percentages were 46.89% and 63.60%. SCM 2811 + The percentage of cells remained similar in all conditions, ranging from 31.51% to 53.52%. On day 20, in most cases T SCM 2811 +The percentage of T cells decreased, and only T cells cultured in the presence of medium alone or in the presence of CD3 / CD28 alone maintained a percentage similar to that at day 15. We next determined what percentage of T SCM The cells were also positive for CD3 and 2811 ( Fig.23 iii) On day 15, T cells in the presence of CD3 / CD28 or in combination with IL-7, IL-15, IL-21 increased compared to the medium-only control or day 0 results. SCM On day 20, T cells were expressed in the presence of CD3 / CD28 or in combination with IL-7, IL-15, and IL-21, compared with the medium-only control or day 0 results. SCM The percentage of cells did not increase.

[0353] These results indicate that CD3 / CD28 stimulation induces 2811 isolated from healthy human donors + Ex vivo expansion of cells. Stimulation of T cells with anti-CD3 / CD28 increased the number of + The frequency of T cells was further increased when IL-7, IL-15, and IL-21 were all added to the culture, and this expansion peaked at 15 days after stimulation. SCM This expansion caused these cells to expand three to nine times.

[0354] Example 19. T cells stimulated with soluble FG2811mG1 stimulate CD4 and CD8 T cell proliferation through Fc cross-linking

[0355] We next investigated whether soluble FG2811mG1 could stimulate CD4 and CD8 T cells when cultured in the presence or absence of splenocytes. The addition of splenocytes should allow Fc crosslinking and stimulate T cell responses. Splenocytes were isolated from HHDII and HHDII / DP4 mice, and pan T cells (CD3 + ), HHDIIT cells and HHDII / DP4 splenocytes were labeled with CFSE. HHDIIT cells were cultured with FG2811mG1, LPS or medium alone in the presence or absence of HHDII / DP4 splenocytes. On day 15, CD4 and CD8 proliferation responses were determined. In the absence of co-culture with splenocytes, only 0.14% CD4T (CFSE low) proliferated in the presence of soluble FG2811mG1, however, this was not higher than the medium alone control (0.16% CFSE 低), so it is just the background level. In the absence of co-culture with spleen cells, only 0.02% CD8 T (CSFE 低 ), which is different from the medium-only control (0% CSFE 低 ) were very similar, so it was just background level. Both CD4 and CD8 T cells showed good proliferation response to LPS (3.34% and 39.56%, respectively). In the case of co-culture with spleen cells, 15.2% CD4 T (CSFE 低 ), 2.33% CD8 T cells (CSFE 低 ), both CD4 and CD8 T cells showed a good proliferation response to LPS, which was enhanced in the presence of PBMCs (59.88% and 54.10%, respectively).

[0356] These results show that when co-cultured in the presence of splenocytes, soluble FG2811mG1 can stimulate CD4 and CD8 proliferation responses (Figure 24). If splenocytes are not added to the culture, T cells cannot proliferate, indicating that Fc cross-linking is the mode of action of 2811mAb. When co-cultured with splenocytes and FG2811mG1, the CD4 T cell population was greater than the CD8 T cell population in T cell expansion (15.2% vs. 2.33%). These results show the potential of 2811mAb in ex vivo expansion of T cells and its mode of action is through Fc cross-linking.

[0357] Implementation

[0358] Other embodiments of the present invention are described below:

[0359] 1. An isolated specific binding member capable of binding to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc).

[0360] 2. The binding member of embodiment 1, wherein the binding member is capable of binding to SSEA-4 on a glycolipid.

[0361] 3. The binding member of any preceding embodiment, wherein the binding member is capable of targeting stem memory T cells (T SCM ).

[0362] 4. The binding member of any preceding embodiment, wherein the binding member is capable of inducing stem memory T cells (T SCM )’s proliferation.

[0363] 5. The binding member of any preceding embodiment, wherein said binding member does not bind to SSEA-3.

[0364] 6. The binding member of any preceding embodiment, wherein the binding member is mAb FG2811.72 or chimeric FG2811.72 (CH2811 / CH2811.72), or a fragment thereof.

[0365] 7. The binding member of any preceding embodiment, wherein said binding member is bispecific.

[0366] 8. The binding member of embodiment 7, wherein the bispecific binding member is additionally specific for CD3.

