Cellular compositions comprising antigen-specific T cells for adoptive therapy and their use.

Abstract

The present invention provides an isolated cell composition suitable for adoptive immunotherapy, as well as methods of manufacturing the cell compositions and methods of treatment with the cell compositions. The composition comprises, in a pharmaceutically acceptable carrier, at least about 10 6 CD8+ T cells specific for target peptide antigen(s). In various embodiments, the composition is predominantly CD8+ T cells, and at least about 20% of the T cells in the composition exhibit a central or effector memory phenotype, providing a robust and durable adoptive therapy of a natural T cell repertoire that has undergone natural selection.

Description

1 / 52 Cellular compositions comprising antigen-specific T cells for adoptive therapy and use. OF THE SAME PRIORITY

[001] This application claims the benefit of the US Provisional Application. 62 / 561,044, filed September 20, 2017, and the benefit of U.S. Provisional Application 62 / 656,679, filed April 12, 2018, each incorporated herein by reference in its entirety. FUNDAMENTALS

[002] Adoptive immunotherapies, such as donor lymphocyte infusions, are used to treat leukemia relapse after hematopoietic stem cell transplantation (HSCT) to enhance graft-versus-leukemia (GVL) effect. These approaches generally take several months to work; and require very large doses of cells, resulting in a substantial risk of graft-versus-host disease (GVHD). See, McLaughlin L, et al., Adoptive T-cell therapies for refractory / relapsed leukemia and lymphoma; current strategies and recent advances. Ther. Adv. Hematol. 2015 vol. 6 (6) 295307.

[003] With current therapeutic options, the outcome for patients with relapsed leukemia, for example, after HSCT, is difficult. Although adoptive cell therapies may provide some benefit, the number of specific target cells that can be supplied is usually insufficient and highly variable, and it is difficult to activate and expand naive T cell populations from ex vivo donor lymphocytes, especially with regard to cancer-specific CTL precursors that are often extremely low and even undetectable in the peripheral blood of healthy individuals. Quintarelli C, et al., Cytotoxic T lymphocytes directed to the preferentially expressed antigens of melanoma (PRAME) target chronic myeloid Petition 870260070676, dated 07 / 16 / 2026, page 7 / 134 2 / 52 leukemia. Blood 2008; 112: 1876-1885. Furthermore, cell therapies, such as chimeric antigen receptor (CAR) T-cell and natural killer cell therapies, tend to induce depleted cellular phenotypes that are not sufficiently robust and / or have limited persistence in vivo, and may exhibit target toxicity outside the tissue. See, Cruz and Bollard, T-cell and natural killer cell therapies for hematological malignancies after hematopoietic stem cell transplantation; enhancing the graft-versus-leukemia effect. Haematologica 2015; 100 (6) 709-719. Additionally, these therapies generally have limited flexibility due to the single target designed.

[004] Cellular compositions are needed to provide more effective and safe adoptive immunotherapy options, including for patients suffering from leukemia or lymphoma (including acute or chronic leukemia), as well as other patients who could benefit from adoptive immunotherapy. In several respects and modalities, the present invention meets these needs. SUMMARY OF THE INVENTION

[005] In various aspects and embodiments, the invention provides a composition of isolated cells suitable for adoptive immunotherapy, as well as methods of manufacturing the cell compositions and methods of treatment with the cell compositions. The composition comprises, in a pharmaceutically acceptable carrier, at least about 10⁶ CD8+ T cells specific for the target peptide antigen(s). In various embodiments, the composition is predominantly CD8+ T cells, and at least about 20% of the T cells in the composition exhibit a central or effector memory phenotype, providing a robust and durable adoptive therapy from a repertoire of natural T cells that has undergone natural selection. The cell composition does not comprise T cells expressing a chimeric antigen receptor or a TCR. Petition 870260070676, dated 07 / 16 / 2026, page 8 / 134 3 / 52 recombinant, and therefore in various modalities, provides an alternative to those technologies that generally produce more depleted T-cell phenotypes, less durable responses, and greater toxicities.

[006] In several embodiments, the cell composition comprises at least about 107 CD8+ T cells specific for the target peptide antigens, or at least about 108, at least about 109, or at least about 1010 CD8+ T cells specific for the target peptide antigens, to provide robust destruction of target cells and long persistence in vivo. For example, for the treatment of acute myeloid leukemia (AML) or myelodysplastic syndrome, the cell composition may comprise T cells specific for the WT1, PRAME, Survivin, and Cyclin A1 peptide antigens.

[007] In several embodiments, the T cells in the composition (and / or T cells specific for target antigens) are at least about 50% core memory or effector T cells, or in some embodiments are at least about 70% core memory or effector T cells, or at least about 80% core memory or effector T cells. In some embodiments, the memory cells are about 25:75 to about 75:25 core memory to effector cells. The cell composition comprises less than about 20% terminally differentiated memory T cells (e.g., Temra cells) and no more than about 20% pure cells. In some embodiments, the cell composition comprises about 5 to about 25% memory stem T cells (TSCMs).This cell phenotype can be created and / or controlled using an enrichment and expansion process with paramagnetic artificial antigen-presenting cells (aAPCs) and a recombinant T cell growth factor cocktail. Petition 870260070676, dated 07 / 16 / 2026, page 9 / 134 4 / 52

[008] In several modalities, the cellular composition is at least 90% CD8+ T cells (e.g., CD3+CD8+ cells). For example, the composition of isolated cells may be characterized by having less than about 10% or less than about 5% CD4+ T cells. When expanding CD8+ T cells ex vivo, CD4+ cells tend to outgrow CD8+ cells and compete for growth signals and are not required for a robust and durable in vivo response.

[009] In several modalities, antigen-specific T cells exhibit a polyfunctional phenotype after activation. For example, after activation, T cells are positive for two or more of the following: intracellular staining for IL-2, IFN-γ production, TNFα production, and CD107A. In several modalities, at least 50% or at least 70% of antigen-specific T cells exhibit at least two of these markers. In several modalities, at least 50% or at least 70% of antigen-specific T cells exhibit at least three of these markers, or in some modalities all four of these markers.

[010] Cell compositions according to various modalities can be prepared by an enrichment and expansion process. In some modalities, enriched CD8+ cells are specific for the target antigen(s) (e.g., tumor-associated antigens or virus-associated antigens). This cell population, even when predominantly pure cells in the source lymphocytes, can be rapidly expanded in culture to reach the cell compositions described here. Enrichment can occur using paramagnetic beads to positively select cell populations, which may have the added advantage of activating pure cells due to the potent magnetic clustering of surface receptors. Petition 870260070676, dated 07 / 16 / 2026, page 10 / 134 5 / 52 T cells. For example, paramagnetic spheres or nanoparticles may contain monomeric or multimeric (e.g., dimeric) HLA ligands that present peptide antigens, along with a co-stimulatory signal on the same or different particles, such as an agonist for CD28 (e.g., a CD28 antibody agonist). In some embodiments, CD28+ cells are also enriched, which may be simultaneous with specific antigen enrichment.

[011] In several modalities, target peptide antigens are tumor- or cancer-associated antigens, including tumor-derived, tumor-specific, and neoantigen antigens. Tumor-associated antigen-specific T cells are generally very rare and, in many cases, undetectable in the peripheral blood of healthy individuals. This is often a distinction observed between virus-specific and tumor antigen-specific T cells.

[012] In some embodiments, the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. For example, at least one peptide antigen may be associated with HIV, hepatitis (e.g., B, C, or D), CMV, Epstein-Barr virus (EBV), influenza, herpesvirus (e.g., HSV 1 or 2 or varicella zoster), and adenovirus. CMV, for example, is the most common viral pathogen found in transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation. Viral activation is known to be implicated in cancer biology.

[013] In other modalities, the cellular composition comprises T cells specific for tumor-associated antigens, with pathogen-associated T cells provided as Petition 870260070676, dated 07 / 16 / 2026, page 11 / 134 6 / 52 spectator cells. Specifically, by enriching CD8+ T cells based on selection of both HLA and anti-CD28 peptides, spectator cells will be enriched and expanded, particularly when using a T cell growth factor cocktail that can drive non-specific expansion of these cells without specific antigen activation. In these modalities, while a large portion of the composition is of T cells specific for the target peptides (e.g., 5% to 75%), the remaining T cells (from about 0.25% to about 25%) provide some reconstitution of the immune system for common pathogens, which is particularly beneficial after transplantation or beneficial in cancers with viral etiology.

[014] Some modalities employ T cell growth factors during expansion, which affect T cell proliferation and / or differentiation.Particularly useful cytokines include MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, and IFN-γ. In these or other modalities, cells are expanded in culture in the presence of one, two, or three cytokines selected from MIP-1β, IL-1β, and IL-6. In some modalities, the cytokines also include IL-10. Cells can be expanded in culture for 1 to 4 weeks, or for 10 to 21 days.

[015] In other respects, the invention provides methods for manufacturing the cell compositions, including by enrichment and expansion with aAPCs, as described herein. Specifically, after depletion of CD4+ cells from lymphocytes of origin (e.g., from a healthy donor), antigen-specific CD8+ T cells are enriched to T cells specific for the peptide target antigens, as well as CD28+ cells in some embodiments. The target cells can be enriched using nanoparticle or microparticle aAPCs, such as paramagnetic particles that activate T cells ex vivo by magnetic field-induced clustering of receptors. Petition 870260070676, dated 07 / 16 / 2026, page 12 / 134 7 / 52 of cell surface. Other materials, including latex or other polymeric particles, can also be used to cluster cell surface receptors (without magnetically induced clustering). Enriched T cells can be rapidly expanded ex vivo, including with the use of reconstituted T cell growth factors (e.g., comprising factors selected from MIP-Iβ, IL-Iβ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ). In some embodiments, cells are expanded in culture in the presence of one, two, or three cytokines selected from MIP-Iβ, IL-Iβ, and IL-6, and optionally IL-10. In some embodiments, the growth factors comprise or consist essentially of IL-2, IL-4, IL-6, IFN-γ, and IFN-1β.

[016] In other respects, the invention provides methods for adoptive cell therapy, including methods for treating a cancer patient and / or patients undergoing allogeneic stem cell transplantation, with or without lymphoma deletion therapy, cytoreductive therapy, immunomodulatory therapy (prior to administration of cell therapy). Cell therapy may also be provided with or without cytokine support after treatment. In some embodiments, the patient has a hematologic cancer, which in some embodiments has relapsed after allogeneic stem cell transplantation. In some embodiments, the patient has acute myeloid leukemia (AML) or myelodysplastic syndrome. For example, in some embodiments, the cell composition comprises T cells specific for the WT1, PRAME, Survivin, and Cyclin A peptide antigens. However, in other embodiments, the cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas.Examples of target peptide antigens are described here.

