Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
  • A61K40/4203—Receptors for growth factors
  • A61K40/4204—Epidermal growth factor receptors [EGFR]
• • A—HUMAN NECESSITIES
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells
  • A61K2039/5156—Animal cells expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
  • A61K2239/11—Antigen recognition domain
  • A61K2239/13—Antibody-based
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
  • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
  • C07K16/3076—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
  • C07K16/3084—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

• the present invention relates to the fields of brain cancer and cell-mediated delivery of cytotoxic agents. More specifically, the invention provides genetically modified migratory interneuronal precursor cells as a delivery vector for the efficacious treatment of different brain malignancies, particularly glioblastoma. Background of the invention Several publications and patent documents are cited throughout the specification in order to describe the state-of-the-art to which this invention pertains.
• Brain tumors are formed from an abnormal mass of tissue in which malignant cells grow and multiply rapidly and uncontrollably.
• the two major types of brain tumors are either primary or metastatic.
• Primary brain tumors include low or high-grade tumors that originate from the tissues of the brain or those surrounding of the brain. According to the study published by a “Cancer Journal of Clinicians”, in 2021, an estimated 83,570 people were diagnosed with primary brain tumors in the U.S. Approximately 29.7% of all primary brain and other CNS tumors are high grade and 70.3% are low grade, such that non-malignant tumors more than twice as common as malignant tumors. However, the high-grade tumors account for most brain tumor related deaths.
• the average annual age-adjusted incidence rate of primary high grade brain and other CNS tumors was 7.08 per 100,000 the one of primary low grade brain and other CNS tumors was 16.71 per 100,000.
• the average annual mortality rate of primary high grade brain tumors for the five-year period between 2013 and 2017 was 4.42 per 100,000 with an average of 16,249 deaths per year.
• the five-year survival rate following diagnosis of a primary high grade brain tumors was 23.5%, while the five-year survival rate following diagnosis of a primary low grade brain tumors was 82.4%.
• the median observed survival in for primary high-grade glioblastoma was the lowest observed at 8 months.
• Glioblastoma is a devastating, high grade neoplasm of the brain with a 5% five-year overall survival 1 .
• High grade gliomas (HGG) like glioblastoma represent the leading cause of cancer-related mortality in the pediatric population 2 and are one of the most recalcitrant tumors to treat in adults as well 3 .
• Glioblastomas have a notoriously immune suppressed tumor microenvironment (TME), where microglia inhibit CD8 T-cell recruitment and activity, thereby evading clearance 4,5 .
• TEE tumor microenvironment
• Novel therapies disrupting or coopting the TME have rapidly advanced with clinical trials ongoing that for example block CD47 (NCT05169944), an immune checkpoint receptor blocking phagocytosis by macrophages 6,7 .
• Cellular therapies are being explored, but so far have not yielded the anticipated results 8 , including Chimeric Antigen Receptor T-cell therapy (CAR-T) which is inherently subject to the immune suppressive TME.
• CAR-T Chimeric Antigen Receptor T-cell therapy
• Secondary brain tumors, or brain metastases are cancers that originate in other parts of the body and spread to the brain. Most common primary tissues of origin are lung, breast, colon, kidney and skin. Brain metastases are always malignant and have very poor prognosis. Although frequent in an adult population, brain metastases are rare in a pediatric population.
• Standard of care therapies for brain tumors include surgical removal, radiation therapy, chemotherapy and targeted therapies. Some high-grade primary brain tumors, like medulloblastoma, can be successfully treated with these standard of care therapies. Surgery is a common treatment for brain tumors, and, especially for low-grade tumors might be the only treatment needed.
• Radiation therapy also called “radiotherapy,” “irradiation,” or simply “radiation”
• Radiation therapy involves the use of x-rays, gamma rays, neutrons, protons, and other sources to kill cancer cells and shrink tumors by damaging their DNA.
• radiation therapy of the brain can lead to negative outcomes later in life. Effective alternatives to radiation could lead to fewer of these side effects.
• Chemotherapy is often part of the standard of care of patients with brain tumors. In some cases, the disease is well-controlled with the use of these agents; however, many high- grade tumors do not respond to chemotherapy.
• Cellular therapies like CAR-T therapy, have revolutionized treatment for a number of cancers. Unfortunately, these therapies have so far shown limited efficacy for brain tumors, in part due to the lack of unique tumor antigens and an immune suppressive microenvironment 8,35 .
• a composition for the treatment of brain cancer is disclosed.
• composition comprises a purified population of genetically engineered post-mitotic migratory interneuron precursor cells (MIPs) chemoattracted to brain cancer cells, wherein said MIPs comprise at least one recombinant nucleic acid expressing a secretable, inhibitory bi-specific antibody having i) a first binding domain which forms a first specific binding pair with a surface ligand on an immune effector cell and ii) a second binding domain which forms a second specific binding pair with a surface ligand on a brain cancer cell.
• MIPs genetically engineered post-mitotic migratory interneuron precursor cells
• the composition also comprises a purified population of immune effector cells comprising the ligand bound by said second binding domain, secretion of said bispecific antibody in proximity of brain cancer cells and immune effector cells linking both types of cells, and causing activation of the immune effector cell, thereby killing any brain cancer cell in proximity thereof.
• the MIP cells are LHX6 + .
• the recombinant nucleic acid present in the genetically engineered cells encodes a bispecific antibody having said first and second binding domains in first and second scFv sequences said scFv sequence being operably linked at coupling sites to a linker peptide.
• the brain cancer is a glioblastoma.
• the brain cancer is a metastatic cancer originating from any tissue other than brain.
• metastatic brain cancers can metastasize from lung, breast, kidney, prostate, bone, skin, colon, thyroid gland, liver, genitourinary tract, pancreas, lymph/blood, neuroblastoma, head and neck.
• the immune effector cell is a CD8+ T cell
• said first ligand binds CD3 on said T cell
• said second ligand binds EGFR on said brain cancer cell when said T cell and said brain cell are in proximity to one another.
• the polynucleotide present in the MIP cells can be delivered in an expression vector, selected from lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno associated virus vectors, adenovirus vectors, pox virus vectors, herpes virus vectors, engineered hybrid viruses, and transposon mediated vectors.
• a purified post-mitotic migratory interneuron precursor cell (MIP) population genetically engineered to express a therapeutic agent is disclosed.
• the genetically engineered MIP the therapeutic agent is cytotoxic.
• the cytotoxic agent is a bispecific antibody which binds brain cancer cells and activates CD8+ T cells. Also disclosed is a method for the treatment of brain cancer, in a patient in need thereof.
• An exemplary method entails administration of an effective amount of any one of the compositions described above into the cranial cavity wherein said T cells and MIPs are administered, said MIPs being chemoattracted to said brain cancer cells and secreting said bispecific antibody in proximity of said brain cancer cells and said T cells, thereby linking both types of cells, said linking causing activation of said T cell, thereby killing any brain cancer cell in proximity thereof.
• the brain cancer is a glioblastoma.
• the brain cancer is a metastatic cancer originating from any tissue other than brain.
• Antigen to be targeted in the method include without limitation, CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, MUC17, HER2, EpCAM, B7H3, EGFR, GD2, CD1640, TRAIL, trimer TRAIL and CD3.
• the composition is administered via a cannula placed anywhere in the brain.
• the composition is administered via repeated dosing though an Ommaya Reservoir directly into the ventricles of the brain.
• the level of at least one of the prognostic biomarkers listed in Table 1 in the patient is assessed prior to treatment, to determine whether the patient is a candidate for the therapy described herein.
• a method for the treatment of brain cancer comprising administration of an effective amount of genetically engineered MIPs directly into the cranial cavity, said MIPs being chemoattracted to said brain cancer cells and secreting a bispecific antibody in proximity of said brain cancer cells and endogenous CD8+ T cells present in the brain, thereby linking both types of cells, said linking causing activation of said endogenous CD8+ T cell, thereby killing any brain cancer cell in proximity thereof.
• another aspect of the invention includes a method for identifying brain cancer patients most likely to benefit from treatment with the compositions described herein.
• An exemplary method comprises detecting in a biological sample one or more MIP migration- associated biomarkers listed in Table 1, and comparing said level of biomarkers with a reference level from subjects who benefitted from said treatment, patients exhibiting equal or higher levels being identified as most likely to benefit from treatment.
• the invention provides a composition comprising GD2-BiTE in a pharmaceutically acceptable carrier for use of the treatment of any cancer expressing a GD2 antigen.
