US20210008113A1 - Methods of making and using guidance and navigation control proteins - Google Patents

Methods of making and using guidance and navigation control proteins Download PDF

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Publication number
US20210008113A1
US20210008113A1 US17/040,519 US201917040519A US2021008113A1 US 20210008113 A1 US20210008113 A1 US 20210008113A1 US 201917040519 A US201917040519 A US 201917040519A US 2021008113 A1 US2021008113 A1 US 2021008113A1
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United States
Prior art keywords
cell
cells
cytotoxic
therapeutic
gnc
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US17/040,519
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Inventor
Yi Zhu
Ole Olsen
Jahan Khalili
Dong Xia
David JELLYMAN
Katrina BYKOVA
Anne-Marie ROUSSEAU
Camilla WANG
Zeren Gao
Hui Huang
Steven K. LUNDY
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Systimmune Inc
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Sichuan Baili Pharmaceutical Co Ltd
Systimmune Inc
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Assigned to SYSTIMMUNE, INC., SICHUAN BAILI PHARMACEUTICAL CO., LTD. reassignment SYSTIMMUNE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BYKOVA, Katrina, GAO, ZEREN, HUANG, HUI, JELLYMAN, David, KHALILI, JAHAN, LUNDY, Steven K., OLSEN, OLE, ROUSSEAU, ANNE-MARIE, WANG, Camilla, XIA, DONG, ZHU, YI
Publication of US20210008113A1 publication Critical patent/US20210008113A1/en
Assigned to SYSTIMMUNE INC., Baili-Bio (Chengdu) Pharmaceutical Co., Ltd. reassignment SYSTIMMUNE INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SYSTIMMUNE, INC., SICHUAN BAILI PHARMACEUTICAL CO. LTD.
Assigned to SYSTIMMUNE, INC., Baili-Bio (Chengdu) Pharmaceutical Co., Ltd. reassignment SYSTIMMUNE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: Baili-Bio (Chengdu) Pharmaceutical Co., Ltd., SYSTIMMUNE INC.
Assigned to SYSTIMMUNE, INC. reassignment SYSTIMMUNE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: Baili-Bio (Chengdu) Pharmaceutical Co., Ltd., SYSTIMMUNE, INC.
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Definitions

  • the present application generally relates to the technical field of Guidance and Navigation Control (GNC) proteins with multi-specific binding activities against surface molecules on both immune cells and tumor cells, and more particularly relates to making and using GNC proteins.
  • GNC Guidance and Navigation Control
  • Cancer cells develop various strategies to evade the immune system.
  • One of the underlying mechanisms for the immune escape is the reduced recognition of cancer cells by the immune system. Defective presentation of cancer specific antigens or lack of thereof results in immune tolerance and cancer progression. In the presence of effective immune recognition tumors use other mechanisms to avoid elimination by the immune system.
  • Immunocompetent tumors create suppressive microenvironments to downregulate the immune response. Multiple players are involved in shaping the suppressive tumor microenvironment, including tumor cells, regulatory T cells, Myeloid-Derived Suppressor cells, stromal cells, and other cell types.
  • the suppression of immune response can be executed in a cell contact-dependent format as well as in a contact-independent manner, via secretion of immunosuppressive cytokines or elimination of essential survival factors from the local environment.
  • Cell contact-dependent suppression relies on molecules expressed on the cell surface, e.g. Programmed Death Ligand 1 (PD-L1), T-lymphocyte-associated protein 4 (CTLA-4) and others (Dunn, Old et al. 2004, Adachi and Tamada 2015).
  • Yervoy binds to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressed on activated T cells and blocks the interaction of CTLA-4 with CD80/86 on antigen-presenting cells thereby blocking the negative or inhibitory signal delivered into the T cell through CTLA-4 resulting in re-activation of the antigen-specific T cell leading to, in many patients, eradication of the tumor.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • bi-specific antibody where the binding domain of an antibody which is specific for a tumor associated antigen, e.g., CD19, is linked to an antibody binding domain specific for CD3 on T cells thus creating a bi-specific T cell engager or BiTe molecule.
  • the FDA approved a bi-specific antibody called Blinatumumab for the treatment of Precursor B-Cell Acute Lymphoblastic Leukemia.
  • Blinatumumab links the single-chain variable fragment (scFv) specific for CD19 expressed on leukemic cells with the scFv specific for CD3 expressed on T cells (Benjamin and Stein 2016).
  • scFv single-chain variable fragment
  • T cells T cells
  • CAR-T chimeric antigen receptor T cells
  • the clinical success of CAR-T therapy has revealed durable complete remissions and prolonged survival of patients with CD19-positive treatment-refractory B cell malignancies (Gill and June 2015).
  • CRS Cytokine release syndrome
  • cytokine storm is considered as the major adverse effect after the infusion of engineered CAR-T cells (Bonifant, Jackson et al. 2016).
  • the onset and severity of CRS seems to be personally specific to the patient.
  • Current options of mitigating CRS are mainly focused on rapid response and management care because the option of controlling CRS prior to T cell infusion is limited.
  • the application provides, among others, methods for generating therapeutic compositions containing a guidance and navigation (GNC) proteins, methods for treating cancer conditions using a guidance and navigation control (GNC) proteins, and therapeutic compositions containing GNC proteins or therapeutic cells having cytotoxic cells coated (or bound) with GNC proteins.
  • GNC guidance and navigation
  • GNC guidance and navigation control
  • the application provides therapeutic compositions.
  • the therapeutic composition comprises a cytotoxic cell, a GNC protein, and a therapeutic cell.
  • the therapeutic cell comprises the GNC protein bound to the cytotoxic cell through the binding interaction with the cytotoxic cell receptor, and the therapeutic cell composition is substantially free exogenous of viral and non-viral DNA and RNA.
  • the therapeutic composition may further comprise a second GNC protein, a second therapeutic cell, or a combination thereof, wherein the second therapeutic cell comprises the cytotoxic cells with the second GNC protein bound thereupon or with both the first and the second GNC proteins bound thereupon.
  • GNC protein includes a cytotoxic binding moiety and a cancer targeting moiety.
  • the cytotoxic binding moiety has a binding specificity to a cytotoxic cell receptor and is configured to activate the cytotoxic cell through the binding with the cytotoxic cell receptor.
  • the cancer targeting moiety has a binding specificity to a cancer cell receptor.
  • the GNC protein includes a binding domain for T-cell receptors.
  • T-cell receptor include without limitation CD3, CD28, PDL1, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, NKp30, CD28H, CD27, CD226, CD96, CD112R, A2AR, CD160, CD244, CECAM1, CD200R, TNFRSF25 (DR3), or a combination thereof.
  • the GNC protein is capable of activating a T-cell by binding the T-cell binding moiety to a T-cell receptor on the T-cell.
  • the GNC protein is capable of activating a T-cell by binding multiple T-cell binding moieties on the T-cell.
  • the GNC protein includes a binding domain for a NK cell receptor.