[017] In some modalities, the patient has an infectious disease or is at risk of an infectious disease. For example, the Petition 870260070676, dated 07 / 16 / 2026, p. 13 / 134 8 out of 52 patients who underwent HSCT are at particular risk of infectious disease due to their immunocompromised status. Infectious diseases that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include AIDS, hepatitis B / C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpes virus infection (including herpes zoster), and adenovirus infection.

[018] Other aspects and modalities will become evident from the detailed description below. DESCRIPTION OF THE FIGURES

[019] FIGURE 1 shows that MART-specific T cells enriched and expanded ex vivo from donor lymphocytes show a polyfunctional phenotype, including intracellular staining for IL-2 (proliferation and memory), IFN-γ (activation of other T cells, memory, MHC upregulation), TNF-α (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity). Most T cells show at least three functional phenotypes.

[020] FIGURE 2 shows that MART- and AML-specific T cells enriched and expanded ex vivo from donor lymphocytes using paramagnetic aAPCs are predominantly central memory (Tcm) and effector memory (Tem) phenotype.

[021] FIGURE 3 shows that antigen-specific T cells can be enriched and expanded in batches. The figure also shows batch enrichment and expansion of T cells specific for the antigenic peptides Pramew0 RHAMM, WT1 and Survivin.

[022] FIGURE 4 shows that the composition with individual stimulation and expansion had consistent levels of antigen-specific T cells in AML. The individual stimulation and expansion process consistently generates ~15% antigen-specific T cells. Petition 870260070676, dated 07 / 16 / 2026, page 14 / 134 9 / 52

[023] FIGURE 5 shows that the simultaneous stimulation / expansion process generates AML-specific T cell frequencies comparable to individual stimulation / expansion. The composition shown prepared by batch stimulation / expansion has ~47% antigen-specific T cells.

[024] FIGURE 6 shows that the generated T cells demonstrate antigen-specific killing of AML tumor cells (THP-1 cell line). AML-specific T cells are targeted for 5 epitopes of WT-1, PRAME and Survivin.

[025] FIGURE 7 shows that the cytokine cocktail used for ex vivo expansion affects the number and phenotype of the resulting cells. The reconstituted T-cell growth factor (TF) includes IL-1β, IL-2, IL-4, IL-6, IL-21, IFN-γ, and MIPI1β.

[026] FIGURE 8 shows the presence of virus-specific spectator T cells on day 7 after specific enrichment and expansion for MART-1.

[027] FIGURE 9 shows the presence of virus-specific spectator T cells on day 14 after specific enrichment and expansion for MART-1.

[028] FIGURE 10 shows the presence of virus-specific spectator T cells on day 14 after AML-specific enrichment and expansion. These cells were largely of a memory phenotype.

[029] FIGURE 11 shows the detection of CMV-specific spectator T cells during the MART-1-specific enrichment and expansion process. The percentage of virus-specific spectator cells remains constant until day 14, while the number and percentage of MART-1-specific T cells increase dramatically.

[030] FIGURE 12 shows the detection of spectator cells Petition 870260070676, dated 07 / 16 / 2026, page 15 / 134 10 / 52 virus-specific cells on day 14 after specific enrichment and expansion of MART-1 using a recombinant T-cell growth factor cocktail (IL-Iβ, IL-2, IL-4, IL-6, IL-21, IFN-γ and MIPI-β), which enhances the expansion of these spectator cells.

[031] FIGURE 13 has two panels (Figure 13A and Figure 13B) showing the specificity and phenotype of Mart-1-specific T cells generated by the enrichment and expansion process using a recombinant T cell growth factor cocktail (IL-2, IL-4, IL-6, IFN-γ, and IL-1β). Mart-1-specific T cells (Figure 13A, right panel) constituted approximately 35% of the culture and showed a core memory (~89%) and effector memory (~9%) phenotype. The total culture showed a phenotype of ~66% core memory and ~32% effector memory. DETAILED DESCRIPTION OF THE INVENTION

[032] In several aspects and embodiments, the invention provides a composition of isolated cells suitable for adoptive immunotherapy, as well as methods of manufacturing the cell compositions and methods of treatment with the cell compositions. The composition comprises, in a pharmaceutically acceptable carrier, at least about 10⁶ CD8+ T cells specific for the target peptide antigen(s). In several embodiments, at least about 20% of the T cells in the composition exhibit a central or effector memory phenotype, providing a robust and durable adoptive therapy. The cell composition does not comprise T cells expressing a chimeric antigen receptor or a recombinant TCR and therefore, in several embodiments, provides an alternative to those technologies that generally produce more depleted T cell phenotypes and less durable responses.

[033] As used herein, the term target peptide antigen(s) or target antigens refers to peptide antigens employed ex vivo. Petition 870260070676, dated 07 / 16 / 2026, page 16 / 134 11 / 52 to enrich and / or expand the desired CD8+ cell population, for example, in conjunction with artificial antigen-presenting cells (aAPCs) or professional antigen-presenting cell platforms (pAPCs) (e.g., dendritic cells). aAPCs or pAPCs are employed to activate and expand CTLs from donor or patient lymphocytes. In some modalities, the target peptide antigens are peptide epitopes loaded onto aAPCs for ex vivo enrichment and expansion of specific CD8+ T cells. Thus, the term specific to the target peptide antigen means that the T cell is an antigen recognized with the target antigen.

[034] In several embodiments, the cell composition comprises at least about 107 CD8+ T cells specific for the target peptide antigens, or at least about 108, at least about 109, or at least about 1010 CD8+ T cells specific for the target peptide antigens, to provide robust destruction of the target cells. In some embodiments, the cell composition contains 1 x 107 to 1 x 109 CD8+ T cells specific for the target antigens, or in some embodiments 5 x 107 to 5 x 108 CD8+ T cells specific for the target antigens. For example, the composition may comprise about 5 x 105 to about 5 x 106 cells per mL, in a volume of 50 to 200 mL. In certain embodiments, the composition volume is <100 mL (e.g., 50 to 100 mL). The cells in the composition, in various modalities, are at least 70% viable and provided in a sterile medium, which may be a cryoprotective medium (e.g., 10% DMSO).

[035] The cells of the composition, which are predominantly CD8+ cytotoxic lymphocytes (CTLs), are also substantially of a central or effector memory phenotype. CTLs generally include the following phenotypic populations: pure memory T stem cells (Tscm), central memory, effector memory, and cells of Petition 870260070676, dated 07 / 16 / 2026, p. 17 / 134 12 / 52 terminally differentiated memory. According to embodiments of the invention, the T cells specific for the target antigens are substantially composed of core memory and effector memory phenotypes. In some embodiments, the T cells specific for target antigens further comprise memory T stem cells (Tscm). The cellular composition thus provides a durable response, including in vivo persistence of antigen-specific T cells for at least about 1 month, or at least about 3 months, or at least about 6 months, or at least about 12 months, or at least about 18 months, or at least about two years in some embodiments.

[036] A pure T cell has differentiated in the bone marrow and undergone positive and negative central selection processes in the thymus. A pure T cell is considered mature and, unlike activated or memory T cells, has not encountered its cognate antigen. Naive T cells can be characterized by the surface expression of L-selectin (CD62L) and the absence of activation markers. In the pure state, T cells are generally quiescent and do not divide. According to this description, pure T cells are defined as CD62L+ and CD45RA+.

[037] Memory T cells include memory stem cells T (Tscm), core memory, and effector memory T cells. Memory T cells have previously responded to their cognate antigen. Upon a second encounter with the cognate antigen, memory T cells can reproduce to mount a faster and stronger immune response. Memory T cells include at least effector and core memory subtypes. Memory T cell subtypes are long-lived and can rapidly expand into a large number of effector T cells after exposure to the cognate antigen.

[038] T-cell memory stem cells (Tscm) are defined here Petition 870260070676, dated 07 / 16 / 2026, page 18 / 134 13 / 52 as CD45RA+ and as having at least two markers (or in some embodiments at least three or all four markers) selected from CXCR3+, CD95+, CD11a+, and CD58+. This memory subpopulation possesses stem cell-like self-renewal capabilities as well as the multipotent capacity to reconstitute both effector and memory T cell subpopulations. Memory T cells (mTs) can represent a small fraction of circulating T lymphocytes (e.g., > 5%) and have the ability to rapidly proliferate and release inflammatory cytokines in response to antigen re-exposure. Consequently, mTs are a subset of the memory T cell subpopulation. The phenotypes of mTs can be created and / or controlled using, as disclosed herein, an enrichment and expansion process with paramagnetic artificial antigen-presenting cells (aAPCs) and a recombinant T cell growth factor cocktail.

[039] According to this description, central memory T cells (Tcm cells) are defined as CD62L+ and CD45RA-. This memory subpopulation is commonly found in lymph nodes and peripheral circulation. Effector memory T cells (Tem cells) are defined as CD62L- and CD45RA-. These memory T cells lack lymph node return receptors and are therefore found in peripheral circulation and tissues. TEMRA stands for terminally differentiated effector memory cells that re-express CD45RA. These cells lack the ability to divide and are CD62L- and CD45RA+.

[040] Tcm cells exhibit a capacity for self-renewal and, according to embodiments of the invention, are important for achieving a long-lasting effect. Tem cells also have some capacity for self-renewal and strongly express genes essential for cytotoxic function. Temra cells also provide a Petition 870260070676, dated 07 / 16 / 2026, p. 19 / 134 14 / 52 robust cytotoxic function, but do not exhibit self-renewal capacity.

[041] Compositions in various modalities comprise CTLs are substantially composed of Tscm, Tcm, and Tem cells to balance the duration of effect against the potent destruction of malignancy or other target cells.

