SSEA-4 binding members

Abstract

The present disclosure relates to the expression of developmentally specific embryonic antigen 4 (SSEA-4) on stem memory T cells (TSCM), which can then be used as a target for isolating, activating, and expanding subsets of TSCM both in vivo and in vitro. It also relates to pharmaceutical antibody compositions that bind to SSEA-4 and target TSCM, and methods for their use. The disclosed antibodies recognize the SSEA-4 glycolipid, induce proliferation of TSCM, and can be used to select this unique population from blood for adoptive T cell transfer of transduced T cell receptor (TCR), transduced chimeric antigen receptor (CAR)-T, or for clinical expansion of cells for hematopoietic stem cell transplantation. Methods of use include, but are not limited to, cancer treatment and diagnosis. An example was related to the antibody designated F2811.72.

Description

[Technical field]

[0001] The present invention relates to stem memory T cells (T SCM ) which can then be used as targets for isolating, activating, and expanding a subset of stem memory T cells (T SCM ) is associated with the expression of stage-specific embryonic antigen 4 (SSEA-4) on T cells. SCM The present disclosure relates to pharmaceutical antibody compositions and methods for their use that target SSEA-4 glycolipids and SCM These can be used to induce proliferation of T cells and select this unique population from blood for clinical expansion for adoptive T cell transfer of transduced T cell receptor (TCR), transduced chimeric antigen receptor (CAR)-T or for cells for hematopoietic stem cell transplantation. Methods of use include, but are not limited to, the development of T cells in patients with cancer or long-term viral infections or following chemotherapy. SCM and methods for use in cancer therapy and diagnosis as an agonist (IgG2) chimeric monoclonal antibody (mAb) for in vivo stimulation of [Background technology]

[0002] SSEAs are globo-series glycolipids, consisting of three species: SSEA-1, SSEA-3, and SSEA-4 (Suzuki et al. 2013). Sialylgalactosyl globosides (sialylGb5Cer, SGG, MSGG) or SSEA-4 are globo-series gangliosides synthesized from SSEA-3 by the enzyme ST3 β-galactoside α-2,3-sialyltransferase 2 (ST3GAL2) (Saito et al. 2003). The expression of these globosides has been defined mainly by mAbs, due to the complexity of purification and the number of genes involved in their synthesis. The main limitation of this approach is that most of these mAbs have low specificity, making the expression of individual globosides difficult to interpret. With this caveat, the expression of SSEA-4 has been defined as follows: SSEA-4 is a component of the glycosynapse of cell membranes. During human preimplantation development, SSEA-4 is first observed in pluripotent cells of the inner cell mass and then disappears after differentiation (Tondeur et al. 2008). After birth, germline stem cells in human testes and ovaries (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 has been identified via immunization of 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).In solid tumors, overexpression of SSEA-4 has been found in glioblastomas (~55% of grade I, ~55% of grade II, ~60% of grade III, and ~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), basaloid lung carcinoma (Gottschling et al. 2013), ovarian epithelial carcinoma (Ye et al. 2010), and oral cancer (Noto et al. 2013). There is much interest in identifying ultra-specific glycan markers that are cancer-associated and / or predictive, and in developing antibodies against the markers for use in the diagnosis and treatment of a broad spectrum of cancers. SSEA-4 is a glycan that is expressed in embryonic stem cells and downregulated in adult stem cells. However, unexpectedly, the inventors found that this expression was conserved in both human and mouse T. SCM It was shown that the T SCM This is the first time that a marker specific to this species has been described.

[0003] Memory T cells (CD4 + and CD8 + Memory T cells (including T cells) are divided into several subsets: SCM , central memory T cells (T CM ), transitional memory T cells (T TM )(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). To generate more potent antitumor cells in human clinical trials, T cells derived from naive central memory lymphocytes or tumor-infiltrating lymphocytes (TILs) have been investigated. SCM Debate continues as to which method should be used to induce cell production (Klebanoff, Gattinoni, and Restifo 2012).

[0004] Human T SCM cells have been described as a population of memory T cells that survive for long periods and share phenotypic similarities with naive T cells (CD45RO - , CCR7 + , CD45RA + , CD62L + , CD27 + , CD28 + , and IL-7Rα + ), and also significantly express CD95, IL-2Rβ (CD122), and CXCR3 (Gattinoni et al. 2011). T SCM cells are a clonally expanded subset of primitive memory T that arise upon antigen stimulation and exhibit significantly high proliferative and reconstituting capacities (Gattinoni et al. 2011).

[0005] Maintenance of long-term persistent immunity is thought to be dependent on T SCM cells, which constitute a small, least differentiated subset of memory T cells and are approximately 2 - 4% of the total CD4 + and CD8 + T cell populations in the blood (Gattinoni et al. 2011; Lugli, Gattinoni, et al. 2013). T SCM cells are postmitotic CD44 low CD62 high CD8 + T cell subsets that express Sca-1 (stem cell antigen 1), CD122, and Bcl-2. First observed in a mouse model of graft-versus-host disease (GVHD) by Zhang et al. (Zhang et al. 2005), who reported a novel subset of these alloreactive CD8 + T cells, which have been shown to have high self-renewal and multipotency, T CM , T EM , and T TEIn humans, one example is the differentiation of naive yellow fever (YF)-specific CD8 cells after vaccination. + It stems from the identification of a T cell population that has been stably maintained for over 25 years and is capable of self-renewal ex vivo (Fuertes Marraco et al. 2015). SCM These cells can be identified by flow cytometry based on the co-expression of several naive markers together with the marker CD95 (Mahnke et al. 2013). The low frequency of these cells limits their detailed characterization, so it is unclear whether they are antigen-specific T SCM There are limited reports on CD8 T cells. For example, less than 1% of total human T cells are CD8 + CD45RA + CCR7 + CD127 + CD95 + Virus-specific T SCM Human CMV-specific T cells SCM T cells can be detected at a frequency of approximately 1 / 10,000 T cells, similar to that observed in other subsets (Schmueck-Henneresse et al. 2015; Di Benedetto et al. 2015). SCM The cells have been shown to reside preferentially in lymph nodes and at lower abundance in the spleen and bone marrow (Lugli, Dominguez, et al. 2013).

[0006] T SCM T cells may play a key role in specific antitumor responses and long-term immune surveillance of tumors (Darlak et al. 2014; Coulie et al. 2014; Martin 2014). SCMT cells are emerging as key players in the maintenance of long-lived T cell memory and are therefore considered an attractive population for use in adoptive transfer-based immunotherapy of cancer. However, the molecular signals regulating their production remain poorly defined. Experiments performed in the context of adoptive immunotherapy have shown that T cells lacking two key transcription factors governing T cell differentiation, T-box transcription factor (T-bet) and Eomesodermin (eomes), fail to induce anti-tumor responses and express markers that are characteristic of T SCM Therefore, it was found that T SCM The antitumor potential of seems to depend more on their further differentiation into effector memory cells than on their intrinsic activity (Li et al. 2013).

[0007] Although adoptive T cell therapy is an effective strategy for cancer immunotherapy, infused T cells are frequently functionally exhausted and, as a result, provide poor prognosis after transplantation into patients. SCM Adoptive transfer of cells overcomes this drawback. SCMThis is because the cells resemble naive T cells, but are remarkably proliferative, survive for long periods, and generate large numbers of effector T cells in response to antigenic stimulation. Adoptive cell therapy using T cells with tumor specificity derived from natural TCRs or artificial CARs has reached late-stage clinical trials. Immunotherapeutic treatment of cancer using T cells expressing CARs is a relatively novel approach in adoptive cell therapy. CAR-T cells have shown remarkable success in transforming certain B-cell malignancies, but to date have achieved less successful responses against solid tumors. This strategy is based on genetically equipping T cells with a novel synthetic receptor consisting of an antibody-like recognition extracellular domain and a T-cell signaling intracellular domain. Direct identification of intact antigen provided by the antibody-derived binding domain of this receptor allows T cells to bypass the major histocompatibility complex (MHC)-mediated antigen recognition restrictions, allowing a given CAR to be used in all patients, regardless of their MHC haplotype. MHC independence provides CAR-T cells with a fundamental antitumor advantage. Because some tumor cells downregulate MHC expression to evade TCR-mediated immune responses (Garrido et al. 1993), T cells engineered to express a CAR of interest can still recognize and eradicate tumor cells. Furthermore, the use of CAR-T cells can broaden the scope of potential tumor targets to epitopes beyond the scope of TCR-based recognition, for example, to include not only proteins but also carbohydrates (Mezzanzanica et al. 1998) and glycolipids (Yvon et al. 2009) in tumor targeting.

[0008] The characterization of T cells selected for expansion and adoptive transfer is critical in determining the persistence of the transferred cells. In the presence of infection or cancer, antigen-specific T cells can proliferate and differentiate into effector T cells, specialized for rapid elimination of pathogens, and memory T cells, which can persist for long periods and protect against disease recurrence. The memory T cell compartment is heterogeneous and includes multiple subsets with unique properties. The immunological spectrum of memory includes T cells that express CD45RA, CCR7, and CD62L, like naive T cells, as well as CD95. SCM Contains cells. SCM The cells are central memory T cells (T CM ) and effector memory T cells (T EM cells), and terminal effector T cells (T TE ), which have a remarkable potential for self-renewal, as shown by stepwise transplantation experiments (Cieri et al. 2013). The contribution of different memory subsets to the maintenance of the overall memory compartment of antigen-specific T cells is discussed in detail in the literature. SCM These have not been fully characterized due to their low frequency, and their detailed characterization has been limited (Schmueck-Henneresse et al. 2015). SCM Produce and grow T cells to fight cancer cells SCM Strategies that allow for cell repopulation need to be fully defined. Cieri and colleagues reported that naive T cells were priming with anti-CD3 / CD28 and low doses of IL-7 and IL-15 to generate large numbers of T cells. SCM They described the production of T cells from naive precursors in vitro. SCM These results suggest that it is possible to generate, expand, and genetically manipulate T cells. However, the expanded cells no longer express CD45RA but do express CD45RO, and thus they are T CM In addition, in vitro produced T SCM The cells exhibited high proliferative capacity after adoptive transfer into immune-deficient mice, a finding consistent with naturally occurring T SCMAmong the known memory T cell populations, T SCM Cell subsets are highly relevant 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 Although T cells may facilitate the clinical development of cell-directed (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; Suresh et al. 2014), the expression of T cells in circulating lymphocytes is unclear. SCM The low cell numbers limit these techniques (Gattinoni and Restifo 2013).

[0009] Altered glycosylation is a characteristic of cancer cells, and several glycan structures are well-known tumor markers (Meezan et al. 1969; Hakomori 2002). These aberrant changes can include a global increase in N-linked glycan branching (Lau and Dennis 2008) and sialic acid content (van Beek, Smets, and Emmelot 1973), the loss or overexpression of specific glycan epitopes (Sell 1990; Hakomori and Zhang 1997; Taylor-Papadimitriou and Epenetos 1994), the persistence of truncated glycans or the appearance of novel glycans (Huang et al. 2013). Indeed, many tumors have increased expression of certain glycolipids, particularly gangliosides, glycosphingolipids (GSLs), and sialic acid attached to glycan chains. Many studies have demonstrated that aberrant glycosylation contributes to the initial oncogenic transformation and plays a key role in inducing tumor invasion and metastasis (Hakomori 2002).Overexpression of a wide range of 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 (Chang et al. 2008).

[0010] Successful cancer immunotherapy relies on the generation of mAbs with good specificity and potent killing. The complexity of the glycome and the altered expression of glycosyltransferases associated with malignant transformation make carbohydrates associated with cancer cells excellent targets (Christiansen et al. 2014; Dalziel et al. 2014; Daniotti et al. 2013; Hakomori 2002). Glycolipids in particular are attractive due to their dense cell surface distribution, mobility, and association with membrane microdomains, all of which contribute to their involvement in a wide range of cell signaling and adhesive properties (Fuster and Esko 2005; Hakomori 2002; Hakomori 2008). However, generating anti-glycolipid antibodies is a challenging task, as glycolipids do not provide T cell help and mAbs are usually low affinity IgMs. Summary of the Invention

[0011] FG2811.72 (also abbreviated as FG2811) mAb is a murine IgG3 mAb generated from mice immunized with the glycosyl-engineered murine fibroblast cell line SSEA-3 / 4-LMTK. Interestingly, FG2811 mAb specifically recognized SSEA-4, which is similar to SSEA-3 in terms of structure, except that it has an additional terminal sialic acid residue. α-2,3-sialyltransferase is encoded by the ST3GAL2 gene and has been suggested to be the major enzyme responsible for the sialylation of SSEA-3 to SSEA-4. FG2811 mAb specifically binds SSEA-4 and does not cross-react with SSEA-3. This is in contrast to the previously derived mAb MC813, which we found also binds SSEA-3 and Forssman, and MC613, which bound SSEA-3 and Globo-H. In contrast to MC813, FG2811 does not bind to erythrocytes, which do not express SSEA-4, suggesting that MC813 binding may be related to the expression of SSEA-3 / Forssmann (Cooling and Hwang 2005). US Patent Publication No. 2010 / 0047827 describes a mAb that binds to SSEA-4, but the applicants show that it not only binds to the terminal disaccharide Neu5Ac(α2-3)Gal that may be expressed by a range of other globosides, but also to SSEA-3 and Globo-H. US Patent Publication No. 2016 / 0289340 discloses several novel anti-SSEA4 binding mAbs that appear to be specific, but in their assays MC813 is also specific for SSEA-4, in contrast to our results. Binding screening of normal blood revealed that FG2811, but not MC813, recognized a small population of lymphocytes, suggesting that T SCM In contrast to the previous SSEA-4 mAb (MC813), which cross-reacts with SSEA-3 and Forssman antigens, the present disclosure demonstrates that T SCM We describe a highly SSEA-4 specific mAb, FG2811, that can stimulate the proliferation and maintenance of SSEA-4.

[0012] In one aspect the invention provides a specific binding member which specifically binds to SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc.

[0013] In a further aspect, the present invention relates to the use of specific binding members of the invention to stimulate stem memory T cells (T SCM The present invention provides a method for identifying such cells by detecting the presence of SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Glc.

[0014] In a further aspect, the present invention relates to the use of specific binding members of the invention to stimulate stem memory T cells (T SCM The present invention provides a method for purifying the cells by detecting the presence of SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc on the SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc.

[0015] In a further aspect, the present invention relates to stem memory T cells (T SCM In a further aspect, the present invention provides a specific binding member capable of targeting stem memory T cells (T SCM In some aspects of the invention, the specific binding member is capable of specifically binding to stem memory T cells (T SCM ) proliferation.

[0016] In a further aspect, the present invention provides a specific binding member which binds to SSEA-4 Neu5Ac(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 ultra-specific.

[0017] In a further aspect, the present invention provides a specific binding member that binds to SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc, said binding member being capable of binding to stem memory T cells (T SCM The present invention provides specific binding members capable of stimulating the proliferation of .

[0018] In a further aspect, the present invention provides a specific binding member that binds to SSEA-4 Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc, said binding member being capable of binding to stem memory T cells (T SCM ) can be activated by a specific binding member.

[0019] In some aspects of the invention, the specific binding members of the invention inhibit the proliferation of 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% stem memory T cells (T SCM ) proliferation.

[0020] In some embodiments of the present invention, stem memory T cells (T SCM Activation of stem memory T cells (T) may be measured by the production of specific markers or by an increase in the functional activity of the cells. In some aspects of the invention, the specific binding members of the invention are capable of measuring the activation of stem memory T cells (T) in the absence of the specific binding member. 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% stem memory T cells (T SCM ) can be activated.

[0021] In some aspects of the invention, the specific binding member has a molecular weight of about 10-8 Affinity (K d ) and may be capable of binding to Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc presented by glycolipids. Specific binding members are approximately -9 Affinity of M (K d ) to those presented by glycolipids. A specific binding member may be capable of binding to those presented by glycolipids at about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Affinity (K d ) may be capable of binding to those presented by glycolipids.

[0022] A further 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 the amino acid sequences of residues 27-38 (CDRH1), 56-65 (CDRH2) and 105-113 (CDRH3) of Figures 2a and 2b.

[0023] A specific binding member of the invention may comprise an amino acid sequence substantially as set out in Figure 2a as 1-126 (VH). In one embodiment of the invention a specific binding member of the invention comprises a binding domain comprising an amino acid sequence substantially as set out in Figure 2a as residues 105-113 (CDRH3). In this embodiment of the invention the specific binding member may further comprise one or both, preferably both, of the binding domains substantially as set out in Figure 2a as residues 27-38 (CDRH1) and residues 56-65 (CDRH2).

[0024] In another aspect, the invention provides a specific binding member comprising one or more binding domains selected from the amino acid sequence of residues 27-38 (CDRL1), 56-65 (CDRL2) and 105-113 (CDRL3) of Figure 2b.

[0025] In one aspect of the invention, the binding domain may comprise the amino acid sequence substantially as set out as residues 105-113 (CDRL3) of the amino acid sequence in Figure 2b. In this embodiment, the specific binding member may further comprise one or both, preferably both, of the binding domains substantially as set out as residues 27-38 (CDRL1) and residues 56-65 (CDRL2) of the amino acid sequence shown in Figure 2b.

[0026] In some embodiments of the invention, the variable heavy and / or light chains may comprise HCDRs 1-3 and LCDRs 1-3 of antibody FG2811. In some embodiments of the invention, the variable heavy and / or light chains may comprise HCDRs 1-3 and LCDRs 1-3 of antibody FG2811 and framework regions of FG2811.

[0027] Specific binding members comprising multiple binding domains of the same or different sequences, or combinations thereof, are included within the scope of the present invention. Each binding domain may be carried by a human antibody framework. For example, one or more framework regions may be substituted with framework regions of a whole human antibody or its variable regions.

[0028] One isolated specific binding member of the invention comprises a sequence substantially as set out as residues 1 to 123 (VL) of the amino acid sequence shown in Figure 2b.

[0029] In some embodiments, a specific binding member having the sequence of the CDRs of Figure 2a may be combined with a specific binding member having the sequence of the CDRs of Figure 2b.

[0030] In one embodiment, a specific binding member comprises a light chain variable sequence comprising one or more (i.e., 1, 2 or 3) of LCDR1, LCDR2 and LCDR3, LCDR1 contains SSVNY, LCDR2 includes DTS LCDR3 includes FQASGYPLT. A light chain variable sequence; A heavy chain variable sequence comprising one or more (i.e., one, two, or three) of HCDR1, HCDR2, and HCDR3, HCDR1 comprises GFSLNSYG, HCDR2 comprises IWGDGST, HCDR3 contains TKPGSGYAF, Heavy chain variable sequence and may include.

[0031] In a further aspect, the invention provides a specific binding member comprising a VH domain comprising residues 1 to 126 of the amino acid sequence of Figure 2a, and a VL domain comprising residues 1 to 123 of the amino acid sequence of Figure 2b.

[0032] 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.

[0033] The present invention also encompasses a specific binding member as described above, wherein the sequences of the binding domains are substantially as set out in Figure 2. Thus, there is provided a specific binding member as described above, wherein one or more binding domains differ from the binding domain depicted in Figure 2 by 1 to 5, 1 to 4, 1 to 3, 2 or 1 amino acid substitutions.