[0367] 9. The binding member of any preceding embodiment, wherein the binding member comprises one or more binding domains selected from Figure 2 The amino acid sequences of residues 27 to 38 (CDRH1), residues 56 to 65 (CDRH2), and residues 105 to 113 (CDRH3) of a.

[0368] 10. The binding member of any preceding embodiment, wherein the binding member comprises one or more binding domains selected from Figure 2 b Amino acid sequences of residues 27 to 38 (CDRL1), residues 56 to 65 (CDRL2), and residues 105 to 113 (CDRL3).

[0369] 11. The binding member of any preceding embodiment, wherein the binding member comprises a light chain variable sequence and a heavy chain variable sequence, the light chain variable sequence comprising one or more of LCDR1, LCDR2 and LCDR3, wherein

[0370] LCDR1 contains SSVNY,

[0371] LCDR2 includes DTS, and

[0372] LCDR3 contains FQASGYPLT; and

[0373] The heavy chain variable sequence comprises one or more of HCDR1, HCDR2 and HCDR3, wherein

[0374] HCDR1 contains GFSLNSYG,

[0375] HCDR2 contains IWGDGST, and

[0376] HCDR3 comprises TKPGSGYAF.

[0377] 12. The binding member of any preceding embodiment, wherein the binding domain is carried by a human antibody framework.

[0378] 13. The binding member of any preceding embodiment, wherein the binding member comprises: Figure 2 a VH domain comprising residues 1 to 126 of the amino acid sequence of Figure 2 b. The VL domain of residues 1 to 123 of the amino acid sequence of

[0379] 14. The binding member of any preceding embodiment, wherein said binding member comprises a human antibody constant region.

[0380] 15. The binding member of any preceding embodiment, wherein the binding member is an antibody, antibody fragment, Fab, (Fab')2, scFv, Fv, dAb, Fd or diabody.

[0381] 16. The binding member of any preceding embodiment, wherein the binding member is a scFv comprising, in the following order: 1) a leader sequence, 2) a heavy chain variable region, 3) a 3xGGGGS spacer, 4) a light chain variable region, and 5) a poly-Ala and a 6xHis tag for purification.

[0382] 17. The binding member of any one of embodiments 1 to 15, wherein the binding member is a scFv comprising, in the following order: 1) a leader sequence, 2) a light chain variable region, 3) a 3xGGGGS spacer, and 4) a heavy chain variable region, optionally further comprising a 5′ or 3′ purification tag.

[0383] 18. The binding member of any preceding embodiment, wherein the binding member is provided in the form of a chimeric antigen receptor (CAR).

[0384] 19. The binding member of embodiment 18, wherein the binding member is a scFv provided in the form of a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) is in a heavy chain-light chain orientation or a light chain-heavy chain orientation.

[0385] 20. The binding member of any one of embodiments 1 to 17, wherein the binding member is provided in the form of an agonist (IgG2) monoclonal antibody.

[0386] 21. The binding member of any one of embodiments 1 to 17, wherein the binding member is provided in the form of an antagonist monoclonal antibody.

[0387] 22. The binding member of any preceding embodiment, wherein said binding member is monoclonal, such as a monoclonal antibody.

[0388] 23. The binding member of any preceding embodiment, wherein the binding member is a human antibody, a humanized antibody, a chimeric antibody or a mosaic antibody.

[0389] 24. An isolated specific binding member capable of binding to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc), which competes with the isolated specific binding member of any one of embodiments 1 to 23.

[0390] 25. A binding member according to any preceding embodiment for use in therapy.

[0391] 26. A binding member according to any one of embodiments 1 to 24 for use in a method for preventing, treating or diagnosing cancer.

[0392] 27. A binding member according to any one of embodiments 1 to 24 for use in treating patients with chronic viral infections.

[0393] 28. A binding member according to any one of embodiments 1 to 24 for use in a method of treating an autoimmune disease, HIV, adult T-cell leukemia or graft-versus-host disease.

[0394] 29. A method of treating or preventing cancer comprising administering a binding member according to any one of embodiments 1 to 24 to a subject in need thereof.

[0395] 30. A method for treating or preventing a patient suffering from a chronic viral infection, comprising administering to a subject in need thereof a binding member according to any one of embodiments 1 to 24.

[0396] 31. A method of treating or preventing an autoimmune disease, HIV, adult T-cell leukemia or graft-versus-host disease, comprising administering a binding member according to any one of embodiments 1 to 24 to a subject in need thereof.