  • NK cell receptor include, without limitation, receptors for activation of NK cell such as CD16, NKG2D, KIR2DS1, KIR2DS2, KIR2DS4, KIR3DS1, NKG2C, NKG2E, NKG2H; agonist receptors such as NKp30a, NKp30b, NKp46, NKp80, DNAM-1, CD96, CD160, 4-1BB, GITR, CD27, OX-40, CRTAM; and antagonist receptors such as KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, NKG2A, NKp30c, TIGIT, SIGLEC7, SIGLEC9, LILR, LAIR-1, KLRG1, PD-1, CTLA-4, CD161.
  • the GNC protein includes a binding domain for a macrophage receptor.
  • macrophage receptor include, without limitation, agonist receptor on macrophage such as TLR2, TLR4, CD16, CD64, CD40, CD80, CD86, TREM-1, TREM-2, ILT-1, ILT-6a, ILT-7, ILT-8, EMR2, Dectin-1, CD69; antagonist receptors such as CD32b, SIRPa, LAIR-1, VISTA, TIM-3, CD200R, CD300a, CD300f, SIGLEC1, SIGLEC3, SIGLEC5, SIGLEC7, SIGLEC9, ILT-2, ILT-3, ILT-4, ILT-5, LILRB3, LILRB4, DCIR; and other surface receptors such as CSF-1R, LOX-1, CCR2, FRP, CD163, CR3, DC-SIGN, CD206, SR-A, CD36, MARCO.
  • the GNC protein includes a binding domain for a dendritic cell receptor.
  • dendritic cell receptor include, without limitation, agonist receptors on dendritic cell such as TLR, CD16, CD64, CD40, CD80, CD86, HVEM, CD70; antagonist receptors such as VISTA, TIM-3, LAG-3, BTLA; and other surface receptors such as CSF-1R, LOX-1, CCR7, DC-SIGN, GM-CSF-R, IL-4R, IL-10R, CD36, CD206, DCIR, RIG-1, CLEC9A, CXCR4.
  • the GNC protein may include a T-cell binding moiety and a cancer-targeting moiety.
  • the T-cell binding moiety has a binding specificity to a T-cell receptor comprising CD3, CD28, PDL1, PDL2, PD1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, CD30, CD27, or a combination thereof.
  • the cancer targeting moiety has a binding specificity to a cancer cell receptor.
  • the cancer cell receptor may include BCMA, CD19, CD20, CD33, CD123, CD22, CD30, ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, as yet to be discovered tumor associated antigens or a combination thereof.
  • the GNC protein may have multi-specific antigen binding activities to the surface molecules of a T cell and a tumour cell.
  • the guidance and navigation control (GNC) protein comprises a binding domain for a T cell activating receptor, a binding domain for a tumor associated antigen, a bind domain for an immune checkpoint receptor, and a binding domain for a T cell co-stimulating receptor.
  • the binding domain for the tumor associated antigen is not adjacent to the binding domain for the T cell co-stimulating receptor.
  • the binding domain for the T cell activating receptor is adjacent to the binding domain for the tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the T cell activating receptor may include without limitation CD3.
  • the T cell co-stimulating receptor may include without limitation 4-1BB, CD28, OX40, GITR, CD40L, ICOS, Light, CD27, CD30, or a combination thereof.
  • the immune checkpoint receptor may include without limitation PD-L1, PD-1, TIGIT, TIM-3, LAG-3, CTLA4, BTLA, VISTA, PDL2, or a combination thereof.
  • the tumor associated antigen may include without limitation ROR1, CD19, EGFRVIII, BCMA, CD20, CD33, CD123, CD22, CD30, CEA, HER2, EGFR, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, or a combination thereof.
  • the tumor associated antigen may be ROR1.
  • the tumor associated antigen may be CD19.
  • the tumor associated antigen may be EGFRVIII.
  • the guidance and navigation control (GNC) protein may be an antibody or an antibody monomer or a fragment thereof.
  • the GNC protein may be a tri-specific antibody.
  • the GNC protein may be a tetra-specific antibody.
  • the GNC protein includes Fc domain or a fragment thereof. Any Fc domain from an antibody may be used.
  • Example Fc domains may include Fc domains from IgG, IgA, IgD, IgM, IgE, or a fragment or a combination thereof.
  • Fc domain may be natural or engineered.
  • the Fc domain may contain an antigen binding site.
  • the GNC protein comprises a bi-specific antibody, a tri-specific antibody, a tetra-specific antibody, or a combination thereof yielding up to eight binding motifs on the GNC protein.
  • GNC proteins may include an immunoglobulin G (IgG) moiety with two heavy chains and two light chains, and at least two scFv moieties being covalently connected to either C or N terminals of the heavy or light chains.
  • the IgG moiety may provide stability to the scFv moiety, and a tri-specific GNC protein may have two moieties for binding the surface molecules on T cells.
  • the guidance and navigation control (GNC) protein may be an antibody.
  • the tumor associated antigen comprises ROR1, CD19, or EGRFVIII.
  • the T cell activating receptor comprises CD3 and the binding domain for CD3 may be linked to the binding domain for the tumor associated (TAA) antigen through a linker to form a CD3-TAA pair.
  • the IgG Fc domain may intermediate the CD3-TAA pair and the binding domain for the immune checkpoint receptor.
  • the immune checkpoint receptor may be PD-L1.
  • the linker may be a covalent bond or a peptide linker.
  • the peptide linker may have from about 2 to about 100 amino acid residues.
  • the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for CD3, the binding domain for EGFRVI, IgG Fc domain, the bind domain for PD-L1, and the binding domain for 41-BB.
  • the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for 4-1BB, the binding domain for PD-L1, IgG Fc domain, the bind domain for ROR1, and the binding domain for CD3.
  • the guidance and navigation control (GNC) protein has a N-terminal and a C-terminal, comprising in tandem from the N-terminal to the C-terminal, the binding domain for CD3, the binding domain for CD19, IgG Fc domain, the bind domain for PD-L1, and the binding domain for 4-1BB.
  • the GNC protein comprises an amino acid having a percentage homology to SEQ ID NO. 50, 52, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110.
  • the percentage homology is not less than 70%. 80%, 90%, 95%, 98% or 99%.
  • the application provides nucleic acid sequences encoding the GNC protein or its fragments disclosed thereof.
  • the nucleic acid has a percentage homology to SEQ ID NO. 49, 51, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109.
  • the percentage homology is not less than 70%. 80%, 90%, 95%, 98% or 99%.
  • the application provides methods for generating a therapeutic composition.
  • the method may include the steps of providing a cell material comprising a cytotoxic cell, incubating the cell material with a first GNC protein to provide an activated cell composition, and formulating the activated cell composition to provide a therapeutic composition.
  • the activated cell composition contains a first therapeutic cell.
  • the first therapeutic cell comprises the first GNC protein bound to the cytotoxic cell through the binding interaction with the first cytotoxic cell receptor.
  • the therapeutic composition is substantially free of exogenous viral and non-viral DNA or RNA.