• the GD2-BiTE sequence is set forth in SEQ ID NO:66.
• SEQ ID NO: 66 comprises modifications that enhance one or both of stability and bioavailability.
• a method for administration of the composition of GD2-BiTE or modified versions thereof to patients comprising direct delivery of said GD2-BiTE through infusion, via a cannula, an Ommaya reservoir, or intravenously for treatment of a brain tumor.
• the GD2-BiTE composition is administered in combination with a T-cell infusion.
• the composition is administered via direct delivery of said GD2- BiTE through infusion, via a cannula, an Ommaya reservoir, or intravenously, for treatment of any tumor expressing GD2 antigen.
• Fig. 1A Overview of how CXCL12 binding activates CXCR4 and its downstream pathways, which include the RAS pathway. Almost immediately after CXCL12 binding, CXCR4 becomes phosphorylated which drives Arrestin binding and CXCR4 degradation. 22
• Fig. 1B Violin plot comparing expression of CXCL12, HMGB1 and MIF in normal cortex and pediatric HGG.
• Fig 1C Violin plot comparing expression of CXCL12, HMGB1 and MIF in normal cortex and pediatric LGG.
• Fig 1D Table of expression of CXCL12, HMGB1 and MIF in breast and lung cancer brain metastases (TPM: transcripts per million) showing substantial higher expression than normal control cortex values.
• FIG. 1E Cortical and Striatal interneuron precursors are derived from MGE residing precursors and migrate to populate the Cortex and Striatum respectively.
• Figures 2A -2B Fig. 2A) An scFv is composed of the linker connected heavy and light variable fragment (V H and V L ) of the variable fragment (Fv) of an antibody. The Fv is responsible for antigen binding.
• Fig. 2B EGFR-BiTE mechanism of action: Binding of the BiTE to EGFR (tumor cell) and CD3 (T-cell) activates the T-cell and stimulates release of perforin and granzyme eliminating the tumor cell. Modified from 29 .
• Figures 3A – 3F Fig.
• Fig. 3C Co-staining for Citrine (green) and LHX6 (red) in differentiated MIPs (plated on Rat cortical feeders) showing that all Citrine positive cells are LHX6 positive i.e., MIP.
• Fig. 3D MIP: IF Staining with MCIP Markers MAP2 and SOX6.
• Fig. 3E In vitro migration assay: Tumor spheres (red) are allowed to form and become encased in a matrigel matrix containing MIP (green).
• MIPs are allowed to migrate for 5 days, and the radius of the migration front is determined. Bottom: Images at day 0 and 5 post plating.
• Fig. 3F Incucyte transwell migration assay: Wells are coated with matrigel and MCIPs are plated on the top well ( ⁇ 20k cells). 24h later a chemoattractant is added to the bottom well and migration is monitored, data is plotted as fold change number of cells on the bottom of the membrane compared to control. Top: migration to chemokines, bottom: migration to conditioned media. 100ng/ml CXCL12, NRG1, IGFPB2, HGF and MIF significantly attract MCIPs (p ⁇ 0.05). MCIPs have a 4-fold increased migration to 8/13 conditioned medias.
• Fig 3H Human MIPs (green) migrate to the site of a glioblastoma within 14 days when injected 2mm apart. Top: MIP injection site and tumor injection site are indicated. MIPs migrate through different paths to the tumor (red, tumor 1746) indicated by green arrows.
• FIG. 4A 2D killing assay (0 and 48h) showing 1746 pHGG cells (red) mixed with BiTE expressing 293T cells and CD8 T-cells. A Cytotox agent stains dead cells fluorescent green.
• Fig. 4B 3058 pHGG sphere (red) mixed with BiTE expressing MIPs and CD8 T-cells (small cells surrounding sphere), show potent tumor killing by EGFR within 48h. No killing was observed with a control BiTE Fig.
• FIG. 4C-D Mice were injected with 3058 tumor cells, BiTE expressing MIPs and T-cells in a single location.
• CD19-BiTE was used as a control.
• Fig. 4C Luminesce signal shows a tumor present with CD19-BiTE MIP controls but not with EGFR- BiTE expressing MIPs.
• Fig. 4E Survival curves of the 3058 xenografts, where 3058 and MIPs were injected in a single location.
• FIG. 6 Western blots showing RAS pathway activation (pERK) in MIPs after exposure to glioblastoma conditioned MIP media, rCXCL12, rMIF, rHMGB1 or rCXCL12/rHMGB1 and showing CXCR4 phosphorylation upon rCXCL12 exposure. Arrows indicate the 3 phosphorylated bands. CXCR4 becomes rapidly phosphorylated (1min) and is degraded over a 15 min time period.
• Figure 7 Schematic overview of our BiTE, where we link the scFv of EGFR to the scFv of CD3.
• the IGK leader sequence drives secretion of the BiME, a HIS tag allows us to purify the BiME.
• transplanted MIPs can migrate in the cerebral cortex of rodents, pigs, ferrets, and primates 14 . At least within rodents, MIPs can also migrate when transplanted outside of the cerebral cortex, including in striatum 15 , cerebellum 16 , and spinal cord 17 . During fetal brain development, MIPs are chemoattracted to migrate long distances from their subcortical origins into the cerebral cortex or striatum (FIG. 1E). Several MIP chemoattractants have been identified, CXCL12 for example activates CXCR4 and CXCR7 on MIPs to induce this migration.
• BiTEs are in essence two antibodies linked together, where one antibody binds to a tumor antigen and the other binds to CD3 on the T-cell, resulting in T-cell activation and elimination of the engaged tumor cell.
• the CD8 T- cells have no innate recognition of the tumor cell as this is provided by the BiTE.
• the in vitro and in vivo data below show that EGFR-BiTE secreting MIPs eliminate EGFR expressing glioblastoma cells.
• local EGFR BiTE secretion has limited systemic toxicity, thereby making EGFR a viable non-unique target.
• several BiTE therapies are FDA approved.
• MIPs can be equipped with a wide range of agents that destroy glioblastoma cells (FIG. 4).
• modified MIPs differentiated from patient or compatible donor or “off the shelf” induced pluripotent stem cells are injected into the margin of the surgical cavity to “clean- up” tumor cells remaining post-surgery.
• CXCL12 is chemoattractive for MIPs.
• MIPs can migrate to glioblastoma in vivo (FIGS. 3G -3H).
• Glioblastoma express a number of proteins that can attract MIPs.
• Factors and biomarkers responsible for MIP-to-glioblastoma migration are identified herein and can be used to advantage to improve migratory capacity of MIPs by over-expressing migration promoting receptors like CXCR4, CXCR7 or by preventing CXCR4 or CXCR7 degradation upon ligand binding.
• compositions and methods provided herein can be used to advantage for the production of novel anti-glioblastoma therapeutic strategies for both forebrain glioblastoma and for other solid tumors of the CNS.
• the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary.
• the terminology used herein is for the purpose of describing particular forms and embodiments only and is not intended to be limiting.
• test compound refers to a chemical, drug or peptide to be tested by one or more screening method(s) as a putative modulator.
• a test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof.
• various predetermined concentrations of test compounds are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar.
• Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
• the terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control.
• the terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
• modulate refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds.
• an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
• a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
• a compound that increases a known activity is an “agonist”.
• One that decreases, or prevents, a known activity is an “antagonist”.
• the term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition or activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level.
• Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.
• preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
• in need of treatment refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.
• subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity.
• the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
• a mammal e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent
• the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
• a patient refers to a subject afflicted with a disease or disorder.
• patient includes human and veterinary subjects.
• treatment and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, associated with brain cancer.
• This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
• this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
• palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
• preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
• supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
• a cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject.
• a “cell” can be a cell from any organism. “Migratory inhibitory interneuron precursor cells” from the cerebral cortex or striatum originate embryonically, in the ventral/subcortical portion of the telencephalic neural tube 9-12 .
• MIP post- mitotic migratory inhibitory interneuron precursors
• MIP migration takes roughly one week in mice and 4-6 weeks in humans 13 and is guided by both chemorepulsant and chemo-attractive factors.
• MIP cells can be used to advantage for killing malignant cells in the brain with minimal adverse side effects.
• immune effector cells refers to cells from the human body that are capable of effecting or enhancing an immune response. These are collected from the human body (harvested) so they can be administered to patients directly into the brain in need thereof.
