KR20210075090A - Methods of Treatment of Immunotherapy-Related Toxicity Using GM-CSF Antagonists - Google Patents

Abstract

Provided is a method of neutralizing and/or eliminating human GM-CSF in a subject in need thereof comprising administering to the subject a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^{k/o CAR-T cell)} do. Also provided is a method for GM-CSF gene inactivation or GM-CSF knockout (KO) in a cell, comprising targeted genome editing or GM-CSF gene silencing. GM-CSF gene inactivated or knocked out , GM-CSF gene inactivated or GM-CSF knockout CAR-T cells (GM-CSF ^k/o CAR-T cells) and/or recombinant GM-CSF antagonist Provided is a method for preventing or reducing immunotherapy-related toxicity, comprising administering to A method of reducing the level of a cytokine or chemokine other than GM-CSF in an individual having immunotherapy-related toxicity comprising administering to the individual a recombinant hGM-CSF antagonist is provided. Also provided is a method of treating or preventing immunotherapy-related toxicity in a subject comprising administering to the subject a CAR T-cell having a GM-CSF gene knockout (GM-CSF ^{k/o CAR-T cell).} do. A method for preventing or reducing blood-brain barrier disruption in an individual treated with immunotherapy, comprising administering to the individual a CAR-T cell (GM-CSF ^{k/o CAR-T cell) having a GM-CSF gene knockout.} A method is also provided.

Description

Methods of Treatment of Immunotherapy-Related Toxicity Using GM-CSF Antagonists

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/567,187, filed October 2, 2017, and U.S. Provisional Application No. 62/729,043, filed September 10, 2018, filed on October 2, 2018 in the United States continuation-in-part of U.S. Application No. 16/149,346, continuation-in-part of U.S. Application No. 16/204,220, filed on November 29, 2018, continuation-in-part of U.S. Application Serial No. 16/248,762, filed January 15, 2019; This is a continuation-in-part of U.S. Application No. 16/283,694, filed February 22, 2019, and this application is filed on PCT Application No. PCT/US2018/053933, filed October 2, 2018, which is incorporated herein by reference. claim priority for

technical field

The present invention provides for neutralizing human GM-CSF in a subject in need thereof and/or comprising administering to the subject a CAR-T cell (GM-CSF ^{k/o CAR-T cell) having a GM-CSF gene knockout.} How to get rid of it. The present invention relates to a method for GM-CSF gene inactivation or GM-CSF knockout (KO) in cells, comprising targeted genome editing or GM-CSF gene silencing.

The present invention also provides for preventing immunotherapy-related toxicity comprising administering to a subject CAR-T cells (GM-CSF ^k/o CAR-T cells) with GM-CSF gene inactivation or GM-CSF knockout. Or to a method for reducing, wherein the GM-CSF gene is inactivated or knocked out by the method.

The present invention relates to a method of reducing blood-brain barrier disruption in a subject treated with immunotherapy comprising administering to the subject a recombinant GM-CSF antagonist. The present invention also relates to a method of preserving blood-brain barrier integrity in a subject treated with immunotherapy comprising administering to the subject a recombinant hGM-CSF antagonist. The present invention relates to a method for reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof comprising administering to the subject a recombinant GM-CSF antagonist. The present invention relates to methods of reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in subjects treated with immunotherapy without the development of immunotherapy-related toxicity. The present invention also relates to methods of reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy in the presence of the occurrence of an immunotherapy-related toxicity. The present invention also relates to a method of reducing cytokine or chemokine levels other than GM-CSF in an individual having the development of an immunotherapy-related toxicity comprising administering to the individual a recombinant GM-CSF antagonist. The present invention also relates to an immunotherapy-related toxicity in a subject comprising administering to the subject a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^k/o CAR-T cell) and a recombinant hGM-CSF antagonist. It relates to a method for treating or preventing Also provided herein is a method of inhibiting or reducing the occurrence and/or severity of an immunotherapy-related toxicity in an individual comprising administering to the individual a recombinant GM-CSF antagonist.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine secreted by a variety of cells including macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts. GM-CSF stimulates the differentiation of granulocytes and monocytes. Monocytes then migrate to tissues and mature into macrophages and dendritic cells. Therefore, the secretion of GM-CSF dramatically increases the number of macrophages. GM-CSF is also involved in the inflammatory response of the central nervous system (CNS) that induces the influx of blood-derived monocytes and macrophages and the activation of astrocytes and microglia. Immune-related toxicities include potentially life-threatening immune responses that arise as a result of the high levels of immune activation that occur with other immunotherapies. Immune-related toxicity is currently a major complication in the application of immunotherapy in cancer patients. Chimeric antigen receptor T (CAR-T) cell therapy has emerged as a new and potentially innovative therapy for cancer treatment. Based on an unprecedented response to B-cell malignancies, two CD19-targeted CAR-T (CART19) cell products were approved by the FDA in 2017. However, the widespread application of CAR-T cell therapy is limited by the emergence of unique and potentially lethal toxicity. These include cytokine release syndrome (CRS) and neurotoxicity (NT). Up to 50% of patients treated with CART19 cells develop grade 3 or higher CRS or NT and multiple deaths have been reported. These toxicities are associated with long-term hospitalization, intensive care unit (ICU) admission, and the long-term effects of NT are unknown. Controlling these CART19 cell-related toxicities is therefore essential to reduce morbidity, mortality, length of hospital stay, ICU hospitalization, necessary supportive care, and significant overhead associated with CAR-T cell therapy. It is clear that an important need remains for methods of preventing and treating immune-related toxicities. An ideal approach would minimize the risk of these life-threatening complications without affecting the efficacy of the immunotherapy, potentially efficacious by, for example, allowing safe dose escalation of immunotherapeutic compounds and/or expansion of T cells. will be able to improve

In one aspect, the present invention provides human GM-CSF in a subject in need thereof, comprising administering to the subject a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^{k/o CAR-T cell).} Methods for neutralizing and/or eliminating are provided.

In another aspect, the present invention provides a method for GM-CSF gene inactivation or GM-CSF knockout (KO) in a cell comprising targeted genome editing or GM-CSF gene silencing.

In a further aspect, the invention provides an immunotherapy-related method comprising administering to a subject a CAR-T cell (GM-CSF ^k/o CAR-T cell) having GM-CSF gene inactivation or GM-CSF knockout. A method of preventing/reducing toxicity is provided, wherein the GM-CSF gene is inactivated or knocked out by the methods described herein.

In one aspect, the invention provides a method of reducing blood-brain barrier disruption in a subject treated with immunotherapy comprising administering to the subject a recombinant GM-CSF antagonist.

In another aspect, the invention provides a method of preserving blood-brain barrier integrity in a subject treated with immunotherapy comprising administering to the subject a recombinant hGM-CSF antagonist.

In another aspect, the present invention provides a method of reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof, comprising administering to the subject a recombinant GM-CSF antagonist.

In another aspect, the present invention is blood in the object treated, the immunotherapy comprises administering to the GM-CSF gene knockout CAR-T cells (GM-CSF ^{k / o} CAR-T cells) with the object- A method of preventing or reducing brain barrier disruption is provided.

In one aspect, the present invention provides a method for reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy comprising administering to the subject a recombinant hGM-CSF antagonist, without the development of immunotherapy-related toxicity. It provides a way to delay. In a related aspect, the invention provides a method for reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in the presence of immunotherapy-related toxicity in a subject treated with immunotherapy comprising administering to the subject a recombinant hGM-CSF antagonist. provide a way

In another aspect, the invention provides a method of reducing the level of a cytokine or chemokine other than GM-CSF in a subject having the development of an immunotherapy-related toxicity comprising administering to the subject a recombinant hGM-CSF antagonist. provided that the level of the cytokine or chemokine is reduced as compared to the level of the subject during the development of an immunotherapy-related toxicity.

In a further aspect, the invention provides a method of treating or preventing immunotherapy-related toxicity in a subject comprising administering to the subject a chimeric antigen receptor-expressing T-cell (CAR-T cell) and a recombinant hGM-CSF antagonist. As such, the CAR-T cell is a cell having a GM-CSF gene 'knockout' (GM-CSF ^k/o CAR-T cell). GM-CSF ^k/o CAR-T cells may be administered in combination with a hGM-CSF antagonist (a recombinant hGM-CSF antagonist).

In one aspect, disclosed herein is a method of inhibiting or reducing the incidence or severity of an immunotherapy-related toxicity in an individual comprising administering to the individual a recombinant hGM-CSF antagonist.

In a related aspect, the immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cytokine administration, cancer vaccine administration, T cell engaging therapies, or any combination thereof.

In another aspect, adoptive cell transfer is a chimeric antigen receptor-expressing T-cell (CAR), a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR) ) modified natural killer cells, or dendritic cells, or combinations thereof. In a related aspect, the monoclonal antibody is an anti-CD3, anti-CD52, anti-PD1, anti-PD-L1, anti-CTLA4, anti-CD20, anti-BCMA antibody, bispecific antibody, or bispecific T-cell a participant (BiTE: bispecific T-cell engager) antibody, or any combination thereof. In a related aspect, the cytokine is IFNα, IFNβ, IFNγ, IFNλ, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL-18 , GM-CSF, TNFα, or a combination thereof.

In another aspect, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises reducing the concentration of one or more inflammation-related factors in the serum, tissue fluid, or CSF of the subject. In a related aspect, the inflammation-associated factor is a C-reactive protein, GM-CSF, IL-1, IL-2, sIL2Ra, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1 (AKA CCL2), MIG, MIP1b, IFNγ, CX3CR1, or TNFα, or any combination thereof. In another aspect, administration of a recombinant GM-CSF antagonist does not decrease the efficacy of the immunotherapy. In another aspect, administration of a recombinant GM-CSF antagonist increases the efficacy of the immunotherapy. In another aspect, administration of the recombinant GM-CSF antagonist occurs prior to, concurrently with, or after immunotherapy. In a related aspect, the recombinant GM-CSF antagonist is a corticosteroid, an anti-IL-6 antibody, tocilizumab, an anti-IL-1 antibody, a cyclosporine, an antiepileptic, a benzodiazepine, acetazolamide, hyperventilation therapy, or hyperventilation therapy. coadministered with hyperosmolar therapy, or a combination thereof.