[0034] The invention also encompasses specific binding members that have the property of binding to the same epitope as the VH and VL sequences depicted in Figure 2. An epitope of an isolated antibody or binding fragment or member thereof is the region of an antigen to which the isolated antibody or binding fragment or member thereof binds. Two antibodies or binding fragments or members thereof bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one isolated antibody or binding fragment or member thereof inhibits the binding of the other by at least 50%, preferably at least 75%, 90%, or even 99%, as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., (Junghans et al. 1990), incorporated herein by reference).

[0035] In a preferred embodiment of the invention, the competing specific binding member competes with an antibody comprising a VH chain having the amino acid sequence of residues 1 to 126 of Figure 2a and a VL chain having the amino acid sequence of residues 1 to 123 of Figure 2b for binding to SSEA-4, Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc bound only to glycolipids.

[0036] Preferably, the competing specific binding member is an antibody, such as a mAb, or any of the antibody fragments described throughout this document.

[0037] After a single prototype mAb, e.g., FG2811 mAb, with the desired properties described herein is isolated, it is easy to generate other mAbs with similar properties by using methods known in the art. For example, the method of (Jespers et al. 1994), incorporated herein by reference as an example, can be used to guide the selection of mAbs with the same epitope and thus similar properties as the prototype mAb. Using phage display, the heavy chain of the prototype antibody is first paired with a repertoire of (preferably human) light chains to select mAbs that bind to glycans, and then the new light chains are paired with a repertoire of (preferably human) heavy chains to select mAbs that bind to (preferably human) glycans with the same epitope as the prototype mAb.

[0038] Included in the present invention are mAbs capable of binding to SSEA-4 bound only to glycolipids, inducing ADCC and / or CDC, and at least 90%, 95%, or 99% identical in the VH and / or VL domains to the VH or VL domains of FIG. 2. The reference to 90%, 95%, or 99% identity may be to the framework regions of the VH and / or VL domains only. In particular, the CDR regions may be identical, while the framework regions may vary by up to 1%, 5%, or 10%. Preferably, such antibodies differ from the sequence of FIG. 2 by substitutions (e.g., conservative substitutions), deletions, or insertions of a small number of functionally insignificant amino acids. In all embodiments of the present invention, the specific binding pair may be an antibody, or an 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 antibody of the invention may be a humanized, chimeric or veneered antibody, or may be a non-human antibody of any species. In one embodiment, the specific binding partner of the invention is the murine antibody FG2811, which comprises the heavy chain as depicted in Figure 2a and the light chain as depicted in Figure 2b.

[0039] Specific binding members of the present invention may carry a detectable or functional label.

[0040] In a further aspect, the invention provides isolated nucleic acids encoding specific binding members of the invention and methods of preparing specific binding members of the invention comprising expressing said nucleic acid under conditions which result in expression of said binding member and recovering said binding member.Included in the invention are isolated nucleic acids encoding specific binding members which are capable of specifically binding to SSEA-4 and which are at least 90%, at least 95%, or at least 99% identical to the sequences provided herein.

[0041] The specific binding members of the invention may be used in methods of treatment or diagnosis of the human or animal body, for example a method of treating a tumour in a patient, preferably a human, comprising the step of administering to said patient an effective amount of a specific binding member of the invention. The invention also provides specific binding members of the invention for use in medicine, preferably for use in the treatment of tumours, and the use of specific binding members of the invention in the manufacture of a medicament for the diagnosis or treatment of a tumour. The tumour may be a gastric, colorectal, pancreatic, lung, ovarian or breast tumour.

[0042] The present invention discloses an antigen that the specific binding member of the present invention binds to.The specific binding member of the present invention can provide SSEA-4 that can be bound, preferably specifically.SSEA-4 can be provided in an isolated form and can be used for screening to develop further specific binding members therefor.For example, a library of compounds can be screened for library members that specifically bind to SSEA-4.

[0043] In a further aspect, the invention provides an isolated specific binding member capable of binding to an SSEA-4-containing glycan (i.e., Neu5Ac(α2 3)Gal(β1 3)GalNAc(β1 3)Gal(α1 4)Gal(β1 4)Glc), preferably of the first aspect of the invention, for use in the diagnosis or prognostic diagnosis of tumors of the stomach, colorectal, pancreas, lung, ovary, and breast.

[0044] In a further aspect of the invention, a method of inducing the proliferation of stem memory T cells (T SCM ) ex vivo, the method comprising the step of contacting the stem memory T cells (T SCM ) with the specific binding member of the present specification, is provided.

[0045] In a further aspect of the invention, a cell culture medium for inducing the proliferation of stem memory T cells (T SCM ) comprising the specific binding member of the invention is provided.

[0046] In a further aspect of the invention, a method of inducing the proliferation of stem memory T cells (T SCM ) in vivo, the method comprising the step of administering the specific binding member of the invention to a subject, is provided.

[0047] In a further aspect of the invention, the binding member of the invention for use in therapy is provided. In a further aspect of the invention, a method of treating a patient, the method comprising the step of administering the specific binding member of the invention to a patient in need thereof, is provided.

[0048] In a further aspect of the invention, the 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 is provided.

[0049] In a further aspect of the invention, a method of treating or preventing cancer, the method comprising the step of administering the specific binding member of the invention to a subject in need thereof, is provided.

[0050] In a further aspect of the invention there is provided a method of treating or preventing a patient with a long term viral infection comprising administering a specific binding member of the invention to a subject in need thereof.

[0051] In a further aspect of the invention there is provided a method of treating or preventing an autoimmune disease, HIV, adult T-cell leukaemia or graft versus host disease comprising administering a specific binding member of the invention to a subject in need thereof.

[0052] In a further aspect of the invention, stem memory T cells (T SCM ) and a specific binding member of the invention, wherein the proliferation rate is greater than that of stem memory T cells (T SCM ) is provided that is at least 10% faster when compared to a corresponding cell culture comprising the

[0053] In some embodiments of the present invention, stem memory T cells (T SCM The proliferation rate of cell cultures comprising stem memory T cells (T) and a specific binding member of the invention is significantly higher than that of stem memory T cells (T) without a specific binding member of the invention. SCM ), 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% faster.

[0054] In a further aspect of the invention, specific binding members of the invention can be used to stimulate stem memory T cells (T SCM A method for purifying stem memory T cells (T) in a cell population, comprising: SCM In some embodiments, the method provides a method, wherein the proportion of cells that are purified using a specific binding member of the invention is at least about 10% higher than in a corresponding cell population that has not been purified using a specific binding member of the invention.

[0055] In some embodiments of the present invention, stem memory T cells (T SCM ) is 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% higher when compared to a corresponding cell population that has not been purified using a specific binding member of the invention.

[0056] In some aspects of the invention, a specific binding member of the invention is an isolated antibody or binding fragment or member thereof.

[0057] The present invention further provides a method for the diagnosis of cancer, comprising using a specific binding member of the present invention to detect SSEA4-containing glycans in a sample from an individual. In some diagnostic methods of the present invention, the pattern of glycans detected by the binding member can be used to stratify the treatment options of an individual.

[0058] These and other aspects of the invention are described in further detail below.

[0059] As used herein, a "specific binding member" is a member of a pair of molecules that have binding specificity for each other. The members of a specific binding pair can be naturally occurring or wholly or partially synthetically produced. One member of the pair has an area on its surface, which may be a protrusion or a cavity, that specifically binds to, and is therefore complementary to, a particular spatial and diametric configuration of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, 5 receptor-ligand, enzyme-substrate. The present invention generally relates to antigen-antibody type reactions, but also to small molecules that bind to antigens as defined herein.

[0060] As used herein, "treatment" includes any regime that can benefit a human or non-human animal, preferably a mammal. Treatment may be in respect of an existing condition or may be preventative (prophylactic treatment).

[0061] As used herein, a "tumor" is an abnormal growth of tissue. It may be localized (benign) or may invade nearby tissues (malignant) or distant tissues (metastatic). Tumors include neoplastic growths that cause cancer, as well as esophageal, colorectal, gastric, breast, ovarian, and endometrial tumors, and cancerous tissues or cell lines, including, but not limited to, white blood cells. As used herein, "tumor" also includes within its scope endometriosis.

[0062] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partially or wholly synthetically produced. The term also encompasses all polypeptides or proteins having a binding domain that is or is homologous to an antibody binding domain. They may be derived from natural sources or they may be partially or wholly synthetically produced. Exemplary antibodies of the invention include immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotypic subclasses; fragments that contain the antigen binding domain, e.g., Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies can be polyclonal or monoclonal. Monoclonal antibodies can be designated as "mAbs."

[0063] It is possible to take monoclonal and other antibodies and use recombinant DNA technology to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such technology may involve introducing DNA encoding the immunoglobulin variable region or CDRs of an antibody into the constant region or constant region plus framework regions of a different immunoglobulin. See, for example, EP 184187, GB 2188638, or EP 239400. Hybridomas or other cells that produce the antibodies may be subject to genetic mutation or other changes that may or may not alter the binding specificity of the antibodies produced.

[0064] Since antibodies can be modified in many ways, the term "antibody" should be interpreted to encompass all specific binding members or substances that have a binding domain with the required specificity. The term thus includes antibodies, antibody fragments, derivatives, functional equivalents, and homologs of humanized antibodies, including all polypeptides that contain an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus, chimeric molecules or equivalents that contain an immunoglobulin binding domain fused to another polypeptide are included. Cloning and expression of chimeric antibodies are described in European Patent Publications 0120694 and 0125023. Humanized antibodies can be modified antibodies that have the variable regions of a non-human, e.g., mouse, antibody and the constant regions of a human antibody. Methods for making humanized antibodies are described, for example, in U.S. Pat. No. 5,225,539.

[0065] It has been shown that fragments of a whole antibody can perform the function of binding to an antigen. Examples of binding fragments are: (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward et al. 1989); (v) an isolated CDR region; (vi) a F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (vii) a single chain Fv molecule (scFv), in which the VH and VL domains form an antigen-binding site by linking them together with a peptide linker that connects the two domains (Bird et al. 1988; Huston et al. 1988); (viii) bispecific single chain Fv dimers (WO 92 / 09965); and (ix) "diabodies," multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; (Holliger, Prospero, and Winter 1993)).

[0066] Diabodies are multimers of polypeptides in which each polypeptide comprises a first domain comprising the binding region of an immunoglobulin light chain and a second domain comprising the binding region of an immunoglobulin heavy chain, and the two domains are linked (e.g., by a peptide linker) but cannot bind to each other to form an antigen-binding site: the antigen-binding site is formed by the binding of the first domain of one polypeptide in the multimer to the second domain of another polypeptide in the multimer (WO 94 / 13804).

[0067] When bispecific antibodies are used, they may be conventional bispecific antibodies that can be produced in a variety of ways (Holliger and Winter 1993), for example prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed without an Fc region using only the variable domains, presumably reducing the effects of anti-idiotypic reactions. Other forms of bispecific antibodies include the single chain "Janusins" described in (Traunecker, Lanzavecchia, and Karjalainen 1991).

[0068] Bispecific diabodies may also be useful because, in contrast to bispecific whole antibodies, they can be readily constructed and expressed in E. coli. Diabodies (and many other polypeptides, e.g., antibody fragments) of appropriate binding specificity can be readily selected from libraries using phage display (WO 94 / 13804). If one arm of the diabody remains constant, e.g., with specificity for antigen X, this allows the creation of libraries in which the other arm is varied and antibodies of appropriate specificity can be selected.

[0069] A "binding domain" is a portion of a specific binding member that contains an area that specifically binds and is complementary to part or all of an antigen. If the binding member is an antibody or an antigen-binding fragment thereof, the binding domain may be a CDR. If the antigen is large, the antibody may only bind to a specific part of the antigen, this part being called an epitope. An antigen-binding domain may be provided by one or more antibody variable domains. An antigen-binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0070] "Specific" is generally used to describe the situation where one member of a specific binding pair does not show any significant binding to molecules other than its specific binding partner, e.g. has less than about 30%, preferably less than 20%, less than 10%, or less than 1% cross-reactivity with any other molecule. The term is also applicable, for example, when 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 can bind to a variety of antigens carrying this epitope. A specific binding member of the present invention exhibits Leu binding in the absence of detectable binding to any other antigen (e.g. any other glycan) when binding is tested by the protocol presented in the "Glycome Analysis" section of the Examples herein. y In some cases, it may be possible to specifically bind to

[0071] "Isolated" refers to the situation where a specific binding member of the invention or a nucleic acid encoding said binding member is preferably according to the invention. Generally, the members and nucleic acids are free or substantially free from naturally associated substances such as other polypeptides or nucleic acids found in their natural environment or the environment in which they are prepared (where said preparation is by recombinant DNA techniques carried out in vitro or in vivo) (e.g. cell culture). The specific binding members and nucleic acids may be formulated with a diluent or adjuvant, or may even be isolated for practical purposes - for example, the members will typically be mixed with gelatin or other carrier when used to coat microtiter plates for use in immunoassays, or mixed with a pharma- ceutically acceptable carrier or diluent when used diagnostically or therapeutically. The specific binding members may be glycosylated, either naturally or by heterologous eukaryotic systems, or they may be unglycosylated (for example when produced by expression in prokaryotic cells).

[0072] "Substantially as set out" means that the amino acid sequences of the invention are identical or significantly homologous to the amino acid sequence described. By "significantly homologous" it is contemplated that 1-5, 1-4, 1-3, 2 or 1 amino acid substitutions can be made in the sequence.

[0073] The present invention also includes within its scope polypeptides having the amino acid sequences presented in Figure 2, polynucleotides having the nucleic acid sequences presented in Figure 2, and sequences having substantial identity thereto, e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 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 (e.g., gaps may be introduced in the first sequence for best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of the two sequences that results in 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 x 100).

[0074] The determination of percent identity between two sequences can be achieved using a mathematical algorithm known to those skilled in the art. One example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990 (Karlin and Altschul 1990), modified as in Karlin and Altschul, 1993 (Karlin and Altschul 1993). The NBLAST and XBLAST programs of Altschul et al., 1990 (Altschul et al. 1990) incorporate such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program (score=100, wordlength=12) to obtain nucleotide 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. To obtain gapped alignments for comparison, Gapped BLAST can be used as described in Altschul et al., 1997 (Altschul et al. 1997). Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Id.). When using BLAST, Gapped BLAST, 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 used for sequence comparison is the algorithm of Myers and Miller, 1989 (Myers and Miller 1989). The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, described in Torellis and Robotti, 1994 (Torelli and Robotti 1994); and FASTA, described in Pearson and Lipman, 1988 (Pearson and Lipman 1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0075] The isolated specific binding members of the invention are capable of binding to the SSEA-4 carbohydrate, which may be the SSEA-4 ceramide or may be on a protein moiety. A binding domain comprising the amino acid sequence substantially as set out as residues 105 to 116 in Figure 2 (CDRH3) and 105 to 113 in Figure 2 may be carried in a structure which allows binding of these regions to the SSEA-4 carbohydrate.

[0076] The structures for carrying the binding domains of the invention may generally be antibody heavy or light chain sequences or substantial portions thereof, in which the binding domains are located at 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 may be determined by reference to http: / / www.imgt.org / . The amino acid sequence substantially as set out as residues 105-116 of Figures 1a and 1b may be carried as the CDR3 of a human heavy chain variable domain or substantial portion thereof, and the amino acid sequence substantially as set out as residues 105-113 of Figure 1c may be carried as the CDR3 of a human light chain variable domain or substantial portion thereof.

[0077] The variable domain may be derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. The CDR3-derived sequences of the present invention may be introduced into a repertoire of variable domains lacking CDR3 regions using recombinant DNA technology. For example, Marks et al., 1992 (Marks et al. 1992) describes a method for generating a repertoire of antibody variable domains in which a consensus primer directed to or adjacent to the 5' end of the variable domain region is used in conjunction with a consensus primer against the third framework region of the human VH gene to provide a repertoire of VH variable domains lacking CDR3. Marks et al., 1992 (Marks et al. 1992) further describes how this repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, CDR3 derived sequences of the invention may be shuffled with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains can be combined with the cognate VL or VH domains to provide a specific binding member of the invention. This repertoire can then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, from which suitable specific binding members can be selected. The repertoire can be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, from which suitable specific binding members can be selected. 4 or more individual members, e.g. 10 6 ~10 8 or 10 10 The committee may consist of members of

[0078] Similar shuffling or combinatorial techniques have also been disclosed by Stemmer, 1994 (Stemmer 1994), who describes the technique in the context of β-lactamase genes, but observes that this approach can be used to generate antibodies. A further alternative is to use random mutagenesis of, for example, the FG2811 VH or VL genes to generate mutations in the entire variable domain, to generate novel VH or VL regions carrying sequences derived from the CDR3 of the invention. Such a technique has been described by Gram et al., 1992 (Gram et al. 1992), using error-prone PCR.

[0079] Another method that can be used is to carry out mutagenesis on the CDR regions of the VH or VL genes. Such techniques are disclosed by Barbas et al., 1994 (Barbas et al. 1994) and Schier et al., 1996 (Schier et al. 1996). A substantial portion of an immunoglobulin variable domain generally includes at least three CDR regions together with their intervening framework regions. This portion may also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N- or C-terminus of the substantial portion of the variable domain may not be normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the invention by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (e.g. in the production of diabodies) or protein tags, as discussed in more detail below.

[0080] The present invention provides specific binding members comprising a pair of binding domains based on the amino acid sequences of the VL and VH regions substantially as depicted in Figure 2, namely amino acids 1-127 of Figure 2 (VH) and amino acids 1-124 of Figure 2 (VL). Single binding domains based on any of these sequences form further aspects of the invention. In the case of binding domains based on the amino acid sequence of the VH region substantially as depicted in Figure 2, such binding domains may be used as targeting agents, since it is known that immunoglobulin VH domains are capable of binding to target antigens in a specific manner. In the case of any of the single chain specific binding domains, these domains may be used to screen for complementary domains capable of forming two-domain specific binding members with in vivo properties as good as or comparable to those of the FG2811 antibody disclosed herein.

[0081] This can be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO 92 / 01047, in which individual colonies containing either H or L chain clones are used to infect the entire library of clones encoding the other chain (L or H), and specific binding members of the resulting double chain are selected by phage display techniques such as those disclosed in this reference. This technique is also disclosed in Marks et al., 1992 (Marks et al. 1992).

[0082] Specific binding members of the invention may further comprise an antibody constant region or part thereof. For example, specific binding members based on the VL region shown in Figure 2a may bind at their C-terminus to an antibody light chain constant domain. Similarly, specific binding members based on the VH region shown in Figure 2 may bind at their C-terminus to all or part of an immunoglobulin heavy chain from any antibody isotype, for example IgG, IgA, IgE and IgM, and isotype subclasses, in particular IgG1, IgG2 and IgG4.

[0083] Specific binding members of the present invention may be used in methods of diagnosis and treatment of tumours in human or animal subjects.

[0084] When used diagnostically, the specific binding members of the invention may be coupled to a detectable label, e.g. 131 I or 99 The antibodies may be labeled with a radioactive label such as Tc, which may be attached to the specific binding members of the invention using conventional chemical techniques known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin that can be detected via binding to a specific cognate detectable moiety, for example labeled avidin.

[0085] Furthermore, the specific binding members of the present invention can be administered alone or in combination with other treatments simultaneously or sequentially, depending on the condition to be treated. Thus, the present invention further provides a product comprising the specific binding member of the present invention and an effective agent as a formulation for simultaneous use, separate use, or sequential use in the treatment of tumors. The effective agent may include chemotherapeutic agents or cytotoxic agents that can act synergistically with the binding members of the present invention, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin, and tamoxifen. Other effective agents may include appropriate doses of analgesics, such as non-steroidal anti-inflammatory drugs (e.g., aspirin, paracetamol, ibuprofen, or ketoprofen) or opiates, such as morphine, or antiemetics.