  • the cell material may include or be derived from PBMC.
  • the first GNC protein may include a first cytotoxic binding moiety and a first cancer targeting moiety.
  • the first cytotoxic binding moiety has a specificity to a first cytotoxic cell receptor and is configured to activate the first cytotoxic cell through the binding with the first cytotoxic cell receptor.
  • the first cancer targeting moiety has a specificity to a first cancer cell receptor.
  • the method may repeat the incubating step by incubating a second GNC protein with the activated cell composition.
  • the second GNC protein comprising a second cytotoxic binding moiety and a second cancer targeting moiety, the second cytotoxic binding moiety has a specificity to a second cytotoxic cell receptor, and the second cancer targeting moiety has a specificity to a second cancer cell receptor.
  • the activated cell composition comprises a second therapeutic cell, and the second therapeutic cell comprises the second GNC protein bound to the cytotoxic cell or the first therapeutic cell through the binding interaction with the second cytotoxic cell receptor.
  • the first and the second cancer-targeting moiety independently has a specificity for CD19, PDL1, or a combination thereof. In one embodiment, the first and the second cytotoxic binding moiety independently has a specificity for CD3, PDL1, 41BB, or a combination thereof.
  • the method may further include the repeated incubating steps by incubating additional GNC proteins with the activated composition.
  • the additional GNC proteins may be a third GNC protein, a fourth GNC protein, etc. to provide addition therapeutic cells, each having the additional protein bound to the cytotoxic cell.
  • the first, second, and the additional GNC protein may be the same or may be different.
  • the therapeutic cells may have one GNC protein, multiple same GNC proteins, or multiple different GNC proteins bound thereupon. In one embodiment, the therapeutic cell may have the first GNC protein bound thereupon. In one embodiment, the therapeutic cell may have both the first and the second GNC proteins bound thereupon. In one embodiment, the therapeutic cell may have the first, the second and the additional GNC proteins bound thereupon.
  • the therapeutic cell comprises the cytotoxic cell having at least one bound GNC protein. In one embodiment, the therapeutic cell comprises the cytotoxic cell having at least 10, 20, 50, 100, 200, 300, 400 bound GNC proteins.
  • the therapeutic composition may include the first therapeutic cell, the first GNC protein, the cytotoxic cell, or a combination thereof.
  • the therapeutic composition may include the second therapeutic cell, the second GNC protein, comprises the first therapeutic cell, the first GNC protein, the cytotoxic cell, or a combination thereof.
  • the therapeutic composition may include additional GNC proteins and additional therapeutic cells.
  • the incubating step may serve to expand the therapeutic cells.
  • expanding the therapeutic cell may include incubating the therapeutic cells with an additional amount of the GNC protein to provide an expanded cell population.
  • the expanded cell population comprises at least 10 2 , at least 103, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 cells per ml.
  • the expanded cell population comprises the GNC bound cell, the GNC protein, the cytotoxic cell, or a combination thereof.
  • a GNC protein in order to deplete PD-1+ T cells, a GNC protein may be added to the expansion culture that redirects killing to PD-1+ T cells therefore resulting in reduction in PD-1+ exhausted T cells.
  • a GNC protein in order to preferentially support PD-1+ T cells, a GNC protein may be added to the expansion culture that relieves checkpoint signaling through PD-1 on T cells therefore resulting in functional improvement of PD-1+ T cells.
  • a GNC protein in order to isolate 4-1BB mediated co-stimulation through 3 rd gen CAR-T, a GNC protein may be added to the expansion culture that redirects killing to 4-1BB+ T cells or resulting in therapeutic composition with controlling level of 4-1BB stimulation in the therapeutic cells, such as CAR-T cells.
  • the cancer targeting moiety has the specificity against B cell, and the therapeutic composition is substantially free of B cell. Therefore, the methods disclosed herein couple the activation and purification functions for the therapeutic cells, which allows the methods to produce B cell free therapeutic composition without the need to introduce any foreign materials (such as beads) nor any foreign genetic materials (such as viral and non-viral DNA or RNA vectors).
  • the ratio of the GNC protein and the cytotoxic cell is at least 30 to 1 when incubating the cell material with the GNC protein.
  • the therapeutic composition may include at least 10 7 cells per ml.
  • the application provides methods for using guidance and navigation control (GNC) proteins for cancer treatment.
  • the method of treating a subject having a cancer comprises providing a cytotoxic cell, combining a GNC protein with the cytotoxic cell to provide a therapeutic cell, optionally expanding the therapeutic cell to provide an expanded cell population, and administering the therapeutic cell or the expanded cell population to the subject.
  • GNC guidance and navigation control
  • the method include the step of providing a cell material comprising a cytotoxic cell, incubating the cell material with a first GNC protein to provide an activated cell composition, wherein the activated cell composition comprises a first therapeutic cell, formulating the activated cell composition to provide a therapeutic composition, wherein the therapeutic composition is substantially free exogenous of viral and non-viral DNA or RNA, and administering the therapeutic composition to the subject.
  • the method may further include the steps of incubating a second GNC protein with the activated cell composition to provide the activated cell composition further comprising a second therapeutic cell. In one embodiment, the method may further include the step of incubating additional GNC proteins with the activated cell composition to provide the activated cell composition further comprising additional therapeutic cells.
  • the method may further comprise isolating the cytotoxic cell from peripheral blood mononuclear cells (PBMC) before providing the cytotoxic cell.
  • the method may further comprise isolating the peripheral blood mononuclear cells (PBMC) from a blood.
  • the blood is from the subject.
  • the blood is not from the subject.
  • the cytotoxic cells may be from the patient that is under treatment or a different individual, such as a universal donor.
  • the cytotoxic cell may be an autologous T cell, an alloreactive T cell, or a universal donor T cell.
  • a GNC protein may be added to the expansion culture that redirects killing to tumor antigens, example tumor antigen may include CD19 for B cell malignancies, Epcam for Breast carcinoma, MCP1 for melanoma.
  • the method includes steps of providing a blood from the subject, isolating peripheral blood mononuclear cells (PBMC) from the blood, isolating a cytotoxic cell from the PBMC, combining a GNC protein with the cytotoxic cell to provide a therapeutic cell, optionally expanding the therapeutic cell to provide an expanded cell population, and administering the therapeutic cell or the expanded cell population to the subject.
  • PBMC peripheral blood mononuclear cells
  • the method further comprises administering additional GNC protein to the subject after administering the therapeutic composition to the subject.
  • the cytotoxic cell may include CD3+ T cell, NK cell, or a combination thereof.
  • the isolating of the cytotoxic cell comprises isolating at least one subpopulation of cytotoxic cells to provide the therapeutic T cells.
  • the subpopulation of cytotoxic cells comprises CD4+ cells, CD8+ cells, CD56+ cells, CD69+ cells, CD107a+ cells, CD45RA+ cells, CD45RO+ cells, CD2+ cells, CD178+ cells, Granzyme+ cells, or a combination thereof.