In another aspect, the immunotherapy-related toxicity comprises a brain disease, injury or dysfunction. In a related aspect, the brain disease, injury or dysfunction comprises CAR-T cell related NT or CAR-T cell related encephalopathy syndrome (CRES). In a related aspect, inhibiting or reducing the occurrence of a brain disease, injury, or dysfunction comprises: headache, delirium, anxiety, tremor, seizure activity, confusion, arousal change, hallucinations, ataxia, ataxia, ataxia, facial nerve palsy in a subject , reducing motor weakness, seizures, nonconvulsive EEG attacks, altered levels of consciousness, coma, endothelial activation, vascular leakage, intravascular coagulation, or any combination thereof. In another aspect, the immunotherapy-related toxicity comprises CAR-T induced Cytokine Release Syndrome (CRS). In a related aspect, inhibiting or reducing the occurrence of CRS includes, but is not limited to, reducing or inhibiting hyperthermia, myalgia, nausea, hypotension, hypoxia or shock, or combinations thereof. In a related aspect, the immunotherapy-related toxicity is life threatening.

In another aspect, the serum concentration of ANG2 or VWF, or the serum ANG2:ANG1 ratio of the subject is reduced. In a related aspect, the subject has a body temperature greater than 38° C., an IL-6 serum concentration >16 pg/ml, or an MCP-1 serum concentration greater than 1,300 pg/ml during the first 36 hours after infusion of said CAR-T cells. . In a related aspect, the subject tends to be predisposed to said brain disease, injury or dysfunction. In a related aspect, the subject has an ANG2:ANG1 ratio in serum greater than 1 prior to infusion of said CAR-T cells.

In another aspect, the immunotherapy-related toxicity comprises hemophagocytic lymphohistiocytosis (HLH) or macrophage-activation syndrome (MAS). In a related aspect, inhibiting or reducing the occurrence of HLH or MAS comprises increasing survival time and/or time to relapse, decreasing macrophage activation, decreasing T cell activation, decreasing the concentration of IFNγ in the peripheral circulation, or GM- in the peripheral circulation. reducing the concentration of CSF, or a combination thereof.

In another aspect, the subject has fever, splenomegaly, cytopenia involving two or more strains, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis, daytime NK-cell activity with or without NK-cell activity, a ferritin serum concentration greater than 500 U/ml, or a soluble CD25 serum concentration greater than 2400 U/ml, or any combination thereof. In a related aspect, the subject tends to acquire HLH or MAS. In a related aspect, the individual carries a mutation in a gene selected from PRF1, UNC13D, STX11, STXBP2 or RAB27A, has reduced perforin expression, or any combination thereof.

In one embodiment, the GM-CSF antagonist is an anti-hGM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody blocks binding of hGM-CSF to the alpha subunit of the hGM-CSF receptor. In another embodiment, the anti-hGM-CSF antibody is a polyclonal antibody. In another embodiment, the anti-hGM-CSF antibody is a monoclonal antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In some embodiments, monoclonal anti-hGM-CSF antibodies, single chain Fvs, and Fabs may be produced in chickens; Chicken IgY is the avian equivalent of mammalian IgG antibodies (Park et al ., Biotechnology Letters (2005) 27:289-295; Finley et al ., Appl. Environ. Microbiol., May 2006, p. 3343-3349). Chicken IgY antibodies have the following advantages: higher avidity, ie, overall binding strength between antibody and antigen, higher specificity (less cross-reactivity with mammalian proteins other than immunogens); High yield and low background in egg yolk (less false positive staining due to structural differences in Fc regions of IgY and IgG). In another embodiment, the anti-hGM-CSF antibody comprises a camelid, eg, a llama-derived single variable domain on a heavy chain antibody lacking a light chain (also called sdAbs, VHH and Nanobodies®); The VHH domain (about 15 kDa) is the smallest known antigen recognition site occurring in mammals with full binding capacity and affinity (equivalent to traditional antibodies) (Garaicoechea et al . (2015) PLoS ONE 10(8): e0133665; Arbabi-Ghahroudi M (2017) Front. Immunol. 8:1589; Wu et al ., Translational Oncology (2018) 11, 366-373). In another embodiment, the antibody fragment is conjugated to polyethylene glycol. In another embodiment, the anti-hGM-CSF antibody has an affinity ranging from about 5 pM to about 50 pM. In another embodiment, the anti-hGM-CSF antibody is a neutralizing antibody. In another embodiment, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In another embodiment, the anti-hGM-CSF antibody is a human antibody. In another embodiment, the anti-hGM-CSF antibody comprises a human variable region. In another embodiment, the anti-hGM-CSF antibody comprises an engineered human variable region. In another embodiment the anti-hGM-CSF antibody comprises a humanized variable region. In another embodiment, the anti-hGM-CSF antibody comprises an engineered human variable region. In another embodiment, the anti-hGM-CSF antibody comprises a humanized variable region.

In one embodiment, the anti-hGM-CSF antibody comprises a human light chain constant region. In another embodiment, the anti-hGM-CSF antibody comprises a human heavy chain constant region. In another embodiment, the human heavy chain constant region is a gamma chain. In another embodiment, the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. In another embodiment, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. In another embodiment, the anti-GM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody.

In one embodiment, the anti-hGM-CSF antibody comprises a heavy chain variable region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V-segment, wherein the J-segment is at least 95% human JH4 (YFDYWGQGTLVTVSS) and wherein said V-segment comprises at least 90% identity to a human germline VH1 1-02 or VH1 1-03 sequence; or a heavy chain variable region comprising a CDR3 binding specificity determinant comprising RQRFPY. In another embodiment, the J segment comprises YFDYWGQGTLVTVSS. In another embodiment, CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In another embodiment, the heavy chain variable region CDR1 or CDR2 may be a human germline VH1 sequence; or both CDR1 and CDR2 may be human germline VH1. In another embodiment, the antibody comprises a heavy chain variable region CDR1 or CDR2, or both CDR1 and CDR2, as shown in the VH region shown in FIG. 1 . In another embodiment, the anti-hGM-CSF antibody has a V _{-segment having the V H} V-segment sequence shown in FIG. 1 . In another embodiment, V _H has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 .

In another embodiment, an anti-hGM-CSF antibody, e.g., an anti-hGM-CSF antibody having a heavy chain variable region as described in the preceding paragraph, comprises a CDR3 binding specificity determinant comprising the amino acid sequence FNK or FNR. and a light chain variable region.

In another embodiment, the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising the amino acid sequence FNK or FNR. In one embodiment, the anti-GM-CSF antibody comprises a human germline JK4 region. In another embodiment, the antibody V _L region CDR3 comprises QQFN(K/R)SPLT. In another embodiment, the anti-GM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. In yet another embodiment, the VL domain comprises a CDR1, CDR2, or both, or the CDR1 and CDR2 region of the V _L shown in FIG. In another embodiment, the V _L region comprises a V segment having at least 95% identity to the VKIIIA27 V-segment sequence as shown in FIG. 1 . In another embodiment, the V _L region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 .

In one embodiment, the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. In another embodiment, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In another embodiment, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. In another embodiment, the GM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody non lipocalin scaffold antibody mimetic, calixarene antibody mimetic, and antibody-like binding peptidomimetic.

In one embodiment, disclosed herein is a method of increasing the efficacy of CAR-T immunotherapy in an individual comprising administering to the individual a recombinant hGM-CSF antagonist, wherein the administration comprises administering to the individual CAR-T immunotherapy. increase the efficacy of In another embodiment, administration of said recombinant hGM-CSF antagonist occurs before, concurrently with, or after said CAR-T immunotherapy. In another embodiment, the increased potency is increased CAR-T cell expansion, decreased number of bone marrow-derived suppressor cells (MDSCs) that inhibit T-cell function, synergy with checkpoint inhibitors, or any thereof include combinations. In another embodiment, said increased CAR-T cell expansion comprises at least a 50% increase as compared to a control. In another embodiment, said increased CAR-T cell expansion comprises at least one-quarter log expansion compared to a control. In another embodiment, said increased cell expansion comprises at least 1/2 log expansion compared to a control. In another embodiment, said increased cell expansion comprises at least one log expansion compared to a control. In another embodiment, said increased cell expansion comprises more than 1 log expansion as compared to a control.

In one embodiment, the hGM-CSF antagonist comprises a neutralizing antibody. In another embodiment, the neutralizing antibody is a monoclonal antibody.

In one embodiment, disclosed herein is a method of inhibiting or reducing the occurrence or severity of a CAR-T related toxicity in an individual comprising administering to the individual a recombinant hGM-CSF antagonist, wherein the administration comprises administering to the individual a CAR -Inhibits or reduces the occurrence or severity of T-related toxicity. In one embodiment, the CAR-T related toxicity comprises NT, CRS, or a combination thereof. In one embodiment, the methods of treatment provided herein prevent and treat NT and CRS in a subject in need thereof. In some embodiments, the CAR-T cell associated NT is reduced by about 50% relative to the decrease in NT in an individual treated with the CAR-T cell and a control antibody. In various embodiments, the recombinant hGM-CSF antagonist is an hGM-CSF neutralizing antibody according to embodiments described herein.

In another embodiment, inhibiting or reducing the occurrence of CRS comprises increasing survival time and/or time to recurrence, decreasing macrophage activation, decreasing T cell activation, or decreasing the concentration of circulating hGM-CSF, or any thereof. include combinations. In another embodiment, the subject has a fever (rigidity, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, enlarged pulse pressure , hypotension, capillary leakage, early cardiac output increase, late cardiac output decrease, D-dimer elevation, hypofibrinogenemia with or without bleeding, azotemia, transaminitis, hyperbilirubin hyperemia, mental status changes, confusion, delirium, overt aphasia, hallucinations, tremors, dysmetria, gait changes, seizures, organ failure, or a combination thereof).

In another embodiment, inhibiting or reducing the occurrence or severity of CAR-T related toxicity comprises preventing the development of CAR-T related toxicity.

In another embodiment, disclosed herein is a method of blocking or reducing GM-CSF expression in a cell comprising knocking out or silencing GM-CSF gene expression in the cell. In one embodiment, blocking or reducing GM-CSF expression is gene-silencing using DNA constructs to activate short interfering RNS (siRNA), CRISPR, RNAi, endogenous RNA interference (RNAi) pathways in animal cells. technology, DNA-induced RNA interference (ddRNAi), or engineered transcription activator-like effector nucleases (TALENs), i.e. custom sequence-specific DNA-binding domains fused to nucleases that cleave DNA in a non-sequence-specific manner. Target genome editing using artificial proteins composed of (Joung and Sander, Nat Rev Mol Cell Biol. 2013 January; 14(1): 49-55, incorporated herein by reference in its entirety). In one embodiment, the cell is a CAR-T cell.

In one embodiment, the subject is a human.