[042] In several embodiments, the T cells in the composition are at least about 30% central memory and effector cells, or at least about 40% central memory or effector cells, or at least about 50% central memory or effector T cells, or in some embodiments are at least about 70% central memory or effector cells, or at least about 80% central memory or effector T cells. In some embodiments, the memory cells are about 10:90 to about 90:10 central memory to effector cells. In some embodiments, the T cells in the composition are about 25:75 to about 75:25 central memory to effector cells. In some embodiments, the memory T cells are about 40:60 to about 60:40 central memory to effector T cells.The cellular composition comprises less than about 20% of terminally differentiated memory T cells (e.g., Temra cells) or less than about 10% or less than about 5% or less than about 4% of terminally differentiated memory T cells in some embodiments. In several embodiments, CD8+ T cells contain no more than about 20% pure cells, or in some embodiments, no more than about 15% pure cells, or no more than about 10% pure cells or no more than about 5% pure cells, or no more than about 4% pure cells, or no more than about 3% pure cells, or no more than about 2% pure cells, or no more than about 1.5%, or no more than about 1% pure cells. Petition 870260070676, dated 07 / 16 / 2026, p. 20 / 134 15 / 52 In various modalities, CD8+ T cells contain from about 5% to about 25% of scm T cells, or in some modalities, from about 5% to about 20% of scm T cells or from about 5% to about 15% of scm T cells.

[043] In several embodiments, the T cells specific for the target antigens are at least about 30% central memory and effector cells, or at least about 40% central memory or effector cells, or at least about 50% central memory or effector T cells, or in some embodiments are at least about 70% central memory or effector cells, or at least about 80% central memory or effector T cells. In some embodiments, these memory cells are about 10:90 to about 90:10 central memory to effector cells. In some embodiments, these T cells are about 25:75 to about 75:25 central memory to effector cells. In some embodiments, the memory T cells are about 40:60 to about 60:40 central memory to effector T cells.Target antigen-specific T cells are less than about 20% terminally differentiated memory T cells (e.g., TEMRA cells) or less than about 10% or less than about 5% or less than about 4% terminally differentiated memory T cells. In various embodiments, target antigen-specific T cells contain no more than about 20% pure cells, or in some embodiments, no more than about 15% pure cells, or no more than about 10% pure cells or no more than about 5% pure cells, or no more than about 2% or 1.5% or 1% pure cells. In several modalities, antigen-specific T cells contain from about 5% to about 25% of scm T cells, or in some modalities, from about 5% to about 20% of scm T cells or from about 5% to about 15% of scm T cells. This. Petition 870260070676, dated 07 / 16 / 2026, p. 21 / 134 The 16 / 52 phenotype can be created by the enrichment and expansion process with cells for artificial paramagnetic antigen presentation (aAPCs).

[044] In several forms, the cellular composition is at least 90% T cells or at least 95% T cells or at least 98% or at least 99% T cells. For the purposes of this description, T cells are characterized as CD3+ cells. T cells are generally CD8+. For example, the composition of isolated cells may be characterized as having less than about 10% or less than about 5% CD4+ T cells or, in some embodiments, less than about 2%, less than about 1.5% or less than about 1% CD4+ T cells. When expanding CD8+ T cells ex vivo, CD4+ cells tend to grow over CD8+ cells and compete for growth signals and are not required for a robust and durable in vivo response.

[045] The presence of polyfunctional CD4+ and CD8+ T cells has been described as correlating with the response to cancer vaccine therapy with peptide neoantigens. Ott PA, et al., An immunogenic personal neoantigen vaccine for patients with melanoma, Nature 547(7662):217-221 (2017). CD4+ and CD8+ T cells are also described as important for mediating the destruction of tumor cells. See, Tran E, Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344, 641-645 (2014); Sahin U, et al., Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer, Nature 547(7662):222-226 (2017). With regard to this description, it is believed that adoptive cell compositions only need to provide substantial numbers of antigen-specific CD8+ T cells for a robust and durable response, and particularly where antigen-specific CD8+ T cells are provided in sufficient numbers and Petition 870260070676, dated 07 / 16 / 2026, p. 22 / 134 17 / 52 are substantially of the central and effector memory phenotype. In several modalities, antigen-specific CD8+ T cells also comprise memory T stem cells.

[046] In several modalities, the cellular composition is substantially CD28+.

[047] In several modalities, antigen-specific T cells exhibit a polyfunctional phenotype after activation. For example, after activation, T cells are positive for two or more of the following: intracellular staining for IL-2, which is a marker for proliferation and memory; IFN-γ production, which activates other T cells and induces memory and MHC upregulation; TNF-α production, a pro-inflammatory marker; and CD107A, which is a marker for granzyme release and cytotoxic activity. In several modalities, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of antigen-specific T cells exhibit at least three of these markers. In several modalities, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of antigen-specific T cells exhibit all four of these markers.In some modalities, polyfunctionality is assessed or quantified using target killing assays, which evaluate the ability of CD8+ cytotoxic T cells to lyse target cells presenting the peptide antigen in complex with MHC.

[048] Cell compositions according to various modalities can be prepared by enriching CD8+ cells that are specific for the target antigen(s) (e.g., tumor-associated antigens or virus-associated antigens). This cell population, even when predominantly pure cells in the source lymphocytes, can be rapidly expanded in culture to reach the cell compositions described here. CD4+ cells Petition 870260070676, dated 07 / 16 / 2026, page 23 / 134 18 / 52 can be depleted (specific pre- or post-antigen enrichment) of lymphocytes using CD4+ cell-depleting microspheres. Specific antigen enrichment of CD8+ cells can occur using paramagnetic beads to positively select cell populations, and which may have the added advantage of activating pure cells due to the potent magnetic clustering of T cell surface receptors. For example, paramagnetic beads or nanoparticles may contain monomeric or multimeric (e.g., dimeric) HLA ligands that present peptide antigens, along with a co-stimulatory signal in some modalities, such as an agonist for CD28 (e.g., a CD28 antibody agonist). Exemplary methods according to these modalities are described in WO 2016 / 044530 and PCT / US2017 / 22663, which are incorporated herein by reference in their entirety.

[049] In some embodiments, CD28+ cells are also enriched, which may be simultaneous with antigen-specific enrichment. CD28 is expressed on T cells and is a co-stimulatory signal necessary for T cell activation and survival. CD28 is the only B7 receptor constitutively expressed on pure T cells. The association of the TCR of a pure T cell with the MHC-antigen complex without CD28 co-stimulation may result in a T cell that is anergic. In some embodiments, CD28+ cells are not enriched, but a CD28 agonist is added in soluble form during the enrichment process or added as a conjugate to non-paramagnetic beads. In some embodiments, CD28 (in conjugated or unconjugated form) is added to cells after antigen-specific enrichment in order to activate the cells for the expansion phase.

[050] In several modalities, specific T cells for Petition 870260070676, dated 07 / 16 / 2026, p. 24 / 134 19 / 52 target antigens (e.g., by virtue of the peptides displayed by aAPCs or pAPCs) are specific for 1 to about 100 target antigens, or 1 to about 75 target antigens, or 1 to about 50 target antigens, or 1 to about 25 target antigens, or 1 to about 20 target antigens, or 1 to about 15 target antigens, or 1 to 10 target antigens, or 1 to 5 target antigens. In several embodiments, there are at least 3, or at least 4, or at least 5 target antigens. The distinct target antigens may include overlapping peptide epitopes in some embodiments. T cells specific for these peptide antigens can be enriched and expanded in batches, allowing for rapid and parallel production of cell compositions. In some forms, the composition contains T cells specific for 5 to 15 or 5 to 10 peptide antigens.The specificity of a T cell with respect to a target peptide antigen in the composition is defined by staining with MHC multimer (e.g., staining with dimer or tetramer), as is well known in the technique.

[051] For example, a cocktail of nano-aAPCs, each aAPC presenting a different and distinct target antigen, can be used to enrich T cells against multiple antigens simultaneously. For example, T cells specific for 2 to 10 antigens can be simultaneously enriched from the lymphocyte source. In this embodiment, numerous different batches of nano-aAPCs, each containing a different MHC peptide, would be combined and used to simultaneously enrich T cells against each of the antigens of interest. The resulting T cell pool would be activated against each of these antigens and expanded together in culture. These antigens may be related to a single therapeutic intervention; for example, multiple antigens present in a single tumor or malignant cell. Petition 870260070676, dated 07 / 16 / 2026, page 25 / 134 20 / 52

[052] Target peptide antigens are generally suitable for presentation by an HLA-A, B or C molecular complex and, in some embodiments, an HLA-A2 molecular complex.

[053] In several modalities, the target peptide antigens are tumor- or cancer-associated antigens, including tumor-derived antigens or tumor-specific antigens. T cells specific for tumor-associated antigens are generally very rare and, in many cases, undetectable in the peripheral blood of healthy individuals. Furthermore, the cells are often of a naive phenotype, particularly when using donor T lymphocytes. See, Quintarelli et al., Cytotoxic T lymphocytes directed to the preferentially expressed antigens of melanoma (PRAME) target chronic myeloid leukemia. Blood 2008; 112: 1876-1885. This is often a distinction observed between virus-specific and tumor antigen-specific T cells.

[054] Tumor-associated antigens or cancer-specific antigens include unique tumor or cancer antigens, expressed exclusively by the tumor or malignant cells from which they derive, shared tumor antigens expressed in many tumors but not in normal adult tissues (oncofetal antigens), and tissue-specific antigens also expressed by the normal tissue from which the tumor arose. Tumor-associated antigens may be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressors, fusion proteins, or oncoviral proteins.

[055] In some modalities, the target peptide antigens include one or more hematologic cancer-associated or derived antigens, such as leukemia, lymphoma, or myeloma. For example, hematologic malignancies may include acute myeloid leukemia, chronic myeloid leukemia, childhood acute leukemia, non-Hodgkin lymphomas, leukemia Petition 870260070676, dated 07 / 16 / 2026, page 26 / 134 21 / 52 acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and cutaneous T-cell-rich lymphoid hyperplasia. In other modalities, target peptide antigens include one or more associated with or derived from a solid tumor, including melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, liver cancer, pancreatic cancer, renal cancer, endometrial cancer, testicular cancer, stomach cancer, oral mucosal dysplasia, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.

[056] A variety of tumor-associated antigens are known in the art. Oncofetal and embryonic antigens include carcinoembryonic antigen and alpha-fetoprotein (generally only highly expressed in developing embryos, but frequently expressed in liver and colon tumors, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, hepatic, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic oncofetal antigen, 5T4 (expressed in gastric carcinoma), alpha-fetoprotein receptor (expressed in various tumor types, particularly breast tumors), and M2A (expressed in germ cell neoplasia).