  • the combining of a GNC protein with the cytotoxic cell comprises incubating the GNC protein with the cytotoxic cell for a period of time from about 2 hours to about 14 days, from about 1 day to about 7 days, from about 8 hours to about 24 hours, from about 4 days to about 7 days, or from about 10 days to about 14 days.
  • the incubating period may be more than 14 days. In one embodiment, the incubating period may be less than 2 hours.
  • the ratio between the GNC protein and the cytotoxic cell is at least 600 to 1, 500 to 1, 400 to 1, 300 to 1, 200 to 1, 100 to 1, or 1 to 1. In one embodiment, the ratio between the GNC protein and the cytotoxic cell is from about 1 to 1, 10 to 1, 100 to 1, or to about 1000 to 1 ratio.
  • the method may further comprise evaluating therapeutic efficacy after the administering step.
  • the evaluating therapeutic efficacy includes checking one or more biomarkers of the cancer, monitoring the life span of the therapeutic cells, or a combination thereof.
  • evaluating therapeutic efficacy comprises checking one or more biomarkers of the cancer, monitoring the life span of the therapeutic cells, or a combination thereof.
  • the biomarker comprises a tumor antigen, release of cytokines e.g., gamma interferon, IL-2, IL-8, and/or chemokines, and/or CD markers on the surface of various cell types e.g., CD69, PD-1, TIGIT, and/or mutated nucleic acid released into the bloodstream by tumors upon death, circulating tumor cells and their associated nucleic acid, or exosome associated nucleic acid, host inflammatory mediators, or tumor derived analytes, or a combination thereof.
  • cytokines e.g., gamma interferon, IL-2, IL-8, and/or chemokines
  • CD markers on the surface of various cell types e.g., CD69, PD-1, TIGIT, and/or mutated nucleic acid released into the bloodstream by tumors upon death, circulating tumor cells and their associated nucleic acid, or exosome associated nucleic acid, host inflammatory mediators, or tumor
  • the biomarker comprises a tumor antigen, tumor-associated apoptotic bodies, small molecule metabolites, release of cytokines, lymphocyte surface marker expression, phosphorylated/dephosphorylated signaling molecules, transcription factors, or a combination thereof.
  • the method disclosed herein is free of the step of transfecting the cytotoxic cell with a DNA vector or a viral vector.
  • the therapeutic cell or the expanded cell population is substantially free of a DNA vector or a viral vector.
  • the method may be used to treat a human subject suffering from cancer.
  • the cancer comprises cells expressing ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD20, CD33, CD123, CD22, CD30, CD19, as yet to be identified tumor associated antigens, or a combination thereof.
  • the method may be used to treat mammals.
  • Example cancers includes without limitation breast cancer, colorectal cancer, anal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, head and neck cancer, nasopharyngeal cancer, skin cancer, melanoma, ovarian cancer, prostate cancer, urethral cancer, lung cancer, non-small lung cell cancer, small cell lung cancer, brain tumor, glioma, neuroblastoma, esophageal cancer, gastric cancer, liver cancer, kidney cancer, bladder cancer, cervical cancer, endometrial cancer, thyroid cancer, eye cancer, sarcoma, bone cancer, leukemia, myeloma or lymphoma.
  • the method may further include administering an effective amount of a therapeutic agent after the administering the therapeutic cell or the expanded cell population to the subject.
  • the therapeutic agent comprises a monoclonal antibody, a chemotherapy agent, an enzyme, a protein, a co-stimulator, or a combination thereof.
  • the co-stimulator is configured to increase the amount of cytotoxic T cells in the subject.
  • the application further provides a solution comprising an effective concentration of the GNC protein.
  • the solution is blood plasma in the subject under treatment.
  • the solution includes the GNC protein bound cells.
  • the solution includes a GNC cluster including a GNC protein, a T-cell bound to the T-cell binding moiety of the GNC protein, and a cancer cell is bound to the caner-targeting moiety of the GNC protein.
  • FIG. 1 shows a GNC protein comprising four antigen-specific binding domains in an antibody structure with targeting specificity to CD19 positive cells;
  • FIG. 2 illustrates that a tetra-specific GNC antibody mediates multi-specific binding between a T cell and a tumor cell
  • FIG. 3 is a flowchart comparing manufacturing processes for GNC-T cell therapy (left) and CAR-T cell therapy (right);
  • FIG. 4 is a diagram showing sources of cell material for preparing GNC-activated therapeutic cell composition
  • FIG. 5 is a diagram showing sources of selected T cells for preparing GNC-activated therapeutic composition
  • FIG. 6 is a diagram showing the preparation of GNC-activated therapeutic T cell composition
  • FIG. 7 is a diagram showing the incubating and formulating steps for preparing the first GNC-activated T cells for GNC-T cell therapy
  • FIG. 8 shows that GNC proteins (SI-35E class) induce IL-2 secretion from PBMC;
  • FIG. 9 shows that GNC proteins (SI-35E class) induce granzyme B secretion from PBMC;
  • FIG. 10 shows that GNC proteins (SI-35E class) induce expression of the activation marker CD69 on CD4+ T cells;
  • FIG. 11 shows that GNC proteins (SI-35E class) induce expression of the activation marker CD69 on CD8+ T cells;
  • FIG. 12 shows that GNC proteins (SI-35E class) induce expression of the activation marker CD69 on CD56+NK cells;
  • FIG. 13 shows that GNC proteins (SI-35E class) induce expression of the marker of cytotoxic degranulation CD107a on CD4+ T cells;
  • FIG. 14 shows that GNC proteins (SI-35E class) induce expression of the marker of cytotoxic degranulation CD107a on CD8+ T cells;
  • FIG. 15 shows that GNC proteins (SI-35E class) induce expression of the marker of cytotoxic degranulation CD107a on CD56+NK cells;
  • FIG. 16 shows that GNC proteins (SI-35E class) activate CD3+ T cells to proliferate
  • FIG. 17 shows that GNC proteins (SI-35E class) activate CD3+ T cells to secrete gamma interferon;
  • FIG. 18 shows that GNC proteins (SI-35E class) activate na ⁇ ve CD8+/CD45RA+ T cells to proliferate;
  • FIG. 19 shows that GNC proteins (SI-35E class) activate na ⁇ ve CD8+/CD45RA+ T cells to secrete gamma interferon;
  • FIG. 20 shows Images of GNC activated cell growth in 6-well G-Rex plates over time
  • FIG. 21 shows the example process of making the therapeutic composition as disclosed thereof (A), and cell viability of PBMC, GET, and GNC-T cells after thawing (B);
  • FIG. 22 shows the result of flow cytometry analyses of PBMC-derived, the first GNC (SI-38E17)-activated therapeutic cell composition (Product A) ( 22 A), the second GNC (SI-38E17)-coated therapeutic cell composition (Product B) ( 22 B), and input PBMC cell material (22C).