• Exemplary immune effector cells as used herein include CD8+ T cells, and NK cells.
• chemoattraction refers to the unidirectional movement of a cell, in response to a chemical gradient of ligands. During chemoattraction, cells move in the direction from a low to a high concentration of the chemoattractant.
• effective amount of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result.
• Anti-tumor efficacy herein refers to an inhibitory effect on tumor growth, particularly brain tumor growth, which can be manifested as slowing, retarding, arresting or even reversing (i.e., shrinking) growth of the tumor.
• sample as used herein means any biological fluid or tissue that contains the biomarkers.
• the most suitable samples for use in the methods and with the compositions are blood samples, including serum, plasma, whole blood, and peripheral blood. It is also anticipated that other biological fluids, such as CNS fluids, saliva or urine may be used similarly. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means.
• change in expression is meant an increased expression level of a selected biomarker, or upregulation of the genes or transcript encoding it in comparison to the reference or control; a decreased expression level of a selected biomarker or a downregulation of the genes or transcript encoding it in comparison to the reference or control; or a combination of certain increased/upregulated and decreased/down regulated biomarkers.
• the degree of change in target expression can vary with each individual and is subject to variation with each population. For example, in one embodiment, a large change, e.g., 2-3 fold increase or decrease in a small number of biomarkers, e.g., from 1 to 9 characteristic biomarkers, is statistically significant.
• target biomarker or “target biomarker signature” as used herein is meant those proteins/peptides or the genes/transcripts encoding same, the expression of which changes (either in an up-regulated or down-regulated manner) characteristically in the presence of brain cancer from that in a healthy individual.
• at least one target biomarker forms a suitable biomarker signature for use in the methods and compositions.
• at least two target biomarkers form a suitable biomarker signature for use in the methods and compositions.
• Specific biomarker signatures can include any combination of brain cancer biomarkers employing at least one biomarker identified herein.
• microarray refers to an ordered arrangement of hybridizable array elements, e.g., primers, probes, ligands, on a substrate.
• the term can also refer to a high grade glioblastoma (HGG) tissue microarray.
• ligand refers to a molecule that binds to a protein or peptide, and includes antibodies and fragments thereof.
• polynucleotide when used in singular or plural form, generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA encoding a sequence of interest.
• polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• polynucleotide specifically includes cDNAs.
• the term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases.
• polynucleotide embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
• sequence of interest can include one or more ancillary sequences that facilitate expression of a nucleic acid of interest, e.g, promoters, leader sequences, polyA tails, and internal ribosome entry sites (IRES).
• ancillary sequences can include sequences that facilitate the cleavage of gene products or encoded RNAs post-expression, including but not limited to recognition sites of cellular or viral proteases or sequence encoding for catalytically active RNA or polypeptide sequences. Also included are sequences encoding functional RNAs, siRNA and shRNA.
• sequences encoding other RNAs that function as guides or co-factors in gene editing processes can be incorporated into the nanoparticle.
• the nucleic acid of interest can also comprise non-coding sequence that facilitates recombination of the DNA of interest with the cellular (host) genome, including but not limited to sequence derived from retroviruses (including lentiviruses), bacteriophages, or cellular genomes.
• Other non-coding sequence to be included include those that facilitate circularization of the DNA cargo post- delivery or promote the maintenance of DNA cargo as an episome post-delivery.
• the nucleic acid of interest can be flanked by sequences corresponding to the inverted terminal repeats (ITRs) of an adenovirus genome, or modified forms of the ITRs. Any of the above-mentioned nucleic acid sequences can be modified to limit recognition by the innate immune response.
• oligonucleotide refers to a relatively short polynucleotide of less than 20 bases, including, without limitation, single-stranded deoxyribonucleotides, single- or double- stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs.
• Oligonucleotides such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms. “Antisense oligonucleotides or strands” are oligonucleotides that are complementary to sense oligonucleotides, pre-mRNA, RNA or sense strands of particular genes and which bind to such genes and gene products by means of base pairing.
• base pairing nucleotides include but are not limited to, uracil, thymine, adenine, cytosine, guanine and hypoxanthine having their respective amino groups protected by acyl protecting groups, 2-fluorouracil, 2- fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil and other modified nucleobases such as 8- substituted purines, xanthines, or hypoxanthines.
• base pairing nucleotides include but are not limited to, expanded-size nucleobases in which one or more benzene rings has been added.
• the antisense oligonucleotide need not base pair with every nucleotide in the sense oligonucleotide.
• a “derivative” of a polypeptide, polynucleotide or fragments thereof means a sequence modified by varying the sequence of the construct, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself.
• “Derivatives” of a gene or nucleotide sequence refers to any isolated nucleic acid molecule that contains significant sequence similarity to the gene or nucleotide sequence or a part thereof.
• “derivatives” include such isolated nucleic acids containing modified nucleotides or mimetics of naturally-occurring nucleotides.
• Immune response modifiers include compounds that possess potent immunomodulating activity including but not limited to antiviral and antitumor activity. Certain IRMs modulate the production and secretion of cytokines.
• certain IRM compounds induce the production and secretion of cytokines such as, e.g., Type I interferons, TNF-alpha, IL- 1, IL-6, IL-8, IL-0, IL-12, MIP-1, and/or MCP-1.
• certain IRM compounds can inhibit production and secretion of certain TH2 cytokines, such as IL-4 and IL-5.
• some IRM compounds are said to suppress IL-1 and TNF (U.S. Pat. No. 6,518,265).
• wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
• variant or “mutant” should be taken to mean the exhibition of qualities that have a pattern that deviates from the wild type or a comprises non naturally occurring components.
• variant refers to a nucleic acid sequence or polypeptide comprising a sequence, which differs (by deletion, insertion, and/or substitution of a nucleic acid or amino acid, an L or D stereoisomer an amino acid, or a non-naturally occurring amino acid) in one or more nucleic acid or amino acid positions differ from that of a wild type nucleic acid or polypeptide sequence.
• non-naturally occurring or “engineered” are used interchangeably and indicate the involvement of the hand of man.
• nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
• linker refers to a connection between two protein coding sequences or their protein products. Linkers comprise a stretch of contiguous nucleic acids or amino acids, which holds at least one cleavage site that enables separation of the genes or their products through cleavage of the linker.
• the linker comprises a cleavage site at its 5′ end and a cleavage site at its 3′ end, or a cleavage site at its N-terminal end and a cleavage site at its C-terminal end.
• the invention also includes polynucleotides encoding the peptides or fusion proteins comprising the protein sequences described herein. Those of skill in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide.
• the polynucleotides may be codon-optimized for expression in a particular cell. Any polynucleotide sequences may be used which encode a desired form of the polypeptides described herein.
• the polynucleotide sequences which encode the polypeptides of the invention represent non-naturally occurring sequences. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.
• the term “codon optimization” refers to changing the codons of a nucleotide sequence without altering the amino acid sequence that it encodes in order to favor expression in a specific species. Codon optimization may be used to increase the abundance of the peptide or protein that the nucleotide sequence encodes since “rare” codons are removed and replaced with abundant codons.
• the phrases “% sequence identity,” “percent identity,” or “% identity” refer to the percentage of residue matches between at least two amino acid sequences aligned using a standardized algorithm. Methods of amino acid sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions.
• Percent identity for amino acid sequences may be determined as understood in the art.
• the structural similarity is typically at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity.
• Polypeptide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
• Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, may be used to describe a length over which percentage identity may be measured.
• a “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
• specific binding pairs are antigens and antibodies, biotin and streptavidin, ligands and receptors and complementary nucleotide sequences. The skilled person is aware of many other examples. Further, the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule.
• the specific binding pair comprises nucleic acid sequences
• they will be of a length to hybridize to each other under conditions of the assay, preferably greater than 10 nucleotides long, more preferably greater than 15 or 20 nucleotides long.
• Vectors and Production Transgenic cells expression said polynucleotides also form an aspect of the invention.
• a transgenic cell may be obtained by introducing a recombinant nucleic acid molecule that encodes a protein of this disclosure.
• the term “recombinant nucleic acid” refers to a polynucleotide that is manipulated by human intervention.
• a recombinant nucleic acid molecule can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature.
• the two or more nucleotide sequences can be operatively linked and, for example, can encode a fusion polypeptide.