[057] Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and specific surface immunoglobulins (expressed in lymphomas). Petition 870260070676, dated 07 / 16 / 2026, page 27 / 134 22 / 52

[058] The products of the mutated oncogene or tumor suppressor gene include Ras and p53, both expressed in many tumor types, Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, myc (associated with lung cancer), ras, p53, non-mutant associated with breast tumors, MAGE-1 and MAGE-3 (associated with melanoma, lung, and other cancers). Fusion proteins include BCR-ABL, which is expressed in chromic myeloid leukemia. Oncoviral proteins include HPV type 16, E6, and E7, found in cervical carcinoma.

[059] Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancers); CD10 (formerly known as common acute lymphoblastic leukemia antigen, or CALLA) or surface immunoglobulin (expressed in B-cell leukemias and lymphomas); the IL-2 receptor alpha chain, T-cell receptor, CD45R, CD4+ / CD8+ (expressed in T-cell leukemias and lymphomas); prostate-specific antigen and prostatic acid phosphatase (expressed in prostate carcinoma); GP 100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S100 (expressed in melanoma); cytokeratins (expressed in various carcinomas); and CD19, CD20, and CD37 (expressed in lymphoma).

[060] Tumor-associated antigens also include altered glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e.g., GM2 and GD2, expressed in melanomas and some brain tumors); blood group antigens, particularly sialylated T and Tn antigens, which may be aberrantly expressed in carcinomas; and mucins, such as CA-125 and CA-19-9 (expressed in ovarian carcinomas) or subglycosyl MUC-1 (expressed in breast and pancreatic carcinomas). Petition 870260070676, dated 07 / 16 / 2026, p. 28 / 134 23 / 52

[061] For example, in some embodiments, one or more target antigens are associated with bladder cancer, such as one or more NY-ESO-1, MAGE-A10, and MUC-1 antigens. In some embodiments, one or more target antigens are associated with brain cancer and may include one or more of the NY-ESO-1, Survivin, and CMV antigens. In some embodiments, one or more target antigens are associated with breast cancer and may include one or more of the MUC-1, Survivin, WT-1, HER-2, and CEA antigens. In some embodiments, one or more target antigens are associated with cervical cancer and may include the HPV antigen. In some embodiments, one or more target antigens are associated with colorectal cancer and may include one or more of the NY-ESO-1, Survivin, WT-1, MUC-1, and CEA antigens. In some modalities, one or more target antigens are associated with esophageal cancer and may include the NY-ESO-1 antigen.In some modalities, one or more target antigens may be associated with head and neck cancer and may include the HPV antigen. In some modalities, the target antigen is associated with kidney or liver cancer and may include the NY-ESO-1 antigen. In some modalities, the target antigen is associated with lung cancer and may include one or more NY-ESO-1, Survivin, WT1, MAGE-A10, and MUC-1 antigens. In some modalities, one or more target antigens are associated with melanoma and may include one or more NY-ESO-1, Survivin, MAGE-A10, MART-1, and GP-100 antigens. In some modalities, one or more peptide antigens are associated with ovarian cancer and may include one or more NY-ESO-1, WT-1, and Mesothelin antigens. In some modalities, one or more target antigens are associated with prostate cancer and may include one or more of the antigens Survivin, hTERT, PSA, PAP, and PSMA. In some modalities, the target antigen is associated with a sarcoma and may include the NY-ESO-1 antigen.In some modalities, one or the other. Petition 870260070676, dated 07 / 16 / 2026, p. 29 / 134 24 / 52 more target antigens are associated with lymphoma and may include the EBV antigen. In some modalities, one or more target antigens are associated with multiple myeloma and may include one or more NY-ESO-1, WT-1, and SOX2 antigens.

[062] In some embodiments, one or more target antigens are associated with acute myeloid leukemia or myelodysplastic syndrome and may include one or more antigens (including 1, 2, 3, 4, or 5 of) Survivin, WT-1, PRAME, RHAMM, PR3, and cyclin A1. In some embodiments, the target antigens include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the target antigens in Table 1 below. Table 1: Target peptide antigens of exemplary AML Antigen Peptide name / position Sequence SEQ ID No: WT-1 126-134 RMFPNAPYL SEQ ID No: 1 235-243 CMTWNQMNL SEQ ID No:2 37-45 VLDFAPPGA SEQ ID No: 3 187-19-19 SEQ ID NYSEQ ID: P100 VLDGLDVLL SEQ ID N°:5 P435 NLTHVLYPV SEQ ID N°:6 P142 SLYSFPEPEA SEQ ID N°:7 P300 ALYVDSLFFL SEQ ID N°:8 P425 SLLQHLIGL SEQ ID N°:9 Survivin ELT 95-104 SEQL LGE ID: 104-113 LDRERAKNKI SEQ ID NO:11 Cyclina A1 227-235 FLDRFLSCM SEQ ID NO: 12 341-351 SLIAAAAFCLA SEQ ID NO: 13

[063] In some embodiments, one or more antigens of Petition 870260070676, of 16 / 07 / 2026, p. 30 / 134 25 / 52 target peptides are neoantigens. For example, in some modalities, patient-specific neoantigens are identified and synthesized to carry aAPCs. In some modalities, between three and ten neoantigens are identified by genetic analysis of patient malignancy (e.g., by nucleic acid sequencing of malignant cells), followed by predictive bioinformatics. In some modalities, the antigens are natural, non-mutated cancer antigens, many of which are known.

[064] In several embodiments, at least one of the target peptide antigens is recognized by a low-frequency precursor T cell. According to these embodiments, the invention enables the rapid activation and expansion of these cells for adoptive therapy.

[065] In some modalities, the target peptide antigens include at least one that is associated with or derived from a pathogen, such as a viral, bacterial, fungal, or parasitic pathogen. For example, at least one peptide antigen may be associated with HIV, hepatitis (e.g., A, B, C, or D), CMV, Epstein-Barr virus (EBV), influenza, herpesvirus (e.g., HSV 1 or 2 or varicella zoster), and adenovirus. CMV, for example, is the most common viral pathogen found in transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation. This is due to the immunocompromised state of these patients, which allows for reactivation of the latent virus in seropositive patients or opportunistic infection in seronegative individuals. In these modalities, the patient may receive adoptive immunotherapy comprising pathogen antigen-specific T cells.The method can lead to the generation of a specific CTL derived from the patient or a suitable donor before the procedure begins. Petition 870260070676, dated 07 / 16 / 2026, page 31 / 134 26 / 52 transplant.

[066] In some embodiments, at least one target antigen is a pathogen-associated antigen, including antigens associated with protozoa, bacteria, fungi (unicellular and multicellular), viruses, prions, intracellular parasites, helminths, and other infectious agents.

[067] Bacterial antigens include antigens of gram-positive cocci, gram-negative bacilli, gram-negative bacteria, anaerobic bacteria, such as organisms of the families Actinomycetaceae, Bacillaceae, Bartonellaceae, Bordetellae, Captophagaceae, Corynebacteriaceae, Enterobacteriaceae, Legionellaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pasteurellaceae, Pseudomonadaceae, Spirochaetaceae, Vibrionaceae and organisms of the genera Acinetobacter, Brucella, Campylobacter, Erysipelothrix, Ewingella, Francisella, Gardnerella, Helicobacter, Levinea, Listeria, Streptobacillus and Tropheryma.

[068] Antigens of protozoan infectious agents include antigens of malaria plasmodium, Leishmania species, Trypanosoma species and Schistosoma species.

[069] Fungal antigens include Aspergillus antigens, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, Sporothrix, organisms of the order Mucorales, organisms that induce choromycosis and mycetoma, and organisms of the genera Trichophyton, Microsporum, and Epidermophyton.

[070] Viral peptide antigens include, among others, those of adenovirus, herpes simplex virus, papillomavirus, respiratory syncytial virus, poxvirus, HIV, influenza virus, EBV, hepatitis, and CMV. Particularly useful viral peptide antigens include HIV proteins such as HIV gag proteins (including, but not limited to, membrane anchoring protein (MA), core capsid protein (CA), and nucleocapsid protein (NC)), HIV polymerase, matrix of Petition 870260070676, dated 07 / 16 / 2026, page 32 / 134 27 / 52 influenza virus (M1) nucleocapsid protein (NP) of the influenza virus protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis E protein (HBeAg), hepatitis B DNA polymerase, hepatitis C antigens and the like.

[071] In some modalities, the target peptide antigens include one or more tumor-associated antigens and one or more virus-associated antigens (such as CMV, EBV, influenza, or Adenovirus) to provide an antitumor response while protecting against common pathogens that complicate recovery after HSCT.

[072] Patients who have undergone HSCT are at particular risk of infectious disease, given their immunocompromised state. The immunocompromised state of these patients allows for reactivation of latent virus in seropositive patients or opportunistic infection in seronegative individuals. For example, post-transplant lymphoproliferative disease (PTLD) occurs in a significant fraction of transplant patients and results from Epstein-Barr virus (EBV) infection. EBV infection is believed to be present in approximately 90% of the adult population in the United States.Active viral replication and infection are controlled by the immune system, but, as in the case of CMV, individuals immunocompromised by transplant therapies lose the controlling populations of T cells, allowing viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematologic and non-hematologic cancers.

[073] In other modalities, the cellular composition comprises T cells specific for tumor-associated antigens, with T cells associated with pathogens provided as bystander cells. Specifically, when enriching for T cells Petition 870260070676, dated 07 / 16 / 2026, page 33 / 134 28 / 52 Based on selection with both peptide-HLA and anti-CD28 complexes, spectator cells will be enriched and expanded, particularly when using a T-cell growth factor cocktail that can drive non-specific expansion of these cells without specific antigen activation. In these modalities, while a large portion of the composition are T cells specific for the target peptides (e.g., 5% to 75%, or 10% to 50%), the remaining T cells provide some reconstitution of the immune system against common pathogenic bacteria, which is particularly beneficial after transplantation. For example, the composition may comprise T cells specific for CMV, EBV, influenza, and Adenovirus. In each case, pathogen-specific T cells may be present from 0.1% to about 4% of the composition.

[074] In several embodiments, the invention involves compositions prepared by enrichment and expansion of antigen-specific CD8+ T cells. The precursor T cells can be obtained from the patient or from an HLA-compatible donor. The source T cells can be fresh or frozen samples. The precursor T cells can be obtained from various sources comprising WBCs, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, spleen tissue, buffy coat fraction, and tumors. In some embodiments, the precursor T cells are obtained from a unit of blood collected from an individual using any number of techniques known to one skilled in the art. For example, precursor T cells from an individual's circulating blood can be obtained by apheresis or leukapheresis.The product of apheresis typically contains lymphocytes, including T cells and precursor T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Leukapheresis is a procedure of... Petition 870260070676, dated 07 / 16 / 2026, page 34 / 134 29 / 52 laboratory in which white blood cells are separated from a blood sample.