[0086] While not wishing to be bound by theory, the properties of the binding members of the present invention that act synergistically with the effective agent to enhance tumor killing may not be due to immune effector mechanisms, but rather may be a direct result of the binding of the binding members to the SSEA-4 glycan bound to the cell surface. Cancer immunotherapy, including antibodies against immune checkpoint molecules, has been shown to be effective in combination with different immuno-oncology treatment modalities against various malignancies.

[0087] The specific binding members of the present invention are usually administered in the form of a pharmaceutical composition, which may contain at least one component in addition to the specific binding member. In addition to the active ingredient, the pharmaceutical composition may contain pharma- ceutically acceptable excipients, diluents, carriers, buffers, stabilizers, or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substances will vary depending on the route of administration, which may be oral or by injection, e.g., intravenous injection. Injection is the primary route for therapeutic administration of the composition, although it is envisaged that delivery via a catheter or other surgical tube may also be used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal, and intramuscular administration. Liquid formulations may be utilized after reconstitution from powder formulations.

[0088] For intravenous injection or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability.Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's solution, lactated Ringer's solution, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as necessary.

[0089] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier or adjuvant, such as gelatin. Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, dextrose, or other sugar solutions or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol, may be included. When the formulation is liquid, it may be, for example, a physiological salt solution, including a non-phosphate buffer of pH 6.8-7.6, or a lyophilized powder.

[0090] The composition can also be administered via microspheres, liposomes, other microparticulate delivery systems, or sustained-release formulations that are placed in certain tissues, including blood.Suitable examples of sustained-release carriers include semipermeable polymer matrices in the form of common articles, such as suppositories or microcapsules.Implantable or microencapsulated sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919; European Patent Publication No. 0058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al. 1983), poly(2-hydroxyethyl-methacrylate). Liposomes containing polypeptides are prepared by well-known methods: DE 3,218, 121A; (Eppstein et al. 1985); (Hwang, Luk, and Beaumier 1980); EP 0052522; EP 0036676; EP 0088046; EP 0143949; EP 0142541; U.S. Patent Publication No. 83-11808; U.S. Patent Nos. 4,485,045 and 4,544,545. Essentially, liposomes are small (about 200-800 angstroms) unilamellar type, in which the lipid content is greater than about 30 mol% cholesterol, and the selected ratio is adjusted for optimal rate of polypeptide leakage. The compositions may be administered in a localized manner to the site of a tumor or other desired site, or may be delivered in a manner that targets tumor or other cells.

[0091] The composition is preferably administered to an individual in a "therapeutically effective amount", which is an amount sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will vary depending on the nature and severity of what is being treated. Prescription of treatment, such as determining dosage, is within the responsibility of general practitioners and other physicians, and will usually take into account the disorder being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to practitioners. The compositions of the present invention are particularly relevant for the treatment of existing tumors, particularly cancer, and for preventing the recurrence of such conditions after initial treatment or surgery. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16 th edition, Oslo, A. (ed), 1980 (Remington 1980).

[0092] 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. Generally, a serum concentration of the polypeptide and antibody that allows for receptor saturation is desired. A concentration of greater than about 0.1 nM is usually sufficient. For example, 100 mg / m 2 This dose of antibody provides a serum concentration of about 20 nM for about 8 days.

[0093] As a rough guideline, antibody doses range from 10 to 300 mg / m 2The antibody fragment may be administered weekly in an amount of 0.1 mg / kg / day. An equivalent dose of the antibody fragment is used at more frequent intervals to maintain serum levels above concentrations that allow saturation of the SSEA4 carbohydrate. The dose of the composition will vary depending on the properties of the binding member, such as its binding activity and plasma half-life in vivo, the concentration of the polypeptide in the formulation, the route, site and rate of administration, the clinical tolerance of the patient involved, the pathology afflicting the patient, etc., and is within the skill of the physician. For example, a dose of 300 μg of antibody per patient per administration is preferred, although doses may range from about 10 μg to 6 mg per administration. Different doses are utilized during a series of successive inoculations. The practitioner may administer an initial inoculation and then boost with a relatively lower dose of antibody.

[0094] The present invention is also directed to an optimized immunization schedule for enhancing a protective immune response against cancer. The present invention provides an immunization schedule for enhancing a protective immune response against cancer.

[0095] Binding members of the invention may be made wholly or partly by chemical synthesis. They may be readily prepared by well-established standard solution-phase or, preferably, solid-phase peptide synthesis techniques (general descriptions of which are widely available (see, for example, JM Stewart and JD Young, 1984 (Stewart and Young 1984); M. Bodanzsky and A. Bodanzsky, 1984 (Bodanzsky and Bodanzsky 1984)), or they may be prepared in solution, by solution-phase methods or any combination of solid-phase, solution-phase and solution chemical techniques, for example by first completing each peptide moiety and then, if desired and appropriate, after removing any protecting groups present, introducing residue X by reaction of the respective carbonate or sulfonic acid or a reactive derivative thereof.

[0096] Another convenient way to produce a binding member according to the invention is to express nucleic acid encoding it by using nucleic acid in an expression system.

[0097] The present invention further provides isolated nucleic acids encoding the specific binding members of the present invention. Nucleic acids include DNA and RNA. In a preferred embodiment, the present invention provides nucleic acids encoding the specific binding members of the present invention as defined above. An example of such a nucleic acid is shown in Figure 2. The skilled artisan can determine substitutions, deletions and / or additions to the nucleic acid that still provide the specific binding member of the present invention.

[0098] The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as described above. The invention also provides recombinant host cells comprising one or more constructs as described above. As mentioned, nucleic acids encoding specific binding members of the invention form an aspect of the invention, as do methods of producing specific binding members comprising expression from the encoding nucleic acid. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the specific binding member may be isolated and / or purified using any suitable technique and then used as appropriate.

[0099] 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 available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, and many others. A common preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see, e.g., Pluckthun, 1991 (Pluckthun 1991). Expression of eukaryotic cells in culture is also available to those skilled in the art as an option for the production of specific binding members. For recent reviews, see, e.g., Reff, 1993 (Reff 1993); Trill et al., 1995 (Trill, Shatzman, and Ganguly 1995).

[0100] Suitable vectors can be selected or constructed, including suitable control sequences, including promoter sequences, transcription termination sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences.Vector can be plasmid, virus, such as "phage" or phagemid, as appropriate.For further details, see, for example, Sambrook et al., 1989 (Sambrook 1989).Many known techniques and protocols for nucleic acid manipulation, mutagenesis, sequencing, introduction of DNA into cells, and gene expression, as well as protein analysis, for example, in the preparation of nucleic acid constructs, are detailed in Ausubel et al., 1992 (Ausubel 1992).

[0101] Thus, a further aspect of the invention provides a host cell comprising a nucleic acid as disclosed herein. A further aspect provides a method comprising introducing the nucleic acid into a host cell. The introduction may use any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus, or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. After the introduction, expression from the nucleic acid may be caused or allowed, for example, by culturing the host cell under conditions for gene expression.

[0102] The nucleic acids of the invention can be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome by standard techniques.

[0103] The present invention also provides a method comprising using a construct as described above in an expression system for expressing a specific binding member or polypeptide as described above.

[0104] Preferred features of each embodiment of the invention are as for each other embodiment mutatis mutandis. Prior art documents referred to herein are incorporated to the fullest extent permitted by law.

[0105] In certain aspects, the disclosure provides a pharmaceutical composition comprising a mAb or binding fragment thereof described herein and a pharma- ceutically acceptable carrier.

[0106] Immunomodulatory mAbs are designed to block key inhibitory pathways that suppress effector T cells (checkpoint blockers) or agonistically require costimulatory immune receptors (immunostimulatory). In this patent, we show that 2811 mAb can stimulate T cell proliferation in vitro and in vivo. Isotype-dependent FcγRIIB engagement has been shown to be essential for immune agonistic mAb activity. These agents stimulate signaling through their target receptors, usually members of the tumor necrosis factor receptor (TNFR) superfamily, and receptor clustering and subsequent downstream signaling are promoted by the interaction of FcγRIIB with mAb Fc. As cancer therapeutics, they are designed to boost tumor immunity by requiring costimulatory receptors such as CD40, 4-1BB, or OX40 on APCs or T effector cells, or to promote apoptosis by stimulating death receptors (DRs) such as DR4, DR5, or Fas (CD95) on cancer cells. In contrast to direct targeting agents, the agonistic activity of these mAbs relies on their properties requiring inhibitory FcγRIIB; mAbs with a high ratio of binding to activating receptors over inhibitory receptors (A:I) (e.g., mouse IgG2a, human IgG1) are largely inactive in preclinical models, while those with low A:I ratios (e.g., mouse IgG1 and hIgG2) are significantly agonistic. Signaling through FcγRIIB is not required to provide activity; rather, FcγRIIB provides a cross-linking scaffold for the mAb to promote TNFR clustering and activation (Beers, Glennie, and White 2016). Therefore, FG2811 mIgG1 may be used in vivo to stimulate TSCM, and plate-bound 2811 hIgG1 or mIgG3 may be used in vitro. Another approach would be to use the hIgG2 isotype.This human isotype has limited binding affinity for FcγRIIB and has the intrinsic property of driving receptor clustering through its unique hinge disulfide configuration (White 2015; Liu 2019; Yu 2020). Upon synthesis, hIgG2 converts into a range of isoforms through rearrangements of disulfide bonds in its hinge and CH1 domains, with more compact and rigid forms exhibiting potent FcγRIIB-independent receptor clustering in vitro and in vivo. Thus, we demonstrate 2811 hIgG2-induced stimulation of TSCM.

[0107] In some aspects, the present invention provides a method for the production of stem memory T cells (T cells) ex vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM In some aspects, the present invention provides a method for isolating stem memory T cells (T) ex vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM In some aspects, the present invention provides a method for expanding stem memory T cells (T) ex vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM The present invention provides methods for isolating and propagating the

[0108] In a specific embodiment, the present invention provides ex vivo stem memory T cells (T SCM In a particular embodiment, the present invention provides a method for isolating and / or expanding stem memory T cells (T) ex vivo using an isolated antibody or binding fragment or member thereof comprising the binding domain of mAb 2811 and any framework region from any mouse or human antibody isotype. SCM The present invention provides methods for isolating and / or propagating the Bacillus subtilis var.

[0109] In some aspects, the present invention provides a method for the generation of stem memory T cells (T cells) in vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM In some aspects, the present invention provides a method for isolating stem memory T cells (T) in vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM In some aspects, the present invention provides a method for expanding stem memory T cells (T) in vivo via binding of an isolated specific binding member of the invention to the SSEA-4 antigen. SCM The present invention provides methods for isolating and propagating the

[0110] In some aspects, the present invention provides a method for the generation of stem memory T cells (T cells) in vivo via binding to the SSEA-4 antigen. SCM In some aspects, the present invention provides an isolated specific binding member of the invention for use in a method for isolating stem memory T cells (T) in vivo via binding to the SSEA-4 antigen. SCM In some aspects, the present invention provides an isolated specific binding member of the invention for use in a method for expanding stem memory T cells (T) in vivo via binding to the SSEA-4 antigen. SCM The present invention provides an isolated specific binding member of the invention for use in a method for isolating and propagating a specific binding member of the present invention.

[0111] In a specific embodiment, the present invention provides a method for the generation of stem memory T cells (T cells) in vivo using mAb 2811 of either mouse or human isotype. SCM In a particular embodiment, the present invention provides a method for isolating and / or expanding stem memory T cells (T) in vivo using an isolated antibody or binding fragment or member thereof comprising the binding domain of mAb 2811 and any framework region from any mouse or human antibody isotype. SCM The present invention provides methods for isolating and / or propagating the Bacillus subtilis var.

[0112] In some embodiments of the present invention, stem memory T cells (T SCM ) refers to increasing cell proliferation and / or promoting cell division.

[0113] In some aspects of the invention, cells are identified or purified by using a binding member of the invention to target or label cells and then applying a cell sorting or cell separation method. In some aspects of the invention, the binding member of the invention may be used in cell sorting or cell separation methods such as Fluorescence Activated Cell Sorting (FACS), Flow Cytometry, Immunomagnetic Cell Separation, Immunodensity Cell Separation, Immunoguided Laser Capture Microdissection (LCM). In a preferred embodiment, the binding member of the invention may be used in Fluorescence Activated Cell Sorting (FACS). In a preferred embodiment, the binding member of the invention may be used in flow cytometry methods.

[0114] In certain aspects, the invention provides methods of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an isolated specific binding member of the invention. In some methods of the invention, the administered binding member stimulates proliferation of the isolated specific binding member of the invention in the subject.

[0115] In certain embodiments, the method provides for the treatment of a cancer selected from the group consisting of brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, bone cancer, skin cancer, cervical cancer, ovarian cancer, and prostate cancer.

[0116] In certain aspects, the invention provides a pharmaceutical composition comprising an isolated specific binding member of the invention for use in a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, in some methods of the invention the administered binding member stimulates proliferation of the isolated specific binding member of the invention in the subject.

[0117] In certain embodiments, the pharmaceutical composition for use in the methods of the present invention treats a cancer selected from the group consisting of brain cancer, lung cancer, breast cancer, oral cancer, esophageal cancer, gastric cancer, liver cancer, bile duct cancer, pancreatic cancer, colon cancer, kidney cancer, bone cancer, skin cancer, cervical cancer, ovarian cancer, and prostate cancer.

[0118] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims.

[0119] As used herein, nomenclature of symbols, graphics, and text for describing glycans and related structures, including, for example, "Symbols Nomenclatures for Glycan Representation" by Ajit Varki et al. (Varki et al. 2009), is well established and understood in the art. [Brief description of the drawings]