• a recombinant nucleic acid molecule also can be based on, but manipulated so as to be different, from a naturally occurring polynucleotide, for example, a polynucleotide having one or more nucleotide changes such that a first codon, which normally is found in the polynucleotide, is biased for chloroplast codon usage, or such that a sequence of interest is introduced into the polynucleotide, for example, a restriction endonuclease recognition site or a splice site, a promoter, a DNA origin of replication, or the like. Any appropriate technique for introducing recombinant nucleic acid molecules into cells may be used.
• constructs refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand.
• Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies.
• Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies.
• a “vector” is capable of transferring gene sequences to target cells.
• vector construct typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
• the term includes cloning and expression vehicles, as well as integrating vectors.
• the constructs and vectors provided herein may be prepared by methods available to those of skill in the art. Notably each of the constructs or expression cassettes claimed are recombinant molecules and as such do not occur in nature.
• the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, and recombinant DNA techniques that are well known and commonly employed in the art. Standard techniques available to those skilled in the art may be used for cloning, DNA and RNA isolation, amplification and purification. Such techniques are thoroughly explained in the literature.
• the terms “heterologous promoter,” “promoter,” “promoter region,” or “promoter sequence” refer generally to transcriptional regulatory regions of a gene, which may be found at the 5′ or 3′ side of the polynucleotides described herein, or within the coding region of the polynucleotides, or within introns in the polynucleotides.
• a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence.
• the typical 5′ promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
• a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
• the disclosed polynucleotides are operably connected to the promoter.
• a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence.
• a promoter is operably linked to a polynucleotide if the promoter is connected to the polynucleotide such that it may affect transcription of the polynucleotides.
• the polynucleotides may be operably linked to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 promoters.
• Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-preferred and tissue-specific promoters.
• the promoter is a constitutive promoter capable of driving high levels of an operably linked protein coding sequence.
• the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid).
• Non-viral vector delivery systems include DNA plasmids, RNA (e.g.
• Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
• Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
• Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam TM and Lipofectin TM ).
• Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
• lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.
• RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
• Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
• Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues. The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells.
• Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
• Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol.
• MiLV murine leukemia virus
• GaLV gibbon ape leukemia virus
• SIV Simian Immuno deficiency virus
• HAV human immuno deficiency virus
• adenoviral based systems may be used.
• Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
• Adeno-associated virus vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
• Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle.
• the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed.
• the missing viral functions are typically supplied in trans by the packaging cell line.
• AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
• Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
• the cell line may also be infected with adenovirus as a helper.
• the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
• labels or “reporter molecules” are chemical or biochemical moieties useful for labeling a nucleic acid (including a single nucleotide), polynucleotide, oligonucleotide, or protein ligand, e.g., amino acid, peptide sequence, protein, or antibody.
• “Labels” and “reporter molecules” include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionucleotides, enzymes, substrates, cofactors, inhibitors, radioactive isotopes, magnetic particles, and other moieties known in the art. “Labels” or “reporter molecules” are capable of generating a measurable signal and may be covalently or noncovalently joined to an oligonucleotide or nucleotide (e.g., a non-natural nucleotide) or ligand.
• a "targeting molecule” or “targeting agent” is a peptide or other molecule that binds to a targeted component).
• the binding affinity of the targeting molecule may be in the range of 1 nM to 1 ⁇ M.
• the targeting molecule may be an antagonist of a receptor on the surface of a targeted cell.
• adjuvant refers to a compound that, with a specific immunogen or antigen, will augment or otherwise alter or modify the resultant immune response. Modification of the immune response includes intensification or broadening the specificity of either or both antibody and cellular immune responses. Modification of the immune response can also mean decreasing or suppressing certain antigen-specific immune responses.
• the adjuvant is a cyclic dinucleotide.
• Suitable antibodies and antibody fragments may be agonistic or antagonistic and include those with anticancer activity and those which modify host cell responses to the cancer, for example: an agonist or antagonistic antibody or antibody fragment may decrease vascularization or normalize vascularization of the tumor.
• agonistic antibodies or other encoded proteins may render the host cell more visible to the host's innate and adaptive immune responses, for example by expressing antigens, danger signals, cytokines or chemokines to attract and activate the same, or by binding to co-stimulatory or checkpoint pathway molecules to enhance adaptive immune responses.
• Registered therapeutic antibodies suitable for use in the methods described herein include without limitation abciximab, adalimumab, alemtzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, cetuximab, certolzumab, daclizumab, denosumab, eculzumab, efalixumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, ofatumumab, palivizumab, panitumumab, ranibizumab, rituximab, tocilizumab, tositumomab and trastuzumab.
• Antibody-binding fragments includes an antibody fragment able to target the antigen with the same, similar or better specificity to the original “antibody” from which it was derived.
• Antibody fragments include: Fab, modified Fab, Fab′, modified Fab′, F(ab′) 2, Fv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online 2(3), 209-217).
• Multi-valent antibodies may comprise multiple specificities e.g. bispecific or may be monospecific (see for example WO 92/22853, WO05/113605, WO2009/040562 and WO2010/035012).
• binding domain refers to a domain which (specifically) binds to (i.e., interacts with or recognizes) a given target epitope or a given target site on a target molecule (antigen), such as, e.g., CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, MUC17, HER2, EpCAM, EGFR, GD2 or CD3.
• a target molecule such as, e.g., CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, MUC17, HER2, EpCAM, EGFR, GD2 or CD3.
• the structure and function of the first binding domain (recognizing, e.g., CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, HER2, EpCAM, GD2, EGFR or MUC17), and preferably also the structure and/or function of the second binding domain (recognizing CD3), is/are based on the structure and/or function of an antibody, e.g., of a full-length or whole immunoglobulin molecule.
• the first and second binding domain are connected with a linker ( Figure 7).
• the structure and function are drawn from the variable heavy chain (VH) and/or variable light chain (VL) domains of an antibody or fragment thereof.
• the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VH region).
• the second binding domain preferably also comprises the minimum structural requirements of an antibody which allow for the target binding.
• the second binding domain comprises at least three light chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3 of the VH region).
• one or more of the antigen binding domains are human or humanized or chimeric.
• the antibody construct comprises a single chain antibody construct.
• An scFv comprises a variable heavy chain, an scFv linker, and a variable light domain.
• the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, the C-terminus of which is attached to the N-terminus of a variable heavy chain (N-vh- linker-vl-C), although the configuration can be switched (N-vl-linker-vh-C).
• the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, the C- terminus of which is attached to the N-terminus of a variable light chain (N-vl-linker-vh-C), although the configuration can be switched (N-vh-linker-v-C).
• Peptide linkers may be used in the context of antigen binding domains and variable domains (VH/VL).
• a peptide linker may link variable domains and/or may be used to fuse a third domain to an antibody construct.
• Peptide linkers used in the context of the disclosure do not comprise polymerization activity.
• Peptide linkers also may be used to attach other domains or modules or regions (such as half-life extending domains) to an antigen binding protein, such as the bispecific antibody constructs described herein.
• suitable peptide linkers are those described in U.S.
• the antibody construct comprises a third domain comprising a “Fc” or “Fc region” or “Fc domain,” which refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
• Fc domain refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N- terminal to these domains.
• Fc may include the J chain.
• the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cy1 (Cy1 ) and Cy2 (Cy2).
• a bispecific antibody construct of the disclosure is preferably based on an IgG antibody (which includes several subclasses, including, but not limited to lgG1, lgG2, lgG3, and lgG4).
• the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat.
• amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcyR receptors or to the FcRn receptor.
• a bispecific antibody construct comprising a first binding domain that binds to a target cell surface antigen and a second binding domain that binds to human CD3 on the surface of a T cell.
• the target cell surface antigen can be any antigen present on a brain cancer cell, preferably a glioblastoma specific antigen.
• the bispecific antibody construct comprises a third domain comprising, in an amino to carboxyl order, hinge-CH2 domain-CH3 domain-linker-hinge- CH2 domain-CH3 domain.
• each of the first and second binding domains comprise a VH region and a VL region.
• the formulations described herein comprise a bispecific antibody construct which binds human CD3 and human EGFR, or human CD3 and human MSLN, or human CD3 and human DLL3, or human CD3 and human FLT3, or human CD3 and human CDH19, or human CD3 and human BCMA, or human CD3 and PSMA, or human CD3 and human CD33, or human CD3 and human CD19, human CD3 and human CD70, or human CD3 and human MUC17, or human CD3 and human CLDN18.2.
• solid matrix refers to any format, such as beads, microparticles, a microarray, the surface of a microtitration well or a test tube, a dipstick or a filter.