[075] Cells collected by apheresis can be washed to remove the plasma fraction and placed in a buffer or medium suitable for subsequent processing steps. The washing steps can be performed by methods known to those skilled in the art, such as the use of a semi-automated continuous flow centrifuge. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca- and Mg-free PBS. Alternatively, undesirable components of the apheresis sample can be removed and the cells directly resuspended in a culture medium.

[076] If desired, precursor T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient.

[077] In certain modalities, leukocytes are collected by leukapheresis and can be subsequently enriched for CD8+ T cells, for example, by depleting the sample of CD4+ cells and / or positively enriching for CD8+ cells. In some modalities, other cell types are depleted, such as NK cells. The CD8-enriched cells can then be further enriched for antigen-specific T cells.

[078] In several embodiments, the sample comprising immune cells (e.g., CD8+ T cells) is placed in contact with an artificial antigen-presenting cell (aAPC) with magnetic properties. Paramagnetic materials have a small, positive susceptibility to magnetic fields. These materials are attracted by a magnetic field, and the material does not retain the magnetic properties when the external field is removed. Petition 870260070676, dated 07 / 16 / 2026, page 35 / 134 30 / 52 Exemplary paramagnetic materials include, without limitation, magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic spheres suitable for magnetic enrichment are commercially available (DYNABEADS™, MACS MICROBEADS™, Miltenyi Biotec). In some embodiments, the aAPC particle is a dextran iron sphere (e.g., dextran-coated iron oxide sphere).

[079] Antigen-presenting complexes comprise an antigen-binding cleft and are generally MHC class I, which can be ligated or tethered to provide dimeric or multimeric MHC. In some embodiments, the MHC is monomeric, but its close association within the nanoparticle is sufficient for avidity and activation. In some embodiments, the MHC is dimeric. Dimeric MHC class I ligands can be constructed by fusion with immunoglobulin heavy chain sequences, which are then associated via one or more disulfide bonds (with or without associated light chains). MHC multimers can be created by direct ligation via peptides or chemical ligands, or they can be multimeric by association with streptavidin via biotin moieties. In some embodiments, antigen-presenting complexes are MHC class I complexes involving fusions with immunoglobulin sequences.

[080] MHC class I molecular complexes with immunoglobulin sequences are described in U.S. Patent 6,268,411, which is incorporated herein by reference in its entirety. These MHC class I molecular complexes can be formed in a conformationally intact manner at the ends of immunoglobulin heavy chains. MHC class I molecular complexes to which antigenic peptides are attached can stably bind to antigen-specific lymphocyte receptors. Petition 870260070676, dated 07 / 16 / 2026, page 36 / 134 31 / 52 (e.g., T cell receptors). In several embodiments, the immunoglobulin heavy chain sequence is not complete, but comprises an Ig hinge region and one or more of the CH1, CH2, and / or CH3 domains. The Ig sequence may or may not comprise a variable region, but where variable region sequences are present, the variable region may be complete or partial. The complex may further comprise immunoglobulin light chains. MHC class I ligands (e.g., HLA-Ig) without variable chain sequences (and without light chain) can be employed with site-directed conjugation for particles, as described in WO 2016 / 105542, which is incorporated by reference in its entirety.

[081] Examples of MHC class I molecular complexes comprise at least two fusion proteins.A first fusion protein comprises a first MHC class I α chain and a first immunoglobulin heavy chain (or a portion thereof comprising the hinge region), and a second fusion protein comprises a second MHC class I α chain and a second immunoglobulin heavy chain (or a portion comprising the hinge region). The first and second immunoglobulin heavy chains associate to form the MHC class I molecular complex, which comprises two MHC class I peptide-binding clefts. The immunoglobulin heavy chain may be the heavy chain of an IgM, IgD, IgG1, IgG3, IgG2e, IgG2a, IgG4, IgE, or IgA. In some embodiments, an IgG heavy chain is used to form MHC class I molecular complexes. If multivalent MHC class I molecular complexes are desired, IgM or IgA heavy chains may be used to provide pentavalent or tetravalent molecules, respectively.

[082] Exemplary class I molecules include HLA-A, HLA-B, Petition 870260070676, dated 07 / 16 / 2026, p. 37 / 134 32 / 52 HLA-C, HLA-E, and these can be used individually or in any combination. In some modalities, the antigen-presenting complex is an HLA-A2 ligand. The term MHC, as used here, can be replaced by HLA in each case.

[083] Immunoglobulin sequences in some modalities are humanized monoclonal antibody sequences.

[084] aAPCs may contain a Signal 2, such as an anti-CD28 ligand. Signal 2 is generally a T cell-affecting molecule, that is, a molecule that has a biological effect on a precursor T cell or on an antigen-specific T cell. In certain embodiments, Signal 2 is a T cell co-stimulatory molecule. T cell co-stimulatory molecules contribute to the activation of antigen-specific T cells. These molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, and antibodies that specifically bind to OX40.In some embodiments, the co-stimulatory molecule (signal 2) is an antibody (e.g., a monoclonal antibody) or a portion thereof, such as F(ab')2, Fab, scFv, or a single-chain antibody or other antigen-binding fragment. In some embodiments, the antibody is a humanized monoclonal antibody or a portion thereof with antigen-binding activity, or it is a fully human antibody or a portion thereof with antigen-binding activity.

[085] Combinations of co-stimulatory ligands that can be employed (in the same or separate nanoparticles) include anti-CD28 / anti-CD27 and anti-CD28 / anti-41BB. The ratios of these co-stimulatory ligands can vary to effect expansion.

[086] The exemplary signal 1 and signal 2 ligands are described in Petition 870260070676, dated 07 / 16 / 2026, p. 38 / 134 33 / 52 document WO 2014 / 209868, which describes ligands with a free sulfhydryl group (e.g., unpaired cysteine), so that the constant region can be coupled to nanoparticle supports with appropriate chemical functionality.

[087] Useful adhesion molecules for nano-aAPCs can be used to mediate the adhesion of nano-aAPCs to a T cell or a T cell precursor. Useful adhesion molecules include, for example, ICAM-1 and LFA-3.

[088] In some embodiments, signal 1 is provided by peptide-HLA-A2 complexes and signal 2 is provided by B7.1-Ig or antiCD28. An exemplary anti-CD28 monoclonal antibody is 9.3 mAb (Tan et al., J. Exp. Med. 1993 177:165), which can be humanized in certain embodiments and / or conjugated to the sphere as a fully intact antibody or an antigen-binding fragment thereof.

[089] Magnetic activation can occur for 2 minutes to 5 hours, or 5 minutes to two hours, followed by expansion in culture for at least 5 days and up to two weeks, or up to 3 weeks in some modalities. In some modalities, magnetic activation occurs for at least 2 minutes but less than 30 minutes (e.g., about 5 or 10 minutes). The resulting CD8+ T cells can be phenotypically characterized to confirm the presence of memory T stem cells (Tscm), as well as a high central and effector memory phenotype.

[090] Some modalities employ T-cell growth factors during expansion, which affect T-cell proliferation and / or differentiation. Examples of T-cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. If desired, cytokines may be present in molecular complexes comprising fusion proteins or may be encapsulated by aAPC or provided in soluble form. Cytokines Petition 870260070676, dated 07 / 16 / 2026, page 39 / 134 34 / 52 particularly useful include MIP-Ιβ, IL-Ιβ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ and CXCL10. In some embodiments, the growth factors include MIP-Ιβ 3, 4, 5, or 6, IL-Ιβ, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, and INF-γ. In these or other embodiments, cells are expanded in culture in the presence of one, two, three cytokines selected from MIP-Ιβ, IL-Ιβ and IL-6 and, optionally, IL-10. In some embodiments, the cells are not cultured in the presence of IL7 and / or IL-21 and / or IL-15. Cells can be expanded in culture for 1 to 4 weeks, such as about two weeks (about 14 days) or about 3 weeks.

[091] In some modalities, cells are expanded in culture in the presence of 4 to 8 cytokines, to achieve a balance between T cell expansion (including antigen-specific T cell expansion), activation, and memory phenotype. In some modalities, cells are expanded in the presence of IL-4. In some modalities, cells are expanded in the presence of IL-4 and IL-6. In some modalities, cells are expanded in the presence of IL-4 and IL-1β. In some modalities, cells are expanded in the presence of IL-4, IL-6, and α-1β. In some modalities, cells are expanded in the presence of IL-2, IL-4, and IL-6. In some modalities, cells are expanded in culture in the presence of IL-2, IL-4, IL-6, IFN-γ, and α-1β. In some modalities, cells are also expanded in the presence of IL-10.

[092] In some forms, the growth factors consist of, or consist essentially of, IL-2, IL-4, IL-6, IFN-γ and IL1β, and optionally IL-10.

[093] In some embodiments, IL-2 is present at the beginning of the culture from 10 to 200 International Units (IU) per mL, as from about 20 to about 100 IU / mL, or about 20 to about 60 IU / mL. In some embodiments, IL-2 is present at the beginning of the culture in about Petition 870260070676, dated 07 / 16 / 2026, p. 40 / 134 35 / 52 from 30 to about 50 lU / mL (e.g., about 40 lU / mL). The IL-2 IU (86 / 500 NIBSC) can be determined using a proliferation assay (e.g., using the CTLL-2 cell line), as described, for example, by Gearing and Bird (1987) Lymphokines and Interferons, A Practical Approach. Clemens, MJ et al. (eds): IRL Press. 295. In some embodiments, IL-2 is present at the beginning of the culture from about 2 to about 25 ng / mL, as well as from about 5 to about 15 ng / mL.

[094] In these or independent modalities, IL-4 is present at the beginning of the culture at 0.2 to 25 International Units (IU) per mL, such as from about 0.5 to about 10 IU / mL, or from about 0.5 to about 5 IU / mL. In some modalities, IL-4 is present at the beginning of the culture at about 1 IU / mL. The IL-4 IU (88 / 656 NIBSC) can be defined using a proliferation assay (e.g., using the TF-1 cell line), as described, for example, by Kitamura T. et al., (1991) IL-1 positively regulates the expression of cytokine receptors in a factor-dependent human hematopoietic cell line, TF-1. Int. Immunol. 3:571-577. In some forms, IL-4 is present at the beginning of the culture at about 0.2 to about 2 ng / mL, as well as at about 0.2 to about 1 ng / mL (for example, about 0.5 ng / mL).