In one embodiment, disclosed herein is an hGM-CSF antagonist for use in a method of inhibiting or reducing the incidence or severity of an immunotherapy-related toxicity in an individual comprising administering to the individual a recombinant hGM-CSF antagonist. . In one embodiment, disclosed herein is a pharmaceutical composition comprising an anti-hGM-CSF antagonist.

1 _{provides exemplary V H} and V _L sequences of anti-GM-CSF antibodies.
2A-2B show the binding of GM-CSF to Ab1 ( FIG. 2A ) or Ab2 ( FIG. 2B ) as determined by surface plasmon resonance analysis (Biacore 3000) at 37°C. Ab1 and Ab2 were captured by anti-Fab polyclonal antibodies immobilized on a Biacore chip. Different concentrations of GM-CSF as indicated were injected onto the surface. Global fit analysis was performed assuming 1:1 interaction using Scrubber2 software.
3A-3B show binding of Ab1 and Ab2 to glycosylated GM-CSF ( FIG. 3A ) and non-glycosylated GM-CSF ( FIG. 3B ). Binding to glycosylated GM-CSF expressed in human 293 cells or non-glycosylated GM-CSF expressed in E. coli was determined by ELISA. Representative results of a single experiment are shown (exp 1). Two-fold dilutions of Ab1 and Ab2 starting at 1500 ng/ml were applied to GM-CSF coated wells. Each point represents the mean ± standard error for triplicate determinations. Sigmoidal curve fit was performed using Prism 5.0 software (Graph Pad).
4A-4B show competition ELISAs showing binding of Ab1 and Ab2 to shared epitopes. ELISA plates coated with 50 ng/well of recombinant GM-CSF were incubated with 50 nM biotinylated Ab2 with varying concentrations of antibody (Ab2, Ab1 or isotype control antibody). Biotinylated antibody binding was assayed using Neutravidin-HRP conjugates. Competition for binding to GM-CSF was for either 1 hour ( FIG. 4A ) or 18 hours ( FIG. 4B ). Each point represents the mean ± standard error for triplicate determinations. Sigmoid curve fitting was performed using Prism 5.0 software (Graph Pad).
5 shows inhibition of GM-CSF-induced IL-8 expression. Various amounts of each antibody were incubated with 0.5 ng/ml GM-CSF and incubated with U937 cells for 16 hours. IL-8 secreted into the culture supernatant was measured by ELISA.
6 shows dose-dependent inhibition of GM-CSF-stimulated CD11b on human granulocytes by anti-GM-CSF antibodies.
7 shows dose-dependent inhibition of GM-CSF-induced HLA-DR on CD14+ human, primary monocytes/macrophages by anti-GM-CSF antibodies.
8 shows the role of GM-CSF (myeloid inflammatory factor) as a key cytokine in stimulating CAR-T-related activity and leukocyte proliferation, which is characteristic of certain leukemias, such as acute myeloid leukemia (AML).
9 shows inhibition of GM-CSF-dependent human TF-1 cell proliferation (human erythroleukemia) by neutralization of human GM-CSF using anti-GM-CSF antibody. KB003 is a recombinant monoclonal antibody designed to target and neutralize human GM-CSF. KB002 is a mouse/human chimeric monoclonal antibody that targets and neutralizes hGM-CSF.
10 is a schematic diagram of a chimeric antigen receptor.
11 shows the results of CAR-T19 therapy showing a high response rate in relapsed refractory ALL. Data show historical outcomes of R/R ALL and outcomes of R/R ALL after CAR-T19 treatment (Maude, et al NEJM 2014).
12 shows evidence showing a significant GM-CSF association for NT. GM-CSF levels are associated with serious adverse events following CAR-T cell treatment. GM-CSF levels precede and regulate cytokines other than IL-15. Elevated GM-CSF is clearly associated with ≥ Grade 3 NT. IL-2 is the only other cytokine with this association.
13 shows the estimated time course of CRS and NT following CD19 CAR-T cell therapy. The onset of symptoms and the severity of CRS depend on the inducer, the type of cancer, the age of the patient, and the degree of immune cell activation. The onset of CAR-T related CRS symptoms generally occurs days to weeks after T-cell infusion, consistent with maximal T-cell expansion. Similar to CRS associated with mAb therapy, CRS associated with adoptive T cell therapy is associated with elevated IFNγ, IL-6, TNFα, IL-1, IL-2, IL-6, GM-CSF, IL-10, IL-8. and IL-5. Although no clear CAR-T cell dose-response relationship exists for CRS, very high doses of T cells can lead to early onset of symptoms.
14 shows that GM-CSF is a major initiator of CAR-T side effects. The figure shows the central role of GM-CSF in CRS and NT. Perforin allows granzyme to cross the tumor cell membrane. CAR-T produced GM-CSF recruits CCR2+ myeloid cells to the tumor site to produce CCL2 (MCP1). CCL2 positively potentiates its own production by recruitment of CCR2+ myeloid cells. IL-1 and IL-6 in myeloid cells form another positive feedback loop with CAR-T by inducing the production of GM-CSF. Phosphatidylserine is exposed as a result of perforin and granzyme cell membrane disruption. Phosphatidyl-serine stimulates myeloid cell production of CCL2, IL-1, IL-6 and other inflammatory effectors. The end result of this self-reinforcing feedback loop is endothelial activation, vascular permeability and ultimately CRS and NT. Moreover, animal model evidence shows that while GM-CSF knockout mice show no signs of CRS, IL-6 knockout mice can still develop CRS. GM-CSF receptor k/o in CCR2+ myeloid cells abrogates the cascade in models of neuroinflammation (Sentman, et al., J. Immunol.; Coxford, et al. Immunity 2015 (43)510-514; Ishii et al. , Blood 2016 128:3358; Teachy, et al. Cancer Discov. 2016 June 6(6): 664-679; Lee, et al., Blood 2016 124:2:188; Barrett, et al., Blood 2016: 128 -654, each of which is incorporated herein by reference in its entirety.
15A-15G show that GM-CSF CRISPR knockout T-cells show reduced expression of GM-CSF, but similar levels of other cytokines and degranulation. a. Generation of GM-CSF knockout CAR-T (see Example 6).
16A-16I show that GM-CSF neutralizing antibodies according to embodiments described herein do not inhibit CAR-T mediated killing, proliferation, or cytokine production, but successfully neutralize GM-CSF (Example 7 Reference).
17A-17B show protocols and results from a mouse model of human CRS. (See Example 5).
18A-18C show CAR-T efficacy in a xenograft model in combination with a GM-CSF neutralizing antibody according to embodiments described herein. GM-CSF neutralizing antibodies were not shown to inhibit CAR-T efficacy in vivo (see Example 8).
19 shows in vitro and in vivo preclinical data showing that GM-CSF neutralizing antibodies according to embodiments described herein did not impair the effect of CAR-T on survival. In the absence of PBMCs, GM-CSF neutralizing antibodies do not interfere with CAR-T cell function in vivo. The survival rates of CAR-T + control and CAR-T + GM-CSF neutralizing antibodies were similar. (See Example 9).
20A-20B show in vitro and in vivo preclinical data showing that GM-CSF neutralizing antibodies according to embodiments described herein increase CAR-T expansion. GM-CSF neutralizing antibody increases CAR-T cancer cell death in vitro. Antibody neutralization of GM-CSF increases proliferation of CAR-T cells in the presence of PBMCs. CAR-T proliferation was increased by GM-CSF neutralizing antibody in the presence of PBMCs (unaffected without PBMCs). The anti-GM-CSF antibody did not inhibit CAR-T degranulation, intracellular GM-CSF production, or IL-2 production (see Example 10).
Figure 21 shows that CAR-T expansion is associated with improved overall response rate. CAR AUC (area under the curve) defined as the cumulative level of CAR+ cells/μL of blood during the first 28 days after CAR-T administration. P values calculated by Wilcoxon rank sum test (Neelapu, et al ICML 2017 Abstract 8) (see Example 11).
22 shows a study protocol for GM-CSF neutralizing antibodies according to embodiments described herein (see Example 12). CRS and NT are assessed daily during hospitalization and during the first 30 days at clinic visits. Eligible individuals to receive GM-CSF neutralizing antibodies on days -1, +1 and +3 of CAR-T treatment. Additional dosing can be considered up to at least day 7 . Tumor assessments to be performed at baseline and at 1, 3, 6, 9, 12, 18 and 24 months. Blood samples (PBMC and serum) on days -5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270 and 360 (see Example 12).
23A-24B show that GM-CSF depletion increases CAR-T cell expansion. 23A shows increased ex vivo expansion of ^{GM-CSF k/o} CAR-T cells compared to control CAR-T cells. 23B shows more robust CAR-T cell proliferation after treatment with GM-CSF neutralizing antibody according to embodiments described herein (see Example 13).
24 shows the safety profile of GM-CSF neutralizing antibodies according to embodiments described herein (see Example 14).
25A-25D show that GM-CSF neutralizing antibody demonstrated a 90% reduction in neuroinflammation in a mouse preclinical model when added to CAR-T cell therapy. 25A shows MRI data (T1 hyperintensity indicative of BBB destruction and neuroinflammation) compared to mouse brain showing signs of neurotoxicity after administration of CAR-T cells and control antibody (top row). Thus, and compared to an untreated (baseline) mouse brain (bottom row), the mouse brain is protected from neuroinflammation following administration of CAR-T cells and a GM-CSF neutralizing antibody according to an embodiment described herein. 25B shows an increase from baseline in mice administered CAR-T cells and a GM-CSF neutralizing antibody according to an embodiment described herein, compared to an increase of slightly over 100% in mice receiving CAR-T cells and a control antibody. There was about a 10% percent increase in brain T1 high-intensity signals. As shown in the comparative graph, a ~10% increase in brain T1 high-intensity signal from baseline in mice administered with CAR-T and GM-CSF neutralizing antibodies was associated with neuroinflammation present in mice receiving CAR-T cells and control antibody. compared to the amount of , a 90% reduction in neuroinflammation, as measured by brain T1 high-intensity signals from baseline.
25C-25D show CAR-T + according to embodiments described herein, compared to untreated mice (having 500,000 to 1.5M leukemia cells) and CAR-T + control antibody (having 15,000 to 100,000 leukemia cells) Treatment with GM-CSF neutralizing antibody resulted in a significant reduction in the number of leukemia cells (reduced to 500 to 5,000 cells) and an improvement in overall disease control (see Example 15).
26A-26I show that GM-CSF blockade helps control CART19 toxicity and improves efficacy. Figure 26A shows that CART19 and lenzilumab treated CART19 are equally effective in survival outcome in high tumor burden NALM6 recurrence model compared to UTD (untransduced T cells) (7-8 mice per group, n=2). . 26B-26D show lenzilumab and anti-mouse GM-CSF antibody-controlled CRS induced weight loss, serum human GM-CSF neutralization, and serum mouse MCP-1 in a primary ALL xenograft CART19 CRS/NT model. (monocyte chemoattractant protein-1) expression decreased (3 mice per group, * p<0.05). Figure 26E shows that lenzilumab and anti-mouse GM-CSF antibody reduced brain inflammation as shown by MRI in primary ALL xenograft CART19 CRS/NT model (3 mice per group, *p<0.05, **p <0.01). 26F-26G show improved efficacy of CART19 + lenzilumab treated mice compared to anti-mouse GM-CSF antibody treated mice in a primary ALL xenograft CART19 CRS/NT model. That is, CART19 + anti-hGM-CSF antibody showed reduced CD19 + brain leukemia burden and decreased proportion of brain macrophages (3 mice per group). 26H shows that CRISPR Cas9 K/O of GM-CSF reduced its expression through intracellular staining and NALM6 stimulation in CART19 and UTD (representative experiment, n=2). 26I shows that, in a high tumor burden NALM6 recurrence model, CART19 and GM-CSF K/O CART19 control tumor burden better than UTD, and GM/CSF K/O CART19 cells control tumor burden slightly better than CART19. improved efficacy (6 mice per group, * p<0.05, **** p<0.0001). Error bars SEM.