  • FIG. 23 shows GNC-T therapeutic cell composition of GET cells and formulated GNC-T cells from G-Rex 100M bioreactor after thawing;
  • FIG. 24 shows the result of RTCC of CHO-ROR1 cells by using GNC (SI-35E class)-coated PBMC cells;
  • FIG. 25 shows kinetics of PBMC-derived, SI-38E17 GNC-activated therapeutic cells on killing precursor B cell leukemia Kasumi over time
  • FIG. 26 shows efficacy of killing Nalm-6, MEC-1, Daudi, and Jurkat cells by using PMBC-derived, SI-38E17 GNC-activated therapeutic cells.
  • FIG. 27 shows the killing of Nalm-6, MEC-1, Daudi, and Jurkat leukemic cells by using PBMC-derived, SI-38E17 GNC-activated therapeutic cells in a spike-in model.
  • the guidance navigation control (GNC) proteins are characterized by their composition of multiple antigen-specific binding domains (AgBDs) and by their ability of directing T cells (or other effector cells) to cancer cells (or other target cells such as bystander suppressor cells) through the binding of multiple surface molecules on a T cell and a tumor cell.
  • GNC proteins are composed of Moiety 1 for binding at least one surface molecule on a T cell and Moiety 2 for binding at least one surface antigen on a cancer cell as shown in TABLE 1.
  • FIG. 1 shows the structure of an example tetra-specific GNC antibody comprising AgBDs for binding to both a T cell expressing CD3, PD-L1, and/or 4-1BB and a target B cell expressing CD19, as illustrated in FIG. 2 .
  • the cytotoxic T cells are regulated by T cell receptor complex proteins, as well as co-stimulation signaling proteins via either agonist receptors or antagonist receptors on their surface.
  • T cell receptor complex proteins such as T cell receptors or antagonist receptors on their surface.
  • multiple AgBDs may compose Moiety 1 and Moiety 2, respectively. Examples of molecules that can be targeted by agonistic or antagonistic binding domains in Moiety 1 and 2 are shown in TABLE 1.
  • the GNC proteins may have at least one linker to link Moiety 1 and Moiety 2.
  • any linker molecule can be used to link two or more AgBDs together either in vitro or in vivo by using complementary linkers of DNA/RNA or protein-protein interactions, including but not limited to, that of biotin-avidin, leucine-zipper, and any two-hybrid positive protein.
  • the linkers may be an antibody backbone structure or antibody fragments, so that GNC protein and GNC antibody may have the same meaning, e.g. the structure of the example tetra-specific GNC antibody in FIG. 1 .
  • GNC proteins or antibodies are capable of directing a T cell to a cancer cell, in vivo or ex vivo, through the binding function of multiple AgBDs ( FIG. 2 ).
  • the T cells may be derived from the same patient or different individuals, and the cancer cell may exist in vivo, in vitro, or ex vivo.
  • the examples provided in the present application enable GNC proteins as a prime agent in a T cell therapy, i.e. GNC-T cell therapy, for activating and controlling cytotoxic T cells ex vivo, prior to adoptive transfer.
  • the present application relates to methods of making GNC-activated therapeutic cell composition.
  • Multiple AgBDs can be divided into Moiety 1 and Moiety 2 due to their interface with a T cell and a cancer cell, respectively (TABLE 1).
  • a GNC protein with two AgBDs may simultaneously bind to a surface molecule, such as CD3 on a T cell, and a tumor antigen, such as ROR1 on a tumor cell, for re-directing the T cell to the tumor cell.
  • a third AgBD for example, one that specifically binds to 41BB
  • a fourth AgBD to a GNC protein for example, one that specifically binds to PD-L1 on a tumor cell, may block the inhibitory pathway of PD-L1 on tumor cells or that is mediated through its binding to PD-1 on the T cells.
  • GNC proteins are constructed to acquire multiple AgBDs specifically for binding unequal numbers of T cell antagonists and agonists, not only to re-direct activated T cells to tumor cells but also to control their activity in vivo (TABLE 2). Therefore, in some embodiments, GNC proteins may be bi-specific, tri-specific, tetra-specific, penta-specific, hexa-specific, hepta-specific, or octa-specific proteins.
  • the application relates to a GNC-T cell therapy where GNC proteins are used to expand the T cells ex vivo prior to adoptive transfer ( FIG. 3 ).
  • the ex vivo priming of autonomous T cells provides the cytotoxic T cells guidance and navigation control.
  • PBMC peripheral blood mononuclear cells
  • specific types of cell populations within PBMC e.g., CD8+, CD45RO+ memory T cells may be isolated and primed ex vivo by GNC proteins.
  • PBMC peripheral blood mononuclear cells
  • CD8+, CD45RO+ memory T cells may be isolated and primed ex vivo by GNC proteins.
  • These expanded cytotoxic T cells can be formulated and infused back to the patient through adoptive transfer. While attacking the cancer in vivo, additional GNC proteins may be infused into the patient for managing the efficacy and lifespan of cytotoxicity.
  • GNC-T cell therapy is different from GNC protein-based immunotherapy, where GNC proteins are directly administered into patients.
  • GNC-T cell therapy does not rule out the direct administration of GNC proteins for managing the efficacy of infused cytotoxic T cells in vivo in a controlled manner.
  • Additional GNC protein can both promote cytolytic activity and encourage T cell proliferation dependent of the configuration of AgBDs.
  • the application relates to the production of therapeutic GNC-T cells.
  • CAR-T therapy cell material, for example patient leukocytes, are collected by apheresis, and a subset of CD3+ T cells is selected and activated to facilitate gene transfer to the cellular material, which is then expanded in number by the introduction of foreign material scaffold for support to the T cell populations, for example, by using anti-CD3/anti-CD28 antibody coated beads.
  • GNC-T cell material does not require the introduction of scaffold impurities for T cell expansion from patient leukocytes.
  • the CAR-T therapy cellular material must undergo the gene transfer that involves the preparation and transfection of CAR-T vector DNA, which results in genetically modifying the genome of the T cells. Furthermore, these genetically modified T cells may undergo another round of T cell expansion before being transferred back into the patient.
  • the random integration of CAR-T vector DNA carries a risk of transformation of the T cells leading to primary leukemogenesis or introduction of the CAR-T vector to leukemia cells increasing the risk of relapse by mechanism of internal sequestration of the CAR target antigen (Zhang, Liu et al. 2017).
  • GNC-T cell therapy has the advantages of not involving the transfection of any vector DNA, therefore there is no risk of genetic modification prior to adoptive transfer, which provides one of the significant advantages and technical improvements over the existing CAR-T therapy.
  • the efficacy of GNC-T cell therapy may be improved when PBMC or different T cell subsets are being primed and activated ex vivo as shown in FIGS. 5 & 6 .
  • Similar approaches have been explored in the use of CAR-T therapy, where selected specific ratios of some subsets of T cells may be transferred back to the patient (Turtle, Hanafi et al. 2016, Turtle, Hanafi et al. 2016).
  • the PBMC of a patient with circulating leukemic cells may profoundly alter the cellular composition and thus affect the suitability of the final therapeutic cellular products.