• the material of the matrix may be polystyrene, cellulose, latex, nitrocellulose, nylon, polyacrylamide, dextran or agarose.
• the following materials and methods are provided to facilitate practice of the invention. The skilled person is aware that other techniques can be employed.
• Western blot Cells were lysed with 1% SDS lysis buffer (1% SDS, 100mM NaCl, 10mM Tris(base) pH7.5). Protein concentration was measured by the Bradford Assay (Bio-Rad).
• SDS lysis buffer 1% SDS, 100mM NaCl, 10mM Tris(base) pH7.5
• Protein concentration was measured by the Bradford Assay (Bio-Rad).
• For immunoblot cells were lysed in boiling 1%SDS lysis buffer, sample buffer was added and samples were boiled for 5 minutes before separation by electrophoresis on 4-12% SDS-PAGE gel in the electrophoresis chamber (Bio-Rad).
• Protein was transferred to nitrocellulose membrane using the same electrophoresis chamber. Non-specific protein binding was prevented by blocking the membrane with the Incept (PBS) blocking solution (Li-Cor). Membrane was incubated at 4 degrees overnight in blocking solution with primary antibodies (pERK, pAKT, total AKT or Vinculin from Cell signaling). After five washes with PBS-T (0.1% Tween-20), the blot was incubated with respective secondary antibodies (Li-COR Rabbit (1:10,000) at room temperature. The Li-Cor Odyssey imager was used for detection according to manufacturer's instruction.
• PBS Incept
• Li-Cor Li-Cor Odyssey imager was used for detection according to manufacturer's instruction.
• hESC Culture and Neural Differentiation hESCs WA-09, HES-3 (NKX2.1::GFP), and iPSCs (C72 and SeV6) were maintained on mouse embryonic fibroblasts and dissociated with accutase (Innovative Cell Technologies) for differentiation or dispase for passaging (Chambers et al., 2009).
• Differentiation media were described previously (Kriks et al., 2011): knockout serum replacer (KSR) and N2 medium for neural induction, and neurobasal medium + B27 (GIBCO) and N2 supplements (Invitrogen) for neuronal differentiation.
• Small-molecule compounds were as follows: XAV939 (2 mM; Stemgent;), LDN193189 (100 nM; Stemgent), SB431542 (10 mM; Tocris Bioscience), purmorphamine (1–2 mM; Calbiochem), ascorbic acid (200 mM), and dibutyryl-cyclic AMP (200 mM; both from Sigma-Aldrich).
• Recombinant growth factors were as follows: SHH (C25II; 50–500 ng/ml), Noggin (125 ng/ml), DKK1 (250 ng/ml), BDNF (10 ng/ml), all from R&D Systems, and FGF2 (5–10 ng/ml; Promega).
• ES or iPSC cells can be used for human treatment.
• iPSC can be prepared using standard protocols as described in Ghaedi et al. in Methods Mol Biol. (2019); 1576: 55–92, which is incorporated herein by reference.
• Modification of stem cells Transgenes can be introduced using a variety of methods as explained above. These include lentiviral transduction, retroviral transduction, piggybac vectors etc. In the example below, a lentiviral construct was employed, with the transgene under the CAG promotor referred to herein as a modified pLEX306 construct. These transgenes serve multiple purposes, including delivering a therapeutic agent or genes/factors/modifications that improve migration.
• BiTEs useful in the present invention, include without limitation,EGFR-BiTE, GD2-BiTE, B7H3-BiTE, IL13RA-BiTE, KDR-BiTE, EGFRvIII-BiTE, HER2-Bite, Epcam-BiTE
• EGFR-BiTE GD2-BiTE
• B7H3-BiTE IL13RA-BiTE
• KDR-BiTE KDR-BiTE
• EGFRvIII-BiTE HER2-Bite
• Epcam-BiTE Epcam-BiTE
• Each of the aforementioned BiTe’s can be synthesized using the methods described herein.
• CXCR4 blocking peptides are also of interest.
• Chemokines that remodel the tumor microenvironment to reduce the immune suppressive nature of these tumors like IL6, IL1b, CSF2, IFN ⁇ , TNF ⁇ can also be used to advantage in the treatment of brain cancer.
• Markers that enhance detection Improving migration, e.g., via introduction of a nucleic acid construct, (e.g., a lentiviral construct) with gene driven by hSYN, EIF2A, CAG, PGK or other promoter which result in: - CXCR4 expression; - CXCR4 S338X expression; - CXCR7 expression; - CXCR7 S335X expression; - CD74 expression; - CD44 expression; - Expression of or more sequences encoding variants of CXCR4 and CXCR7 (e.g., splice variants), said sequences lacking or comprising modifications to prevent degradation of sequence alterations that alter degradation of these receptors upon ligand binding.
• a nucleic acid construct e.g., a lentiviral construct
• variants of CD74 and CD44 e.g., (splice variants) - shRNA targeting CXCR7 (also known as ACKR3, shRNAs ordered from Sigma Aldrich: (TRCN0000014512, TRCN0000358300, TRCN0000363139, TRCN0000363205, TRCN0000378566) - CRISPR can also be used to modify the endogenous CXCR4 or knock out CXCR7: a) CXCR4 S338X generation by CRISPR sequence: CTCTCCAAAGGAAAGCGAGG (SEQ ID NO: 2), PAM: TGG; CRISPR from IDT technologies (Hs.HC9.BLWL3886.AA), with the following sequence /AltR1/rCrU rCrUrC rArArArGrG rArArArGrGrGrUrUrArA
• HLA class II histocompatibility antigen gamma chain isoform c [Homo sapiens] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFS ILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMR MATPLLMQALPMGALPQGPMQNATKYGNMTEDHVMHLLQSHWNWRTRLLG WV*(SEQ ID NO:16) >CD74-2 NP_001020330.1 HLA class II histocompatibility antigen gamma chain isoform a [Homo sapiens] MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFS ILVTLLLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPK
• hESC- derived NKX2.1::GFP+ cells were isolated by FACS and carefully injected (5,000 cells) into the periventricular region of the MGE using an oocyte microinjector (Nanoject II; Drummond Scientific). After 2–6 days, cultures were fixed and immunostained. For cell counting, zone 1 was defined as the region from the ventricular zone of the lateral ganglionic eminence extending to the pallial-subpallial border, and zone 2 was defined as the region from the lateral cortex through the neocortex to the cortical hem.
• beads loaded with recombinant ligand can be placed on brain slices of day 13.5-day old mouse embryos of the Lhx6-GFP reporter strain (Tg(Lhx6- EGFP)BP331Gsat) 48 to determine if they attract MIPs.
• MIPs are GFP positive (Lhx6 promotor expression).
• Coronal telencephalic slices 250 mm were prepared as described previously (Anderson et al., 2001) and maintained in neurobasal medium/B27 with 5 ng/ml FGF2 (Promega). Beads loaded with BSA or recombinant ligand are placed at specific locations on the section for 12h.
• the Anti-EGFR.BiTE and anti-CD19.BiTE were synthesized and cloned into a third- generation lentiviral plasmid backbone under the regulation of the CAG promotor.
• BiTEs were designed against wild-type EGFR and CD19 with both sequences flanked by an Ig ⁇ leader peptide and a His-tag element ( Figure 7).
• Individual anti-EGFR and anti-CD3 scFv sequences were as previously described.
• the sequences of CD19 BiTE including the anti-CD3 scFv, as well as the anti-EGFR scFv, were obtained from publicly available sequences for blinatumomab and cetuximab, respectively. Ribosomal skip sites were incorporated at appropriate locations.
• MIPs 5000 RFP+ brain tumor cells are plated in 100ul (N2AA media, see MIP differentiation protocol) in a low attachment round bottom well plate (MS-9096UZ), are spun down 5min at 150g and are allowed to form a sphere overnight. Subsequently, 50k MIPs are overlayed on top of the brain tumor sphere mixed in 50% Matrigel. MIP migration to the brain tumor sphere is tracked using the Incucyte live imager over 3-7 days. Everything is repeated in triplicate.
• 2D monolayer Brain tumor cells (10,000 cells, expressing nuclear RFP) are plated in their specific brain tumor media together with bispecific engager expressing MIPs (2,000 cells, targeting or control engager) in triplicate. The following day 50k T-cells are added and each well is imaged every 8h. The number of brain tumor cells is counted (2D assay) using the Incucyte software.