27A-27D show that in vitro GM-CSF neutralization enhances CAR-T cell proliferation in the presence of monocytes and does not impair CAR-T cell effector function. 27A shows CAR- producing hGM-CSF in vitro, as analyzed by multiplex after 3 days of culture with CART19 in medium alone, or with CART19 co-cultured with NALM6, compared to isotype control treatment. The neutralization of lenzilumab (hGM-CSF neutralizing antibody) of T cells is shown. n=2 experiments, 2 replicates per experiment, representative experiments shown, *** p<0.001 between lenzilumab and isotype control treatment, t test, mean ± SEM. 27B graphically shows that hGM-CSF neutralizing antibody treatment did not inhibit the proliferative ability of CAR-T cells, as analyzed in CSFE flow cytometry analysis of live CD3 cells. n=3 donors, 2 replicates per donor, representative experiments at 3 day time points shown, ns p>0.05 between lenzilumab and isotype control treatment, t test, mean ± SEM. Alone: refers to CART19 in medium alone, refers to MOLM13:CART19 + MOLM13, refers to PMA/ION:CART19 + 5ng/ml PMA and 0.1ug/ml ION, refers to NALM6:CART19 + NALM6. 27C graphically shows lenzilumab, which enhances proliferation of CART19 by neutralization of hGM-CSF compared to isotype controls treated with CART19 when co-cultured with human monocytes. n=3 donors at day 3, 2 replicates per donor, **** p<0.0001, mean ± SEM. 27D graphically shows that lenzilumab treatment did not inhibit the cytotoxicity of CART19 or non-transduced T cells (UTDs) when incubated with NALM6. n=3 donors, 2 replicates per donor, representative experiments at 48 h time points shown, ns p>0.05 between lenzilumab and isotime control treatment, t test, mean ± SEM.
28A-28F demonstrate that GM-CSF neutralization in vivo enhances CAR-T cell anti-tumor activity (ie, tumor cell killing) in a xenograft model. 28A shows the experimental outline: NSG mice were injected with CD19 + luciferase + cell line NALM6 (1x10 ^{6 cells per mouse IV).} After 4-6 days, mice were imaged, randomized, and received an equivalent number of total cells ^{of 1-1.5x10 6 CAR-T19 or control UTD cells with lenzilumab or control IgG the next day (10 mg/Kg,} IP administration daily for 10 days from the day of CAR-T injection). Mice underwent serial bioluminescence imaging to assess disease burden from day 7 after CAR-T cell injection and tracked overall survival. Tail vein bleeding was performed 7-8 days after CAR-T cell injection. Figure 28B shows lenzilumab neutralization of CAR-T generated serum hGMCSF in vivo compared to isotype control treatment, as analyzed by hGM-CSF singleplex. n=2 experiments, 7-8 mice per group, representative experiments, analyzed with serum at day 8 after CAR-T cells/UTD injection, *** p<0.001 between lenzilumab and isotype control treatment, t test, mean ± SEM. 28C graphically depicts that lenzilumab treated CAR-T is equally effective in controlling tumor burden in vivo compared to isotype control treated CAR-T treatment in a high tumor burden recurrent xenograft model of ALL. . 7 days post CAR-T injection, n=2 experiments, 7-8 mice per group per group, representative experiments shown, ***p<0.001, *p<0.05, ns p>0.05, t test, mean ± SEM . Figure 28D shows the mouse image of Figure 28C. Figure 28E shows the experimental outline: NSG mice were injected with blasts derived from ALL patients (1x10 ⁶ cells per IV mouse). When mice were continuously bled and CD19+ cells were >1/uL, mice were randomized to receive 2.5x10 ⁶ CART19 with lenzilumab or control IgG (10 mg/Kg, starting on the day of CAR-T injection, 10 IP provided daily for days). Mice were assessed for disease burden and overall survival followed from day 14 after CAR-T cell injection with continuous tail vein bleeding. 28F shows that lenzilumab treatment with CAR-T therapy was more of tumor burden over time in a primary acute lymphoblastic leukemia (ALL) xenograft model, compared to isotype control treatment with CAR-T therapy. Graph that results in continuous control. 6 mice per group, **p<0.01, *p<0.05, ns p>0.05, t test, mean ± SEM.
29A-29E demonstrate that GM-CSF CRISPR knockout CAR-T cells exhibit reduced expression of GM-CSF, similar levels of key cytokines and chemokines, and enhanced antitumor activity. 29A shows that CRISPR Cas9 GM-CSF ^k/o CART19 exhibits reduced GM-CSF production compared to wild-type CART19, but other cytokine production and degranulation are not inhibited by GM-CSF gene disruption. CART19 and GM-CSF ^K/O CART19 stimulated with NALM6, n=3 experiments, 2 replicates per experiment, *** p<0.001, * p<0.05, ns p>0.05, GM-CSF ^k/o CART19 and CAR19 comparison, t test, mean ± SEM. 29B shows that GM-CSF ^k/o CAR-T reduced serum human GM-CSF in vivo compared to CAR-T treatment, as analyzed by multiplex. ****p<0.0001, ***p<0.001 between ^{GM-CSF k/o} CART19 and wild-type CART19, 5-6 mice per group (4-6 mice at bleeding time 8 days after CAR-T cell injection); t test, mean ± SEM. 29C ^{shows that GM-CSF k/o} CART19 in vivo improves overall survival compared to wild-type CART19 in a high tumor burden recurrent xenograft model of ALL using the NALM6 cell line. 5-6 mice per group, **p<0.01, log-rank. 29D-29E show that human (FIG. 29D) and mouse (FIG. 29E) cytokines and chemokines from serum multiplex other than hGM-CSF ^{show no statistical difference between GM-CSF k/o} CART19 and wild-type CART19. , which also suggests that important T-cell cytokines and chemokines are not depleted by reducing GM-CSF expression. 5-6 mice per group (4-6 mice at bleeding), **** p<0.0001, t test.
30A-30D show patient-derived xenograft models for neuroinflammation and cytokine release syndrome. 30A ^{shows the experimental outline: Mice received 1-3x10 6} primary blasts derived from the peripheral blood of patients with primary ALL. Mice were monitored for engraftment for ~10-13 weeks via tail vein bleeding. When serum CD19+ cells were ≧10 cells/uL, mice received CART19 (2-5× ^{10 6} cells) as indicated and started antibody treatment for a total of 10 days. To measure the health of the mice, body weight was measured daily. Mouse brain MRI was performed 5-6 days after CART19 injection, tail vein bleeding for cytokines/chemokines was performed, and T cell analysis was performed 4-11 days after CART19 injection, in two independent experiments. 30B shows that the combination of CART19 and GM-CSF neutralizing is equally effective as an isotype control antibody in combination with CART19 in controlling the CD19+ burden of ALL cells. Representative experiment, 3 mice per group, 11 days after CART19 injection, *p<0.05 between GM-CSF neutralization + CART19 and isotype control + CART19, t test, mean ± SEM. FIG. 30C shows brain MRI data suggesting that CART19 therapy shows T1 enhancement, brain blood-brain barrier disruption and possible edema. 3 mice per group, 5-6 days after injection of CART19, representative images. 30D shows human CD3 cell infiltration in the brain of high tumor burden primary ALL xenografts treated with CART19 compared to untreated PDX controls. 3 mice per group, representative images.
31 shows the altered canonical pathway in the brain from patient-derived xenografts after treatment with CART19 cells.
Red boxes indicate upregulation of genes in CART19 + isotype control treated mice compared to untreated patient-derived xenografts.
32A-32D show that in vivo GM-CSF neutralization ameliorates CRS after CART19 treatment in a xenograft model . 32A shows that lenzilumab and anti-mouse GM-CSF antibody prevent CRS induced weight loss compared to mice treated with CART19 and an isotype control antibody. 3 mice per group, 2-way anova, mean ± SEM. Figure 32B shows that human GM-CSF was neutralized in patient-derived xenografts treated with lenzilumab and mouse GM-CSF neutralizing antibody. 3 mice per group, ***p<0.001, *p<0.05, t test, mean ± SEM. Figure 32C shows that the human cytokine/chemokine heat map (sera collected 11 days after CART19 injection) shows cytokine and chemokine increases typical of CRS after CART19 treatment. GMCSF neutralization results in significant reductions of several cytokines and chemokines, including bone marrow-associated cytokines and chemokines, compared to mice treated with CART19 and an isotype control antibody, as indicated in the panel. 3 mice per group, serum at 11 days post injection of CART19, *** p<0.001, ** p<0.01, * p<0.05, mice treated with GM-CSF neutralizing antibody and isotype controls receiving CAR-T cell therapy. Comparison of treated mice, t test. Figure 32d shows that the mouse cytokines / chemokines heat map (CART19 the serum collected 11 days after injection) in a CRS after CART19 treated mouse shows a typical increase in the cytokines and chemokines. GM-CSF neutralization results in significant reduction of several cytokines and chemokines, including several myeloid differentiation cytokines and chemokines, compared to CART19 treatment with control antibody as indicated in the panel. 3 mice per group, serum at 11 days post CART19 injection, *p<0.05, comparing GM-CSF neutralizing antibody treated mice with isotype control treated mice receiving CAR-T cell therapy, t test.
33A-33D show that in vivo GM-CSF neutralization ameliorated neuroinflammation after CART19 treatment in a xenograft model. 33A-33B show gadolinium-enhanced T1-high-intensity signal (cubic mm) MRI, wherein GM-CSF neutralization reduced brain inflammation, reduced blood-brain barrier disruption, and possible edema compared to isotype control (A). show that it has helped Summary images, (33B) 3 mice per group, **p<0.01, *p<0.05, 1-way ANOVA, mean ± SD. Figure 33C shows that human CD3 T cells were present in the brain after treatment with CART19 therapy. GM-CSF neutralization resulted in a trend to decrease CD3 infiltration in the brain as analyzed by flow cytometry in the brain hemispheres. 3 mice per group, mean ± SEM. 33D shows that CD11b+ bright macrophages were reduced in the brain of mice that received GM-CSF neutralization during CAR-T treatment compared to isotype controls during CAR-T treatment as analyzed by flow cytometry in brain hemispheres. 3 mice per group, mean ± SEM.
34A-34B show generation of GM-CSF ^{k/o CART19 cells.} 34A shows an experimental overview; Figure 34B shows the gRNA sequence and primer sequence for the generation of GM-CSF ^{k/o CART19.} To generate GM-CSF ^k/o CART19 cells, gRNA was cloned into a Cas9 lentiviral vector under the control of the U6 promoter and used for lentiviral production. T cells derived from normal donors were stimulated with CD3/CD28 beads and double transduced with CAR19 virus and CRISPR/Cas9 virus 24 hours later. CD3/CD28 magnetic bead removal was performed on day 6, and GM-CSF ^k/o CART19 cells or control CART19 cells were cryopreserved on day 8.
35 shows a flowchart for the procedure used for RNA sequencing. Binary base call data was converted to fastq using Illumina bcl2fastq software. Adapter sequences were removed using Trimmomatic and quality checked using FastQC. The latest human (GRCh38) and mouse (GRCm38) reference genomes were downloaded from NCBI. Genomic index files were generated using STAR and paired end reads were mapped to the genome for each condition. HTSeq was used to generate expression coefficients for each gene and DeSeq2 was used to calculate differential expression. Gene ontology was evaluated using Enrichr.
Figure 36 shows that lenzilumab + CAR-T cell treated mice have similar survival compared to isotype control antibody + CAR-T cell treated mice in a high tumor burden recurrent xenograft model of ALL. n=2 experiments, 7-8 mice per group, representative experiments are shown, **** p<0.0001, *** p<0.001, * p<0.05, log rank.
37 shows representative TIDE sequences for identifying genomic alterations in GM-CSF CRISPR Cas9 knockout CAR-T cells. n=2 experiments, representative experiments are shown.
38 shows that in vivo GM-CSF knockout CAR-T cells show slightly improved control of tumor burden compared to wild-type CAR-T cells in a high tumor burden recurrent xenograft model of ALL. Days post injection of CAR-T cells listed on the x-axis, 5-6 mice per group (2 remaining in UTD group on day 13), representative experiments are shown, **** p<0.0001, * p<0.05, 2-way ANOVA, mean ± SEM.
39 shows a patient-derived xenograft model for neuroinflammation and CRS using CART19+ anti-hGM-CSF antibody treatment. High tumor burden primary ALL xenograft treated CART19 + anti-hGM-CSF antibody treatment shows human CD3 cell infiltration in the brain compared to untreated PDX control ( FIG. 30D ) ( FIG. 39 ). 3 mice per group, representative images.
40A-40B show that BBB integrity is preserved and neuroinflammation is significantly reduced after CAR-T and lenzilumab therapy. FIG. 40A shows confocal microscopy clearly showing in high-resolution images that the BBB is significantly impaired after CAR-T therapy, and CAR-T and lenzilumab therapy show maintenance of BBB integrity.
40B is a view of Santomasso, BD et al., published on OnlineFirst on June 7, 2018 and incorporated by reference in its entirety; Adopted from DOI: 10.1158/2159-8290.CD-17-1319, it shows high levels of protein in CSF (shown in data from Santomasso), indicative of BBB disruption and protein leakage into the CNS.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be more readily understood by reference to the following detailed description, which forms a part of this disclosure. This disclosure is not limited to the particular products, methods, conditions, or parameters described and/or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to cover the claimed disclosure. It should be understood that it is not intended to limit