[0120] [Figure 1] Schematic diagram of the production of SSEA-3 and SSEA-4 glycans from lactosylceramide (LC). LMTK mouse fibroblast cell line was transduced with A4GALT, B3GALNT1, and B3GALT5 genes to generate α-1,4-galactosyltransferase, β-1,3-N-acetylgalactosaminyltransferase, and β-1,3-galactosyltransferase, respectively, and glycans were sequentially added to LC to produce SSEA-3 and SSEA-4 glycans. [Diagram 2] Amino acid and nucleotide sequences of FG2811 IgG3 heavy and kappa light chain variable regions and mIgG1, hIgG1, and hIgG2 constant regions. (A) Nucleotide and amino acid sequences of mature FG2811 heavy chain variable region showing framework regions (FR) 1-3 and complementarity determining regions (CDR) 1-3. (B) Nucleotide and amino acid sequences of mature FG2811 kappa chain variable region showing framework regions (FR) 1-3 and complementarity determining regions (CDR) 1-3. (C) Germline aligned nucleotide and amino acid sequences of mIgG1. (D) Germline aligned nucleotide and amino acid sequences of hIgG2. (E) Germline aligned nucleotide and amino acid sequences of hIgG1. [Diagram 3] Binding patterns of 2811 mouse IgG (IgG1 and IgG3) isotypes versus chimeric IgG (IgG1 and IgG2) isotypes to SSEA-3 / 4-LMTK cells. Binding of FG2811mG3, FG2811mG1, CH2811hG1, CH2811hG2, MC813 (anti-SSEA-4 mAb; mouse IgG1), MC631 (anti-SSEA-3 mAb; rat IgM), FG88.7 (anti-Lewisa / c / x mAb; mouse IgG3), anti-mouse secondary and tertiary antibodies alone, anti-human secondary and tertiary antibodies alone, and media alone to SSEA-3 / 4-LMTK cells was assessed by flow cytometry. The results are presented as geometric mean (Gm) values. [Figure 4]Evaluation of the Specificity of FG2811mG3 for SSEA-4 (A) Binding of FG2811mG3 mAb to lipid antigens as evaluated by HPTLC. Thin layer chromatography analysis of 1) wild-type LMTK and 2) SSEA-3 / 4-LMTK cell membrane lipid extracts using 5 μg / ml of i) FG2811mG3 mAb, ii) MC631 mAb, and iii) MC813 mAb. (B) FG2811mG3 mAb bound to SSEA-3 / 4-LMTK cell surface antigens. Binding of 5 μg / ml of i) secondary antibody alone, 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 presented as Gm values. (C) Reactivity of FG2811mG3 mAb with glycan antigens (SSEA-3, SSEA-4, Globo-H, and Forssman) coupled to HSA. Binding of 5 μg / ml of i) FG2811mG3, ii) MC813, iii) MC631, iv) M1 / 87, and v) KM93 mAb to glycans coupled to HSA was evaluated by ELISA. MC631 (anti-SSEA-3 and SSEA-4), MC813 (anti-SSEA-4), M1 / 87 (anti-Forssman), and KM93 (anti-sialyl-Lewisx) were included as positive control mAbs. 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 vs. control, ANOVA followed by Bonferroni's multiple comparison test, GraphPad Prism 6). (D) Binding of FG2811mG3 mAb to the Consortium for Functional Glycomics array (CFG, core H, version 5.1). Sp means the length of the spacer between glycans on the slide. [Diagram 5]Evaluation of FG2811mG3 affinity to antigen. (A) Binding kinetics of FG2811mG3 mAb to SSEA-3 / 4-LMTK cell membrane lipid antigen was tested using SPR (Biacore X). (B) ELISA of SSEA-3 / 4-LMTK cell membrane lipid. A range of concentrations of FG2811mG3 mAb were incubated in microwells coated with SSEA-3 / 4-LMTK cell membrane lipid. The EC50 value (6.8×10-10M) was obtained via nonlinear regression on log-transformed data (GraphPad Prism 6). (C) SSEA-3 / 4-LMTK cell surface binding. SSEA-3 / 4-LMTK cells were incubated with a range of concentrations of FG2811mG3 mAb and cell binding was analyzed by flow cytometry. Kd values ​​were generated by fitting the background subtracted data to a one site specific binding model (GraphPad Prism 6). Representative binding curves from three independent experiments are shown. [Figure 6] Binding of FG2811mG3 antibody to a panel of human cancer cell lines. (A) Antibody binding to brain cancer cell lines (U251, KNS42, DAOY, SF188, U87, and UW2283). Binding of antibodies FG2811mG3, MC813, mouse IgG3kappa isotype control, and secondary antibody alone (no primary antibody) to brain cancer cell lines at 5 μg / ml was assessed by flow cytometry and results are presented as Gm values. (B) Antibody binding to ovarian (SKOV3, IGROV1, and OVCAR-5), breast (T47D, MCF7, DU4475, and HCC1187), and colorectal (Colo205 and HCT15). Binding of antibodies FG2811mG3, anti-HLA-A, B, C (W6 / 32), and secondary antibody alone (no primary antibody) to a panel of cancer cell lines at 5 μg / ml was assessed by flow cytometry and results were presented as Gm values. [Figure 7]Cytotoxic activity of the FG2811mG3 antibody (A) ADCC killing of cancer cells by the FG2811mG3 mAb. Dose-dependent ADCC activity of the FG2811mG3 mAb on SKOV3 cells and T47D cells. Cancer target cells labeled with 51Cr were co-incubated with increasing concentrations of the FG2811mG3 mAb (0.003 - 10 μg / ml) and human PBMCs (target cells:PBMCs; 100:1). 51Cr released into the supernatant was measured and expressed as a percentage of the total 51Cr released with 10% Triton-X. Anti-CD55 mAb (791T / 36) was used as a negative control mAb. Significance compared to the PBMC control was established by ANOVA followed by Bonferroni's multiple comparison test in GraphPad Prism 6 (***, P < 0.001 vs. control). (B) CDC killing of cancer cells by the FG2811mG3 mAb. Dose-dependent CDC activity of the FG2811mG3 mAb on SKOV3 cells and T47D cells. Cancer target cells labeled with 51Cr were co-incubated with increasing concentrations of the FG2811mG3 mAb (0.003 - 10 μg / ml) and human serum. 51Cr released into the supernatant was measured and expressed as a percentage of the total 51Cr released with 10% Triton-X. Anti-CD55 mAb (791T / 36) was used as a negative control mAb. Significance compared to the PBMC control was established by ANOVA followed by Bonferroni's multiple comparison test in GraphPad Prism 6 (*, P < 0.005; ***, P < 0.001 vs. control). (C) FG2811mG3 induced direct cell death of cancer cells at 37°C. Uptake of propidium iodide (PI) after exposure to the mAb was evaluated by flow cytometry analysis. SSEA-3 / 4-LMTK cells were incubated with 30 μg / ml of the FG2811mG3 mAb at 37°C. Hydrogen peroxide (H2O2) and medium alone were included as positive and negative controls, respectively. (D) Phase contrast images of cancer cells treated with FG2811mG3. Images (magnification ×10) show SSEA-3 / 4-LMTK cells, SKOV3 cells, and LMTK cells after incubation for 72 hours with 30 μg / ml of the FG2811mG3 mAb and medium alone. [Figure 8] Normal erythrocyte binding. (A) Evaluation of binding of FG2811mG3 mAb to erythrocytes of healthy donors by flow cytometry. Binding of erythrocytes by FG2811mG3 mAb was compared to 791T / 36 positive control mAb (anti-CD55 mAb) by flow cytometry. Both mAbs were used at 10 μg / ml. Isotype control mAb and medium alone were used as negative controls. Results are representative of five donors. (B) Hemagglutination assay. Agglutination of erythrocytes by FG2811mG3 mAb at various concentrations (0.625-10 μg / ml) was compared to 791T / 36 and a positive control antibody of the anti-blood group. PBS was used as a negative control. Results are representative of five donors. [Figure 9] Binding of FG2811mG1 to human blood cells. (A) FG2811mG1 bound to PBMCs of whole blood from healthy donors. Binding of FG2811mG1, MC813, mouse IgG1 isotype control antibody (isotype ctrl), OKT3 (anti-CD3), 198 (anti-CEACAM6), and FITC secondary antibody specific for anti-mouse IgG Fc alone (no primary antibody) to whole blood from healthy donors was assessed by indirect immunofluorescence staining and flow cytometry analysis. All mAbs were used at 5 μg / ml. Results shown are representative of whole blood from seven different healthy donors. Results are shown in dot plots and histograms. (B) Phenotyping of PBMCs. Sequential panels depicting the flow cytometry gating strategy used to phenotype CD3+FG2811mG3+ PBMCs; gates were drawn for analysis on CD3+FG2811mG3+ cells; CD3+FG2811mG3+ cells were confirmed for expression of CD45RA and CD45RO. CD45RA+, CD45RA+RO+, and CD45RO+ cells were further confirmed for expression of CD62L, CD95, and CCR-7 markers. [Figure 10]CH2811hG1-enriched and CD122 / CD95-enriched naïve T cells from four healthy donors (BD3, BD13, BD61, BD96) were transcriptionally profiled using bulk RNAseq. (A) Venn diagram showing common genes between two sets of differentially expressed (DE) genes obtained by comparing naïve CD8 T cells (GSE83808) with CH2811hG1-enriched and CD122 / CD95-enriched naïve T cells, respectively. The 2227 common genes identified were analyzed for stemness signatures using StemChecker (Pinto et al. 2015). Statistically significant enrichment of genes associated with stem cell subsets and significantly enriched targets of transcription factors associated with stemness are shown in the table. (B) Heatmap and hierarchical clustering (Euclidean distance) of CH2811hG1-enriched and CD122 / CD95-enriched transcriptome profiles based on 257 overlapping genes from ESCs and 113 overlapping genes from HSCs (both from table A), respectively. There is no clear separation of the two enriched populations, suggesting commonality in their stemness profiles. (C) Heatmap (>2-fold, p<0.001) based on (i) DE genes between CD8+TSCMs and TNs from Gattinoni et al., 2011 (Gattinoni et al. 2011) and (ii) a subset of transcription factor, effector function, exhaustion, and homing adhesion genes shown to be associated with effector differentiation from Pilipow et al., 2018 (Pilipow et al. 2018). Donors 1-6 were derived from GSE114765, CD8 / CD4 naive, and memory datasets were derived from GSE23321. [Figure 11]CH2811hG1 antibody induced PBMC proliferation. (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) medium, and cells were harvested on day 11 to confirm proliferation of CD4 and CD8 T cells. The percentage of proliferation of specific T cell populations was assessed via CSFE dye dilution analysis. Results are representative of two donors. (B) Summary of CD4 and CD8 PBMC proliferation. [Figure 12] CH2811hG1 antibody induced T cell proliferation. (A) Confirmation of T cell purity and CSFE labeling. 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 confirmed by staining T cells with anti-CD3 antibody, and CSFE labeling was confirmed in the FITC channel. (B) Plate-bound CH2811hG1 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) medium, and cells were harvested on days 7, 11, and 14 to confirm proliferation of CD4 and CD8 T cells. The percentage of proliferation of specific T cell populations was assessed via CSFE dye dilution analysis. Results are representative of four donors. (C) Summary of i) total T cell proliferation, ii) CD4 T cell proliferation, and iii) CD8 T cell proliferation from four healthy donors (BD61, BD2, BD3, BD26) assessed by CSFE dye dilution analysis. (D) Percentage of CH2811hG1 stimulated CD4 T cells at a specific number of divisions after 11 days in vitro. T cells were loaded with CSFE dye and stimulated with i) anti-CD3, ii) CH2811hG1, or iii) medium on day 0. On day 11, CSFE profiles were analyzed by flow cytometry. The number of cell divisions is represented by the square boxes and the percentage of cells that have undergone a specific number of divisions is represented above the square boxes. [Figure 13]Assessment of TCR repertoire clonotypes in CH2811hG1 stimulated T cells. T cell repertoires were detected from RNA extracted from CSFEhigh and low CH2811hG1 stimulated T cells from two donors on day 19 (BD3) and day 14 (BD26), respectively. The diversity of the TCR repertoire is shown in a tree map (where each rounded rectangle represents a unique entry:VJ-uCDR3, and the size of the spot indicates the relative frequency). (A) Plot of the diversity of CFSEhigh (B) and CFSElow (C) TRA chains, CFSEhigh (D) and CFSElow (E) TRB chains of CH2811 stimulated T cells. The higher the diversity of the sample, the closer the solid line is to the dashed line. This line is constructed into a curve describing the overall diversity of the sample, where the "complete" diversity is the black dashed line (each unique clonotype received an equal read, i.e. no clonal expansion or dominant clones). [Figure 14] Kinetics of individual cytokine / chemokine responses. Pure T cells isolated from four healthy donors (BD61, BD2, BD3, BD26) were stimulated with CH2811hG1 (5 μg / ml) on day 0. Unstimulated cells (medium) were included as a negative control. Supernatants were collected on days 7, 11, and 14 and assessed for concentrations (pg / ml) of IFNγ, TNFα, IL-8, IL-10, IL-2, IL-5, IL-17A, IL-7, and IL-21. Individual dots represent different donors. Comparative analysis of cytokine / chemokine results between CH2811hG1 stimulated and unstimulated T cells was performed by applying an unpaired Student's t-test, from which P values ​​were calculated (***, P<0.0001; **, P<0.01; *, P<0.05; GraphPad Prism 6). [Figure 15]T cells stimulated with CH2811hG1 remained viable in vitro for more than 2 months. (A) T cells were stimulated on day 0 with plate-bound CH2811hG1 (5 μg / ml) or anti-CD3 antibody (0.005 μg / ml) or medium. On day 35, under light microscopy, all cells stimulated with i) anti-CD3 antibody and ii) medium were dead except for iii) CH2811hG1 stimulated cells (magnification ×20). (B) Phenotypic analysis of viable CH2811hG1 stimulated T cells on day 35. Sequential panels depicting the flow cytometry gating strategy used to phenotype FG2811mG3+ cells. Gates are drawn for analysis of i) FG2811mG3+ and ii) FG2811mG3− cells; these were confirmed for expression of CD3 and CD122. CD3+ cells were further confirmed for expression of CD45RA, CD45RO, CD62L, and CD95 markers (results are representative of one donor). (C) CH2811hG1 stimulated T cells were still viable in vitro at day 35 and maintained proliferation potential. At day 33, viable 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, and ii) at day 70, CH2811hG1 stimulated cells were still viable and showed significant T cell proliferation (magnifications ×10 and ×20). (D) IL-7 and IL-21 may be important autonomous cytokines for long-term in vitro survival of CH2811hG1 stimulated T cells. Representative cytokine / chemokine expression levels (pg / ml) in T cells restimulated with i) CH2811hG1 and ii) anti-CD3 / CD28. T cells were stimulated with CH2811 on day 0, then restimulated with CH2811hG1 on days 33 and 64, or with anti-CD3 / CD28 antibodies on day 33.Supernatants were collected on days 7, 11, 14, 39, 54, and 70 and assessed for concentrations (pg / ml) of IFNγ, IL-10, IL-17A, IL-2, IL-21, IL-5, IL-7, IL-8, and TNF-α. Triangles and arrows represent days of restimulation with 2811 and CD3 / CD28 antibodies, respectively. [Figure 16] Expression of SSEA-4 in mouse immune cells. HHDII / DP4 mice were euthanized and the 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 cytometry analysis. [Figure 17] FG2811mG1 induced phenotypic TSCM cells in C57 / B6 mice. (A) On day 16, the total cell numbers of splenocytes from groups A and B were calculated using trypan blue exclusion analysis. (B) On day 16, splenocytes from individual mice from groups A and B were stained with CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies and evaluated using flow cytometry analysis. (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. On day 24, splenocytes were stained with CD3, CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies and evaluated using flow cytometric analysis. (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. [Figure 18]Tscm cells phenotyped directly ex vivo from HHDII mice and HHDII / DP4 mice. Naïve HHDII mice and HHDII / DP4 mice were sorted, splenocytes were harvested, stained with CD3, CD44, CD62L, SCA-1, and CH2811hG2-PeCy7 antibodies, and evaluated using flow cytometry analysis. (A) Representative flow cytometry plots of ex vivo directly stained splenocytes from HHDII mice. (B) Summary of the results of phenotyping of splenocytes isolated from HHDII mice. (C) Representative flow cytometry plots of ex vivo directly stained splenocytes from HHDII / DP4 mice. (D) Summary of the results of phenotyping of splenocytes isolated from HHDII / DP4 mice. (E) Summary of the results of phenotyping of CD4 T cells and CD8 T cells isolated from HHDII / DP4 mice. [Figure 19] Mouse splenocytes proliferate in response to FG2811mG1 and FG2811hG1 bound to plates. Naïve HHDII mice were sorted, splenocytes were harvested, treated to be rich in pan T cells, and labeled with CFSE. Next, CFSE-labeled T cells were plated in wells containing plate-bound 2811 mouse IgG1 (5 μg / mL) or human IgG1 (5 μg / ml) or anti-CD3 (1 μg / ml) and incubated at 37°C. On days 7, 12, and 14, cells were sampled, stained with anti-CD4 and anti-CD8, and analyzed by flow cytometry. (A) Representative flow cytometry plots of T cells that proliferated in response to FG2811mG1 and FG2811hG1 on day 12. (B) Total percentage of cells that proliferated in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3 on day 12. (C) Total percentage of CD8 T cells that proliferated in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3 on day 12. (D) Total percentage of CD4 T cells that proliferated in response to plate-bound FG2811mG1, FG2811hG1, or anti-CD3 on day 12. [Figure 20]Anti-CD3 and anti-CD28 induce ex vivo expansion of cells with stem cell-like properties from naive HHDII mice that drive the expansion of effector memory cells. Naive HHDII mice were sorted, and splenocytes were harvested, enriched for panT cells, and labeled with CFSE. CFSE-labeled T cells were then plated into wells containing anti-CD3 and anti-CD28 (1 μg / ml each) and incubated at 37°C. On days 7, 12, and 14, cells were sampled, stained with anti-CD4 and anti-CD8, and analyzed by flow cytometry. (A) On days 11, 15, and 20, cells were harvested and stained with CH2811hG2-PeCy7 and / or anti-CD3. (i) Percentage of 2811+ cells among CD3+ cells, (ii) Percentage of CFSElow among CD3+ cells, (iii) Number of 2811+ cells (×104 per mL), (iv) Number of CD3+ T cells (×105 per mL), (n=2 wells). (B) Representative flow cytometry plots of splenocytes stained 11 days after stimulation with CD3 / CD28. Cells were stained with anti-CD3, CD44, CD62L, and CH2811hG2-PeCy7 and assessed using flow cytometry analysis. (C) Total numbers of 2811+ effector memory T cells, central memory T cells, effector T cells, and naive T cells were determined 11 days after stimulation with CD3 / CD28 (n=2 wells). (D) Eleven days after CD3 / CD28 stimulation, the percentages of 2811+ effector memory T cells, central memory T cells, effector T cells, and naive T cells were determined (n=2 wells). [Figure 21]Human 2811hG2 and mouse 2811mG1 induce the ex vivo proliferation of cells with stem cell-like properties derived from naive HHDII / DP4 mice that drive the proliferation of effector memory cells. Naive HHDII / DP4 mice were sorted, spleen cells were collected, processed to be rich in panT cells, and labeled with CFSE. Next, the CFSE-labeled T cells were plated in wells containing soluble human IgG2 (5 μg / mL) or mouse IgG1 2811 Ab (5 μg / mL) or anti-CD3 (1 μg / ml) and CD28, with or without AKTi, thereby stimulating and incubating the cells at 37°C. On days 11 and 15, the cells were sampled, stained with anti-CD3, CD44, CD62L, SCA 1, and CH2811hG2-PeCy7, and evaluated using flow cytometry analysis. (A) Representative flow cytometry plots of T cells that proliferated in response to FG2811mG1 and 2811hG2 on day 11 (n = 2 wells). (B) Eleven days after stimulation with CD3 / CD28, FG2811mG1, or 2811hG2, the total numbers of 2811+ effector memory T cells, central memory T cells, effector T cells, and naive T cells were determined (n = 2 wells). (C) Eleven days after stimulation with CD3 / CD28, FG2811mG1, or 2811hG2, the percentages of 2811+ effector memory T cells, central memory T cells, and effector T cells were determined (n = 2 wells). [Figure 22]Anti-CD3 and CD28 induce ex vivo proliferation of 2811+ cells isolated from healthy donors. PBMCs were isolated from buffy coats and processed to enrich for PanT cells, and approximately 2x106 cells per well of a 24-well plate were incubated for 20 days in the presence of anti-CD3 / CD28 with or without additional cytokines (IL-7 or IL-21). On days 15 and 20, cells were sampled and stained with CD45RA, CD62L, CD122, CD95, CD3, CCR7, and 2811hG Pe-Cy7. (A) Representative flow cytometry plots (n=2 wells) phenotyping T cells that expanded in response to stimulation with anti-CD3 / CD28 on day 20. (B) (i) Percentage of 2811+CD3+ T cells, (ii) total number of 2811+ cells (×104 / mL, n=2 wells) 15 and 20 days after stimulation with anti-CD3 / CD28. [Diagram 23] The frequency of Tscm cells is increased by stimulation with anti-CD3 / CD28. PBMCs were isolated from buffy coats, processed to enrich for PanT cells, and approximately 2x106 cells per well of a 24-well plate were incubated in the presence of anti-CD3 / CD28 for 20 days. On days 15 and 20, cells were harvested and Tscm were stained for expression of CD3+CD45RA+CCR7+CD95+CD122low. Percentage of Tscm cells in the CD3 T cell population (i), percentage of Tscm cells that are also 2811+ (ii), percentage of Tscm 2811+CD3+ cells (iii). [Figure 24]Soluble FG2811mG1 can stimulate mouse T cells through Fc cross-linking. Splenocytes were isolated from HHDII and HHDII / FDP4 mice and processed to enrich for pan T cells from splenocytes harvested from HHDII mice. HHDIIpanT cells and HHDII / DP4 splenocytes were CFSE-labeled. CFSE-labeled T cells were cultured alone or mixed at a 1:1 ratio with HHDII / DP4 splenocytes in the presence of soluble FG2811mG1, controls including media alone (negative control) and LPS (positive control). (A)(i) Representative flow cytometry plots of T cells cultured in the presence of FG2811mG1, LPS, or media alone at day 15. CD4 and CD8 T cell populations of expanded (CFSElow) and non-expanded (CFSEhigh) cells are shown. (ii) Representative flow cytometry plots of T cells cultured with splenocytes in the presence of FG2811mG1, LPS, or media alone on day 15. CD4 and CD8 T cell populations are shown for proliferated (CFSElow) and non-proliferated (CFSEhigh) cells. (B) Summary of CD4 and CD8 T cell proliferative responses when cultured with FG2811mG1, LPS, or media alone with or without splenocytes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0121] method Extraction of plasma membrane glycolipids Pellet of SSEA-3 / 4-LMTK cells (5 × 10 7 Cells (1000 cells) were resuspended in 500 μl of mannitol / HEPES buffer (50 mM mannitol, 5 mM HEPES, pH 7.2, both Sigma) and passed 30 times each through three needles (23G, 25G, 27G). 2was added to the cells and passed through three needles 30 times each as 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, then resuspended in 1 ml of chloroform and incubated at room temperature for 30 minutes with rotation. The sample was then spun at 1,200g for 10 minutes to remove precipitated proteins. The supernatant containing the cell membrane glycolipids was collected and stored at -20°C.

[0122] Preparation of liposomes SSEA-3 / 4-LMTK cell membrane (pm) glycolipid extract (5 x 10 7 Liposomes were generated by mixing 100 μl of lipids (equivalent to 1000 cells) in various ratios with lipids [cholesterol, dicetyl phosphate (DCP), phosphatidylcholine (PC) and α-GalCer] at a total concentration of 10 mg in a round-bottom flask (Table 2). The lipid mixture was then dried at 60 °C using a rotary evaporator until the solvent evaporated leaving a thin film of homogenous lipid on the walls of the flask. After the flask was cooled to room temperature, 100 μl of sterile PBS was added. The opening of the flask was covered with parafilm and then immersed in an ultrasonic bath for 10 min to produce liposomes (all work with chloroform and methanol was performed in a fume hood).

[0123] Immunization Protocol BALB / c mice were 6-8 weeks old (Charles River, UK). Prior to immunization, normal mouse serum (NMS) was collected via tail bleeding and stored at -20°C to serve as a negative control. Mice were transfected with SSEA-3 / 4-LMTK cells (1 × 10 6Individual cells / immunization / mice) were immunized intraperitoneally (i.p.). Seven days after the second immunization and then every 7 days thereafter, antiserum was collected via bleeding from the tail and screened for antibody responses to IgG and IgM. After obtaining a high titer IgG response, the animals were boosted intravenously (i.v.) with SSEA-3 / 4-LMTK cells (1×10 5 Individual cells / immunization / mice) and sacrificed 5 days later.