[0397] 32. A method of enhancing a protective immune response against cancer, comprising administering to a subject in need thereof a binding member according to any one of embodiments 1 to 24.

[0398] 33. The method of embodiment 32, wherein the binding member is prepared for administration together with an additional immunogenic agent, optionally wherein the immunogenic agent is a cancer vaccine.

[0399] 34. The method of embodiment 33, wherein the binding member and the additional immunogenic agent are prepared for simultaneous or sequential administration.

[0400] 35. A binding member for use according to embodiment 25 or 26, or a method according to embodiment 29, wherein the cancer is pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer or lung cancer.

[0401] 36. A binding member for the use of embodiment 25, 26 or 35, or a method of embodiment 28 or embodiment 31, wherein the binding member is administered alone or in combination with other treatments, or is formulated for administration alone or in combination with other treatments.

[0402] 37. A nucleic acid comprising a sequence encoding a binding member according to any one of embodiments 1 to 24.

[0403] 38. The nucleic acid according to embodiment 37, wherein the nucleic acid is a construct in the form of a plasmid, a vector, a transcription or an expression cassette.

[0404] 39. A recombinant host cell comprising a nucleic acid according to embodiment 37 or embodiment 38.

[0405] 40. A method for diagnosing cancer, comprising: using a binding member of any one of embodiments 1 to 24 to detect the glycolipid-linked glycan SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) in a sample from an individual.

[0406] 41. A method according to embodiment 40, wherein the pattern of glycans detected by the binding member is used to stratify treatment options for individuals.

[0407] 42. A pharmaceutical composition comprising a binding member according to any one of embodiments 1 to 24 and a pharmaceutically acceptable carrier.

[0408] 43. The pharmaceutical composition according to embodiment 42, further comprising at least one or other pharmaceutically active substance.

[0409] 44. The pharmaceutical composition according to embodiment 42 or embodiment 43, which is used for treating cancer.

[0410] 45. The pharmaceutical composition of embodiment 42 or embodiment 43, which is used to treat patients with chronic viral infections.

[0411] 46. ​​The pharmaceutical composition of embodiment 42 or embodiment 43, which is used to treat autoimmune diseases, HIV, adult T-cell leukemia or graft-versus-host disease.

[0412] 47. In vitro induction of stem memory T cells (T SCM ) proliferation method, comprising causing the stem memory T cells (TSCM ) is contacted with a binding member according to any one of embodiments 1 to 24.

[0413] 48. Used to induce stem memory T cells (T SCM ) in a cell culture medium in which the cell culture medium proliferates, the cell culture medium comprising a binding member according to any one of embodiments 1 to 24.

[0414] 49. In vitro induction of stem memory T cells (T SCM ) method for proliferation, comprising administering to a subject a binding member according to any one of embodiments 1 to 24.

[0415] 50. Identification of stem memory T cells (T SCM ) method, which is performed by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on a cell using a binding member according to any one of embodiments 1 to 24.

[0416] 51. A method for purifying stem memory T cells (TSCM) by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on cells using a binding member according to any one of embodiments 1 to 24.

[0417] 52. The method of embodiment 50 or 51, wherein said identification or purification is performed in vivo or ex vivo.

[0418] 53. The method of embodiment 50 or 51, wherein the binding member is used to label stem memory T cells (T SCM ) for purification.

[0419] 54. A binding member substantially as herein described, optionally with reference to the accompanying drawings.