Immunotherapy-related toxicity

Those skilled in the art will understand that the term “immunotherapy-related toxicity” refers to a spectrum of inflammatory symptoms due to high levels of immune activation. Different types of toxicity are associated with different immunotherapy approaches.

In some embodiments, the immunotherapy-related toxicity is capillary leak syndrome, heart disease, respiratory disease, CAR-T cell associated encephalopathy syndrome (CRES), neurotoxicity, colitis, convulsions, cytokine release syndrome (CRS), cytokines Storm, decreased left ventricular ejection fraction, diarrhea, disseminated intravascular coagulation, edema, encephalopathy, rash, gastrointestinal bleeding, gastrointestinal perforation, hemophagocytic lymphohistiocytosis (HLH), hepatitis, hypertension, pituitary , immune-related adverse events, immunohepatitis, immunodeficiency, ischemia, hepatotoxicity, macrophage-activated syndrome (MAS), pleural effusion, pericardial effusion, pneumonia, polyarthritis, occipital reversible encephalopathy syndrome (PRES), pulmonary hypertension, thromboembolism and trans including transaminitis.

Although the various types of toxicity differ in pathophysiology and clinical manifestations, they are commonly C-reactive proteins, GM-CSF, IL-1, IL-2, sIL-2Ra, IL-5, IL-6, IL-8, IL- 10, IP10, IL-15, MCP-1 (AKA CCL2), MIG, MIP-1b, IFNγ, CX3CR1, or an increase in inflammation-related factors, such as TNFα. Those of skill in the art will recognize that in some embodiments the term “inflammatory-associated factor” includes molecules, small molecules, peptides, gene transcripts, oligonucleotides, proteins, hormones and biomarkers that are affected during inflammation. Those skilled in the art will recognize that the systems affected during inflammation include up-regulation, down-regulation, activation, inactivation or molecular modification of any kind. Serum concentrations of inflammation-related factors, such as cytokines, can be used as indicators of immunotherapy-related toxicity and can be expressed as -fold increase, percent increase, net increase, or rate of change in cytokine level or concentration. The concentration of inflammation-related factors in body fluids other than serum can also be used as an indicator of immunotherapy-related toxicity. In some embodiments, an absolute cytokine level or concentration above a certain level or concentration may be indicative of an individual suffering from or about to experience immunotherapy-related toxicity. In another embodiment, an absolute cytokine level or concentration at a particular level, e.g., a level or concentration normally found in a control subject, may be indicative of a method of inhibiting or reducing the occurrence of an immunotherapy-related toxicity in an individual. have. One of ordinary skill in the art will recognize that the term “cytokine level” can include a measurement of a concentration, a measurement of a fold change, a measurement of a percent (%) change, or a measurement of a rate change. In addition, methods for measuring cytokines in blood, cerebrospinal fluid (CSF), saliva, serum, urine and plasma are well known in the art.

Several approaches have been elaborated to classify types of neurotoxicity and manage them accordingly. These classifications include fever, hypotension, hypoxia, organ toxicity, cardiac dysfunction, respiratory dysfunction, gastrointestinal dysfunction, liver dysfunction, renal dysfunction, coagulopathy, seizure presence, intracranial pressure, muscle tone, exercise performance, ferritin levels, and It is based on clinical and biological symptoms such as hemophagocytosis Similarly, each type of neurotoxicity can be graded according to its severity. Table 1A (excerpted from Cellular Therapy Implementation: the MDACC Approach, P. Kebriaei, Feb. 24, 2017) discloses a method for grading neurotoxicity into grades 1, 2, 3, and 4 according to severity. However, some of the aforementioned symptoms are not generally associated with neurotoxicity (Lee, et al., Blood 2014; 124:188-195, which is incorporated herein by reference in its entirety).

Table 1A: Method for grading neurotoxicity - Adverse Event Criteria (CTCAE)

Patients with a body temperature of 38.9 °C or higher, an IL-6 serum concentration of 16 pg/ml or higher, or an MCP-1 (AKA CCL2) serum concentration of 1,343.5 pg/ml or higher during the first 36 hours after immunotherapy infusion have a higher risk of severe neurotoxicity higher (Gust, et al. Cancer Discov. 2017 Oct 12).

CRS is a serious condition and life-threatening adverse reaction due to abnormal cytokine regulation and severe inflammation. Symptoms may include, but are not limited to, fever, irregular heartbeat and breathing, nausea, vomiting, and seizures. CRS can be graded, for example, by evaluating the symptoms and their severity as follows: Grade 1 CRS: fever, systemic symptoms; Grade 2 CRS: hypotension - hypoxia in response to body fluids or low-dose pressure - in response to <40% O ₂ , long-term toxicity; Grade 2; Grade 3 CRS: hypotension—requires multiple compressions or high-dose compressions, hypoxia—requires ≥40% O ₂ , organ toxicity—grade 3, grade 4 transaminitis; Grade 4 CRS: mechanical respiration, organ toxicity—grade 4, excluding transaminitis (Lee, et al., Blood 2014; 124:188-195, which is incorporated herein by reference in its entirety).

CRES can be graded, for example, by combining neurological assessment with other parameters such as papillary edema, CSF open pressure, imaging assessment, seizures, and ataxia. Methods for grading CRES are described in Neelapu et al., Nat Rev Clin Oncol. 15(1):47-62 (2018) (Epub 2017 Sep 19), which is incorporated herein by reference in its entirety. Table 1B (excerpted from Neelapu et al., Nat Rev Clin Oncol. 15 (1):47-62 (2018)) shows the classification of grades 1, 2, 3, and alc 4 according to the severity of CRES. method is disclosed.

Table 1B: CRES grading method. In CARTOX 10, points are assigned for each of the following tasks performed correctly: year, month, city, hospital and orientation to the President/Prime Minister of the country of residence (1 point each); Name three objects (1 point each); writing standard sentences; It counts backwards from tens to 100.

Symptoms of NT, CRS and CRES include encephalopathy, headache, delirium, anxiety, tremor, seizure activity, confusion, arousal changes, decreased level of consciousness, hallucinations, speech disorders, aphasia, ataxia, ataxia, facial nerve paralysis, motor weakness, seizures, Nonconvulsive EEG attacks, cerebral edema and coma may be included. CRES is a C-reactive protein, GM-CSF, IL-1, IL-2, sIL2Ra, IL-5, IL-6, IL-8, IL-10, IP10, IL-15, MCP-1, MIG, MIP1b, It is associated with elevated concentrations of circulating cytokines such as IFNγ, CX3CR1, and TNFα.