[0124] Production of mAb Isolation of splenocytes - The mice were euthanized and the spleens were removed. After washing with 5 ml of serum-free medium (RPMI 1640) using a 25-gauge needle, the spleens were gently agitated with sterile forceps to recover splenocytes. 5 ml of splenocytes were collected into a sterile 25-ml common tube and excess fat and connective tissue were discarded. The total volume of the liquid containing splenocytes was increased to 25 ml with serum-free medium (RPMI 1640) and centrifuged at 100 g for 10 minutes. This supernatant was removed, 1 ml of medium and splenocytes were left, and then resuspended in 5 ml of serum-free medium (RPMI 1640) and counted using a hemocytometer with trypan blue staining for viability assessment.

[0125] Fusion of NS0 myeloma cells and splenocytes - The washed splenocytes were in a 25-ml common tube at a ratio of 1:10 with healthy NS0 myeloma cells (NS0:splenocytes; 1×10 7 :1×10 8The cells were combined (1000 x 1000 cells) and centrifuged at 317 g for 5 min. The supernatant was aspirated and the combined cell pellet was gently and slowly resuspended in 800 μl of polyethylene glycol (PEG) for 1 min. The cell mixture was gently stirred for 1 min, after which 1 ml of serum-free medium (RPMI 1640) was added for 1 min with continued stirring. An additional 20 ml of serum-free medium (RPMI 1640) was added for 1 min with continued stirring. The cell mixture was then centrifuged at 317 g for 5 min, the supernatant was removed, and the cell mixture was resuspended in 15 ml of hybridoma medium [500 ml of hybridoma serum-free medium (Gibco): 10 ml of HT (hypoxanthine thymidine) supernatant (50× Hybri-Max; Sigma): 31 μl (31 μg) of methotrexate (1 mg / ml; Sigma): 25 ml of hybridoma cloning factor (Opti-Clone II; MP): 50 ml of filtered NS0 spent medium]. The cell suspension was evenly seeded throughout a 96-well flat-bottom plate and incubated in a cell culture incubator (5% CO 2 ) at 37°C.

[0126] Generation of CH2811hG1 Total RNA was purified using Trizol (Invitrogen, Paisley, UK) according to the manufacturer's protocol at 5 × 10 6 The FG2811 hybridoma cells were used to prepare the cDNA. 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 regions were then cloned into the hIgG1 / κ dual expression vector pDCOrig-hIgG1 (Metheringham et al. 2009) and the sequences were confirmed by sequencing.

[0127] Characterization of mAbs 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, then pipetted into the development tube of a mouse mAb isotyping test kit (AbD Serotec, Kidlington, UK) and incubated at room temperature for 30 seconds. The tube was vortexed briefly to ensure that the colored microparticle solution was completely resuspended. One isotyping section was placed in the tube for 5-10 minutes with the plain red end of the section at the bottom of the tube. The results were interpreted by identifying blue bands appearing above the class or subclass window of the section and one letter of the kappa or lambda window, representing the heavy and light chain composition of the mAb.

[0128] Purification of mouse mAbs - 2 liters of Spent hybridoma serum-free medium (Invitrogen Scotland, UK) was collected and 0.2% sodium azide (Sigma) was added. The spent medium was then filtered through Whatman filter paper and then using a 0.2 μm steritop filter (Sigma). A HiTrap Protein G HP antibody purification column (GE Healthcare) was used for purification as recommended by the manufacturer. The mAb binding buffer consisted of PBS-Tris pH 7.0 and the mAbs were eluted using Tris-Glycine pH 12.0. Fractions containing IgG mAbs were collected, pH neutralized using 10 M HCl, dialyzed overnight against PBS, then aliquoted and stored at -80°C.

[0129] Production of transient mAbs - FG2811mG1, CH2811hG1, and CH2811hG2 mAbs were obtained by transient transfection of Expi293™ cells using the ExpiFectamine™ 293 Transfection kit (Gibco, Life Technologies). HEK293 cells (100 ml, 2×10 6 cells / ml) in suspension were transfected with 100 μg of plasmid DNA, and conditioned medium was harvested on day 7 after transfection.

[0130] Tumor cell lines Cell lines were maintained by regular replacement of the complete culture medium and by splitting to maintain logarithmic growth. All cell lines were periodically confirmed for mycoplasma concentration and authenticated using short tandem repeat (STR) profiling (Table 1). [Table 1] Table 1: Cancer cell lines. GBM: Glioblastoma multiforme

[0131] Antibody binding to cancer cell lines and mouse fibroblasts 1×10 5Cells were incubated with 50 μl of primary antibody (various concentrations) for 1 h at 4° C. Cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 min. The supernatant was discarded and 50 μl of FITC-conjugated anti-mouse / anti-human or biotin-conjugated anti-mouse / anti-human IgG / IgM Fc-specific antibodies (Sigma) diluted 1 / 100 in RPMI 10% FCS were used as secondary antibodies. Cells were incubated in the dark at 4° C. for 1 h. Cells were washed with 200 μl of RPMI 10% FCS and spun at 100 g for 5 min. Biotinylated secondary antibodies were detected using 50 μl of streptavidin-FITC (Sigma) or Strep-PeCy7 (eBioscience) diluted 1 / 100 in RPMI 10% FCS. Cells were washed with 200 μl RPMI 10% FCS and spun at 100 g for 5 min. 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).

[0132] Antibody binding to whole blood 50 μl of healthy donor whole blood was incubated with 50 μl of primary antibody for 1 hour at 4° C. Blood was washed with 150 μl of RPMI 10% NBCS and spun at 100 g for 5 minutes. The supernatant was discarded and cells were incubated with 50 μl of FITC-conjugated anti-mouse / anti-human or biotin-conjugated anti-mouse / anti-human IgG Fc-specific antibody (Sigma; 1 / 100 in RPMI 10% NBCS) as secondary antibody for 1 hour at 4° C. in the dark, then washed with 150 μl of RPMI 10% NBCS and spun at 100 g for 5 minutes. Biotinylated secondary antibodies were detected using 50 μl of streptavidin-FITC (Sigma; 1 / 100 in RPMI 10% NBCS) or streptavidin-PE-Cy7 (eBioscience; 1 / 100 in RPMI 10% NBCS). Cells were incubated for 1 h at 4° C. in the dark, then washed with 200 μl of RPMI 10% NBCS and spun at 100 g for 5 min. After discarding the supernatant, red blood cells were lysed using 50 μl / well Cal-Lyse (Invitrogen, Paisley, UK) followed by 500 μl / well distilled water. Blood was then spun at 100 g for 5 min, the supernatant was discarded and cells were resuspended in 500 μl PBS. Samples were analyzed on an FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

[0133] TLC analysis of glycolipid binding LMTK and SSEA-3 / 4-LMTK cell membrane lipid samples were blotted onto silica plates and developed twice in chloroform (Sigma) / methanol (Sigma) / distilled water (60:30:5 by volume), followed by two developments in hexane (Sigma):diethyl ether (Sigma):acetic acid (Sigma) (80:20:1.5 by volume). Dried plates were sprayed with 0.1% polyisobutyl methacrylate (Sigma) in acetone. After air drying, plates were blocked with PBS 2% (w / v) BSA for 1 h at room temperature and incubated overnight at 4° C. with primary antibodies diluted in PBS 2% (w / v) BSA. Plates were then washed three times with PBS and incubated with secondary antibodies specific for anti-mouse IgG Fc conjugated to biotin (Sigma) diluted 1 / 1000 in PBS 2% (w / v) BSA for 1 h at room temperature. The plates were then washed again with PBS and 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 three more times with PBS and air-dried in the dark. Lipid bands were visualized using a LICOR Odyssey scanner.

[0134] Glycan ELISA (coupled to HSA) 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-Lewisx (SLex) glycan-HSA conjugates, resuspended in PBS (Elicityl, Crolles, France), blocked with 200 μl / well of PBS 5% (w / v) BSA for 1 h 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), plates were read at 450 nm using a Tecan Infinite F50.

[0135] Erythrocyte binding assay Healthy donor red blood cells were washed three times with PBS and resuspended in 10 times the packed cell volume of PBS. 50 μl of washed red blood cells were then incubated with 50 μl of primary antibody at 37° C. for 1 hour. Cells were washed with 150 μl of PBS and spun at 100 g for 5 minutes. The supernatant was discarded and 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. Cells were incubated in the dark at 37° C. for 1 hour, then washed with 150 μl of PBS and spun at 100 g for 5 minutes. The supernatant was discarded and cells were resuspended in 500 μl of PBS. Samples were analyzed by FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

[0136] Red blood cell hemagglutination assay 4 ml of normal donor whole blood was collected in heparin tubes (Becton Dickinson). Whole blood was transferred to a sterile 15 ml conical tube and washed with sterile PBS. The washed blood was centrifuged at 100 g for 5 min. The supernatant was aspirated. The washing step was repeated twice. After the final wash, the blood cell pellet was diluted with sterile PBS to make 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 96-well U-bottom plate. On top of the red blood cells, primary antibody was added at 50 μl / well and incubated at room temperature for 1 hour or until red blood cells agglutinated.

[0137] Monoclonal antibody affinity analysis Kinetic parameters of FG2811mG3 mAb binding to SSEA-4-containing liposomes were determined by surface plasmon resonance (SPR, Biacore 3000, GE Healthcare). L1 sensor chips (GE-healthcare) were preconditioned with 40 mM octyl D-glucoside and then coated with SSEA4-containing liposomes (6000 RU) and loosely bound liposomes were removed with a short pulse of NaOH (10 mM). A reference flow cell was treated in the same way, except that liposomes lacking SSEA-4 were used. In both flow cells, the degree of coverage was almost complete, as injection of HSA (0.1 mg / ml) induced only a slight increase in RU (50-60 RU). After stabilization of the signal from both flow cells, increasing concentrations (0.3 nmol / L to 200 nmol / L) of FG2811mG3 mAb were injected, followed by post-cycle regeneration (10 mM glycine, pH 1.5). Binding curves were fitted to a 1:1 (Langmuir) binding model using BIAevaluation 4.1.

[0138] Glycome analysis of FG2811mG3 (Consortium for Functional Glycomics) To determine the fine specificity of the FG2811mG3 antibody, the antibody was labeled with FITC and sent to the Consortium for Functional Glycomics, where it was screened against over 600 natural and synthetic glycans (core H group, version 5.1). Synthetic and mammalian glycans containing amino linkers were printed onto microscope slides activated with N-hydroxysuccinimide (NHS) to form amide bonds. After incubating the printed slides with 5 μg / ml of the antibody for 1 h at room temperature, binding was detected with goat anti-mouse IgG conjugated to Alexa488. Slides were then dried and scanned, and the screening data was compared to the Consortium for Functional Glycomics database.

[0139] CSFE T cell proliferation assay Isolation of PBMCs Whole blood (buffy coat) was obtained from the National Blood Service (Sheffield) or collected from healthy donors into syringes containing lithium heparin (1000 units / ml; Sigma H0878). Whole blood was diluted 1:1 with RPMI 1640, layered on top of lymphocyte separation medium (Histopaque-1077; Sigma) and then centrifuged at 800g for 25 minutes without brake. After centrifugation, plasma was collected from the top, buffy coat-derived PBMCs. PBMCs were washed twice with RPMI 1640 and spun at 317g for 5 minutes. PBMCs were counted and cells were prepared for T cell isolation.

[0140] Isolation of pure T cells All 1×10 7PBMCs were resuspended in 40 μl of chilled MACS buffer [PBS 1% (v / v) FCS 1% (v / v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665 and 0.5M pH 8 EDTA: Invitrogen). Then, 10 μl of PanT cell biotin antibody (Miltenyi) was added to every 1 × 10 7 10 cells and incubated for 5 min at 4°C in the dark. 30 μl of chilled MACS buffer was added to every 1×10 cells. 7 After addition of 20 μl of PanT cell microbeads (Miltenyi) to every 1 × 10 cells, 7 The cells were added to the 100 μg / ml PBS and incubated at 4° C. in the dark for 10 min. The cells were added to an LS column (Miltenyi) and the flow-through fraction containing CD3 purified T cells was collected. Cells retained on the column were non-T cells.

[0141] Filling with CSFE Purified T cells were washed with RPMI 1640 and the cells were counted. Cells were spun at 317 g for 5 min and the supernatant was removed. All 1×10 7 Purified T cells were resuspended in 1 ml of PBS 10% FCS. CSFE was dissolved in 18 μl of DMSO (Invitrogen) followed by 1.8 ml of PBS 10% FCS. 110 μl of diluted CSFE was then added to every 1 × 10 7 T cells and incubated for 5 min at room temperature in the dark. CSFE-loaded cells were washed with PBS 10% FCS and then resuspended in complete medium (RPM1640 2 (v / v)% Hepes 1 (v / v)% L-glutamine 1 (v / v)% penicillin-streptomycin) 10% donor plasma at 1 x 106 cells / ml. Cells (2 x 10 in 2 ml) were incubated for 5 min at room temperature in the dark. 6Individuals) were added to each well of a 24-well plate pre-coated with CH2811hG1 antibody (5 μg / ml), FG2811mG1 (5 μg / ml), CH2811hG2 antibody (5 μg / ml), or containing anti-CD3 antibody (OKT 3; 0.005 μg / ml), anti-CD3e Ab (1 μg / mL, eBioscience, 16-0031-85), and anti-CD28 Ab (1 μg / ml eBioscience 16-0281-85), or medium alone. Cells were harvested on days 7, 11, and 14 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 CH2811hG2-PeCy7 (in tissue, 1:50 dilution), and then analyzed using a MACSQ flow cytometer (MACSQUANT analyser 10).

[0142] Luminex [Milliplex Map Kit - Human High-Sensitivity T Cell Magnetic Bead Panel (96-well plate assay)] The 9-well format assay was performed in a filter plate as recommended by the manufacturer. A total of 200 μl of wash buffer was added to each well of a 96-well filter plate (Millipore). The plate was sealed and mixed for 10 minutes at room temperature on a plate shaker. The wash buffer was removed by inverting the plate and tapping on a paper towel. Then, 25 μl of each standard, control, and sample (culture supernatant from the CSFE proliferation assay) was added to each well, 25 μl of serum matrix was added to each standard and control well, and 25 μl of assay buffer was added to each sample well. The working bead mixture was vortexed immediately before use. Then, 25 μl of mixed beads was added to each well. The plate was then sealed, covered with aluminum foil, and incubated for 16-18 hours at 4°C with agitation (500-800 rpm) on a plate shaker. After incubation, the plate was rested on the handheld magnet for 60 seconds, then the liquid was removed from the plate by inverting and tapping on a paper towel. Each time, the plate was washed twice with 200 μl of wash buffer. After the second wash, the bottom of the plate was dried by tapping on a paper towel, and 25 μl of detection antibody was added to each well. The plate was then sealed, covered with aluminum foil, and incubated for 1 hour at room temperature with agitation on a plate shaker. Then, 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 rested on the handheld magnet for 60 seconds, then the liquid was removed from the plate by inverting and tapping on a paper towel. Each time, the plate was washed twice with 200 μl of wash buffer. Then, 150 μl of sheath fluid (Luminex) was added to each well. Beads were resuspended for 5 min on a plate shaker and read on a Bio-Plex 3D instrument (Bio-Rad, Hercules, Calif.), which was set to collect at least 50 beads per analyte.Raw data was measured as mean fluorescence intensity (MFI).

[0143] Isolation of naive T cells Whole blood was collected from normal donors into a syringe containing lithium heparin (1000 units / ml; Sigma H0878). Whole blood was diluted 1:1 with RPMI 1640, layered on top of lymphocyte separation medium (Histopaque-1077; Sigma) and centrifuged at 800 g for 25 min without brake. After centrifugation, plasma was collected from the top layer of PBMCs from the buffy coat layer. PBMCs were washed twice with RPMI 1640 and spun at 317 g for 5 min. PBMCs were counted and the cells were ready for isolation of naive T cells. All 1 × 10 7 PBMCs were resuspended in 40 μl of chilled MACS buffer [PBS 1% (v / v) FCS 1% (v / v) EDTA] (PBS: Sigma D8537; FCS: Sigma F9665 and 0.5M pH 8 EDTA: Invitrogen). Then, 10 μl of naive PanT cell biotin antibody (Miltenyi) was added to every 1 × 10 7 10 cells and incubated for 5 min at 4°C in the dark. 30 μl of chilled MACS buffer was added to every 1×10 cells. 7 10 cells, then 20 μl of naive PanT cell microbeads (Miltenyi) were added to every 1 × 10 7 The cells were added to the 100-well plate and incubated at 4°C in the dark for 10 min. The cells were added to an LS column (Miltenyi) and the flow-through containing CD3 purified T cells was collected. Cells retained on the column were non-T cells. Naïve T cells were stained with CH2811hG1 or a combination of CD95 / CD122 antibodies for 30 min. The cells were washed with MACS buffer and proceeded to cell sorting. CH2811hG1+ and CD95 / CD122+ cells were sorted into RNA protect (QIAGEN) and stored at -80°C.

[0144] Sample extraction and quality control Eight T cell samples were placed in an RNA protection reagent. The entire sample volume was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The extracted RNA samples were evaluated for quantity and integrity using a NanoDrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and an Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany) combined with an Eukaryote RNA Pico Bioanalyser chip, respectively. The samples exhibited low-level degradation with RNA integrity numbers (RINs) of 7.4 - 10 and an average yield of 110 ng.

[0145] cDNA synthesis Full-length cDNA molecules were generated from 1 ng of total RNA per sample using the SMART-Seq® v4 Ultra™ Low Input RNA Kit for Sequencing (Clontech, Mountain View, CA, USA). The amount of cDNA was measured using a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and the quality was confirmed using an Agilent 2200 Tapestation and a high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples exhibited sufficient amounts of cDNA, and the molecular sizes were in the range of 400 - 10,000 bp.

[0146] Library preparation and RNA sequencing Sequencing libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina Inc., Cambridge, UK), with 150 pg of cDNA input per sample. A final PCR amplification of 11 cycles was performed. The final libraries were quantified and qualified using a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and an Agilent 2200 Tapestation with high-sensitivity D1000 screentape (Agilent Technologies, Waldbronn, Germany). Equimolar amounts of each sample library were pooled together for sequencing using the Illumina NextSeq® 500 Mid-output kit to generate 75 bp paired-end reads.

[0147] Differential expression analysis 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). Mapping was performed using default parameters and reads were counted with GeneCounts. Differential expression analysis (DE) of 2811 and similarly CD95 / CD122 enriched T cells was performed using the CD4 and CD8 naive T cell derived datasets from GSE114765 using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple test correction (Pilipow et al. JCI insight 2018) to establish FDR (FDR<0.05). Genes common between the DE sets of T cells (two in CD8 and two in CD4) were identified using Venny 2.1 and used as input for the StemChecker database to identify a “stemness” signature (Pinto et al. 2015 ).