  • a high level of circulating leukemic blast cells greater that 10% of WBC
  • the percentage of leukemic cells in the PBMC derived from a patient may be reduced by using cell fractionation methods.
  • These methods may include steps involving density gradient separation, or immunofluorescent cell separation or fluorescent activated cells sorting, immunomagnetic cell separation, or microfluidic flow chambers methods. These methods may be preceded by or follow centrifugation, cell washing, incubation, or temperature modulation. These methods may utilize non-cellular substrates (magnetic beads, Plastic, polymers), modification of non-cellular substrates (protein, antibodies, charge state), antibody treatment, multiple antibody treatments, multi-specific antigen binding proteins and cell surface antigen-based cell coupling. These methods may use enzymatic digestion or, ionic chelation, or mechanical agitation or cell vessel rotation. The method for reduction of leukemic blasts may utilize antibody drug conjugates, or leukemia sensitizing agents. The method may consist of a combination of these approaches.
  • a tetra-specific antibody is produced and used as the GNC protein.
  • the tetra-specific antibody/GNC protein comprises 4 different binding domains linked by antibody fragments as its backbone.
  • a second binding domain is specific for a tumor associated antigen, including but not limited to ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA, CD19, CD20, CD33, CD123, CD22, CD30, and a third and fourth binding domains are specific for two distinct immune checkpoint modulators such as PD-L1, PD-L2, PD-1, OX40, 4-1BB, GITR, TIGIT, TIM-3, LAG-3, CTLA4, CD40L, VISTA, ICOS, BTLA, Light, etc.
  • a tumor associated antigen including but not limited to ROR1, CEA, HER2, EGFR, EGFRvIII, LMP1, LMP2A, Mesothelin, PSMA, EpCAM, glypican-3, gpA33, GD2, TROP2, BCMA
  • GNC protein-mediated GNC-T cell therapy over conventional CAR-T therapies include, but are not limited to, first, that inclusion of an IgG Fc domain may confer the characteristic of a longer half-life in serum compared to a bi-specific BiTe molecule; second, that inclusion of two binding domains specific for immune checkpoint modulators may inhibit the suppressive pathways and engage the co-stimulatory pathways at the same time; third, that cross-linking CD3 on T cells with tumor associated antigens re-directs and guides T cells to kill the tumor cells without the need of removing T cells from the patient and genetically modifying them to be specific for the tumor cells before re-introducing them back into the patient, also known as chimeric antigen receptor T cells (CAR-T) therapy; and fourth, that GNC protein-mediated antibody therapy or T cell therapy does not involve genetic modification of T cells, the latter of which may carry the risk of transforming modified T cells to clonal expansion, i.e. T cell leukemia.
  • CAR-T chimeric antigen
  • the examples of GNC proteins are classes of tetra-specific GNC antibodies, of which 4 AgBDs are covalently linked using an IgG antibody as its backbone ( FIG. 1 ). From the N-terminal of this protein, the first scFv is linked to the Fab domain of the constant domains C H 1, 2, and 3 of IgG antibody which is then linked to another scFv at the C-terminal. Because each of the scFv domains display independent binding specificity, linking of these AgBDs does not need to be done using the constant domains of an IgG antibody.
  • a GNC protein can directly bind to tumor-associated antigen (TAA) and engage the host endogenous T cells to kill tumor cells independent of tumor antigen presentation by MHC to the antigen specific T cell receptors ( FIG. 2 ).
  • TAA tumor-associated antigen
  • CD19 is a TAA targeting CD19 positive B cells and tumor cells.
  • PD-L1 is an example of the immune checkpoint modulating component for tetra-specific GNC antibodies that may overcome the immunosuppressive tumor microenvironment and fully activate the exhausted T cells within the tumor microenvironment.
  • the SI-35E class comprises targets an anti-human CD3 binding domain (SEQ IDs 1-4), an anti-human PD-L1 (SEQ IDs 5-12), an anti-human 4-1BB (SEQ IDs 13-24), and targets a human ROR1 (SEQ IDs 25-32), i.e. a TAA.
  • the classes of SI-38E and SI-39E target CD19 (SEQ IDs 47-50) and EGFR (SEQ ID 51-54), respectively.
  • AgBDs were converted to scFv and VLVH for placement at the N-terminal Domain 1 (D1) or scFv and VHVL for placement at the C-terminal Domains 3 (D3) and 4 (D4) of the GNC protein.
  • All scFv molecules described herein contain a 20 amino acid flexible gly-gly-gly-gly-ser (G4S) X4 linker that operably links the VH and VL, regardless of the V-region orientation (LH or HL).
  • the remaining position in the tetra-specific GNC antibody, Domain 2 (D2), consists of an IgG1 heavy chain, VH-CH1-Hinge-CH2-CH3, and its corresponding light chain, VL-CL, which can be either a kappa or lambda chain.
  • D1 and D2 are genetically linked through a 10 amino acid (G4S) ⁇ 2 linkers, as are D2, D3 and D4 resulting in a contiguous ⁇ 150 kDa heavy chain monomer peptide.
  • G4S 10 amino acid
  • the final symmetric tetra-specific GNC peptide can be purified through the IgG1 Fc (Protein A/Protein G) and assayed to assess functional activity.
  • Heavy and light chain gene “cassettes” were previously constructed such that V-regions could be easily cloned using either restriction enzyme sites (HindIII/NhelI for the heavy chain and HindIII/BsiWI for the light chain) or “restriction-free cloning” such as Gibson Assembly (SGI-DNA, La Jolla, Calif.), Infusion (Takara Bio USA) or NEBuilder (NEB, Ipswich, Mass.), the latter of which was used here.
  • restriction enzyme sites HindIII/NhelI for the heavy chain and HindIII/BsiWI for the light chain
  • “restriction-free cloning” such as Gibson Assembly (SGI-DNA, La Jolla, Calif.), Infusion (Takara Bio USA) or NEBuilder (NEB, Ipswich, Mass.), the latter of which was used here.
  • the tetra-specific GNC antibodies can be produced through a process that involves design of the intact molecule, synthesis and cloning of the nucleotide sequences for each domain, expression in mammalian cells and purification of the final product.
  • nucleotide sequences were assembled using the Geneious 10.2.3 software package (Biomatters, Auckland, NZ) and broken up into their component domains for gene synthesis (Genewiz, South Plainsfield, N.J.).
  • SI-35E18 (SEQ ID 65 and 67) was split into its component domains where the anti-41BB scFv, VL-VH, occupies D1, anti-human PD-L1 clone PL230C6 occupies D2 (Fab position), anti-human ROR1 Ig domain-specific clone 323H7 VHVL scFv occupies D3, and anti-human CD3 scFv, VHVL, occupies the C-terminal D4.
  • nucleotides were appended to each of the domains depending on their position in the larger protein so that each domain overlaps its flanking domains by 20-30 nucleotides which direct site-specific recombination, thus genetically fusing each domain in a single gene assembly step. Due to the high number of homologous regions in the tetra-specific nucleotide sequence, the N-terminal domains 1 and 2 are assembled separately from the C-terminal D3 and D4. The N- and C-terminal fragments were then assembled together in a second NEBuilder reaction. A small aliquot was transformed into E.