The cytokine concentration gradient between serum and CSF observed under normal conditions is reduced or lost during CRES. In addition, CAR-T cells and high protein concentrations were observed in the CSF of patients and correlated with the severity of the condition. All of these indicate blood-brain barrier dysfunction after immunotherapy. The increased vascular permeability can be explained in part by the increased ANG2 concentration and the increased ANG2:ANG1 ratio in neurotoxic patients. ANG1 induces endothelial cell arrest, whereas ANG2 induces endothelial cell activation and microvascular permeability. Patients with increased endothelial activation prior to immunotherapy were reported to be more likely to develop neurotoxicity . Gust, et al. Cancer Discov. 2017 Oct 12).

Hemophagocytic lymphohistiocytosis (HLH) involves severe hyperinflammation due to the uncontrolled proliferation of benign lymphocytes and macrophages that secrete large amounts of inflammatory cytokines. In some embodiments, HLH may be classified as one of the cytokine storm syndromes. In some embodiments, HLH develops after systemic infection, immunodeficiency, malignancy, or strong immunological activation, such as immunotherapy. In some embodiments, the term “HLH” is interchanged with the terms “hemophagocytic lymphohistiocytosis,” “hemophagocytic syndrome,” or “hemophagocytic syndrome,” which all have the same characteristics and meanings. can be used negatively.

Primary HLH includes heterogeneous autosomal recessive disorders. Patients with homozygous mutations in one of several genes exhibit loss of function in proteins involved in cytolytic granule exocytosis. In some embodiments, HLH may be present in infancy with minimal or no triggers. Secondary HLH or acquired HLH occurs after strong immunological activation, such as systemic infection, immunodeficiency, underlying malignancies, or those occurring with immunotherapy. Both forms of HLH are characterized by overwhelming activation of normal T lymphocytes and macrophages, which in the absence of treatment leads to clinical and hematological changes and death.

In some embodiments, HLH is a viral infection, EBV, CMV, parvovirus, HSV, VZV, HHV8, HIV, influenza, hepatitis A, hepatitis B, hepatitis C, bacterial infection, gram-negative rod, mycoplasma spp. and Mycobacterium tuberculosis, parasitic infections, Plasmodium spp., Leishmania spp., Toxoplasma spp., fungal infections, Cryptococcal spp., Candidal spp. and Pneumocystis spp.

Macrophage-activation syndrome (MAS) includes conditions involving uncontrolled activation and proliferation of macrophages and T lymphocytes with marked increases in circulating cytokine levels such as IFNγ and GM-CSF. MAS is closely related to secondary HLH. MAS symptoms include hyperthermia, hepatic splenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation, phagocytosis, hypofibrinemia, hyperferritinemia and hypertriglyceridemia.

CRS involves a non-antigen-specific immune response similar to that found in severe infections. CRS is characterized by some or all of the following symptoms: stiffness, malaise, fatigue, loss of appetite, myalgia, arthralgia, nausea, vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, enlargement Hypertension, hypotension, capillary leakage, increased cardiac output (initial), potentially decreased cardiac output (late), elevated D-dimer, hypofibrinogenemia with or without bleeding, azotemia, transaminitis , hyperbilirubinemia, headache, altered mental status, confusion, delirium, difficulty finding words or apparent frank aphasia, hallucinations, tremors, dysmetria, gait changes, seizures, multiple organ failure. Deaths were also reported. Severe CRS has been reported in up to 60% of patients receiving CAR-T19.

Cytokine storms consist of an immune response that consists of a positive feedback loop between cytokines and leukocytes, resulting in very high levels of various cytokines. The term “cytokine storm” may be used interchangeably with the terms “cytokine cascade” and “hypercytokinemia”, both of which have the same properties and meanings. In some embodiments, the cytokine storm is characterized by IL-2 release and lymphatic proliferation. Cytokine storms cause potentially life-threatening complications including cardiac dysfunction, adult respiratory distress syndrome, neurotoxicity, renal and/or hepatic failure, and coagulation within disseminated blood vessels.

As mentioned, CAR-T cell therapy is currently limited by the risk of life-threatening neurotoxicity and CRS. Despite active management, all CAR-T respondents experience some degree of CRS. Up to 50% of patients treated with CD19 CAR-T have Grade 3 or higher CRS or neurotoxicity. GM-CSF levels and T-cell expansion are the factors most associated with grade 3 or higher CRS and neurotoxicity.

Reducing or eliminating CRS and neurotoxicity in immunotherapy, such as CAR-T cell therapy, is of great value and it is important to determine what triggers or exacerbates the CAR-T signature inflammatory response. Although many cytokines, signaling molecules and cell types are involved in this pathway, GM-CSF is the only cytokine that appears to be central to the pathway. Normally undetectable in human serum, it is central to a circulating positive feedback loop that triggers inflammation to the extreme of cytokine storm and endothelial cell activation. Neurotoxicity and cytokine storm are not the result of the simultaneous release of cytokines, but rather the continuum of inflammation initiated by GM-CSF, resulting in the trafficking and recruitment of myeloid cells to the tumor site.

These myeloid cells perpetuate the inflammatory cascade by producing cytokines observed in CRS and neurotoxicity.

GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor)

As used herein, "granulocyte macrophage-colony stimulating factor" (GM-CSF) refers to a small native glycoprotein having an internal disulfide bond with a molecular weight of about 23 kDa. In some embodiments, GM-CSF refers to human GM-CSF. In some embodiments, GM-CSF refers to non-human GM-CSF. In humans, it is encoded by a gene located within a cytokine cluster on human chromosome 5. The sequences of human genes and proteins are known. This protein has an N-terminal signal sequence and a C-terminal receptor binding domain (Rasko and Gough In: The Cytokine Handbook, A. Thomson, et al, Academic Press, New York (1994) pages 349-369). The structure is similar to that of interleukin, but the amino acid sequence is not. GM-CSF is produced in response to multiple inflammatory mediators by mesenchymal cells present in the hematopoietic environment and surrounding inflammatory sites. GM-CSF can stimulate the production of neutrophil granulocytes, macrophages and mixed granulocyte-macrophage colonies in myeloid cells and can stimulate the formation of eosinophil colonies in fetal liver progenitors. GM-CSF can also stimulate some functional activity in mature granulocytes and macrophages. GM-CSF, a cytokine present in the bone marrow microenvironment, recruits inflammatory monocyte-derived dendritic cells, stimulates secretion of high levels of IL-6 and CCL2/MCP-1, and induces a feedback loop, resulting in more monocytes and inflammatory dendritic cells. Recruit cells to the site of inflammation.

As mentioned, CRS involves an increase in several cytokines and chemokines, including IFN-γ, IL-6, IL-8, CCL2 (MCP-1), CCL3 (MIP1α) and GM-CSF (Teachey, D. et al. (June 2016), Cancer Discovery , CD-16-0040; Morgan R., et al., (April 2010), Molecular Therapy ). IL-6, one of the major inflammatory cytokines, is not produced by CAR-T cells (Barrett, D. et al. (2016), Blood ). Instead, it is produced by myeloid cells that recruit to the site of the tumor. GM-CSF mediates this recruitment, leading to the production of chemokines that activate myeloid cells and migrate them to the tumor site. Elevated GM-CSF levels serve as predictive biomarkers for CRS and indicators of its severity. More than a critical component of the inflammatory cascade, GM-CSF is a key initiator responsible for both CRS and NT. As described herein, in vivo studies using a murine model indicate that genetic silencing of GM-CSF prevents cytokine storm while maintaining CAR-T efficacy. GM-CSF knockout mice have normal levels of INF-γ, IL-6, IL-10, CCL2 (MCP1), CCL3/4 (MIG-1) in vivo and do not develop CRS (Sentman, M.- L., et al (2016), The Journal of Immunology , 197(12), 4674-4685.). The GM-CSF knockout CAR-T model recruits fewer NK cells, CD8 cells, myeloid cells and neutrophils to the tumor site compared to GM-CSF + CAR-T.

The term "soluble granulocyte macrophage-colony stimulating factor receptor" (sGM-CSFR) refers to a non-membrane-bound receptor that binds to GM-CSF but does not transmit a signal when bound to a ligand. do.

As used herein, a "peptide GM-CSF antagonist" refers to a GM-CSF to reduce (partially or completely) or block (partially or completely) signal transduction that may result from the binding of GM-CSF to a cognate receptor expressed in a cell. refers to a peptide that interacts with CSF or its receptor. GM-CSF antagonists act by reducing the amount of GM-CSF ligand available to bind to the receptor (e.g., an antibody that increases clearance of GM-CSF upon binding to GM-CSF) or the ligand is GM-CSF Binding to the CSF or receptor may prevent binding to that receptor (eg, a neutralizing antibody). GM-CSF antagonists may also include other peptide inhibitors, which may include polypeptides that partially or completely inhibit signal transduction by binding to GM-CSF or its receptor. The peptide GM-CSF antagonist is, for example, an anti; It may be a natural or synthetic GM-CSF receptor ligand that antagonizes GM-CSF or other polypeptides. An exemplary assay for detecting GM-CSF antagonist activity is provided in Example 1. In general, peptide GM-CSF antagonists such as neutralizing antibodies have an EC50 of 10 nM or less.

"Purified" GM-CSF antagonist, as used herein, refers to a GM-CSF antagonist that is substantially or essentially free of commonly associated components as found in nature. For example, a GM-CSF antagonist, such as an anti-GM-CSF antibody purified from blood or plasma, is substantially free of other blood or plasma components such as other immunoglobulin molecules. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The main species present in the formulation, the protein, is substantially purified. Typically, "purified" means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure relative to its naturally occurring component.

antibody

As used herein, an “antibody” is defined functionally as a binding protein and structurally defined as comprising an amino acid sequence recognized by one of ordinary skill in the art as derived from the framework region of an immunoglobulin-encoding gene of an animal producing the antibody. refers to protein. An antibody may consist of one or more polypeptides substantially encoded by an immunoglobulin gene or fragment of an immunoglobulin gene. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes as well as numerous immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to contain a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light (VL) and variable heavy (VH) chains refer to these light and heavy chains, respectively.