[0148] Transcriptional profiling using RNAseq Eight T cell samples (four CH2811+, four CD122 / CD95+) were sorted with RNA protection reagent. The entire sample volume was extracted using the Qiagen RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Extracted RNA samples were assessed for quantity and integrity using a NanoDrop 8000 spectrophotometer V2.0 (ThermoScientific, USA) and an Agilent 2100 Bioanalyser combined with a Eukaryote RNA Pico Bioanalyser chip (Agilent Technologies, Waldbronn, Germany), respectively. Samples exhibited low levels of degradation with RINs (RNA integrity numbers) of 7.4-10 and an average yield of 110 ng. Full-length cDNA molecules were generated from 1 ng of total RNA per sample using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech, Mountain View, CA, USA). The quantity of cDNA was measured using a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and the quality was checked using an Agilent 2200 Tapestation and high-sensitivity D5000 screentape (Agilent Technologies, Waldbronn, Germany). All samples exhibited sufficient amounts of cDNA, with molecular sizes ranging from 400 to 10,000 bp. Sequencing libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina Inc., Cambridge, UK), where 150 pg of cDNA was input per sample. A final PCR amplification of 11 cycles was performed.The final libraries were quantified and qualified using a Qubit® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and an Agilent 2200 Tapestation with high-sensitivity D1000 screentape (Agilent Technologies, Waldbronn, Germany). Equimolar amounts of each sample library were pooled together for sequencing using the Ilumina NextSeq® 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 (Esembl assembly GRCh37(hg19)) using STAR (version 2.5.1b). Mapping was performed using default parameters and reads were counted with GeneCounts.

[0149] Differential expression analysis (DE) of CH2811 and similarly CD95 / CD122 enriched transcriptional profiles was performed using the edgeR package (version 3.22) followed by Benjamini-Hochberg multiple test correction to establish FDR (FDR<0.05) using the datasets from CD4 and CD8 naive T cells from GSE83808 (Hosokawa et al. 2017). Genes common between the DE sets of T cells (two in CD8 and two in CD4) were identified using Venny 2.1 and used as input for the StemChecker database to identify overlaps with gene sets related to "stemness" signatures as well as hematopoietic stem cells (HSCs) and embryonic stem cells (ESCs) (Pinto et al. 2015). The distribution of significantly enriched genes is presented via heatmap analysis (https: / / software.broadinstitute.org / morpheus / ). Concurrently, the distribution of genes associated with Tscm and effector T differentiation in CH2811 and CD95 / CD122 enriched profiles compared to published naïve CD4 and CD8 T cells and memory CD4 and CD8 T cells (GSE23321) and activated naïve CD8 T cells (GSE114765) was visualized using http: / / bioinformatics.sdstate.edu / idep / .

[0150] Mouse studies C57BL / 6J mice (Charles River), HHDII / HLA-DP4 (DP*0401) mice (EM:02221, European Mouse Mutant Archive), and HHDII mice (Pasteur Institute), aged 8–12 weeks, were used. All work was carried out under a project license approved by the Home Office. Six mice were randomly divided into two groups (groups A and B) and the investigators were not blinded. On day 0, mice in group A were immunized via the intraperitoneal route (ip) with endotoxin-free FG2811mG1 mAb (250 μg / mouse). Mice in group B served as a non-immunized control group. Spleens were harvested for analysis on day 16, and splenocytes were then pooled together within the sample groups and restimulated in the presence or absence of plate-bound FG2811mG1 antibody (5 μg / ml). On days 24, 27, and 30, splenocytes were harvested from the cultures for analysis using anti-CD3, CD4, CD8, CD44, CD62L, SCA-1, and CH2811hG1 antibodies.

[0151] Dyeing Mouse Test Naïve HHDII DP4 mice were used. All work was carried out under a project license approved by the Ministry of Home Affairs. Spleen, mesenteric lymph nodes, inguinal lymph nodes, bone marrow, and blood samples were collected from naïve mice for analysis. Tissues were incubated with CH2811hG2-PeCy7 (intracellular, 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).

[0152] Working Example The invention will now be further described with reference to the following examples and the accompanying drawings.

[0153] Example 1. Preparation of FG2811.72 Generation and characterization of FG2811 mAb BALB / c mice were immunized intraperitoneally (ip) and boosted intravenously (iv) with the SSEA-3 / 4-expressing cell line (SSEA-3 / 4-LMTK) for 3 months. The 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). The cell line possesses endogenous sialyltransferases that add sialic acid to the termini of SSEA-3 glycans to produce SSEA-4 (Figure 1).

[0154] To generate anti-SSEA-4 specific mAb, splenocytes from immunized mice were fused with myeloma NS0 cells. After several rounds of screening and limiting dilution cloning, anti-SSEA-4 mAb, FG2811mG3, was obtained.

[0155] SSEA-3 and SSEA-4 are known to be globo-series glycolipids. To confirm that FG2811mG3 mAb recognizes cell surface glycolipid antigens on SSEA-3 / 4-LMTK cells, high performance thin layer chromatography (HPTLC) analysis of SSEA-3 / 4-LMTK cell membrane lipid extracts and immunostaining with FG2811mG3 mAb were performed (Figure 4A). MC631 mAb (a commercially available anti-SSEA-3 mAb) and MC813 mAb (a commercially available anti-SSEA-4 mAb) were included as comparisons, and wild-type LMTK cells were used as a negative control cell line. FG2811mG3 and MC631 mAb stained glycolipids expressed in SSEA-3 / 4-LMTK cells but not wild-type LMTK cells. FG2811mG3 mAb showed highly specific glycolipid staining, whereas MC631 stained three different glycolipid antigens, suggesting that MC631 mAb cross-reacts with the other two glycolipid antigens expressed in SSEA-3 / 4-LMTK cells. MC813 was unable to stain glycolipids from both SSEA-3 / 4-LMTK and LMTK cells, which may possibly be due to its own low 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 (mouse IgG3; Gm: 20.77) and MC813 (mouse IgG1; 8.77) mAbs (Figure 4B). Secondary antibody alone (Gm: 0.34) was used as a negative control. Next, ELISA assays were performed to screen FG2811mG3 mAb against SSEA-3, SSEA-4, Globo-H, Forssmann, and sialyl-Lewisx glycans coupled to HSA (Figure 4C). Results from ELISA showed that FG2811mG3 mAb was specific for SSEA-4 glycan (1.2 OD units), and M1 / 87 mAb was specific for Forssmann glycan (1.1 OD units). In contrast, both MC631 and MC813 cross-reacted with other glycans.MC631 mAb recognized SSEA-3 (1.0 OD unit), SSEA-4 (1.0 OD unit), and Globo-H (0.5 OD unit) glycans. MC813 mAb bound to SSEA-3 (0.8 OD unit), SSEA-4 (1.2 OD unit), and Forssman (0.7 OD unit). FG2811mG3 was screened by the Consortium for Functional Glycomics (CFG) against over 600 natural and synthetic glycans to determine its fine specificity, and only FG2811mG3 bound to SSEA-4 glycans, confirming its specificity for SSEA-4 (Figure 4D).

[0156] The binding kinetics of FG2811mG3 mAb to the SSEA-3 / 4-LMTK cell membrane lipid antigen was examined using surface plasmon resonance (SPR; Biacore X). Fitting of the binding curves revealed a fast binding rate (approximately 10 5 1 / Ms) and slow dissociation rate (approximately 10 -4 Strong and distinct functional affinity (K d Approximately 2×10 -9 M) (Figure 5A). This was due to the ELISA-derived EC 50 Value(EC 50 =6.8×10 -10 M) (Fig. 5B) and the functional binding activity of the cells (K d =5×10 -9 M) (Figure 5C).

[0157] Example 2. FG2811 antibody sequence DNA sequencing revealed that FG2811mG3 mAb belongs to the IGHV2-3*01 heavy chain and IGKV4-63*01 light chain gene families (Figure 2A and B). Mutational assessment revealed nine nucleotide differences between the FG2811 heavy chain and germline sequences, resulting in five changes in amino acid residues. Similarly, there were seven nucleotide differences between the FG2811 kappa chain and germline sequences, resulting in five changes in amino acid residues. The nature and pattern of mutations suggest somatic hypermutation and affinity mutation.

[0158] The heavy and light chain variable regions of FG2811 were cloned into expression vectors for mouse IgG1, human IgG2 and IgG1 (Figure 2C-E), transfected into HEK293 cells and the antibodies were purified with protein G. mIgG3, mIgG1, hIgG1 and hIgG2 mabs bound to the SSEA3 / 4 LMTK cell line (Figure 3).

[0159] Example 3. Binding of 2811 to a panel of human cancer cell lines Overexpression of SSEA-4 has been reported in glioblastoma cancer cell lines, as defined by MC813 mAb. Therefore, a panel of brain cancer cell lines was assessed for SSEA-4 expression by flow cytometry analysis using both FG2811mG3 and MC813 mAb at 5 μg / ml (Figure 6A). Mouse IgG3κ isotype control and medium alone (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 binds weakly to DAOY (Gm:50) and UW2283 (Gm:36), but did not bind to other cancer cell lines. In contrast, MC813 bound weakly to U251 (Gm:27) and U87 (Gm:38), strongly to DAOY (Gm:169) and UW2283 (Gm:152), and did not bind to KNS42 and SF188. Due to the low specificity of the MC813 antibody, this result suggests that SSEA-4 expression can only be found in DAOY and U251 cell lines, and this 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 bound strongly to SKOV3 (Gm:203), moderately to T47D (Gm:95) and MCF7 (Gm:76), weakly to IGROV1 (Gm:41) and OVCAR-5 (Gm:87), and did not bind to DU4475 (Gm:26), HCC1187 (Gm:22), Colo205 (Gm:13), and HCT15 (Gm:22).

[0160] Example 4. Cytotoxicity of 2811 mAb The properties of FG2811mG3 mAb in inducing tumor cell death via ADCC were investigated (Figure 7A). Human PBMCs were used as a source of effector cells, and SKOV3 and T47D cells served as target cells. The number of cells killed by FG2811mG3 mAb was measured after 18 hours of incubation at 37°C. Ovarian cancer cells SKOV3 (EC50 :10 -10 M) were susceptible to killing by FG2811mG3 mAb in a concentration-dependent manner, showing up to 66% cell lysis. Despite FG2811mG3 mAb binding to T47D, this mAb was unable to induce killing of T47D cells via ADCC, suggesting that this killing effect was dependent on the expression level of SSEA-4. CDC is known to be an important mechanism involved in the elimination of tumor cells in vivo. We assayed the characteristics of SKOV3 and T47D cells killed by CDC induced by FG2811mG3 mAb in the presence or absence of human serum as a source of complement (Figure 7B). FG2811mG3 mAb inhibited the killing of SKOV3 (EC 50 :10 -9M) Up to 48% cell lysis was shown. FG2811mG3 also did not induce killing of T47D cells via CDC. To investigate whether the FG2811mG3 mAb can induce direct killing of tumor cells, a PI uptake assay was performed using SSEA-3 / 4-LMTK cells and SKOV3 cells with 30 μg / ml of FG2811mG3 mAb (Figure 7C). Hydrogen peroxide and medium alone were included as positive and negative controls, respectively. The FG2811mG3 mAb induced 74.7% PI uptake in SSEA-3 / 4-LMTK and weakly induced 28.6% in SKOV-3 cells. To confirm that the PI assay reflects cell death of truly proliferating cells, the cell viability of SSEA-3 / 4-LMTK cells and SKOV3 cells treated with 30 μg / ml of FG2811mG3 mAb was evaluated under an optical microscope (Figure 7D). Wild-type cells of LMTK and cells treated with medium alone (RPMI) were used as negative controls. SSEA-3 / 4-LMTK cells were observed to aggregate within seconds of adding the FG2811mG3 mAb. However, this phenomenon did not develop when SKOV3 cells and LMTK cells were incubated with the FG2811mG3 mAb. SSEA-3 / 4-LMTK cells and SKOV3 cells treated with FG2811mG3 showed evidence of growth inhibition after 72 hours of addition of the mAb. The FG2811mG3 mAb showed no effect on LMTK cells. Cells incubated with medium alone showed no growth inhibition and reached 100% confluence with some cell death over a 72-hour incubation period.

[0161] Example 5. Staining of 2811 with Red Blood Cells Both SSEA-3 and SSEA-4 were reported to be expressed on red blood cells of most people. Therefore, the binding of FG2811mG3 mAb at a range of concentrations (10 μg / ml) to red blood cells from five donors was evaluated by flow cytometry (Figure 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 red blood cells from all five donors. Further hemagglutination assay confirmed that FG2811mG3 mAb (0.625-10 μg / ml) did not agglutinate red blood cells from the five donors. In contrast, 791T / 36 mAb and anti-blood serum antibodies agglutinated red blood cells from all donors. PBS was used as a negative control (Figure 8B).

[0162] Example 6. Stem memory T cells (T SCM 2811 bonds for T SCM The discovery of SSEA-4 cells and the fact that it is a stem cell marker led to the discovery of 2811 mAb in T SCM This led to the hypothesis that FG2811mG1 mAb may recognize cells in healthy donors. Whole blood samples were collected from seven healthy donors (BD3, BD13, BD18, BD27, BD38, BD96, BD31) and stained with FG2811mG1 mAb (Figure 9A). MC813 mAb was included as a comparison; mouse IgG1 isotype control antibody and secondary antibody alone (no primary antibody) were used as negative controls; 198 antibody (anti-CEACAM6) and OKT3 antibody (anti-CD3) were used as positive control antibodies for granulocytes and PBMCs, respectively. FG2811mG1 mAb stained a small population of peripheral blood mononuclear cells (PBMCs) in the seven healthy donors, ranging from 0.8 to 2.3%. MC813 mAb, which recognizes the SSEA3, SSEA4, and Forssman antigens, did not stain any blood cells across the seven donors. 198 mAb stains granulocytes, and OKT3 mAb stains CD3 + Secondary antibodies and medium alone did not show any cell staining.+ To investigate whether PBMCs are TSCM cells, PBMCs were collected from two healthy donors, co-stained with FG2811, CD3, CD122, CD45RA, CD45RO, CD62L, and CD95 antibodies, and analyzed by multiparameter flow cytometry (Figure 9B, Table 2). + First, all T cells in the 2811 + The population was identified. 2811 + The frequency of CD45 cells ranged from 0.32 to 0.41% across the two donors. Expression of CD45RA and CD45RO markers was then examined using CD3 + 2811 + The CD3 + 2811 + T cells express CD45RA + cells (37.5~38.6%), CD45RO + cells (38–47.8%) and CD45RA + RO + Finally, the expression of CD62L, CD95, and CCR-7 was compared with that of CD45RA + , CD45RO + , and CD45RA + RO + The majority of CD45RA + (88.7-90%), CD45RA + RO + (79.5–89.6%), and CD45RO + (64.5-67.1%) were CD62L + and CD45RA + 27.6–59.7% of cells, CD45RA + RO + 29.5–85.7% of cells, and CD45RO + 81.2–86.1% of cells were CD95 + and CD45RA + 56.1–78.9% of cells, CD45RA + RO + 51.4–78.1% of cells, and CD45RO +53.7–61.2% of cells express CCR-7 + These results were consistent with those of 2811 / CD45RA. + The cells are T SCM Cells, 2811 / CD45RA + RO + T cells are activated SCM 2811 / CD45RO + T cells are activated SCM Cells or T CM This suggests that it may be a cell.

[0163] [Table 2] Table 2. Phenotyping of PBMCs. PBMCs were isolated from two healthy donors (BD13 and BD38) and stained with a panel of antibodies (CD3, FG2811, CD45RA, CD45RO, CD62L, CD95, and CCR-7). PBMC phenotypes were determined using flow cytometry and results are presented as the percentage of positive cells.

[0164] In a hierarchical model of human T cell differentiation, after antigen priming, naive T cells (T N ) are stem memory T cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), and terminally differentiated effector T cells (T TE / TEMRA). These T cell subsets are distinguished by the combinatorial expression of different markers (Table 3) (Gattinoni et al. 2017).

[0165] [Table 3] Table 3: Hierarchical model of human T cell differentiation

[0166] Example 7. RNA Sequencing of 2811-Positive T Cells By transcriptome analysis, we identified the putative T SCM We investigated the degree of association between 2811+ T cells and 2811+ T cells (Gattinoni et al. 2017). Markers CD95 and CD122 (IL-2Rβ) were identified as T N Cells and T SCM The CD45RO marker distinguishes between T cells and other memory T cell subtypes. SCM Therefore, naive T cells were isolated from four healthy donors using the Pan naive 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 stained with CH2811hG1 or a combination of CD95 / CD122 to identify 2811+ cells and putative T SCMCells were isolated, respectively. Differential gene expression (DE) analysis using RNA sequencing of CH2811hG1 and CD95 / CD122-enriched T cells as well as datasets from CD8 natural T cells showed that 2227 (44%) of 5,036 genes significantly up- or down-regulated in SSEA-4 positive (CH2811) cells were shared with DE genes up- or down-regulated in CD95 / CD122 positive T cells, suggesting the existence of substantial overlapping genes between these two populations (Figure 10A). Significantly, of the shared genes, 257 overlapped with a set of embryonic stem cell genes, 103 with hematopoietic stem cells, and 78 with embryonic carcinoma (Figure 10A), implying that SSEA-4 is indeed associated with a subset of T cells with stem cell-like behavior. The distribution profile of the former two overlapping gene sets in our dataset is shown using heatmap analysis. Furthermore, the distribution of TSCM and effector differentiation genes in our CH2811hG1 and CD95 / CD122 enriched T cell profile and comparison with CD8 / CD4 naive and memory T cells and activated CD8 natural T cells ("donor") are also shown (Figure 10B). Hierarchical clustering shows a clear separation of CH2811hG1 and CD95 / CD122 samples, suggesting that they are similar to each other compared to true naive / memory T cells or activated naive T cells and may represent distinct subsets of T cells with stem cell-like behavior (Figure 10C).

[0167] Example 8. T cell proliferation and expansion The costimulation paradigm allows NCells require engagement of both T cell receptor (TCR) signal 1 and costimulatory signal 2 for full activation leading to proliferation and differentiation. However, a subclass of antibodies specific for CD28, known as CD28 superagonists, can fully activate T cells without further stimulation of the TCR, unlike conventional CD28 antibodies. We investigated whether CH2811hG1 mAb can induce proliferation of CD4 and CD8 T cells. First, PBMCs were isolated from two healthy donors (BD3 and BD18), labeled with CSFE, and then stimulated with antibodies using 5 μg / ml plate-bound CH2811hG1 mAb, which showed proliferation of CD4 T cells (13-20%) and CD8 T cells (2-31%) on day 11 (Figure 11A-B). PBMCs stimulated with PHA and medium alone were positive and negative controls.

[0168] To rule out that this was due to Fc activation of antigen-presenting cells, purified T cells (96% pure; Fig. 12A) were isolated from four healthy donors, labeled with CSFE, and then stimulated with 5 μg / ml of plate-bound CH2811hG1 mAb. + 8–18% of T cells and CD8 + Three to seven percent of T cells proliferated, suggesting that this proliferation was not mediated by Fc interactions (Figure 12B-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 that underwent cell division was different, as the majority of SSEA4-positive cells underwent at least four cell divisions within 14 days (96%), whereas only 55% of CD3-stimulated cells underwent four cell divisions (Figure 12D).

[0169] Example 10. Clonotypic assessment of TCR repertoires The clonality of CH2811IgG1 stimulated T cells was assessed from two donors (BD3 and BD26) to determine the TCR repertoire, perform fully automated multiplex PCR, and generate TCRα (TRA) and TCRβ (TRB) chain libraries for next generation sequencing (NGS) analysis of unique CDR3 (uCDR3). Tree plot analysis (Figure 13) revealed the presence of several relatively dominant clonotypes in the CSFElow populations of both donors. The diversity of the non-expanded populations was 18.9 and 12.8 for the TRA and TRB chains, respectively. As expected, the diversity of the 2811 stimulated populations was 3 and 3.3 for the TRA and TRB chains, respectively. This diversity was low, suggesting that these cells represent antigen-experienced cells.