[095] In these or independent modalities, IL-6 may be present at the beginning of culture from 10 to 200 International Units (IU) per mL, as from about 25 to about 100 IU / mL, as from 25 to 75 IU / mL. In some modalities, IL-6 is present at the beginning of culture at about 40 to about 60 IU / mL (e.g., about 50 IU / mL). The IL-6 IU (89 / 548 NIBSC) can be defined using a proliferation assay (e.g., using the B9 cell line), as described, for example, by Gaines-Das RE and Poole S. (1993) The international standard for interleukin-6. Evaluation in an international collaborative study. J. Immunol. Methods 160: 147-153. In some Petition 870260070676, dated 07 / 16 / 2026, p. 41 / 134 In 36 / 52 modalities, IL-6 is present at the beginning of the culture at about 0.2 to about 10 ng / mL, as well as at about 0.2 to about 5 ng / mL (e.g., about 0.5 to 2 ng / mL).

[096] In these or independent modalities, interferon gamma (INF-γ) may be present at the beginning of the culture from 10 to 200 International Units (IU) per mL, as from about 20 to about 100 IU / mL, as from 20 to 60 IU / mL. In some modalities, INF-γ is present at the beginning of the culture at about 30 to about 50 IU / mL (e.g., about 40 IU / mL). The IU of INF-γ (87 / 586 NIBSC) can be defined using an antiviral assay (e.g., with EMC-infected HeLa cells), as described for example in Meager A. (1987) in Lymphokines and interferons, a Practical Approach. Clemens, MJ, et al. (eds): IRL Press. 129. In some forms, INF-γ is present at the beginning of the culture at about 0.5 to about 20 ng / mL, as well as at about 1 to about 10 ng / mL (e.g., 1 to 5 ng / mL).

[097] ^-1β may be present at the beginning of the culture from 5 to 100 International Units (IU) per mL, such as from about 10 to about 50 IU / mL, such as from about 10 to about 30 IU / mL. In some embodiments, ^-1β is present at the beginning of the culture at about 10 to about 20 IU / mL (e.g., about 15 IU / mL). The IU of ^-1β (86 / 680 NIBSC) can be defined using a proliferation assay (e.g., using D.10.G4.1 cells), as described for example by Poole, S. and Gaines-Das, RE (1991) International standards for interleukin-1 alpha and interleukin-1 beta. Evaluation in an international collaborative study. J. Immunol. Methods 142:1-13. In some forms, ^-1β is present at the beginning of the culture at about 0.2 to about 5 ng / mL, as well as from about 0.2 to about 2 ng / mL, or from about 0.2 to about 1 ng / mL.

[098] In several embodiments, cells are cultured in the presence of a growth factor cocktail comprising or Petition 870260070676, dated 07 / 16 / 2026, p. 42 / 134 37 / 52 consists of IL-2, IL-4, IL-6, IFN-γ, and IL-1β. In some embodiments, the relative activity (defined by the respective IU) of IL-2 and IFN-γ is from about 0.5:1 to about 1:0.5 (e.g., about 1:1). In these or independent embodiments, the relative activity (defined by the respective IU) of IL-2 and IL-6 is from about 0.5:1 to 1:0.5. In these or independent embodiments, the relative activity of IL-1β with respect to IL-2, IL-6, and / or IFN-γ (defined by the respective IUs) is from 1:4 to 1:2 (e.g., about 1:3). In these or independent modalities, the relative activity of IL-4 with respect to IL-2, IL-6 and / or IFN-γ (defined by the respective IUs) is 1:30 to 1:60. In these or independent modalities, the relative activity of IL-4 with respect to ^-1β (defined by the respective IUs) is approximately 1:5 to approximately 1:25, as well as approximately 1:10 to approximately 1:20.

[099] In some embodiments, the specific activity of each growth factor (IL-2, IL-4, IL-6, IFN-γ and β-1β) at the beginning of the culture (in IUs) can be shown as a percentage when the total IUs of all growth factors in the culture are considered to be 100%. For example, in some embodiments, the percentage of each growth factor in the culture may be as follows: 20% to 40% IL-2 (e.g., 20 to 30% IL-2); 0.5% to 5% IL-4 (e.g., 1 to 3% IL-4); 25% to 50% IL-6 (e.g., 30 to 40% IL-6); 20% to 40% IFN-γ (e.g., 20 to 30% IFN-γ); and 5% to 20% IL-1p (e.g., 5 to 15% IL-1p).

[0100] aAPC nanoparticles can be made of any material, and the materials can be suitably selected for the desired magnetic property and may comprise, for example, metals such as iron, nickel, cobalt, or rare earth metal alloys. Paramagnetic materials also include magnesium, molybdenum, lithium, tantalum, and iron oxide. Suitable paramagnetic spheres for Petition 870260070676, dated 07 / 16 / 2026, page 43 / 134 38 / 52 Enrichment materials (including cells) are commercially available and include iron dextran spheres, such as iron oxide spheres coated with dextran. In aspects of the invention where magnetic properties are not required, nanoparticles can also be made of non-metallic or organic materials (e.g., polymers), such as cellulose, ceramics, glass, nylon, polystyrene, rubber, plastic, or latex. An example of a material for the preparation of nanoparticles is poly(lactic-co-glycolic acid) (PLGA) or PLA and its copolymers, which can be employed in conjunction with these embodiments. Other materials, including polymers and copolymers that can be employed, include those described in PCT / US2014 / 25889, which is incorporated herein by reference in its entirety.

[0101] In several embodiments, the particle has a size (e.g., average diameter) within about 10 to about 500 nm, or within about 40 to about 400 nm, or within about 100 nm to 400 nm. For magnetic clustering, it is preferable that the nanoparticles have a size in the range of 10 to 250 nm or 20 to 100 nm in some embodiments. The receptor-ligand interactions at the cell-nanoparticle interface are not well understood. However, nanoparticle binding and cell activation are sensitive to membrane spatial organization, which is particularly important during T cell activation, and magnetic fields can be used to manipulate cluster-bound nanoparticles to enhance activation. For example, T cell activation induces a persistently enhanced nanoscale TCR clustering state, and nanoparticles are sensitive to this clustering in a way that larger particles are not.

[0102] Furthermore, the interactions of nanoparticles with TCR clusters can be explored to enhance the Petition 870260070676, dated 07 / 16 / 2026, page 44 / 134 39 / 52 receptor triggering. T cell activation is mediated by the aggregation of signaling proteins, with signaling clusters hundreds of nanometers in diameter initially forming at the periphery of the T cell-APC contact site and migrating inward. As described herein, an external magnetic field can be used to enrich antigen-specific T cells (including pure rare cells) and to drive the aggregation of magnetic nano-aAPCs bound to the TCR, resulting in the aggregation of TCR aggregates and enhanced activation of naive T cells. Magnetic fields can exert sufficiently strong forces on paramagnetic particles, but are biologically inert, making them a powerful tool for controlling particle behavior. Paramagnetic nano-aAPC bound T cells are activated in the presence of an externally applied magnetic field.Nano-aAPCs are magnetized and attracted to the field source and to nearby nanoparticles in the field, inducing bead and therefore TCR aggregation to enhance aAPC-mediated activation.

[0103] Activation chemistries can be used to enable the specific and stable attachment of molecules to the surface of nanoparticles. Several methods can be used to attach proteins to functional groups. For example, the common crosslinking agent glutaraldehyde can be used to connect amine groups of proteins to the surface of aminated nanoparticles in a two-step process. The resulting linkage is hydrolytically stable. Other methods include the use of crosslinkers containing n-hydrosuccinimide (NHS) esters that react with amines in proteins, crosslinkers containing active halogens that react with proteins containing amine, sulfhydryl, or histidine, crosslinkers containing epoxides that react with amine or sulfhydryl groups, Petition 870260070676, dated 07 / 16 / 2026, page 45 / 134 40 / 52 conjugation between maleimide and sulfhydryl groups and the formation of aldehyde protein groups by periodic oxidation of the pendant sugar moieties followed by reductive amination.

[0104] The ratio of specific ligands when used simultaneously on the same or different particles can vary to increase the effectiveness of the nanoparticle in presenting antigen or co-stimulatory ligand. For example, nanoparticles can be coupled to HLA-A2-Ig and anti-CD28 (or other signal 2 ligands) in a variety of ratios, such as about 30:1, about 25:1, about 20:1, about 15:1, about 10:1, about 5:1, about 3:1, about 2:1, about 1:1, about 0.5:1, about 0.3:1; approximately 0.2:1, approximately 0.1:1, or approximately 0.03:1. In some embodiments, the ratio is 2:1 to 1:2. The total amount of protein coupled to the supports may be, for example, approximately 250 mg / mL, approximately 200 mg / mL, approximately 150 mg / mL, approximately 100 mg / mL, or approximately 50 mg / mL of particles.Because effector functions, such as cytokine release and growth, may have different requirements for signal 1 versus signal 2 than T cell activation and differentiation, these functions can be determined separately.

[0105] In certain embodiments, aAPCs are paramagnetic particles in the 50 to 150 nm range, with a PDI (size distribution) less than 0.2 or in some embodiments less than 0.1. aAPCs can have a surface charge of 0 to -10 mV, as well as about -2 to -6 mV. aAPCs can have from 10 to 120 ligands per particle, as well as from about 25 to about 100 ligands per particle, with ligands conjugated to the particle through a free cysteine ​​introduced into the Fc region of the immunoglobulin sequences. The particles may contain a ratio of about 1:1 of HLA:anti-CD28 dimer, which may be present in the same or different particle populations. Nanoparticles provide expansion Petition 870260070676, dated 07 / 16 / 2026, page 46 / 134 41 / 52 potent cognathic T cells, although they do not exhibit stimulation of non-cognathic TCRs, even with passive loading of the peptide antigen. The particles are stable in lyophilized form for at least two or three years.

[0106] After enrichment and expansion, the antigen-specific T cell component of the sample will be at least about 5% or at least about 10% or at least about 15% or at least about 20% or at least about 25% of the antigen-specific T cells. In addition, these T cells usually exhibit a memory phenotype (including central and effector memory, as well as memory T stem cells). From the original patient-isolated sample, antigen-specific T cells in various modalities are expanded (in about 7 days) from about 100-fold to about 10,000-fold, such as at least about 100-fold or at least about 200-fold. After 2 weeks, antigen-specific T cells are expanded at least 1,000 times, or at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, or at least about 5,000 times in various modalities.In some modalities, antigen-specific T cells are expanded more than 5,000-fold or more than 10,000-fold after two weeks. After one or two weeks of expansion, at least about 10⁶, or at least about 10⁷, or at least about 10⁸, or at least about 10⁹ antigen-specific T cells are obtained.