The term “antibody” includes antibody fragments that retain binding specificity. For example, there are many well-characterized antibody fragments. Thus, for example, pepsin cleaves the antibody C-terminus to the disulfide bond of the hinge region to yield F(ab')2, which is itself a dimer of Fab, a light chain linked to VH-CH1 by a disulfide bond. F(ab')2 can be reduced under mild conditions to break disulfide bonds in the hinge region, converting (Fab')2 dimers to Fab' monomers. A Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed discussion of other antibody fragments, see Fundamental Immunology, W.E.Paul, ed., Raven Press, N.Y. (1993)). While various antibody fragments are defined in the context of digestion of intact antibodies, those skilled in the art will recognize that fragments can be synthesized de novo chemically or using recombinant DNA methodologies. Accordingly, the term antibody as used herein also includes antibody fragments produced by modification of whole antibodies or synthesized using recombinant DNA methodologies.

Antibodies include dimers such as VH-VL dimers, VH dimers or VL dimers, single chain antibodies (antibodies present as single polypeptide chains), for example single chain Fv antibodies (sFv or scFv). wherein the variable heavy chain and the variable light region are linked together (either directly or via a peptide linker) to form a continuous polypeptide. Single chain Fv antibodies are covalently linked VH-VL heterodimers that can be expressed from nucleic acids comprising VH- and VL-coding sequences linked directly or joined by peptide coding linkers (eg, Huston, et al. al. Proc. Nat. Acad. Sci. USA , 85:5879-5883, 1988). The VH and VL are each linked by a single polypeptide chain, but the VH and VL domains are non-covalently linked. Alternatively, the antibody may be another fragment, such as a disulfide-stabilized Fv (dsFv). Other fragments can also be generated, including using recombinant techniques. scFv antibodies and several other naturally aggregated but chemically separated light and heavy polypeptide chains that are chemically separated in the antibody V region into molecules that fold into a three-dimensional structure substantially similar to that of the antigen binding site in the antibody V region. structures and known to those of ordinary skill in the art (see, eg, US Pat. Nos. 5,091,513, 5,132,405 and 4,956,778). In some embodiments, the antibody is expressed on phage or produced by recombinant techniques using vectors in which the chain is secreted into a soluble protein, e.g., scFv, Fv, Fab, (Fab')2, or the chain is secreted into a soluble protein. Antibodies produced by recombinant techniques using vectors that are Antibodies for use in the present invention may also include diantibodies and mini-antibodies.

Antibodies of the invention also include heavy chain dimers, such as antibodies from camelids. Since the VH region of camelid heavy chain dimer IgG does not need to undergo hydrophobic interaction with the light chain, the region of the heavy chain in contact with the light chain is generally changed to a camelid hydrophilic amino acid residue. The VH domain of heavy chain dimer IgG is called the VHH domain. Antibodies for use in the present invention include single domain antibodies (dAbs) and nanobodies (see, eg, Cortez-Retamozo, et al., Cancer Res . 64:2853-2857, 2004).

As used herein, “V-region” refers to an antibody variable region domain comprising segments of framework 1, CDR1, framework 2, CDR2 and framework 3, which segments are heavy and light chain V during B-cell differentiation. -contains CDR3 and framework 4 added to the V-segment as a result of rearrangement of region genes. As used herein, “V-segment” refers to the region of the V-region (heavy or light chain) encoded by a V gene. The V-segment of the heavy chain variable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes of the present invention, the V-segment of the light chain variable region is defined as extending through FR3 to CDR3.

As used herein, the term “J-segment” refers to a subsequence of an encoded variable region comprising the C-terminal portions of CDR3 and FR4. The endogenous J-segment is encoded by the immunoglobulin J-gene.

As used herein, "complementarity determining region (CDR)" refers to the three hypervariable regions of each chain that intersect the four "framework" regions established by the light and heavy chain variable regions. CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are generally referred to as CDR1, CDR2 and CDR3, are numbered sequentially from the N-terminus, and are also identified by the chain in which the particular CDR is located. Thus, for example, the V _H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas the V _L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of the antibody, i.e. the combined framework regions of the constituent light and heavy chains, serve to place and align the CDRs in three-dimensional space.

The amino acid sequences of CDRs and framework regions can be determined using various definitions known in the art, such as, for example, Kabat, Chothia, international ImMunoGeneTics database (IMGT) and AbM (e.g., Johnson et al., supra ; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883 ; Chothia C. et al, 1992, structural repertoire of the human VH segments J. Mol Biol 227, 799-817;.... al-Lazikani et al, J.Mol.Biol 1997, 273 reference 4). Definitions of antigen binding sites are also described in Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. , 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1:29(1):207-9 (2001); MacCallum et al , Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol. , 262 (5), 732-745 (1996); and Martin et al , Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al , Methods Enzymol ., 203, 121-153, (1991); Pedersen et al , Immunomethods , 1, 126, (1992); and Rees et al , In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996)

"Epitope" or "antigenic determinant" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from both contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of proteins. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, more typically at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996). see

The term “binding specificity determinant” or “BSD”, as used in the context of the present invention, refers to the minimum contiguous or non-contiguous amino acid sequence in the CDR region required to determine the binding specificity of an antibody. In the present invention, the minimal binding specificity determinant is within some or the entire length of the CDR3 sequences of the heavy and light chains of the antibody.

As used herein, “anti-GM-CSF antibody” or “GM-CSF antibody” are used interchangeably to refer to an antibody that binds to GM-CSF and inhibits GM-CSF receptor binding and activation. Such antibodies can be identified using any number of art-recognized assays that assess GM-CSF binding and/or function. For example, binding assays such as ELISA assays that measure inhibition of GM-CSF binding to alpha receptor subunits can be used. Cell-based assays for GM-CSF receptor signaling, such as assays that determine the proliferation rate of GM-CSF-dependent cell lines in response to limited amounts of GM-CSF, and assays such as IL-8 production in response to GM-CSF exposure It is conveniently used as an assay to measure cytokine production.

As used herein, "neutralizing antibody" refers to an antibody that binds to GM-CSF and inhibits signal transduction by the GM-CSF receptor or prevents binding of GM-CSF to the receptor.

As used herein, “human granulocyte macrophage-colony stimulating factor” (hGM-CSF) refers to a small naturally occurring glycoprotein having an internal disulfide bond with a molecular weight of about 23 kDa; The source and target of GM-CSF are humans; As such, anti-hGM-CSF antibodies as described in the embodiments herein bind only human and primate GM-CSF but not mouse, rat and other mammalian GM-CSF. The hGM-CSF antibody neutralizes human GM-CSF as described in the embodiments herein. In some embodiments, the human hGM-CSF is encoded by a gene located within a cytokine cluster of human chromosome 5. The sequences of human genes and proteins are known. This protein has an N-terminal signal sequence and a C-terminal receptor binding domain (Rasko and Gough In: The Cytokine Handbook, A. Thomson, et al. , Academic Press, New York (1994) pages 349-369) three-dimensional The structure is similar to that of interleukin, but the amino acid sequence is not. GM-CSF is produced in response to several inflammatory mediators present in the hematopoietic environment and surrounding inflammatory sites. GM-CSF can stimulate the production of neutrophil granulocytes, macrophages and mixed granulocyte-macrophage colonies in myeloid cells and can stimulate the formation of eosinophil colonies in fetal liver progenitors. GM-CSF can also stimulate some functional activities in mature granulocytes and macrophages and inhibits apoptosis of granulocytes and macrophages.

The term “equilibrium dissociation constant” or “affinity” abbreviation (K _D ) means the dissociation rate constant (k _d , time ⁻¹ ) divided by the association rate constant (k _a , time ⁻¹ M ⁻¹ ). The equilibrium dissociation constant can be determined using any method known in the art. The antibody of the present invention is a high affinity antibody. Such antibodies have a monovalent affinity that is better than (less than) about 10 nM and often better than about 500 pM or better than about 50 pM as determined by surface plasmon resonance analysis performed at 37°C. Thus, in some embodiments, an antibody of the invention has an affinity (measured using surface plasmon resonance) of less than 50 pM, typically less than about 25 pM, or even less than 10 pM.

In some embodiments, an anti-GM-CSF antibody of the invention has ^{less than about 10 -4} s ^-1 GM, preferably less than 5 x 10 ^-5 s ^-1 , and most preferably less than 10 ^-5 s ^-1 GM. It has a slow dissociation rate with a dissociation rate constant (kd) determined by surface plasmon resonance analysis at 37° C. for monovalent interactions with -CSF.

As used herein, a “chimeric antibody” refers to (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen binding site (variable region) is different or altered in a class, effector function and/or species, or novel in a chimeric antibody. refers to an immunoglobulin molecule linked to a constant region of an entirely different molecule that imparts a property, for example an enzyme, a toxin, a hormone, a growth factor, a drug, etc.; or (b) a variable region or portion thereof, wherein the variable region or portion thereof has different or altered antigenic specificity; or an immunoglobulin molecule altered, replaced or exchanged with the corresponding sequence from another species or another antibody class or subclass.

As used herein, “humanized antibody” refers to an immunoglobulin molecule in the CDRs of a donor antibody grafted onto human framework sequences. Humanized antibodies may also include residues of donor origin in the framework sequences. A humanized antibody may also comprise at least a portion of a human immunoglobulin constant region. Humanized antibodies may also contain residues that are not found in the recipient antibody or in the imported CDR or framework sequences. Humanization can be performed using methods known in the art (eg, Jones et al., Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al. al., Science 239:1534-1536, 1988; Presta, Curr. Op. Struct. Biol. 2:593-596, 1992; US Patent No. 4,816,567), "superhumanizing" (Tan et al. , J. Immunol. 169: 1119, 2002) and techniques such as "resurfacing" (eg, Staelens et al., Mol. Immunol . 43: 1243, 2006; and Roguska et al. al., Proc. Natl. Acad. Sci USA 91: 969, 1994).

A “HUMANEERED®” antibody in the context of the present invention refers to an engineered human antibody that has the binding specificity of a reference antibody. Engineered human antibodies for use in the present invention have immunoglobulin molecules that contain minimal sequence derived from a donor immunoglobulin. In some embodiments, the engineered human antibody may retain only minimal essential binding specificity determinants from the CDR3 region of a reference antibody. Typically, the engineered human antibody binds a DNA sequence encoding a binding specificity determinant (BSD) from the CDR3 region of a heavy chain of a reference antibody to a human VH segment sequence and binds a light chain CDR3 BSD of a reference antibody to a human VL segment sequence manipulated by "BSD" refers to the CDR3-FR4 region, or a portion of this region that mediates binding specificity. Thus, binding specificity determinants are the minimum essential binding specificity determinants of CDR3-FR4, CDR3, CDR3 (representing any region smaller than CDR3 that confer binding specificity when present in the V region of an antibody), the D segment (with respect to the heavy chain region) , or other regions of CDR3-FR4 that confer the binding specificity of the reference antibody. Methods of engineering human antibodies are described in US Patent Application Publication No. 20050255552 and US Patent Application Publication No. 20060134098.