[0170] Example 11. Kinetics of individual cytokine / chemokine responses TSCM cells have been shown to have high proliferation capacity, are self-renewing and multipotent, and they can differentiate into other T cell subsets. + T SCM We hypothesized that FG2811 cells could proliferate and self-renew in vitro without any supplemental cytokines. First, we demonstrated that FG2811 cells could be stimulated with CH2811hG1 antibody and self-renewed in vitro. + To identify cytokines released by TSCM cells, we then + T SCMWe aimed to design a method that could be used to expand the cells and maintain this stemness. T cells were purified from four healthy donors and stimulated with plate-bound CH2811hG1 mAb, and supernatants were collected on days 7, 11, and 14 and assessed for cytokine or chemokine release. Unstimulated cells (medium only) were used as a negative control. Secretion of nine cytokines / chemokines (IFNγ, IL-10, IL-17A, IL-2, IL-21, IL-5, IL-7, IL-8, and TNFα) was assayed using a multiplex cytokine assay (Luminex technology) (Figure 14). Upon stimulation with CH2811hG1 mAb, the chemokine IL-8 was strongly upregulated, whereas more modest levels of TNFα, IL-10, and IL-5 were detectable from days 7 to 14.

[0171] As shown by trypan blue exclusion, unstimulated and anti-CD3 stimulated T cells did not survive beyond 14 days in culture, and only CH2811hG1 stimulated T cells survived beyond 14 days (Figure 15A). On day 35, viable CH2811hG1 stimulated T cells were harvested 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 (Figure 15B). Of the 32.47% viable cells, 3% were FG2811 + The remaining 97% is FG2811 - It was. FG2811 + The cells (Figure 15B(i)) were 99% CD3 + and CD122 + Of these, 60% were CD45RA / RO double positive (CD45RA / RO + ) and 30% were CD45RA + It was. CD45RA / RO + and CD45RA + Both cells were CD62L + and CD95 +These suggested that these were TSCM cells. + The population may be activated TSCM cells, whereas CD45RA + may be more naive-like TSCM cells. - The population (Figure 15B(ii)) was 99% CD3 + However, only 34% were CD122 + It was. FG2811 - CD3 + The population was 62% CD45RO + and 17% were CD45RA. + CD62L was responsible for 49% of FG2811 - CD3 + CD45RO + CD95 is expressed in FG2811 cells - CD3 + CD45RO + It was expressed in 79% of cells. CD45RO / CD62L / CD95 triple positive cells (CD45RO / CD62L / CD95 + ) is activated T SCM Cells or T CM The cells may be CD45RO / CD95 + The cells may be TEM or TEMRA. + The population has more CD62L + cells (approximately 76%), but + The cells were few (approximately 28%). CD45RA / CD62L / CD95 + The cells are T SCMThese results suggest that CH2811hG1 stimulation maintains T cells with "stem cell-like" and memory characteristics in culture for a long period of time, and that they can differentiate into other T cell types. The proliferation potential of these live cells was evaluated 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, the cells restimulated with anti-CD3 / CD28 antibodies had formed T cell blasts (Figure 15C(i)), and most of the T cells restimulated with CD3 / CD28 had died, with only a few live cells remaining. In contrast, cells restimulated twice with CH2811 antibodies were still viable on day 70, and many showed obvious proliferation (Figure 15C(ii)). Supernatants from these two cultures were collected on days 39, 54, and 70 and screened for cytokines and chemokines (Figure 15D). In cultures restimulated with CH2811hG1, the levels of other cytokines / chemokines gradually decreased from day 14 to undetectable levels by day 70, while the levels of IL-7 and IL-21 gradually increased (Figure 15D(i)). This result indicates that IL-7 and IL-21 are the major cytokines in the FG2811 cultures. + T SCM IL-7 may play an important role in T cell autonomy. SCM It is known that IL-21 provides important instructive signals for the formation of T cells (Cieri et al. 2013), and IL-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 were increased, suggesting that there is a variation of activation of different T cell subsets (Figure 15D(ii)). For example, Th1 cells are characterized by secretion of IL-2, IFNy, and TNFα, Th2 secrete IL-5, Th17 secrete IL-17A and IL-21, and regulatory T cells (Treg) secrete IL-10 (Raphael et al. 2015).

[0172] Example 12. FG2811+T in mice SCM Cell Identification Next, we investigated the expression of SSEA-4 in mouse splenocytes, mesenteric lymph node cells, and inguinal lymph node cells using CH2811hG1 antibody ( FIG. 16 ). These results showed that CH2811hG1 antibody stained 0.5% of splenocytes, 0.37% of mesenteric lymph node cells, and 0.52% of inguinal lymph node cells.

[0173] Example 13. FG2811 (mouse IgG1) induces phenotype T in C57B / 6J mice SCM Induce cells. To determine the agonistic effect of FG2811mG1 on T cells in vivo, a group of three mice (group A) were immunized ip with 250 μg FG2811 on day 0. Three non-immunized mice were included as a control group (group B). On day 16, mice from both groups were euthanized and spleens were harvested. The total number of splenocytes from group A was higher compared to mice in group B, 7 × 10 7 ~1×10 8 cells and 3.9 x 10 7 ~6.2×10 7 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 (Figure 17B). CH2811hG1 mAb was used to detect anti-CD4 and CD8 for T cells, CD44 and CD62L for subsets of T and B cells, and SCA-1 for stem cell-like cells (among other markers for hematopoietic stem cells and mouse T SCM SSEA-4, a marker used to identify IL-1 cells, and CD11b and F4 / 80 in macrophages. + Splenocytes were identified. 2811 in group A mice + (0.97-1.2%), CD62L +(5.51–10.83%), and CD62 + CD44 + The frequency of CD4 cells in group A (8.74–15.03%) was lower compared to group B (1.62–1.74%, 17.39–19.2%, and 18.4–27.34%, respectively). + , CD8 + , CD19 + , CD11b + , F4 / 80 + , and CD11b + F4 / 80 + The difference in the percentage of cells is that mice with A3 have fewer CD8 + The effects of immunization with FG2811mG1 antibody on the immune response to 2811 were minimal, except for the T cell population (Table 4). + Induce cell proliferation and differentiation, and produce naïve-like cells in vivo (2811 + , SCA-1 + , and CD62L + ) reduction.

[0174] [Table 4] Table 4: Summary of the frequency of different immune cell subsets in splenocytes of groups A and B on day 16

[0175] Spleen cells from each group were pooled together and then cultured in the presence (A+2811 and B+2811) or absence (A-2811 and B-2811) of FG2811mG1 mAb bound to plates at 5 μg / ml. Subsequently, on days 24, 27, and 30, these cells were harvested, stained with FG2811, CD3, CD4, CD8, CD44, CD62L, SCA-1, CD11b, F4 / 80, and CD19 antibodies, and analyzed by multi-parameter flow cytometry (Figures 17C-D). On day 24, spleen cells from group A were restimulated with or without FG2811mG1 mAb (A+2811 and A-2811), forming small cell populations and large cell populations as shown by the FSC / SSC (forward and side scatter) profile. In contrast, spleen cells from group B restimulated with or without FG2811mG1 mAb (B+2811 and B-2811) did not yield a large population (Figure 17C). The large population continued to persist in the cultures of A+2811 and A-2811 spleen cells until day 30 (Figure 17D). The large cell population consisted mainly of CD3 moderate-high (CD3 mo-hi )CD4 high (CD4 hi ) and CD8 high (CD8 hi ) T cells, and the small cell population consisted of CD3 low-moderate (CD3 lo-mo )CD4 low (CD4 lo ) and CD8 low (CD8 lo ) T cells.

[0176] In a hierarchical model of mouse T cell differentiation, after antigen priming, naive T cells (T N ) progressively differentiate into stem memory T cells (T SCM ), central memory T cells (T CM ), and effector memory T cells (T EM ). These subsets of T cells are distinguished by the combinatorial expression of different markers (Table 5).

[0177]

Table 5

[0178] The phenotypic analysis showed that the CD3 population in the A+ / -2811 cultures mainly consisted of T cells with the phenotype of CD44 mo-hi CD62L - CD62L (T + and / or T N ; 28.8 - 32.49%) and T cells with the phenotype of CD44 SCM CD62L + CD62L (T + ; 37.92 - 41.08%), followed by a small fraction (1.72 - 2.26%) of cells with the CD44 CM CD62L + CD62L phenotype (T - / T EF ; approximately 27.8%). In contrast, the CD3 population in all cultures mainly consisted of T cells with the phenotype of CD44 EM CD62L - CD62L (T - / T lo-mo ; 66.09 - 70.83%), followed by the phenotype of CD44 + CD62L - CD62L (26.77 - 32.17%). The percentages of T EF / T EM cells and T - CD62L - cells were 0.08 - 0.24% and 1.5 - 2.29%, respectively. In addition to T cells, a large cell population also contained CD19 N and / or T SCM cells, and T CM cells, and these were not present in the small cell population at all. Only CD19 hi cells were detected in the small cell population. Interestingly, CD11b + CD62L + SCA-1 + cells, and all of these were not present in the small cell population. Only CD19 lo cells were detected in the small cell population. Interestingly, CD11b + F4 / 80 +The percentage of macrophage population was significantly reduced in the A+ / -2811 group. Stimulation of splenocytes from splenocyte B+2811 cultures of non-immunized mice with FG2811mG1 antibody in vitro did not result in the formation of these large populations even by day 30, indicating that the production of this cell population is an in vivo effect of immunization with FG2811 antibody.

[0179] Example 14. Identification of Tscm cells in HHDII mice and HHDII transgenic mice To determine the frequency of Tscm cells in HHDII (Figure 18A and B) and HHDII / DP4 mice (Figure 18C-E), splenocytes were harvested from naive mice, stained with CH2811hG2-PeCy7, anti-CD3, CD4, CD8, CD44, CD62L, and SCA-1, and then analyzed by multiparameter flow cytometry. CH2811hG2 mAb was used to detect anti-CD4 and CD8 for T cells, CD44 and CD62L for a subset of T cells, and SCA-1 for stem cell-like cells (among other markers for hematopoietic stem cells and mouse T SCM SSEA-4 (a marker used to identify cells) + Splenocytes were identified. In HHDII mice, 10.88% of the cells were 2811+CD3+ cells, which represents 1.85 × 10 per ml. 5This translated to 10.6% of the CD3+ population, and furthermore, 24.61% of the CD3+ population were Tscm cells (Figure 18B). The 2811+ population in HHDII mice (10.88%) was higher than the frequency previously observed in C57 / B6 mice (2.42-3.60%). Furthermore, analysis of the phenotype of the 2811+ population in HHDII mice (Figure 18B) showed that 33.38% were CD44+CD62L- and 47.98% were CD44+CD62L+. We also determined the percentage of 2811+ cells that expressed the stem cell marker SCA-1, which may define Tscm cells in these mice when used in conjunction with other markers (CD44-CD62L+). The majority of 2811+SCA-1+ cells also expressed CD44, suggesting that they are antigen-experienced.

[0180] In HHDII / DP4 mice, 12.01% of the cells were 2811+CD3+ cells, which represents 0.91 × 10 5 This translated to 100 cells, furthermore 6.98% of the CD3+ population were 2811+ as well (Figure 18C and D). The 2811+ population in HHDII / DP4 mice (12.01%) was similar to the frequency in HHDII mice, and similarly, higher than the frequency previously observed in C57 / B6 mice (2.42-3.60%). Furthermore, analysis of the phenotype of the 2811+ population in HHDII / DP4 mice (Figure 18D) showed that 12.09% were CD44+CD62L- and 77.15% were CD44+CD62L+. We also determined the percentage of 2811+ cells that expressed the stem cell marker SCA-1. Also, the majority of 2811+SCA-1+ cells express CD44 (75.51% CD44+CD62L+, 30.03% CD44+CD62L-).

[0181] A more detailed phenotypic analysis was performed on T cell populations from HHDII / DP4 mice, looking at expression of 2811 in subsets of CD4 and CD8 T cells, as well as the exhaustion marker Tim3. The percentage of CD4+ T cells in HHDII / DP4 mice was 14.30%, but the percentage of CD8+ T cells was very low, with only 0.50% CD8+ cells (Figure 18E), with 9.47% of CD4 T cells being 2811+ and 10.14% of CD8 T cells being 2811+. Expression of the exhaustion marker, Tim3, was low in CD4+2811+ (0.42%) and CD8+2811+ (0.34%) cells, consistent with their stem cell characteristics.

[0182] Example 15. Plate-bound human (IgG1) and mouse (IgG1) 2811 induced ex vivo proliferation of mouse splenocytes. We investigated whether plate-bound CH2811hG1 and FG2811mG1 mAbs could induce proliferation of CD4 and CD8 T cells. Splenocytes were harvested from naive HHDII mice, processed to enrich for pan T cells (CD3+), labeled with CFSE, and then antibody-stimulated with plate-bound CH2811hG1 mAb or FG2811mG1, anti-CD3 was used as a positive control, and media was used as a negative control (Figure 19A). The proliferative responses of CD3, CD4, and CD8 T cell populations were determined on days 7, 12, and 14 (Figure 19B-D). The results showed that CD3, CD4, and CD8 T cells proliferated in response to stimulation with plate-bound CH2811hG1 and FG2811mG1 mAbs that was comparable to or slightly superior to media controls. On day 7, 2.72% of CD3 T cells proliferated in response to FG2811mG1 and 2.24% proliferated in response to CH2811hG1, 2.47% of CD8 T cells proliferated in response to FG2811mG1 and 1.46% proliferated in response to CH2811hG1, and 1.32% of CD4 T cells proliferated in response to FG2811mG1 and 0.96% proliferated in response to CH2811hG1. On day 12, the proliferative responses to plate-bound CH2811hG1 and FG2811mG1 increased: in CD3 T cells, 6.46% proliferated in response to FG2811mG1 and 6.27% proliferated in response to CH2811hG1; in CD8 T cells, 6.21% proliferated in response to FG2811mG1 and 3.33% proliferated in response to CH2811hG1; and in CD4 T cells, 5.79% proliferated in response to FG2811mG1 and 2.82% proliferated in response to CH2811hG1.On day 14, the proliferative responses to plate-bound CH2811hG1 and FG2811mG1 were further increased, with 10.07% of CD3 T cells proliferating in response to FG2811mG1 and 8.7% proliferating in response to CH2811hG1, 7.87% of CD8 T cells proliferating in response to FG2811mG1 and 6.61% proliferating in response to CH2811hG1, and 7.29% of CD4 T cells proliferating in response to FG2811mG1 and 5.15% proliferating in response to CH2811hG1. These results demonstrate that mouse splenocytes proliferate ex vivo in response to plate-bound CH2811hG1 and FG2811mG1 mAbs.

[0183] Example 16. Anti-CD3 and CD28 induce ex vivo proliferation of 2811+ cells from HHDII mice. We investigated whether anti-CD3 and anti-CD28 could induce proliferation of 2811+ cells isolated from HHDII mice. Splenocytes were harvested from naive HHDII mice, processed to enrich for panT cells (CD3+), labeled with CFSE, and then stimulated with anti-CD3 and anti-CD28 (1 μg / mL). The proliferative response of the 2811+ population was determined on days 11, 15, and 20 using CH2811hG2-PeCy7 mAb ( FIG. 20A ). The percentage of 2811+CD3+ T cells was increased by stimulation with anti-CD3 and anti-CD28 by day 11, with 61.2% of T cells being 2811+, and by day 15 this further increased to 69.84%, but by day 20 the percentage of 2811+ T cells had decreased to 57.58%. Also, the decrease in the percentage of 2811+ cells observed at day 20 correlated with a decrease in cell viability accompanied by a drop in the total number of T cells and 2811+ T cells (Figure 20Aiii and iv). The percentage of 2811+ cells in the medium only control was 10% directly ex vivo, which increased to 20-30% at days 11 and 15, but also decreased at day 20.

[0184] Phenotypic analysis was performed on 2811+ cells expanded by stimulation with anti-CD3 and anti-CD28. On day 11, staining was performed using CH2811hG2-PeCy7, anti-CD3, CD44, and CD62L (Figure 20B). The subsets of T cells identified were effector memory T cells (T EM ), and central memory T cells defined as CD44+CD62L+ (T CM ), and effector T cells defined by CD44-CD62L- (T EFF ), and naive T cells defined as CD44-CD62L+ (T N Phenotyping results on day 11 (FIG. 20C) showed that stimulation with anti-CD3 and CD28 inhibited 2811+T EM (Average value 67.35 × 10 3 ), T CM (Average value 61.15 × 10 3 ), T EFF (Average value 141 x 10 3 ), and T N (Average value 16.45 × 10 3 Stimulation with anti-CD3 and anti-CD28 pushed the phenotype of 2811+ cells towards a more effector T cell phenotype (Figure 20D). EFF The percentage of cells was 47.7% (mean value), compared with T CM Cells and T EM The percentage of cells had decreased to a lower percentage than unstimulated cells (medium alone).

[0185] These results show that anti-CD3 and anti-CD28 induce ex vivo expansion of 2811+ cells from HHDII mice. Stimulation with anti-CD3 and anti-CD28 resulted in an increase in the number and percentage of 2811+ cells 11-15 days after stimulation. 2811+ T cells (T CM , T N , T EM , T EFF), but this stimulation pushed the T cells towards a more effector T cell phenotype.

[0186] Example 17. Human (IgG2) and mouse (IgG1) 2811 induced ex vivo proliferation of splenocytes from HHDII / DP4 mice. We investigated whether plate-bound CH2811hG2 and FG2811mG1 mAbs could induce proliferation of CD4 and CD8 T cells. Splenocytes were harvested from naive HHDII mice, processed to enrich for pan T cells (CD3+), labeled with CFSE, and then antibody stimulated with plate-bound CH2811hG2, FG2811mG1, anti-CD3 / CD28 (+ / -AKTi) was used as a positive control, and medium was used as a negative control. The proliferative response of the CD3 T cell population was determined on days 11, 15, and 20 (Figure 21A). These results showed that 2811+CD3 T cells proliferated in response to stimulation with plate-bound CH2811hG2 and FG2811mG1 mAbs. At day 11, 8.73% of 2811+CD3+ T cells proliferated in response to FG2811mG1 and 50.47% proliferated in response to CH2811hG2, at day 15, 20.48% of 2811+CD3+ T cells proliferated in response to FG2811mG1 and 40.55% proliferated in response to CH2811hG2, and by day 20, the percentages had decreased slightly to 21.41% of 2811+CD3+ T cells proliferated in response to FG2811mG1 and 35.13% proliferated in response to CH2811hG2. The same increase in 2811+ cells was seen when looking at the percentage of 2811+ cells, 2811+CD3+ cells, and total number of 2811+ cells. Stimulation with anti-CD3 / CD28 with or without AKTi also induced proliferation of 2811+ cells, where 80% of 2811+CD3+ T cells proliferated by stimulation with CD3 / CD28 at each time point, and this percentage was reduced to 60% by the addition of AKTi, which is slightly toxic to the cells. These results show that CH2811hG2 induced proliferation of 2811+ cells at all time points upon stimulation, and the same was seen with FG2811mG1, albeit to a lesser extent.