• Brain tumor cells (10,000 cells, expressing nuclear RFP) are plated in their specific brain tumor media together with bispecific engager expressing MIPs (2,000 cells, targeting or control engager) in triplicate in round bottom well plates (MS-9096UZ), are spun down 5min at 150g and are allowed to form a sphere overnight. The following day 50k T-cells are added and each well is imaged every 8h. The number of brain tumor cells is inferred by the strength of the RFP signal using the Incucyte software. T-cells were obtained from the Penn Immunology Core.
• Transwell migration assay 20k interneurons are plated in the top well of a transwell migration plate (Sartorius, 4582) coated with growth factor depleted Matrigel (1/50 dilution for 3h), chemokines or conditioned media is added 24h post plating and transwell migration is determined 3-5 days later using the Incucyte Live Imager. Conditioned media is generated by incubating 2 million cells in interneuron media for 48h.
• In vivo mouse assays 1 Eight to 10-week-old Nu/J mice were anesthetized with 2.5% isoflurane, placed into the stereotactic frame, then injected sequentially with 500,000 tumor cells.
• mice were allowed to establish for 2 weeks, then mice underwent a second surgical procedure with stereotactic injection of 200,000 MIPs a total of 2 mm apart from initial injection site. One to two weeks later the animals are sacrificed and stained for MIP markers (GFP or NCAM) to assess how well the MIPs migrated to the tumor. 2) In vivo killing assays: a) co-injection of MIPs, tumor cells and T-cells: Eight to 10-week-old Nu/J mice were anesthetized with 2.5% isoflurane, placed into the stereotactic frame, then injected with 500,000 brain tumor cells, of 200,000 MIPs and 2 million activated T-cells in a single location.
• MIP markers GFP or NCAM
• T-cells were obtained from the Penn immunology core and activated using standard protocols (found on the world wide web at thermofisher.com/order/catalog/product/11161D).
• T-cells were obtained from the Penn immunology core and activated using standard protocols (found on the world wide web at thermofisher.com/order/ catalog/product/11161D).
• CXCR4 is one of the most important receptors for MIP migration. Multiple CXCR4 ligands are expressed in brain tumors. The methods described below can be used to advantage to identify ligands which are most effective at modulating migration and cell killing. Identification of pro-migratory ligands/biomarkers expressed in brain tumors. a) In vitro by western blot: MIPs are growth factor starved for 8h and subsequently treated with recombinant ligand (MIF, CXCL12, HMGB1).
• MIF recombinant ligand
• MIPs are harvested 0, 5, 15 and 60 minutes post exposure. CXCR4 pathway activation is then evaluated by western blot using pERK antibodies (assessing RAS-MAPK pathway activation)
• MIF, CXCL12 and/or HMGB1 can also be overexpressed in in brain tumor cells lacking expression of these ligands and migration (LN319) using suitable nucleic acid constructs (e.g., lentiviral or AAV expression vectors). Migration capacity in these modified brain tumor cells can then be assessed in the migration assay described.
• c) In vivo migration assay Cell with knocked down/knocked out of MIF, CXCL12 or HMGB1 (shRNAs or CRISPR) will be used in our in vivo migration assay described herein. 2) Patient selection: Biomarkers so identified can be used to select patient. For example, if MIF is identified as a marker, MIF levels in blood should correlate with expression levels of MIF in the tumor.
• MIPs can be coated with ferrofluid particles to facilitate imaging during MRI. The skilled person is aware of the many protocols suitable for generation of interneurons.
• the protocol recommends withdrawing all morphogenic signaling molecules from the culture starting on D22.
• Plating Matrigel -coated dishes Starting at Day 22, can use the following procedure to passage cells: 1. Dissociate interneuron spheres in 0.05% trypsin + 100 mM trehalose 2. Incubate for 5 min at 37°C, then triturate to fully dissociate the spheres 3. Add equal volume of N2AA media with 0.1 ⁇ M SAG and 10% FBS to neutralize Trypsin, supplemented with Turbo DNase o (for 2 U/mL concentration, add 1 ⁇ L of DNase per mL of media) 4.
• the Clear Point® Neuro Navigation platform can be used for safe and effective delivery of the components of the brain cancer cell killing agents described here in humans.
• the ClearPoint® Neuro platform enables stereotactic navigation and delivery to the brain.
• Applications include, without limitation, electrode insertion, placement of laser catheters, biopsy, and delivery of approved therapeutics in the brain, including biosimilars, gene and stem cell therapies.
• T-cells or MIPs can also be administered with repeated dosing though an Ommaya Reservoir directly into the ventricles of the brain.
• An ommaya reservoir is in essence a catheter connected to a soft plastic dome-shaped reservoir. The catheter is placed in the ventricles of the brain, the reservoir is placed under the skin on top of the skull.
• Example I The present inventors have discovered that MIPs migrate to glioblastoma tumors both in vitro and in vivo. Moreover, we have shown that MIPs are able to express and secrete bispecific engagers that activate the local immune system to eliminate glioblastoma. Our data show that EGFR is a potential epitope to target glioblastoma.
• CXCL12 stromal derived factor 1; SDF1
• SDF1 stromal derived factor 1
• Fig 1A CXCR7 signaling pathway 22
• expression of CXCL12 enhances the invasiveness of many glioblastomas 23,24 by activating CXCR4 on the vascular endothelium and microglia/macrophages.
• the present inventors experts in interneuron development, and glioblastoma treatment, realized that MIPs transplanted into the vicinity of those CXCL12-expressing glioblastomas should migrate to the tumor.
• MIP could be used to advantage as a therapeutic delivery vehicle for cytotoxic agents for the killing of glioblastoma and other brain cancer cells.
• CXCL12 a large percentage of glioblastoma also highly express the CXCR4 and CXCR7 ligands HMGB1 25 and MIF 26 (Fig 1B).
• HMGB1 can also bind with CXCL12 to CXCR4 and CXCR7, additionally it has been reported that MIF needs the presence of the CD74 receptor to bind CXCR4 or CXCR4.
• MIF also binds the CD44/CD74 complex.
• Many low-grade gliomas and brain metastases also secrete CXCL12, HMGB1 and MIF (FIGS.
• BiTEs are bispecific monoclonal antibodies where the single-chain variable fragment 27 (scFv) of an anti-CD3 antibody is bound to the scFv of a targeting antibody 28,29 , in essence creating an adaptor linking T-cells to a tumor antigen of choice 29,30 (Fig 2A,B). This engagement induces an immune response against the bound tumor cell. Without the BiTE, the T- cell does not recognize nor respond to cells expressing the target.
• BiTEs have a short half-life; hence, they will only engage with local T-cells at the BiTE production site. This allows for use of BiTEs to target antigens that are not unique to the tumor yet still retain a low toxicity profile as was for shown for an EGFR-BiTE 31 . Importantly, multiple BiTE clinical trials are ongoing, and the FDA has approved Blinatumomab, a CD33-BiTE used for refractory or recurrent B-ALL and teclistamab-cqyv, a B-cell maturation antigen directed BiTE for adult relapsed or refractory multiple myeloma.
• MIPs can migrate to small clusters of tumor cells and are thus ideally suited to target and eradicate those small lesions post-surgery.
• Modified MIPs differentiated from patient or donor or “off-the-shelf” iPSCs available from commercial sources, can be injected into the margin of the surgical cavity to clear residual tumor cells. “Off-the-shelf” cells have been suggested for use in CAR-T cellular therapy 34 as well.
• ES embryonic stem cell
• MIPs can be derived from the donor, ES cells or iPSCs.
• iPSC and ES derived MIPs have similar migratory capacities.
• Several iPSC based cellular therapies are currently in clinical trials.
• HLA-matched MIPs will be employed.
• a kill switch can also be engineered into the MIPs to minimize undesirable adverse effects.
• the non-unique tumor antigen EGFR is targeted for our bispecific engager experiments.
• BiTEs can also be created with target validated, unique glioblastoma antigens, including for example EGFR vIII, IL13RA2, HER2, EpCAM, and GD2.
• Cellular therapies like CAR-T therapy, have revolutionized treatment for a number of cancers. Unfortunately, these therapies have so far shown limited efficacy for brain tumors, in part due to the lack of unique tumor antigens and an immune suppressive microenvironment 8,35 .
• MIPs are post-mitotic neural precursors capable of long-distance migration in the embryonic human brain via chemo-attractants such as CXCL12 20,21 .
• MIPs as a viable cellular therapy for brain cancer, particularly glioblastoma, and independently, that we can engage resident or supplied T-cells to clear brain cancer. Additionally, our investigation sheds light on new chemoattractants that also play a role in MIP migration during embryonal brain development.