[0107] Suitable incubation conditions (culture medium, temperature, etc.) include those used to culture T cells or T cell precursors, as well as those known in the art for inducing the formation of antigen-specific T cells using DC or artificial antigen-presenting cells.

[0108] Cellular composition can be administered to patients Petition 870260070676, dated 07 / 16 / 2026, page 47 / 134 42 / 52 by any appropriate routes, including intravenous infusion, intra-arterial administration, intralymphatic administration, and intratumoral administration.

[0109] In some modalities, the patient receives immunotherapy with one or more checkpoint inhibitors before (or optionally after) receiving adoptive cell transfer. In several modalities, the checkpoint inhibitor(s) target one or more CTLA-4 or PD-1 / PD-L1, which may include antibodies against such targets, such as monoclonal antibodies or portions thereof, or humanized or fully human versions thereof. In some modalities, checkpoint inhibitor therapy comprises ipilimumab or Keytruda (pembrolizumab).

[0110] In some modalities, the patient receives approximately 1 to 5 rounds of adoptive immunotherapy (e.g., one, two, three, four, or five rounds). In some modalities, each administration of adoptive immunotherapy is given concurrently with, or after (e.g., approximately 1 day to approximately 1 week after), a round of checkpoint inhibitor therapy. In some modalities, adoptive immunotherapy is given approximately 1 day, approximately 2 days, approximately 3 days, approximately 4 days, approximately 5 days, approximately 6 days, or approximately one week after a dose of checkpoint inhibitor. In some modalities, the patient receives only a single administration of the cell composition.

[0111] In some respects, the invention provides methods for personalized immunotherapy against cancer. The methods are performed using aAPCs to identify the antigens to which the patient will respond, followed by administration of the aAPC loaded with the appropriate peptide for the patient, or followed by enrichment and expansion of antigen-specific T cells ex vivo.

[0112] Whole genome sequencing has dramatically changed Petition 870260070676, dated 07 / 16 / 2026, page 48 / 134 43 / 52 our understanding of cancer biology. Cancer sequencing has yielded important data on the molecular processes involved in the development of many human cancers. Driver mutations have been identified in key genes involved in pathways that regulate three major cellular processes: (1) cell fate, (2) cell survival, and (3) genome maintenance. Vogelstein et al., Science 339, 1546-58 (2013).

[0113] Whole genome sequencing also has the potential to revolutionize our approach to cancer immunotherapy. Sequencing data can provide insights into both shared and personalized targets for cancer immunotherapy. In principle, mutant proteins are foreign to the immune system and are putative tumor-specific antigens. In fact, sequencing efforts have defined hundreds, if not thousands, of potentially relevant immune targets. Limited studies have shown that T-cell responses against these neo-epitopes can be found in cancer patients or induced by cancer vaccines. However, the frequency of such responses against a specific cancer and the extent to which these responses are shared among patients are not well known.One of the main reasons for our limited understanding of tumor-specific immune responses is that current approaches to validating potentially immunologically relevant targets are cumbersome and time-consuming.

[0114] Although central tolerance cancels out cell responses T cell-versus-autoproteins, oncogenic mutations induce neo-epitopes against which T cell responses can form. Catalogs of mutations derived from whole-exome sequencing provide a starting point for identifying these neo-epitopes. Using HLA-binding prediction algorithms Petition 870260070676, dated 07 / 16 / 2026, page 49 / 134 44 / 52 (Srivastava, PLoS One 4, e6094 (2009)), it was predicted that each cancer may have up to 7 to 10 neo-epitopes. A similar approach estimated hundreds of tumor neo-epitopes. Such algorithms, however, may have low accuracy in predicting T cell responses, and only 10% of the predicted HLA-binding epitopes are expected to bind in the context of HLA (Lundegaard C, Immunology 130, 309-18 (2010)). Thus, the predicted epitopes must be validated regarding the existence of T cell responses against these potential neo-epitopes.

[0115] In certain modalities, the nano-aAPC system is used to screen for neo-epitopes that induce a T-cell response in a variety of cancers or in a particular patient's cancer. Cancers can be genetically analyzed, for example, by whole-exome sequencing.

[0116] A list of candidate peptides can be generated from the overlap of nine amino acid windows in mutated proteins. All nine AA windows containing a mutated amino acid and 2 non-mutated controls from each protein will be selected. These candidate peptides will be computationally evaluated for MHC binding using a consensus of MHC binding prediction algorithms, including Net MHC and the stabilized matrix method (SMM). The Nano-aAPC and MHC binding algorithms were developed primarily for the HLA-A2 allele. The sensitivity cutoff of the consensus prediction can be adjusted until a tractable number of mutation-containing peptides (~500) and non-mutated control peptides (~50) are identified.

[0117] In exemplary embodiments, the cell composition comprises, in a pharmaceutically acceptable carrier: at least 90% CD8+ T cells and less than 5% CD4+ T cells; at least 106 CD8+ T cells specific for 1 to 10 tumor-associated target peptide antigens and CD8+ T cells specific for Petition 870260070676, dated 07 / 16 / 2026, page 50 / 134 45 / 52 bacterial, viral, and / or fungal pathogens, in which at least 30% of CD8+ T cells are central memory and effector memory T cells with a ratio of 25:75 to 75:25, with less than 10% of CD8+ T cells being terminally differentiated T cells. In some embodiments, at least 50% of CD8+ T cells specific for tumor-associated target peptide antigens are central memory and effector memory T cells with a ratio of 25:75 to 75:25, and with less than 10% of CD8+ T cells being terminally differentiated T cells. In some embodiments, the cellular composition further comprises approximately 5% to approximately 20% memory T stem cells (Tscm), or approximately 5% to approximately 15% memory T stem cells.

[0118] The cell composition further comprises a pharmaceutically acceptable carrier suitable for intravenous infusion and which may be suitable as a cryoprotectant. In the example of a carrier, it is DMSO (e.g., about 10%). Cell compositions may be supplied in unit vials or bags and stored frozen until use. Unit doses may comprise from about 5 x 10⁵ to about 5 x 10⁶ cells per mL, in a volume of 50 to 200 mL. In certain embodiments, the volume of the composition is <100 mL (e.g., from 50 to 100 mL).

[0119] In some respects, the invention provides a method for treating a cancer patient, comprising administering the cellular composition described herein to a patient in need.

[0120] In some modalities, the patient has a hematologic cancer, which in some modalities has recurred after allogeneic stem cell transplantation. In some modalities, the patient has acute myeloid leukemia (AML) or myelodysplastic syndrome.

[0121] Other cancers that can be treated according to this Petition 870260070676, dated 07 / 16 / 2026, page 51 / 134 46 / 52 descriptions include cancers that historically elicit poor immune responses or have a high recurrence rate. Examples of cancer include various types of solid tumors, including carcinomas, sarcomas, and lymphomas. In various modalities, the cancer is melanoma (including metastatic melanoma), colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, testicular cancer, stomach cancer, oral mucosal dysplastic, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma. In various modalities, the cancer is stage I, stage II, stage III, or stage IV. In some modalities, the cancer is metastatic and / or recurrent and / or unresectable.

[0122] In some modalities, the patient is refractory to chemotherapy and / or checkpoint inhibitor therapy.

[0123] In some modalities, the patient also receives low-dose cytokine therapy, which can improve persistence and in vivo response.

[0124] In some modalities, the cancer is a hematologic neoplasm, including leukemia, lymphoma, or myeloma. For example, hematologic malignancy may be acute myeloid leukemia, chronic myeloid leukemia, acute childhood leukemia, non-Hodgkin lymphomas, acute lymphocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and cutaneous T-cell-rich lymphoid hyperplasia. In an exemplary modality, the patient has a hematologic cancer, such as acute myeloid leukemia (AML) or myelodysplastic syndrome, and in some Petition 870260070676, dated 07 / 16 / 2026, page 52 / 134 In 47 / 52 modalities, the patient relapsed after allogeneic stem cell transplantation. In some modalities, the therapy does not induce GVHD.

[0125] In some modalities, in addition to allogeneic stem cell transplantation, the patient also undergoes lymphoma deletion therapy, cytoreductive therapy, or immunomodulatory therapy (before the administration of cell therapy). In some modalities, cell therapy may also be provided with or without cytokine support after treatment.

[0126] In some modalities, the patient has an infectious disease or is at risk of an infectious disease. For example, patients who have undergone HSCT are at particular risk of infectious disease, given their immunocompromised state. Infectious diseases that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include AIDS, hepatitis B / C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpes virus infection (including herpes zoster), and adenovirus infection. CMV, for example, is the most common viral pathogen found in transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation.This is due to the immunocompromised state of these patients, which allows for the reactivation of the latent virus in seropositive patients or opportunistic infection in seronegative individuals. In these modalities, the patient may receive adoptive immunotherapy comprising T cells specific for pathogen antigens. The method may lead to the generation of virus-specific CTLs derived from the patient or from an appropriate donor before the start of the transplant procedure.

[0127] PTLD occurs in a significant fraction of transplant patients and results from infection with the Epstein-Barr virus (EBV). Petition 870260070676, dated 07 / 16 / 2026, page 53 / 134 48 / 52 EBV infection is believed to be present in approximately 90% of the adult population in the United States. Active viral replication and infection are controlled by the immune system, but, as in cases of CMV, individuals immunocompromised by transplant therapies lose the controlling T-cell populations, allowing viral reactivation. This represents a serious impediment to transplant protocols. EBV may also be involved in tumor promotion in a variety of hematologic and non-hematologic cancers.

[0128] Other aspects and embodiments of the invention will be apparent to those skilled in the art. EXAMPLES

[0129] Antigen-specific T cells were enriched and expanded from donor cells isolated by leukapheresis. The cells were depleted of CD4+ cells by negative selection with CD4 microspheres. The resulting cells were enriched for antigen-specific T cells by incubation with paramagnetic nanoparticles (dextran-coated iron oxide nanoparticles, approximately 80–200 nm in diameter). The nanoparticles have surface-conjugated dimeric HLA ligands (presenting the target peptide antigen) as well as an agonistic anti-CD28 monoclonal antibody. The dimeric HLA ligand contains two HLA-A2 domains, comprising the peptide-binding clefts, each fused with an arm of the Ig hinge region. The dimeric HLA-A2 ligands are co-expressed with β2-microglobulin. The aAPC linkers and constructs are disclosed in documents WO 2016 / 044530 and WO 2016 / 105542, which are incorporated herein by reference in their entirety.