  • coli DH10b (Invitrogen, Carlsbad, Calif.) and plated on TB+carbenicillin 100 ug/ml plates (Teknova, Hollister, Calif.) and incubated at 37° C. overnight. Resultant colonies were selected and 2 mL overnight cultures inoculated in TB+carbenicillin. DNA was prepared (Thermo-Fisher, Carlsbad, Calif.) from overnight cultures and subsequently sequenced (Genewiz, South Plainsfield, N.J.) using sequencing primers (Sigma, St. Louis, Mo.) flanking each domain. All DNA sequences were assembled and analyzed in Geneious.
  • SI-38E17 targeting human CD19 (SEQ IDs 47-50)
  • multiple AgBDs carry an anti-human 4-1BB (scFv 466F6, SEQ IDs 17-20) as well as an anti-human PD-L1 (scFv PL221G5 SEQ IDs 9-13), and an anti-human CD3 binding domain (SEQ IDs 1-4).
  • the methods and procedures for producing this tetra-specific antibody were the same.
  • GNC proteins are composed of Moiety 1 for binding at least one surface molecule on a T cell and Moiety 2 for binding at least one surface antigen on a cancer cell (TABLE 1A).
  • the tetra-specific GNC antibodies can be used to directly engage the body's endogenous T cells to kill tumor cells independent of tumor antigen presentation by MHC to the antigen specific T cell receptors. This is in contrast to therapies based solely on immune checkpoint blockade, which have been limited by antigen recognition.
  • the immune checkpoint modulating component may be constructed as a part of tetra-specific GNC antibodies, which may provide benefits similar to that in a standard checkpoint blockade therapy.
  • TABLE 1B shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with NK cell binding domains.
  • TABLE 1C shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with macrophage binding domains.
  • TABLE 1D shows the example compositions of functional moieties (Moiety 1 and Moiety 2) and antigen binding domain in GNC proteins with dendritic cell binding domains.
  • GNC proteins are constructed to acquire multiple AgBDs specifically for binding unequal numbers of T cell antagonists and agonists. In this way, GNC proteins may re-direct activated T cells to tumor cells with certain levels of control of their activity in vivo (TABLE 2). Therefore, GNC proteins may be bi-specific, tri-specific, tetra-specific, penta-specific, hexa-specific, hepta-specific, or even octa-specific proteins.
  • three classes of tetra-specific GNC antibodies i.e. SI-39E, SI-35E, and SI-38E, were created to enable GNC-T cell therapy, of which antibody domains and its specificity is listed in TABLE 3.
  • the structures of tetra-specific GNC antibodies targeting EGFRvIII (SI-39E), ROR1 (SI-35E), and CD19 (SI-38E) are listed in TABLE 4.
  • the SI-35 class listed in Table 4 were tested for their ability to activate and induce proliferation of different cell types, such as CD4+ and/or CD8+ T cells and/or CD56+ natural killer cells (NK) within PBMC.
  • the tetra-specific GNC antibodies were prepared at 2 ⁇ final concentration and titrated in 1:10 serial dilutions across 6 wells of a 96 well plate in 200 ul of RPMI+10% FBS.
  • Human PBMC were purified by standard Ficoll density gradient from a “leukopak” which is an enriched leukapheresis product collected from normal human peripheral blood.
  • the PBMC and serially titrated GNC proteins were combined by adding 100 ⁇ L of PBMC (100,000), and 100 ⁇ L of each antibody dilution to each well of the assay.
  • the assay plate was incubated at 37° C. for approximately 72 hours and then the contents of each assay well were harvested and analyzed by FACS for the number of CD4+ T cells, CD8+ T cells, and CD56+NK cells.
  • Cells were harvested from each well and transferred to a new 96 well V-bottom plate then centrifuged at 400 ⁇ g for 3 minutes. Supernatant was transferred to a 96 well plate for analysis of IL-2 and Granzyme B.
  • Cells were re-suspended in 200 ⁇ L of 2% FBS/PBS of FACS antibodies and incubated on ice for 30 minutes. The plate was centrifuged at 400 ⁇ g for 3 minutes and the supernatant was aspirated. This wash step was repeated once more and then the cells were re-suspended in 100 ⁇ L 2% FBS/PBS and analyzed on a BD LSR FORTESSA.
  • cytotoxic degranulation marker CD107a (LAMP-1) was induced by all GNC proteins tested except those lacking binding at positions 2 and 4 on CD4+( FIG. 13 ), CD8+( FIG. 14 ), but less consistently on CD56+( FIG. 15 ) in the culture.
  • 3 of the GNC proteins (SI-35E42, SI-35E43, and SI-35E46) induced expression of CD69 on CD4+ T cells, CD8+ T cells, and CD56+NK cells, which correlated well with the level of IL-2 and granzyme B secretion ( FIGS. 8 and 9 ) induced by these GNC.
  • Proliferation and production of gamma interferon was measured from cultures of CD3+ or na ⁇ ve CD8+ T cells (70,000 cells/well) stimulated for 5 days with a panel of SI-35 class antibodies.
  • Human CD3+ or CD8+CD45RA+na ⁇ ve T cells were enriched from peripheral blood mononuclear cells from a normal donor using the EasySepTM Human CD3+ or Na ⁇ ve CD8+ T Cell Isolation Kits (StemCell Technologies) as per the manufacturer protocols. The final cell populations were determined to be >98% CD3+ or CD8+CD45RA+ T cells by flow cytometry. Proliferation in the culture was measured after stain with Alamar blue (ThermoFisher Cat. No.
  • FIG. 18 Proliferation of na ⁇ ve CD8+CD45RA+ T cells was more sensitive to the presence or absence of 4-1BB binding domain compared to total CD3+ T cells as shown by addition of soluble anti-4-1BB monoclonal antibody to the culture in which 4-1BB binding on the GNC was absent. A similar pattern was found for secretion of gamma interferon from the na ⁇ ve CD8+ T cells ( FIG. 19 ).
  • GNC-activated and -coated T cells at clinically significant dosage of 10E9 was achieved after 7 days culture.
  • Human PBMC were isolated from LRS cone leukocytes by standard Ficoll density gradient from leukopaks which are enriched leukapheresis product collected from normal human peripheral blood. After collection the cells were frozen at ⁇ 80° C. and then later thawed before putting in culture. Using the G-Rex plate and bioreactor culture systems, the growth of SI-38E17 GNC-stimulated PBMC cultures was monitored for up to 14 days.
  • the culture medium consisted of RPMI 1640, 10% fetal calf serum, 1% non-essential amino acids, 1% GlutaMax, 0.6% glutamine-alanine supplement, 15 ng/mL human IL-2, and 1 nM GNC protein.