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Sequence Listing <110> skansel gmbh <120> SSEA-4 binding members <130> P126328WO <140> PCT / EP2020 / 074878 <141> 2020-09-04 <150> GB1912882.6 <151> 2019-09-06 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 351 <212> DNA <213> Artificial sequence <220> <223> Mature FG2811 heavy chain variable region nucleotide sequence <400> 1 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln acatgcactg tctcagggtt ctcattaaac agttatggtg taagctgggt tcgccagcct 120 Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Ser Tyr Gly Val Ser Trp ccaggaaagg gtctggaatg gctgggagta atatggggtg acgggagcac aaattatcat 180 Pro Glu Lys Gly Leu Glu Trp Leu Gly Glu Val Tyr Gly Val Thr Gly tcagctctca tgtccagact gcgcatcagt aaggacaact ccaagagaca agttttctta 240 Ser Ser Ser Met Ser Arg Thr Ala Ile Ser Lys Asp Asn Pro Lys Arg aaactgaaca gtctgcaaac tgatgacaca gccacgtact actgtaccaa accgggctcg 300 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Gln ggctacgcgt ttgcttactg gggccaaggg actctggtca ctgtctctag 351 Gly Tyr Arg Phe Ala Tyr Gly Ala Lys Gly Thr Leu Val Thr Val Ser <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of mature FG2811 heavy chain variable region <400> 2 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Asn Ser Tyr 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Met 50 55 60 Ser Arg Leu Arg Ile Ser Lys Asp Asn Ser Lys Arg Gln Val Phe Leu 65 70 75 80 Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Thr 85 90 95 Lys Pro Gly Ser Gly Tyr Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Mature FG2811 κ-chain variable region nucleotide sequence <400> 3 gaaaatgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga aaaagtcacc 60 atgacctgca gtgccagttc aagtgttaat tacatgcact ggtaccagca gaagtcaagc 120 acctccccca aactctggat ttatgacaca tccaaactgg cttctggagt cccaggtcgc 180 ttcagtggca gtgggtctgg aaactcttac tctctcacga tcaggaccat ggaggctgaa 240 gatgttgcca cttatttctg ttttcaggcg agtgggtacc cgctcacgtt cggtggtggg 300 accaagctgg agctgaaa 318 <210> 4 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of mature FG2811 kappa chain variable region <400> 4 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Arg Thr Met Glu Ala Glu 65 70 75 80 Asp Val Ala Thr Tyr Phe Cys Phe Gln Ala Ser Gly Tyr Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 5 <211> 972 <212> DNA <213> Artificial sequence <220> <223> mIgG1 nucleotide sequence <400> 5 gctaaaacga cacccccatc tgtctatcca ctggcccctg gttctgctgc ccaaactaac 60 tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120 tggaactctg gttccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180 ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240 acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300 gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360 cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420 gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480 gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540 agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600 aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660 aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720 agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780 aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840 tacttcgtct acagcaagct caatgtgcag aagagcaact gggaggcagg aaatactttc 900 acctgctctg tgttacatga gggcctgcac aaccaccata ctgagaagag cctctcccac 960 tctcctggta aa 972 <210> 6 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> mIgG1 Amino Acid Sequence <400> 6 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 1 5 10 15 Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 65 70 75 80 Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 85 90 95 Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 100 105 110 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 115 120 125 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 130 135 140 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 145 150 155 160 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 165 170 175 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 180 185 190 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 195 200 205 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 210 215 220 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 225 230 235 240 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 245 250 255 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 260 265 270 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 275 280 285 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 290 295 300 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 305 310 315 320 Ser Pro Gly Lys <210> 7 <211> 978 <212> DNA <213> Artificial sequence <220> <223> hIgG2 nucleotide sequence <400> 7 gcttccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg caccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300 aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540 gtggtcagcg tcctcaccgt cgtgcaccag gactggctga acggcaagga gtacaagtgc 600 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960 tccctgtctc cgggtaaa 978 <210> 8 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> hIgG2 Amino Acid Sequence <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 9 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> hIgG1 nucleotide sequence <400> 9 gcttccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 10 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> hIgG1 Amino Acid Sequence <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 11 <211> 321 <212> DNA <213> Artificial sequence <220> <223> Mouse kappa constant region nucleotide sequence <400> 11 cgtgcagatg ctgcaccaac tgtatcgatc ttcccaccat ccagtgagca gttaacatct 60 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240 cgacataaca gctatacctg tgaggccact cacaagacat ctacttcacc cattgtcaag 300 agcttcaaca ggaatgagtg t 321 <210> 12 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Mouse kappa constant region amino acid sequence <400> 12 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 13 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Human κ Constant Region Nucleotide Sequence <400> 13 cgtacggtag cggccccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc caccagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of human κ constant region <400> 14 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 15 <211> 5 <212> PRT <213> Artificial sequence <220> <223> LCDR1 amino acid sequence <400> 15 Ser Ser Val Asn Tyr 1 5 <210> 16 <211> 3 <212> PRT <213> Artificial sequence <220> <223> LCDR2 amino acid sequence <400> 16 Asp Thr Ser 1 <210> 17 <211> 9 <212> PRT <213> Artificial sequence <220> <223> LCDR3 amino acid sequence <400> 17 Phe Gln Ala Ser Gly Tyr Pro Leu Thr 1 5 <210> 18 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HCDR1 amino acid sequence <400> 18 Gly Phe Ser Leu Asn Ser Tyr Gly 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> HCDR2 amino acid sequence <400> 19 Ile Trp Gly Asp Gly Ser Thr 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 amino acid sequence <400> 20 Thr Lys Pro Gly Ser Gly Tyr Ala Phe 1 5