[0130] The cells were incubated in the presence of paramagnetic aAPC, then in the presence of a magnetic field for about Petition 870260070676, dated 07 / 16 / 2026, page 54 / 134 49 / 52 of 5 minutes. The cells associated with the particles were then recovered and expanded ex vivo for various time periods (usually 1 to 2 weeks). Expansion was performed in the presence of growth factors. During a two-week culture period, growth factors were added on days 1 and 7. The cells were re-stimulated with aAPCs on day 7.

[0131] Antigen-specific T cells were also batch-enriched and expanded. For example, FIGURE 3 shows batch-enriched and expanded peptides specific for AML Prame100 RHAMM, WT1, and Survivin. On day 7, the cells contain 1.4% specific for Prame, 1.8% specific for RHAMM, 7.0% specific for WT1, and 2.3% specific for Survivin. The total antigen-specific T cell component is 12.5% ​​in this modality. T cells were characterized by tetramer staining.

[0132] FIGURE 4 shows that the composition with individual stimulation and expansion for two weeks had consistent levels of antigen-specific T cells from AML. The individual stimulation and expansion process consistently generates ~15% antigen-specific T cells.

[0133] FIGURE 5 shows that the simultaneous stimulation / expansion process generates AML-specific T cell frequencies comparable to individual stimulation / expansion. The composition shown prepared by batch stimulation / expansion has ~47% antigen-specific T cells.

[0134] FIGURE 6 shows that the generated T cells demonstrate antigen-specific killing of AML tumor cells (THP-1 cell line). AML-specific T cells are targeted to 5 epitopes of WT-1, PRAME, and Survivin. From 1 to 100 (target-to-effector ratio), ~40% of the target cells were killed.

[0135] As shown in FIGURE 7 the cytokine cocktail used Petition 870260070676, dated 07 / 16 / 2026, page 55 / 134 50 / 52 for ex vivo expansion can impact the number and phenotype of the resulting cells.

[0136] The cells were further characterized by their phenotype: naive (CD62L+, CD45RA+), central memory (CD62L+, CD45RA-), effector memory (CD62L-, CD45RA-), and terminally differentiated memory (CD62L-, CD45RA+). MART1- and AML-specific T cells enriched and expanded ex vivo from donor lymphocytes are predominantly central memory and effector memory phenotypes. See FIGURE 2. Particularly for AML peptides, in three representative experiments, pure cells were present in 3.82%, 14.2%, and 14.8%. Terminally differentiated memory cells were present in 3.82%, 3%, and 6.7%. Meanwhile, the central memory and effector component of antigen-specific cells was 92.3%, 82.8%, and 78.52%.

[0137] The cells were characterized by activation phenotype, namely, staining for IL-2 (proliferation and memory), IFN-γ (activating other T cells, memory, MHC upregulation), TNF-α (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity). See FIGURE 1. As shown, most cells have 3 or even 4 functions. For example, 32.5% of the cells produce IL-2 and IFN-γ after activation, and 94.2% of the cells produce TNF-α and CD107a after activation.

[0138] Viral antigen-specific spectator cells were further quantified by tetramer staining. FIGURE 8 shows the presence of virus-specific spectator T cells on day 7 after MART-1-specific enrichment and expansion. FIGURE 9 shows the presence of virus-specific spectator T cells on day 14 after MART-1-specific enrichment and expansion. These cells are also largely of central memory and effector phenotype. FIGURE 10 shows the presence Petition 870260070676, dated 07 / 16 / 2026, page 56 / 134 Figure 11 shows the detection of CMV-specific spectator T cells during the MART-1-specific enrichment and expansion process. The percentage of virus-specific spectator T cells remains constant until Day 14 (between 0.5 and 1%), while the number and percentage of MART-1-specific T cells increase dramatically.

[0139] FIGURE 12 shows the detection of virus-specific spectator cells on Day 14 after specific enrichment and expansion of MART-1 using a recombinant T-cell growth factor cocktail (IL-Iβ, IL-2, IL-4, IL-6, IL-21, IFN-γ and MIPI-β), demonstrating maintenance and expansion of virus-specific spectator T cells targeting multiple epitopes in addition to Adeno, CMV, EBV and influenza.

[0140] As shown in Figure 13A and Figure 13B, Mart-1 specific T cells were generated by the enrichment and expansion process, in the presence of the following cytokines during expansion: IL-2, IL-4, IL-6, IFN-γ and IL-β. The composition of this cytokine cocktail is shown in Table 1. Table 1: Cytokine cocktail for the expansion phase Cytokines Specific activity in final culture media (IU / mL) Specific activity in 50X stock solution (IU / mL) IL-2 40 2000 IL-4 2.5 125 IL-6 50 2500 IFNY 40 2000 IL-Iβ 15 750

[0141] In this experiment, 6.74 x 109 CD8+ lymphocytes from a donor Petition 870260070676, dated 07 / 16 / 2026, page 57 / 134 52 / 52 healthy cells were enriched as previously described. After enrichment, there were 2.81 x 10⁸ cells in total. On day 14 of expansion, there were 5.28 x 10⁸ total cells, showing a 1.88-fold expansion of the total cells. These expanded cells on day 14 were ~35% specific for MART-1 and approximately 94% viable (Figure 13B). The MART-1 specific cells were expanded approximately 2776 times, assuming that about 1 in 10⁵ precursor cells were MART-1 specific.

[0142] In the total culture assessment, T cells exhibited a phenotype of approximately 66% central memory and approximately 32% effector memory. Less than 2% of the cells were pure, and the amount of TEMRA cells was insignificant. Furthermore, MART-1-specific cells had approximately 89% central memory and approximately 9% effector memory, with less than 2% pure cells and a negligible number of TEMRA cells. Petition 870260070676, dated 07 / 16 / 2026, page 58 / 134

Claims

1 / 4 CLAIMS 1. Isolated cell composition comprising T cells that are activated and expanded ex vivo from a lymphocyte source and suitable for adoptive immunotherapy, characterized in that it comprises, in a pharmaceutically acceptable carrier: at least 108 CD8+ T cells specific for one or more target peptide antigens, wherein at least 10% of CD8+ T cells in the composition are specific for a target peptide antigen, and wherein at least 50% of T cells in the composition exhibit a core memory or effector memory phenotype, and the composition comprises T cells exhibiting a memory T stem cell phenotype; and wherein the composition does not comprise T cells expressing a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR);wherein the composition is produced ex vivo by enriching CD8+ T cells specific for the target peptide antigens from lymphocytes of positive selection origin with a population of paramagnetic artificial antigen-presenting cells (aAPCs) possessing a co-stimulatory ligand and an MHC class I ligand presenting the target peptide antigens, and by expanding the CD8+ T cells specific for the target peptide antigens in the presence of the aAPC population and IL-2, IL-4, IL-6, IFN-γ and IL-1β.

2. Isolated cell composition according to claim 1, characterized in that the CD8+ T cells are specific for 1 to 10 target peptide antigens.

3. Isolated cell composition, according to claim 1 or 2, characterized in that the target peptide antigens are tumor-associated antigens, or in which one or more target peptide antigens are bacterial, viral, fungal or parasitic antigens. Petition 870260070676, dated 07 / 16 / 2026, page 59 / 134 2 / 4 4. Isolated cell composition, according to any one of claims 1 to 3, characterized in that it comprises CD8+ T cells specific for at least one, two, three, four or five target peptide antigens.

5. Isolated cell composition, according to any one of claims 1 to 4, characterized in that the cell composition is at least 90% T cells.

6. Isolated cell composition, according to any one of claims 1 to 4, characterized in that the cell composition comprises at least 15% of CD8+ T cells specific for the target peptide antigens.

7. Isolated cell composition, according to claim 3, characterized in that the cell composition further comprises CD8+ T cells specific for bacterial, viral and / or fungal pathogens, or optionally in that the CD8+ T cells specific for bacterial, viral or fungal pathogens include T cells specific for influenza, CMV, EBV and / or adenovirus antigens.

8. Isolated cell composition according to any one of claims 1 to 7, characterized in that the T cells are at least 70% central memory and effector T cells, or the T cells are at least 80% central memory and effector T cells.

9. Isolated cell composition, according to claim 8, characterized in that the T cells specific for one or more target antigens are at least 50% central and effector memory T cells, or the T cells specific for one or more target antigens are at least 60% central and effector memory T cells, or the T cells specific for one or more target antigens are at least 70% central and effector memory T cells, or the T cells specific for one or more target antigens are at least 80% central and effector memory T cells.

10. Isolated cell composition, according to any one of claims 1 to 9, characterized in that the core memory and effector T cells are 10:90 to 90:10 core memory to effector cells, or the core memory and effector T cells are 25:75 to 75:25 core memory to effector cells, or the core cells and effector memory cells are 40:60 to 60:40 core memory to effector T cells.

11. A composition of isolated cells, according to any one of claims 1 to 10, characterized in that the T cells are less than 10% terminally differentiated, and less than 10% naive cells.

12. Isolated cell composition, according to any one of claims 1 to 11, characterized in that it comprises from 5% to 25% of T memory stem cells.

13. Isolated cell composition, according to any one of claims 1 to 12, characterized in that the cell composition is less than 10% CD4+ T cells.

14. Isolated cell composition, according to any one of claims 1 to 13, characterized in that the aAPCs comprise a costimulatory ligand that is a CD28-binding ligand.

15. Isolated cell composition, according to any one of claims 1 to 14, characterized in that the enrichment is magnetic enrichment with paramagnetic aAPCs and in which the cells and aAPCs are incubated in the presence of a magnetic field for at least one minute.

16. Isolated cell composition, according to Petition 870260070676, dated 07 / 16 / 2026, page 61 / 134 4 / 4 claim 15, characterized in that antigen-specific T cells are expanded without the use of a magnetic field.

17. Isolated cell composition, according to any one of claims 1 to 16, characterized in that one or more target peptide antigens are selected from peptide epitopes of Survivin, WT-1, PRAME, Cyclin A1 and PR3.

18. Use of isolated cell composition, as defined in any one of claims 1 to 17, characterized in that it is for the preparation of an adoptive immunotherapy to treat cancer in a patient in need thereof, wherein the target peptide antigens are tumor-associated antigens. Petition 870260070676, dated 07 / 16 / 2026, p. 62 / 134

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