[0187] Next, phenotypic analysis was performed on 2811+ cells expanded by stimulation with anti-CD3 / CD28 (+ / -AKTi), CH2811hG2, and FG2811mG1 mAbs. On day 11, staining was performed using CH2811hG2-PeCy7, anti-CD3, CD44, and CD62L (Figure 20B). The subsets of T cells identified were effector memory T cells (T EM ), and central memory T cells defined as CD44+CD62L+ (T CM ), and effector T cells defined by CD44-CD62L- (T EFF ), and naive T cells defined as CD44-CD62L+ (T N The phenotyping results on day 11 (FIG. 21B) showed that stimulation with CH2811hG2 or FG2811mG1 mAb reduced the expression of 2811+T EM The total number of cells was 62.8 × 10 3 5.24 × 10 cells stimulated with FG2811mG1 3 It was shown that the number of cells increased to 2811+T CM The total number of erythrocytes was 6.95 × 10 after stimulation with CH2811hG2. 3 1.8 × 10 cells were stimulated with FG2811mG1 3 It increased to 2811+T cells. EFF The total number of erythrocytes was 29.05 × 10 after stimulation with CH2811hG2. 3 7.02 × 10 cells stimulated with FG2811mG1 3 It grows to 2811+T cells. N The total number of cells was only slightly increased, by 0.61 × 10 3 (medium only 0.07×10 3 ) and not with FG2811mG1. We also observed the percentage of 2811+ T cells with stimulation with anti-CD3 / CD28, FG2811mG1, and CH2811hG2 (Figure 21C), and 2811+ T cells EFFThe percentage of cells was 34.82% with stimulation by FG2811mG1, 57.59% with stimulation by CH2811hG2, and 47.36% with stimulation by anti-CD3 / CD28. These results indicate that in T cells derived from HHDII / DP4 mice, there is less bias towards an effector phenotype of T cells when compared to the results obtained from HHDII mice (Figure 20D). Also, the percentage of subsets of T CM and T EM was higher compared to HHDII mice.

[0188] These results show that splenocytes from HHDII / DP4 mice proliferate ex vivo in response to plate-bound CH2811hG2 and FG2811mG1 mAbs, which results in an increase in the number of 2811+ effector memory T cells, central memory T cells, effector T cells, and naive T cells, in addition to an increase in the total number of 2811+ CD3+ T cells. The magnitude of the ex vivo proliferative response to CH2811hG2 is greater compared to the response to FG2811mG1, thus resulting in a greater number of 2811+ cells.

[0189] Example 18. Anti-CD3 and CD28 induce ex vivo proliferation of 2811+ cells from healthy donors. TSCM cells have been shown to have high proliferation potential, are self-renewing and multipotent, and they can further differentiate into other T cell subsets. We investigated whether stimulation with anti-CD3 / CD28 or addition of different cytokines could induce ex vivo proliferation of Tscm cells isolated from healthy donors. PBMCs were isolated from four healthy donors (buffy coats), processed to enrich for panT cells, and T cells were cultured in the presence of anti-CD3 / CD28, IL-7, IL-15, or IL-21. At days 15 and 20, phenotypic analysis was performed using anti-CD3, CD45RA, CD45RO, CD62L, CD95, CD122, and CCR7, and the expression of different markers used to identify T cell populations is listed in Table 6.

[0190] [Table 6] Table 6: Phenotypic markers of human T cell populations

[0191] Phenotypic analysis was performed on CD3+ T cells expanded by stimulation with anti-CD3 / CD28 or with the addition of IL-7, IL-15, and IL-21 added in a range of combinations. Staining was performed on days 15 and 20 (Figure 22A). Phenotyping results on day 15 (Figure 22B) show that stimulation with CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21 increases the percentage of 2811+CD3+ cells, which also correlates with an increase in the total number of 2811+ and CD3+ cells. On day 15, the percentage of 2811+CD3+ T cells stimulated with anti-CD3 / CD28 was 19.64% and 23.94%, which was higher than T cells cultured in the presence of cytokines alone (without stimulation with CD3 / CD28). The addition of IL-7 / IL-21 or IL-7 / IL-15 / IL-21 in combination with stimulation with anti-CD3 / CD28 slightly improved the percentage of 2811+CD3+ cells. The percentage of 2811+CD3+ T cells increased to 23.8% and 27.4% when cells were cultured in the presence of CD3 / CD28, IL-7 / IL-21, and with the addition of IL-15 the percentage increased to 31.53%. The increase in the percentage of 2811+CD3+ T cells also correlated with an increase in the total cell number, with 80×10 4 At day 20, the percentage of 2811+CD3+ was reduced to 16.45% and 17.56% when cultured with CD3 / CD8, IL-7 / IL-21 / IL-15, down from 31.53% on day 15. The decrease in the percentage of 2811+CD3+ T cells also correlated with a decrease in the total number of 2811+ T cells.

[0192] Further detailed phenotypic analysis was performed on T cells from two donors to identify Tscm cells in T cells cultured in the presence of CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21. The frequency of Tscm cells in humans is low, with the percentage of Tscm in four healthy donors ranging from 0.64% to 3.48%. We investigated whether Tscm could expand in the presence of CD3 / CD28 alone or in combination with IL-7, IL-15, and IL-21 (Figure 23). The greatest expansion of Tscm cells was observed when T cells were cultured in the presence of anti-CD3 / CD28 in the presence of IL-7 / IL-21 (3.51% and 6.32% Tscm) or with IL-7, IL-15, IL-21 (3.62% Tscm), which was a 5-fold expansion of Tscm cells in one donor and an 8-fold expansion in the second donor. On day 20, the percentage of Tscm cells expanded further when T cells were cultured in the presence of anti-CD3 / CD28 in the presence of IL-7 / IL-21 (14.84% and 11.33% Tscm) or with IL-7 / IL-15 / IL-21 (13.67%), which was a 3-fold expansion of Tscm cells in one donor and a 9-fold expansion in the second donor (compared to the media control on day 20). Next, we determined what percentage of Tscm cells were also positive for 2811 (Figure 23ii). On day 0, the percentage of Tscm cells that were 2811+ was 46.89% and 63.60%. On day 15, the percentage of Tscm 2811+ cells was still similar between all conditions, these were in the range of 31.51-53.52%. On day 20, the percentage of Tscm 2811+ cells had dropped in most conditions, with only T cells cultured in the presence of media alone or CD3 / CD28 alone maintaining a percentage similar to the results on day 15. Next, we determined what percentage of Tscm cells were also positive for CD3 and 2811 (Figure 23iii).At day 15, the percentage of Tscm cells in the presence of CD3 / CD28 or in the presence of CD3 / CD28 in combination with IL-7, IL-15, IL-21 did not increase compared to the media only control or the results at day 0. At day 20, the percentage of Tscm cells in the presence of CD3 / CD28 or in the presence of CD3 / CD28 in combination with IL-7, IL-15, IL-21 did not increase compared to the media only control or the results at day 0.

[0193] These results show that stimulation with CD3 / CD28 induced ex vivo expansion of 2811+ cells isolated from healthy human donors. Stimulation of T cells with anti-CD3 / CD28 increased the frequency of 2811+ cells, and this expansion was further increased when IL-7, IL-15, and IL-21 were all added to the cultures, with the expansion peaking 15 days after stimulation. Stimulation of T cells with combined anti-CD3 / CD28 expanded the Tscm population, resulting in a 3- to 9-fold expansion of these cells.

[0194] Example 19. Soluble FG2811mG1 stimulated T cells stimulate CD4 and CD8 T cell proliferation via Fc cross-linking. Next, we investigated whether soluble FG2811mG1 could stimulate CD4 and CD8 T cells when cultured in the presence or absence of splenocytes. The addition of splenocytes allows cross-linking of Fc and stimulates T cell responses. Splenocytes were isolated from HHDII and HHDII / DP4 mice, HHDII splenocytes were processed to enrich for panT cells (CD3+), and HHDII T cells and HHDII / DP4 splenocytes were labeled with CFSE. HHDIIT cells were then cultured with or without HHDII / DP4 splenocytes in addition to FG2811mG1, LPS, or medium alone. On day 15, CD4 and CD8 proliferative responses were determined. In the absence of co-culture with splenocytes, only 0.14% of CD4 T cells proliferated in the presence of soluble FG2811mG1 (CFSE low ), which was compared with the medium-only control (0.16% CFSElow ), and thus merely background levels. In the absence of co-culture with splenocytes, only 0.02% of CD8 T cells proliferated in the presence of soluble FG2811mG1 (CFSE low ), which is a medium only control (0% CFSE low ) and thus merely background levels. Both CD4 and CD8 T cells showed good proliferative responses to LPS (3.34% and 39.56%, respectively). In the presence of co-culture with splenocytes, 15.2% of CD4 T cells proliferated in the presence of soluble FG2811mG1 (CFSE low ), and 2.33% of CD8 T cells proliferated in the presence of soluble FG2811mG1 (CFSE low ), both CD4 and CD8 T cells showed good proliferative responses to LPS enhanced in the presence of PBMC (59.88% and 54.10%, respectively).

[0195] These results indicate that soluble FG2811mG1 can stimulate CD4 and CD8 proliferative responses when co-cultured in the presence of splenocytes (Figure 24). T cells did not proliferate when splenocytes were not added to the culture, indicating that Fc cross-linking is the mode of action of 2811 mAb. In the CD4 T cell population, T cell proliferation was greater when co-cultured with splenocytes and FG2811mG1 compared to CD8 T cells (15.2% vs 2.33%). These results indicate the potential of 2811 mAb to expand T cells ex vivo and its mode of action is via Fc cross-linking.

[0196] Embodiment Further embodiments of the present invention are described below.

[0197] 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).

[0198] 2. A binding member according to embodiment 1 which is capable of binding to SSEA-4 on a glycolipid.

[0199] 3. Stem memory T cells (T SCM ) .

[0200] 4. Stem memory T cells (T SCM 3. The binding member according to any preceding embodiment, which is capable of inducing proliferation of

[0201] 5. A binding member according to any of the preceding embodiments which does not bind to SSEA-3.

[0202] 6. A binding member according to any of the preceding embodiments which is mAb FG2811.72 or chimeric FG2811.72 (CH2811 / CH2811.72), or a fragment thereof.

[0203] 7. A binding member according to any of the preceding embodiments which is bispecific.

[0204] 8. The binding member according to embodiment 7, wherein said bispecific binding member is further specific for CD3.

[0205] 9. A binding member according to any of the preceding embodiments, comprising one or more binding domains selected from the amino acid sequence of residues 27-38 (CDRH1), 56-65 (CDRH2) and 105-113 (CDRH3) of Figure 2a.

[0206] 10. A binding member according to any of the preceding embodiments, comprising one or more binding domains selected from the amino acid sequence of residues 27-38 (CDRL1), 56-65 (CDRL2) and 105-113 (CDRL3) of Figure 2b.

[0207] 11. The binding member comprises a light chain variable sequence comprising one or more of LCDR1, LCDR2, and LCDR3, LCDR1 contains SSVNY, LCDR2 includes DTS LCDR3 includes FQASGYPLT. A light chain variable sequence; A heavy chain variable sequence comprising one or more of HCDR1, HCDR2, and HCDR3, HCDR1 comprises GFSLNSYG, HCDR2 comprises IWGDGST, HCDR3 contains TKPGSGYAF, Heavy chain variable sequence and 2. The binding member of any preceding embodiment, comprising:

[0208] 12. A binding member according to any of the preceding embodiments, wherein said binding domain is carried by a human antibody framework.

[0209] 13. A binding member according to any of the previous embodiments, wherein said binding member comprises a VH domain comprising residues 1 to 126 of the amino acid sequence of Figure 2a, and / or a VL domain comprising residues 1 to 123 of the amino acid sequence of Figure 2b.

[0210] 14. A binding member according to any of the previous embodiments, wherein said binding member comprises a human antibody constant region.

[0211] 15. A binding member according to any of the preceding embodiments, wherein said binding member is an antibody, an antibody fragment, Fab, (Fab')2, scFv, Fv, dAb, Fd, or diabody.

[0212] 16. A binding member according to any of the previous embodiments, wherein said binding member is an 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) poly-Ala and 6xHis tags for purification.

[0213] 17. A binding member according to any of embodiments 1 to 15, wherein the binding member is an 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, and optionally further comprising a 5' or 3' purification tag.

[0214] 18. A binding member according to any of the preceding embodiments, provided in the form of a chimeric antigen receptor (CAR).

[0215] 19. A binding member according to embodiment 18, wherein said binding member is an scFv provided in the form of a chimeric antigen receptor (CAR) in either a heavy chain-light chain or a light chain-heavy chain orientation.

[0216] 20. A binding member according to any of embodiments 1 to 17 provided in the form of an agonistic (IgG2) monoclonal antibody.

[0217] 21. A binding member according to any of embodiments 1 to 17 provided in the form of an antagonistic monoclonal antibody.

[0218] 22. A binding member according to any of the preceding embodiments, wherein the binding member is monoclonal, such as a monoclonal antibody.

[0219] 23. The binding member according to any of the preceding embodiments, wherein said binding member is a human antibody, a humanized antibody, a chimeric antibody, or a veneered antibody.

[0220] 24. An isolated specific binding member which is capable of specifically binding to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) and which competes with an isolated specific binding member embodied in any one of embodiments 1 to 23.

[0221] 25. A binding member according to any of the preceding embodiments for use in therapy.

[0222] 26. A binding member according to any of embodiments 1 to 24 for use in a method for preventing, treating or diagnosing cancer.

[0223] 27. A binding member according to any of embodiments 1 to 24 for use in a method of treating a patient with a long-term viral infection.

[0224] 28. A binding member according to any of embodiments 1 to 24 for use in a method of treating an autoimmune disease, HIV, adult T-cell leukaemia, or graft-versus-host disease.

[0225] 29. A method of treating or preventing cancer, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need thereof.

[0226] 30. A method for treating or preventing a patient with a long-term viral infection, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need thereof.

[0227] 31. A method for treating or preventing an autoimmune disease, HIV, adult T-cell leukaemia or graft-versus-host disease, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need thereof.

[0228] 32. A method of enhancing a protective immune response against cancer, comprising administering a binding member according to any of embodiments 1 to 24 to a subject in need thereof.

[0229] 33. The method of embodiment 32, wherein the binding member is prepared for administration together with a further immunogenic agent, optionally a cancer vaccine.

[0230] 34. The method of embodiment 33, wherein the binding member and further immunogenic agent are arranged for simultaneous or sequential administration.

[0231] 35. A binding member for use according to embodiment 25 or 26 or a method according to embodiment 29, wherein said cancer is pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer or lung cancer.

[0232] 36. A binding member for use according to embodiment 25, 26 or 35, or a method according to embodiment 28 or embodiment 31, wherein said binding member is administered or prepared for administration alone or in combination with other treatments.

[0233] 37. A nucleic acid comprising a sequence encoding a binding member according to any one of embodiments 1 to 24.

[0234] 38. The nucleic acid according to embodiment 37, which is a construct in the form of a plasmid, vector, transcription cassette, or expression cassette.

[0235] 39. A recombinant host cell comprising a nucleic acid according to embodiment 37 or 38.

[0236] 40. A method for the diagnosis of cancer, comprising the step of using a binding member embodied in any of embodiments 1 to 24 to detect the glycan SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) bound to a glycolipid in a sample from an individual.

[0237] 41. The method of embodiment 40, wherein the pattern of glycans detected by the binding member is used to stratify treatment options for an individual.

[0238] 42. A pharmaceutical composition comprising a binding member according to any of embodiments 1 to 24 and a pharma- ceutically acceptable carrier.

[0239] 43. The pharmaceutical composition of claim 42, further comprising at least one or other pharma- ceutical active (agent).

[0240] 44. A pharmaceutical composition according to embodiment 42 or embodiment 43 for use in the treatment of cancer.

[0241] 45. A pharmaceutical composition according to embodiment 42 or 43, for use in the treatment of patients infected with a virus for a long period of time.

[0242] 46. ​​The pharmaceutical composition according to embodiment 42 or embodiment 43, for use in the treatment of an autoimmune disease, HIV, adult T-cell leukemia, or graft-versus-host disease.

[0243] 47.Ex vivo stem memory T cells (T SCM ) proliferation, comprising inducing proliferation of stem memory T cells (T SCM 25. A method comprising the step of contacting a binding member according to any one of embodiments 1 to 24.

[0244] 48. A stem memory T cell (T cell) comprising a binding member according to any one of embodiments 1 to 24. SCM ) cell culture medium for inducing proliferation.

[0245] 49. In vivo stem memory T cells (T SCM 25. A method of inducing proliferation of a cell line comprising administering to a subject a binding member according to any one of embodiments 1 to 24.

[0246] 50. Stem memory T cells (T SCM by detecting the presence of SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) on the endothelial cell.

[0247] 51. Stem memory T cells (T SCMA method for purifying said cells by detecting the presence of SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) on the chromatin.

[0248] 52. The method according to embodiment 50 or 51, wherein said identification or purification is carried out in vivo or ex vivo.

[0249] 53. The binding member can be used to isolate stem memory T cells (T SCM 53. The method of embodiment 51 or 52, wherein said labeling step comprises labeling said first or second antibody.

[0250] 54. A binding member substantially as described herein, optionally with reference to the accompanying drawings.

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Claims

1. It can specifically bind to SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) and inhibits stem memory T cells (T ＳＣＭ ) , The antibody is The following CDRs: LCDR1 containing SSVNY, LCDR2 containing DTS, and LCDR3 containing FQASGYPLT and a light chain variable sequence comprising: The following CDRs: HCDR1 comprising GFSLNSYG, HCDR2 comprising IWGDGST, and HCDR3 containing TKPGSGYAF and a heavy chain variable sequence comprising: Including, An antibody or an antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof of claim 1 which is bispecific.

3. An antibody or antigen-binding fragment thereof described in claim 2, which is further specific for CD3.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the CDRs are carried by a human antibody framework.

5. A VH domain comprising the amino acid sequence QVQLKESGPGLVAPSQSLSITCTVSGFSLNSYGVSWVRQPPGKGLEWLGVIWGDGSTNYHSALMSRLRISKDNSKRQVFLKLNSLQTDDTATYYCTKPGSGYAFAYWGQGTLVTVSS, and / or The VL domain includes the amino acid sequence ENVLTQSPAIMSASPGEKVTMTCSASSSSVNYMHWYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTIRTMEAEDVATYFCFQASGYPLTFGGGTKLELK The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising:

6. The antibody or antigen-binding fragment thereof described in any one of claims 1 to 5, which is a Fab, (Fab')2, scFv, Fv, dAb, Fd, or diabody.

7. An antibody or antigen-binding fragment thereof described in any one of claims 1 to 6, which is a human antibody, a humanized antibody, a chimeric antibody, or a veneered antibody.

8. A composition for use in therapy comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7.

9. A composition for use in a method for preventing, treating, or diagnosing cancer, comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7.

10. 10. The composition of claim 9, wherein the method further comprises administering an additional immunogenic agent, optionally a cancer vaccine.

11. A nucleic acid comprising a sequence encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

12. A kit for use in a method for diagnosing cancer, comprising an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, the method comprising a step of using the antibody or antigen-binding fragment thereof to detect the glycan SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) bound to glycolipids in a sample from an individual.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

14. Ex vivo stem memory T cells (T ＳＣＭ ) proliferation, comprising inducing proliferation of stem memory T cells (T ＳＣＭ 8. A method comprising contacting a polypeptide comprising the steps of:

15. A stem memory T cell (T cell) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7. ＳＣＭ ) cell culture medium for inducing proliferation.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 is used to infect stem memory T cells (T ＳＣＭ A method for identifying stem memory T cells (TSCM) by detecting the presence of SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) on T cells.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 is used to infect stem memory T cells (T ＳＣＭ A method for purifying stem memory T cells (TSCM) by detecting the presence of SSEA-4 (Neu5Ac(α2-3)Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Gal(β1-4)Glc) on T cells.

Images