• MIPs as cellular therapies for brain tumors: Even though MIPs are currently in clinical trials for the treatment of epilepsy, the use of MIPs as delivery vector for anti-tumor agents has not yet been explored. Notably, MIPs can be equipped with a range of anti-tumor agents beyond the ones suggested in this proposal. These include, without limitation, BiTEs targeting EGFR, EGFRvIII, HER2, GD2, EpCAM, B7H3, TME modulating agents (IL6, IL1 ⁇ , CSF2, IFN ⁇ , TNF ⁇ ,), markers that enhance tumor detection, e.g., fluorescent markers) and other peptides like the CXCR4 blocking peptide or other therapeutic agents.
• BiTEs targeting EGFR, EGFRvIII, HER2, GD2, EpCAM, B7H3, TME modulating agents IL6, IL1 ⁇ , CSF2, IFN ⁇ , TNF ⁇ ,
• markers that enhance tumor detection e.g., fluorescent markers
• other peptides like the CXCR
• LN229, LN319 and U87 Three (LN229, LN319 and U87) are grown in serum-containing media and form a monolayer, TM31HGG is grown in glioma stem cell media. Luciferase is expressed in the 5 lines (1746, 3058, LN229, LN319 and U87) that form tumors in vivo, allowing for live imaging.
• Lung and breast cancer cell lines The lung and breast cancer cell lines HCC1395, BT549 and HCC1806 (all grown as a monolayer in RPMI + 10% FBS + 1% Pen/Strep + 1X glutaMAX) will be used for evaluation of metastatic brain tumors.
• Migratory Interneuron Precursor cells derived from an LHX6-citrine human ES line The present study assesses MIP differentiated from human ES lines (WA09 line: NIH approved and HES3 line) and from mouse MIPs harvested from 13.5-day old embryos. For human use, MIPs differentiated from the NIH approved human ES line WA09 or iPSCs will be employed. A major subgroup of MIPs express LHX6 after cell cycle exit 42 . Therefore, the LHX6 locus was targeted in human (WA09) ES cells by the Anderson lab (and others) to express fluorescent Citrine (close to GFP in wavelength and recognized by a GFP antibody).
• Citrine 43,44 (Fig 3A-C).
• This Citrine marker is a marker employed in the lab and will not be engineered into MIPs that will be given to patients, we can use NCAM to positively identify MIP identity.
• the MIP differentiation process yields MIPs between days 24 and 40 post differentiation start 45 .
• MIPs can be flow sorted using the Citrine marker which also allows us to identify the MIP in in vivo assays by immunohistochemical staining.
• iPSC derived MIPs will be preferred for use in a clinical setting, WA09 ES cell line derived MIPs were utilized herein, since both migrate and differentiate similarly.
• FIG. 3D shows staining of MIPs with neuronal markers MAP2 and SOX6.
• Incucyte Live Imaging System In vitro live imaging was performed using the Incucyte® S3 Live-Cell imager (Essen bioscience), a fluorescence microscope embedded in the incubator, that images each well of a plate with high frequency (every 1-8 hours). This frequent imaging gives us high-quality data and the ability to track MIP migration or glioblastoma killing 46 .
• MIPs potently migrate to glioblastoma in vitro: MIPs harvested from both mouse embryonic brains and MIPs differentiated from human ES cells potently migrate to glioblastoma spheres in our spheroid migration assay (Fig 3E).
• glioblastoma cells are plated in a round bottom well plate and are allowed to form a sphere overnight. Subsequently, 50k MIPs (green) are overlayed on top of the glioblastoma sphere mixed in 50% Matrigel. MIP migration to the glioblastoma sphere is tracked using the Incucyte live imager over 3-7 days (Fig 3F). MIPs migrate to the majority of glioblastoma cell lines (5/7) evaluated (robust migration: 1746, LN229, U87; moderate migration: 1763, 3058; no/limited migration: 1769, LN319).
• MIPs potently and significantly migrate to 8/13 conditioned high grade glioma medias evaluated (defined as >4x migration versus control), where 20k cells were plated in the top well of a transwell migration plate coated with a 1/50 dilution of growth factor depleted Matrigel. (Fig 3F).
• MIPs potently migrate to glioblastoma in vivo: Crucially, our data also shows that MIPs, derived from mouse embryonic brains or differentiated from human ES cells, transplanted in the forebrain, robustly and aggressively migrate to the margin of glioblastoma within 14 days (FIGS. 3G, 3H). Tantalizingly, MIPs also wrap around micro-lesions, underscoring the sensitivity and specificity of the MIP migration process (FIG. 3G). The ability of MIPs to hone into patches of glioblastoma cells that migrated away from the main tumor should enhance cell killing for the successful treatment of glioblastoma in the clinic.
• Glioblastoma cells (10,000 cells, expressing nuclear RFP) are plated together with bispecific engager expressing cells (2,000 cells, targeting or control engager) in triplicate. The following day 50k T-cells are added and each well is imaged every 8h. The number of glioblastoma cells is counted (2D assay) or inferred (spheroid assay) using the Incucyte software. The bispecific engagers were assessed in a 2-stage process, where our engagers are initially expressed in easy to manipulate 293T cells, which allows us to quickly assess the engager’s ability to eliminate glioblastoma in vitro.
• Bispecific engager MIP cells are generated by infecting our LHX6 ES line with lentivirus, delivering the bispecific engager under a CAG promotor, selecting transduced cells with puromycin and subsequently differentiating these ES cells to MIPs.
• Figure 4A and B shows representative images of potent EGFR-BiTE induced killing of glioblastoma cell lines. Importantly, 4/7 cell lines show very potent EGFR-BiTE induced killing, 2/7 show moderate killing, and line 1/7 showed no killing.
• EGFR-BiTEs potently kill glioblastoma in vivo:
• glioblastoma cells are injected into the cortex of immunocompromised mice (Nu/J).
• the tumor is allowed to establish, and interneurons are injected at 2mm distance from the tumor margin.
• a canula is implanted into the ventricles, and repeated T-cell dosing (weekly) 2 weeks post interneuron injection is performed.
• T-cells are added and each well is imaged every 8h.
• the number of glioblastoma cells is counted (2D assay) or inferred (spheroid assay) using the Incucyte software.
• the bispecific engagers were assessed in a 2-stage process, where our engagers are initially expressed in easy to manipulate 293T cells, which allows us to quickly assess the engager’s ability to eliminate glioblastoma in vitro. When successful glioblastoma killing is observed, we repeat our killing assay with bispecific engager expressing MIPs.
• Bispecific engager MIP cells are generated by infecting our LHX6 ES line with lentivirus, delivering the bispecific engager under a CAG promotor, selecting transduced cells with puromycin and subsequently differentiating these ES cells to MIPs.
• FIGS. 4F and 4G show the killing induced by a GD2 BiTE of tumor 1769. Three of the 4 lines showed potent killing and include 1763 and 1746.
• HMGB1 and MIF bind CXCR4 and CXCR7 as well 25,26 . Both play a role in the chemoattraction of other CXCR4 expressing cells (dendritic cells, CD4+ T-cells and macrophages) 25,38 .
• CXCR4 expressing cells dendritic cells, CD4+ T-cells and macrophages
• MIF and HMGB1 could contribute to MIP-to-glioblastoma migration as they are highly expressed in many glioblastoma (FIG. 1B) and other brain tumors. These factors have not been identified as chemoattractants of MIPs during development.
• CXCL12 is one of the most prominent MIP chemoattractants 20,21,47 . MIPs migrate to a source of CXCL12 in vitro. Additionally, when Li et al. 48 placed CXCL12 loaded beads on brain slices of 13.5-day old mouse embryos, they observed MIP migration to these beads within 12h 48 (Fig. 5). Upon binding of CXCL12 to CXCR4, CXCR4 becomes phosphorylated and activates a number of downstream pathways including the RAS pathway.
• phosphorylated CXCR4 binds Arrestin which stimulates degradation of CXCR4 through endocytosis 22,49 (Fig 1A). If the C-terminal end of CXCR4 is truncated (S338X), Arrestin can no longer bind and CXCR4 remains active on the cell surface, enhancing CXCR4 signaling and enhancing effects of CXCL12 driven migration. 50,51 Other factors/ligands that guide/support MIP migration are NRG1, HGF, BDNF and GDNF.