  • the 6-well G-Rex cultures tolerated seeding densities of 25-100 million PBMC/well for six days, which greatly exceeded recommended amounts, but was tolerated by the cells in the system with a single 50% medium change on day 7. Clustering of cells was indicative of their activation in the culture ( FIG. 20 ). At least 250 million cells from one leukapheresis donor were seeded into two G-Rex 100M bioreactors and cultured in 1 liter of culture medium for seven days. The larger volume of medium allowed the culture to continue without needing to exchange the culture medium. Cell yield in each of the 100M bioreactors was between 1.2-1.4 billion cells with greater than 88% viability.
  • the cells from the bioreactor were harvested as the first GNC-activated therapeutic cell composition, which were optionally concentrated using LOVO Automated Cell Processing System (Fresenius Kabi).
  • One sample (Product B) was exposed to 1 nM SI-38E17, which is identical to the first GNC in this case for preparing a second GNC-activated therapeutic cell composition, potential for being used to target treat patients harboring CD19 positive malignancies ( FIG. 21A ).
  • the second GNC-activated therapeutic cells were washed twice before eluting to a final volume of 54 mL in a sterile processing bag.
  • the other sample (Product A) was only exposed to the first GNC protein during the culture phase and not re-exposed during processing in the LOVO system ( FIG. 21A ).
  • Cells were removed from bags, mixed 1:1 with CryoStor CS10 reagent, and frozen to ⁇ 80° C. The processed cells were thawed and compared to the thawed unstimulated PBMC from the same donor before culture.
  • GNC-expanded T cell (GET) culture was >75% and was not affected by exposure to additional GNC reagent (GNC-T, Product B) during processing ( FIG. 21B ).
  • the mean diameter of the cells increased during culture, indicative of cell activation.
  • Flow cytometry was performed on the input PBMC cell material and the two formulations after thawing using a multi-color panel of antibodies to stain for: live/dead (e780), CD45, TCR ⁇ / ⁇ , CD56, CD4, CD8, CD14, TCR ⁇ / ⁇ , and CD20.
  • FIG. 22C summaries the total number and percentage of each subpopulation of cells. Compared to the input PBMC cell material, while the total number of leukocytes increased from 250 to 1000 millions or four-fold, the total number of each subpopulation of T cells was vastly increased by 55-fold for ⁇ / ⁇ T cells, 45-fold for CD4+ T cells, and 78-fold for CD8+ T cells.
  • This example illustrates a number of advantages of GNC-T cells in comparison to CAR-T cell preparations.
  • the cell composition of the starting material was fresh PBMC from the donor and did not need to be pre-selected for particular subsets of cells or require addition of feeder cells or synthetic beads.
  • the GNC protein was 100% non-nucleotide biological material, and did not require the transfer of RNA or DNA into the cells, or transfection with a viral vector.
  • the GNC-induced expansion yielded a therapeutic dose in 9 days, compared to the average of 40 days for CAR-T cell expansion.
  • the resulting cells were devoid of B cells and highly enriched for activated CD4+ and CD8+ T cells that had potent killing potential against their specific targets.
  • the GNC therapeutic composition was viable and bioactive upon thaw from ⁇ 80° C. Together these advantages are expected to significantly lower waiting times, costs and issues related to infrastructure and training related to CAR-T cell therapy. Improvements in the purity, safety and quantity of the end product will be of significant benefit to the patient.
  • GNC SI-35 class proteins Six of the GNC SI-35 class proteins listed in Table 4 were tested for the ability to activate PBMC for redirected T cell cytotoxicity (RTCC) activity against a human ROR1-transduced CHO cell line ( FIG. 24 ).
  • GNC proteins were prepared at 2 ⁇ final concentration and titrated 1:3 across 10 wells of a 96 well plate in 200 ul of RPMI+10% FBS.
  • the PBMC and serially titrated antibodies were combined by adding 100 ⁇ L of PBMC (200,000), and 100 ⁇ L of each antibody dilution to each well of the assay. The assay plate was incubated at 37° C.
  • CHO-ROR1 target cells 5 ⁇ 10e6, were labeled with CFSE (Invitrogen, #C34554) at 0.5 ⁇ M in 10 mL of culture media for 20 minutes at 37° C.
  • the CHO-ROR1 cells were washed 3 times with 50 mL of culture media before resuspending in 10 mL, counted again and then 5,000 CFSE-labeled CHO-ROR1 cells were added to each well of GNC-activated PBMC. Cells were incubated for another 72 hours and then the contents of each assay well were harvested and analyzed for the number of CFSE-labeled target cells remaining.
  • all of the GNC proteins tested directed RTCC activity with SI-35E42, SI-35E43, and SI-35E46 being the most potent in reducing the number of CHO-ROR1 cells in the well.
  • PBMC from a healthy donor were labeled with GNC protein SI-38E17 at 10-fold serial doses ranging from 0.01 to 100 nM for 30 minutes at 37° C. and then washed prior to culture.
  • the GNC SI-38E17 targets the CD19 antigen expressed on B cell surfaces, and therefore, the Kasumi-2 precursor B cell leukemia line was chosen as a target cell.
  • the Kasumi-2 cell used was transduced to express green fluorescence protein (GFP) and therefore the presence of tumor cells was tracked by measuring the average green fluorescence in 4 images/well collected 9 times over a six-day period.
  • the effector:target (E:T) ratios were escalated by adding GNC-labeled PBMC in a serial 2-fold dilution of 5,000 (1:1) to 160,000 (32:1) cells to duplicate wells. As shown in FIG. 25 , Kasumi-2 cells increased in number in the wells that had from 1:1 to 8:1 E:T ratios of unlabeled PBMC.
  • Daudi-Red cells were serially diluted 10-fold in a range from 200,000 to 20 cells and then mixed 1:1 with 1 million PBMC to create samples of 10%, 1.0%, 0.1%, 0.01% and 0.001% tumor cells, which were then analyzed by flow cytometry ( FIG. 27 ).
  • tumor cells were harvested from a 15 day 6-well G-Rex culture of 1 nM GNC-expanded T cells that had been spiked with 10%, 1% or 0.1% of NALM-6, MEC-1, Daudi, or Jurkat (all NucRed-transduced) tumor cells at time 0 and analyzed using the same flow cytometry settings as above. Tumor cells were reduced to less than 0.001% in all conditions with the exception of the culture in which the MEC-1 tumor line was spiked in at 10% were 44 cells were detected. In this condition the MEC-1 cells were reduced to ⁇ 0.01% in the culture.
  • TABLE 1A Composition of example GNC proteins with T cell binding domains.
  • Bi-specific CD3 ROR1 Tri-specific CD3 ROR1 PD1 Tetra-specific CD3 ROR1 PD1 41BB Penta-specific CD3 ROR1 PD1 41BB LAG3 Hexa-specific CD3 ROR1 PD1 41BB LAG3 TIM3 Hepta-specific CD3 ROR1 PD1 41BB LAG3 TIM3 TIGIT Octa-specific CD3 ROR1 PD1 41BB LAG3 TIM3 TIGIT CD28

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