Claims

1. An isolated antibody or antigen-binding fragment capable of specifically binding to and targeting stem memory T cells (T SCM ), wherein the antibody or antigen-binding fragment comprises a light chain variable region and a heavy chain variable region, The light chain variable region comprises: LCDR1 composed of SSVNY, LCDR2 composed of DTS, and LCDR3 composed of FQASGYPLT, The heavy chain variable region comprises: HCDR1 composed of GFSLNSYG, HCDR2 composed of IWGDGST, and HCDR3 composed of TKPGSGYAF.

2. The antibody or antigen-binding fragment according to claim 1, wherein The antibody or antigen-binding fragment is capable of binding to SSEA-4 on the glycolipid.

3. The antibody or antigen-binding fragment according to claim 1 or 2, wherein The antibody or antigen-binding fragment can induce stem memory T cells (T SCM )’s proliferation.

4. The antibody or antigen-binding fragment according to claim 1 or 2, wherein The antibody or antigen-binding fragment does not bind to SSEA-3.

5. The antibody or antigen-binding fragment according to claim 1 or 2, wherein The antibody or antigen-binding fragment is mAb FG2811.72 or chimeric FG2811.72 (CH2811 / CH2811.72), or a fragment thereof.

6. The antibody or antigen-binding fragment according to claim 1 or 2, wherein: The antibodies or antigen-binding fragments are bispecific.

7. The antibody or antigen-binding fragment according to claim 6, wherein The bispecific antibody or antigen-binding fragment is additionally specific for CD3.

8. The antibody or antigen-binding fragment according to claim 1 or 2, wherein: The CDRs are carried by a human antibody framework.

9. The antibody or antigen-binding fragment according to claim 1 or 2, wherein: The antibody or antigen-binding fragment comprises: a VH domain, wherein the VH domain comprises the following amino acid sequence: QVQLKESGPG LVAPSQSLSI TCTVSGFSLNSYGVSWVRQP PGKGLEWLGV IWGDGSTNYH SALMSRLRIS KDNSKRQVFL KLNSLQTDDT ATYYCTKPGSGYAFAYWGQG TLVTVSS; and / or A VL domain comprising the following amino acid sequence: ENVLTQSPAI MSASPGEKVTMTCSASSSVN YMHWYQQKSS TSPKLWIYDT SKLASGVPGR FSGSGSGNSY SLTIRTMEAE DVATYFCFQASGYPLTFGGG TKLELK.

10. The antibody or antigen-binding fragment according to claim 1 or 2, wherein: The antibody or antigen-binding fragment is Fab, (Fab')2, scFv, Fv, dAb, Fd or diabody.

11. The antibody or antigen-binding fragment according to claim 1 or 2, wherein: The antibody or antigen-binding fragment is a human antibody, a humanized antibody, a chimeric antibody or a mosaic antibody.

12. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing medulloblastoma, ovarian cancer, breast cancer or leukemia.

13. A nucleic acid comprising a sequence encoding the antibody or antigen-binding fragment according to any one of claims 1 to 11.

14. Use of the antibody or antigen-binding fragment of any one of claims 1 to 11 for the preparation of a medicament for diagnosing cancer, wherein the cancer is selected from medulloblastoma, ovarian cancer, breast cancer or leukemia.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

16. In vitro induction of stem memory T cells (T SCM ) proliferation method, the method comprising causing the stem memory T cells (T SCM ) is contacted with the antibody or antigen-binding fragment according to any one of claims 1 to 11.

17. Used to induce stem memory T cells (T SCM ) a cell culture medium for proliferation, the cell culture medium comprising the antibody or antigen-binding fragment according to any one of claims 1 to 11.

18. Identification of stem memory T cells (T SCM ) method, which is carried out by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on cells using the antibody or antigen-binding fragment according to any one of claims 1 to 11.

19. Purification of stem memory T cells (T SCM ) method, which is carried out by detecting the presence of SSEA-4Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on cells using the antibody or antigen-binding fragment according to any one of claims 1 to 11.

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