• CXCR4 ligands highly express CXCR4 ligands: As noted above, the CXCL12-CXCR4 axis is one of the best characterized MIP chemoattractant pathways. Analysis of the CBTN and GTex databases show that 34% of pediatric glioblastoma express higher levels of CXCL12 than the surrounding cortex (Fig 1B, Table 1). In recent years other CXCR4 ligands have been identified (HMGB1 and MIF).
• MIF 187TPM vs the 95 th percentile normal cortex expression of 10.2TPM.
• CD74 isoforms (sequences provided above) could drive MIP migration to MIF expressing tumors.
• expression of CD44 alone or in combination with CD74, CXCR4 and/or CXCR7 could drive MIP migration to glioblastoma more potently.
• Ligands/Receptors which Induce MIP Migration Ligand Fold vs pHGG 80th receptor cortex perc (TPM) Genbank numbe are CCL2 NM_002982; WNT5A NM_001256105, NM_001377271, NM_001377272, NM_003392; CCL3 NM_002983; NTN1 NM_004822; SEMA3A NM_006080; HGF NM_000601; NM_001010931; NM_001010932; NM_001010933; NM_001010934; GPNMB NM_001005340; NM_002510; BMP2 NM_001200; PGF NM_001207012; NM_001293643, NM_002632; HMGB2 NM_001130688; NM_001130689; NM_002129; IL32 NM_001012631; TGFB1 NM_000
• Glioblastoma express ligands of migratory receptors expressed on MIPs: Glioblastoma secrete ligands/ factors that induce migration of various cell types, including those that create an immune suppressive TME.
• MIP-to-glioblastoma migration We analyzed publicly available pHGG (133 tumors, CBTN, pedcibioportal.org), interneuron 52 and normal cortex (Gtex) expression data. Receptors that play a role in cellular migration and are expressed in MIPs were identified. Subsequently, we evaluated if their ligand expression is at least 2-fold higher than median expression in normal cortex in at least 20% of pHGG samples.
• Table 1 lists 25 MIP-to- glioblastoma chemoattractants/mitogens which satisfy these criteria, their respective receptors expressed on MIPs, the signaling pathway they activate and the fold change expression (80 th percentile in tumor vs median expression in normal cortex).
• Our preliminary results show that treating MIPs with recombinant ligands as expected activates downstream signaling pathways.
• Fig 3D With our in vitro spheroid migration assay (Fig 3D), how well a glioblastoma sphere attracts MIPs can be quantified by identifying the migration front of guided MIPs. Each ligand that was able to activate their signaling pathways in MIPs can then be transiently knocked down in 3 glioblastoma cell lines using siRNAs (On-Target smartpool from Horizon Discovery) and any changes in MIP migration assessed (in triplicate). Non-targeting siRNAs can be used as a negative control. Ligand knockdown can be further evaluated with qPCR. Ligands that significantly reduce MIP-to-glioblastoma migration will be further analyzed in our in vivo assay.
• Ligands in a glioblastoma cell line can be knocked out using CRISPR, and, or RNAi silencing, and evaluated when MIPs are no longer attracted to the tumors in vivo. Following knockout of the desired target, cells will be single cell cloned and knockout of the target gene can be validated with qPCR and genomic sequencing. Of note, we have already generated single cell knock-out lines of CXCL12 and in vivo MIP migration can be evaluated. For each ligand that passes our criteria, we can inject three single cell knockout and control clones in duplicate (6 control and 6 experimental mice) and evaluate MIP migration in our in vivo migration assay.
• MIPs (200k) will be injected 2mm from the tumor injection site and allowed to migrate for 2 weeks, as shown in Fig. 3F. Animals will be sacrificed, and the number of MIPs (Citrine expression) can be quantified in serial sections present at the tumor. Any ligand that significantly reduces the number (2-fold) of MIPs that arrive within 2 weeks at the tumor site are implicated as biomarkers for MIP-to-glioblastoma attraction. Finally, the identified ligands can be expressed under 3 different promotors that induce varying levels of expression (promotor PGK: low, EIF1a: medium CAG: high expression) in the LN229 glioblastoma cell line that fails to attract MIPs.
• promotors that induce varying levels of expression
• the LN229 cell line expresses low levels of CXCL12 and MIF.
• the in vivo migration assay can be performed with these lines in triplicate (for each promotor both ligand and a control) and the number of MIPs which reach the tumor in 14 days determined. While inducible promoters could be employed, specific promotors are preferred, as it would be difficult to attain and maintain specific and constant expression levels through tetracycline administration to the brain in vivo.
• IHC staining can be performed on our pediatric HGG Tissue Microarray (TMA) (76 tumors) to evaluate the number of gliomas that are positive for the ligand and classify the expression level, e.g., as low, medium, or high.
• TMA Tissue Microarray
• a TMA of the orthotopic xenograft lines can also be generated to match expression level to chemoattraction. While IHC staining is not ideal for estimating/comparing expression levels; it does enable comparison of protein expression levels of the ligands. Furthermore, IHC used in initial screenings of patient biopsies to determine candidacy for therapy. Expanding the patient population that can be helped with a MIP therapy: CXCR4 and CXCR7 play an essential role for MIP guidance. We have shown that CXCR4 ligands are highly expressed in glioblastoma and that exposure of MIPs to recombinant CXCR4 ligands activates the RAS-MAPK pathway.
• CXCR4 or CXCR7 prevents its degradation and leads to aberrant/ elevated MIP migration in mice.
• MIP with modified or over-expressed CXCR4 or CXCR7 will become more sensitive to CXCR4 ligands and will more rapidly migrate to glioblastoma.
• MIF glioblastoma highly expressed CXCR4 and CXCR7 ligands
• CD74 CD74 and CD44 receptors together.
• CAG promotor drives high expression of a transgene in both ES cells and differentiated MIPs.
• transgenes in the ES cells and were able to differentiate these to MIP that retained their migratory capacity.
• the same approach can be used to express CXCR4 or a control LACZ construct in ES cells prior to MIP differentiation.
• CRISPR technology will be employed (nuclear electroporation of gRNA, Integrated DNA Technologies) to endogenously modify the CXCR4 receptor in the ES cells by introducing a homozygous stop mutation (S338X) that has been shown to prevent degradation of CXCR4 50,51 .
• single cell clone ES cells can be isolated and differentiated into MIPs (3 clones). Three control single cell clones will be generated using a control gRNA. Of note, we have successfully introduced endogenous mutations in other genes using this technology.
• the migratory capacity of the modified MIPs can be evaluated in the following assays: (1) Western blot kinetic experiment: The modified MIPs can be contacted with CXCR4 ligands (CXCL12, HMGB1, MIF) and a western blot kinetic experiment performed to assess prolonged RAS-MAPK pathway activation and CXCR4 phosphorylation (0, 1, 5, 10, 15, 30 and 60 min exposure).
• the migratory capacity of modified and control MIPs will be determined in triplicate for all of our glioblastoma cell lines.
• MIPs expressing sequences for modification which include CXCR4 variants, CXCR4 S338X variant, CXCR7 variant, CXCR7 S352X variants, CD44 variants, CD74 variants and other sequences listed throughout this application.
• Glioblastoma multiforme (GBM) An overview of current therapies and mechanisms of resistance. Pharmacol Res. 2021;171:105780. doi: 10.1016/j.phrs.2021.105780.
• Badie B Schartner J, Prabakaran S, Paul J, Vorpahl J. Expression of Fas ligand by microglia: possible role in glioma immune evasion. J Neuroimmunol. 2001;120(1-2):19-24. doi: 10.1016/s0165-5728(01)00361-7. PubMed PMID: 11694315. 5.
• Lam RS O'Brien-Simpson NM, Holden JA, Lenzo JC, Fong SB, Reynolds EC. Unprimed, M1 and M2 Macrophages Differentially Interact with Porphyromonas gingivalis. PLoS One. 2016;11(7):e0158629. doi: 10.1371/journal.pone.0158629. PubMed PMID: 27383471; PMCID: PMC4934774.
• Schulz D Severin Y, Zanotelli VRT, Bodenmiller B.
• Macrophage Migration Inhibitory Factor Promotes the Interaction between the Tumor, Macrophages, and T Cells to Regulate the Progression of Chemically Induced Colitis-Associated Colorectal Cancer. Mediators Inflamm. 2019;2019:2056085. doi: 10.1155/2019/2056085. PubMed PMID: 31360118; PMCID: PMC6652048. 39.

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