As used herein, the term “human antibody” refers to an antibody that is substantially human from the human immune system, ie, having FR regions and often CDR regions. Thus, the term includes humanized and humaneered antibodies as well as antibodies isolated from mice reconstituted with the human immune system and antibodies isolated from display libraries.

The term “heterologous” when used in reference to a portion of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, eg, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Similarly, heterologous proteins often refer to two or more subsequences that are not found in the same relationship to each other in nature.

For example, the term "recombinant" when used in reference to a cell, nucleic acid, protein or vector indicates that the cell, nucleic acid, protein or vector has been modified by introduction of a heterologous nucleic acid or protein or alteration of a native nucleic acid or protein, or said indicates that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell or otherwise expresses a native gene that is aberrantly expressed, underexpressed or not expressed at all. The term "recombinant nucleic acid" herein generally refers to a nucleic acid originally formed in vitro, generally by manipulation of the nucleic acid using, for example, polymerases and endonucleases, in a form not normally found in nature. In this way, operable linkage of different sequences is achieved. Accordingly, both isolated nucleic acids in linear form or expression vectors formed in vitro by ligating DNA molecules that are not normally linked are considered recombinant for the purposes of the present invention. It is understood that once a recombinant nucleic acid has been made and reintroduced into a host cell or organism, it will be replicated non-recombinantly, ie using the host cell's in vivo cellular machinery rather than in vitro manipulation; However, such nucleic acids once produced recombinantly are subsequently replicated non-recombinantly but are still considered recombinant for the purposes of the present invention. Similarly, a "recombinant protein" is a protein made using recombinant technology, ie, through expression of a recombinant nucleic acid.

The term “specifically (or selectively) binds” or “specifically (or selectively) immunoreactive” refers to a binding reaction in which an antibody binds to an antigen of interest. In the context of the present invention, an antibody typically binds an antigen, for example GM-CSF, with an affinity of 500 nM or less and has an affinity of 5000 nM or more for other antigens.

The term “identical” or “identity” (%) in relation to two or more polypeptide (or nucleic acid) sequences is used for manual alignments and visual inspections or using the BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below. When comparing and aligning identical or identical amino acid residues (or nucleotides) in a specified proportion (i.e., with maximum correspondence across a window of comparison or specified region), about 60% identity over a specified region, preferably is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity). more than one sequence or subsequence (see, eg, NCBI website). Hereinafter, such sequences are said to be "substantially identical." Also, "substantially identical" sequences include sequences with deletions and/or additions and sequences with substitutions and natural, eg, polymorphic or surrogate variants and artificial variants. As described below, preferred algorithms can account for gaps and the like. Preferably, the protein sequence identity exists over a region that is at least about 25 amino acids in length, more preferably over a region that is 50-100 amino acids in length or over the length of the protein.

As used herein, a "comparison window" refers to a portion of a number of consecutive positions typically selected from the group consisting of 20 to 600, typically about 50 to about 200, and more typically about 100 to about 150. refers to, wherein after optimal alignment of the two sequences, the sequence can be compared to a reference sequence of the same number of contiguous positions. Methods for aligning sequences for comparison are well known in the art. Methods for aligning sequences for comparison are well known in the art. For example, Smith & Waterman's local homology algorithm [Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch [J. Mol. Biol. 48:443 (1970), Pearson & Lipman's method of similarity investigation [Proc. Nat'l. Acad. Sci. USA 85:2444 (1988)], by computerized runs of these algorithms [GAP, BESTFIT, FASTA and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.] or by manual alignment and Optimal alignment of sequences for comparison can be performed by visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with an antibody raised against the second polypeptide. Thus, a polypeptide is typically substantially identical to a second polypeptide, eg, the two peptides differ only by conservative substitutions.

Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity are described in Altschul et al., Nuc. Acids Res. 25: 3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215: 403-410 (1990), the BLAST and BLAST 2.0 algorithms. BLAST and BLAST 2.0 are used in conjunction with the parameters described herein to determine percent sequence identity to nucleic acids and proteins of the invention. (for nucleotide sequences) The BLASTN program uses as default a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses a wordlength of 3 as default and an expectation (E) of 10, and the BLOSUM62 score matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)). uses an alignment of 50 (B), an expectation of 10 (E), M=5, N=-4 and a comparison of both strands.

The terms “isolated,” “purified,” or “biologically pure” mean a material that is substantially or essentially free of components that normally accompany it when it is found in its natural state. Analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography are commonly used to measure purity and homogeneity. The main species present in the formulation, the protein, is substantially purified. In some embodiments the term "purified" indicates that the protein produces substantially one band in the electrophoretic gel. Preferably, this means that the protein is at least 85% pure, more preferably at least 95% pure and most preferably at least 99% pure.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding natural amino acids, and to natural amino acid polymers, amino acid polymers containing modified residues, and non-natural amino acid polymers.

The term “amino acid” refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to natural amino acids. Natural amino acids are amino acids encoded by the genetic code and amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Amino acid analogs are compounds having the same basic chemical structure as a natural amino acid, for example, an α carbon, a carboxyl group, an amino group and an R group bonded to a hydrogen, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. means Such analogs may have modified R groups (eg, norleucine) or modified peptide backbones, but may retain the same basic chemical structure as a native amino acid. Amino acid mimetics refers to chemical compounds that have a structure different from the general chemical structure of amino acids, but function similarly to natural amino acids.

Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, one may refer to a nucleotide by its generally accepted one-letter code.

"Conservatively modified variants" apply to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to a nucleic acid encoding the same or substantially identical amino acid sequence, or substantially identical or related, e.g., naturally contiguous sequences, if the nucleic acid does not encode an amino acid sequence. it means. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at any position where an alanine is designated as a codon, the codon can be changed to another codon of that designated codon without altering the encoded polypeptide. Such nucleic acid mutations are "silent mutations" which are a type of conservatively modified mutations. In addition, any nucleic acid sequence herein that encodes a polypeptide represents a silent variation of the nucleic acid. One of ordinary skill in the art, in certain contexts, in a nucleic acid, each codon (except AUG and TGG, where AUG is usually the only codon for methionine, and TGG is usually the only codon for trytophan) can be modified to produce a functionally identical molecule. will recognize that there is Thus, usually silent variations of a nucleic acid encoding a polypeptide are implicit with respect to the product of expression in the sequence described, but not with respect to the actual probe sequence.

With respect to amino acid sequences, one skilled in the art would recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide or protein sequence that alter, add or delete a single amino acid or a small proportion of amino acids in the encoded sequence are "conservatively modified It will be understood that "variants", wherein an alteration replaces an amino acid with a chemically similar amino acid. Conservative substitution tables and substitution matrices such as BLOSUM providing functionally similar amino acids are well known in the art. Such conservatively modified variants further exist and do not exclude polymorphic variants, interspecies homologues and alleles of the present invention. Common conservative substitutions for each other include (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), trytophan (W); (7) serine (S), threonine (T); and (8) cysteine (C), methionine (M) (see, eg, Creighton, Proteins (1984)).

Methods of preventing or treating immunotherapy-related toxicity

In some embodiments, disclosed herein are methods of inhibiting immunotherapy-related toxicity in an individual. In some embodiments, provided herein is a method of reducing the incidence of immunotherapy-related toxicity in an individual. In some embodiments, disclosed herein are methods of neutralizing hGM-CSF. In some embodiments, the method comprises administering to the subject a recombinant hGM-CSF antagonist. In some embodiments, the method comprises hGM-CSF gene silencing. In some embodiments, the method comprises a hGM-CSF gene knockout. Gene silencing and gene knockout methods are well known to those skilled in the art, and include RNA interference (RNAi), CRISPR, short interfering RNS (siRNA), DNA-induced RNA interference (ddRNAi), engineered transcription activator-like effector nuclease (TALEN) or other Targeted genome editing using suitable techniques may include, without limitation.

Gene editing technology to knock out GM-CSF: GM-CSF ^k/o

In some embodiments, gene editing techniques are used to knock out the expression of GM-CSF. Genome editing is performed by delivering endonucleases (including Fok1 or Cas9) that cleave DNA into site-specific segments of the genetic code.

Endonucleases cleave DNA that triggers endogenous DNA repair mechanisms.

Those skilled in the art will recognize that any method of achieving site-specific chromosomal DNA cleavage that triggers endogenous DNA repair results in the same targeted genomic modification. The target genome modification will not differ depending on whether the site specificity is determined by the RNA guide, DNA guide or DNA binding protein, or by the endonuclease used to achieve DNA cleavage. Examples of RNA induced site specificity include, without limitation, CRISPR/Cas9. Examples of DNA induction site specificity include, without limitation, flap endonuclease 1 (FEN-1). Examples of DNA binding proteins used to achieve site specificity include zinc-finger proteins (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases including ARCUS, meganucleases, and the like. not limited

Other methods that may be used for gene silencing are well known to those of skill in the art and may include, without limitation, RNA interference (RNAi), short interfering RNS (siRNA), DNA-induced RNA interference (ddRNAi).

In one aspect, the present invention provides a method for GM-CSF gene inactivation or GM-CSF knockout (KO) in a cell comprising targeted genome editing or GM-CSF gene silencing. In one embodiment of the methods provided, the targeted genome editing comprises an endonuclease, wherein the endonuclease is a Fok1 restriction enzyme or flap endonuclease 1 (FEN-1). In another embodiment, the endonuclease is Cas9 CRISPR related protein 9 (Cas9). In some embodiments, the GM-CSF gene is inactivated by CRISPR/Cas9 which targets and edits GM-CSF in exon 1, exon 2, exon 3 or exon 4. In a further embodiment, the GM-CSF gene inactivation comprises a CRISPR/Cas9 target and edits the GM-CSF in exon 3. In one embodiment, the GM-CSF gene inactivation comprises a CRISPR/Cas9 target in exon 1 and edits the GM-CSF. In one embodiment, the GM-CSF gene inactivation comprises multiple CRISPR/Cas9 enzymes, wherein each Cas9 enzyme targets and edits a different sequence of GM-CSF in exon 1, exon 2, exon 3 or exon 4 . In certain embodiments, GM-CSF gene inactivation comprises biallelic CRISPR/Cas9 targeting and knockout/inactivation of the GM-CSF gene. In another aspect of a provided method for GM-CSF gene inactivation or GM-CSF knockout (KO), the method further comprises treating the primary T cell with valproic acid to enhance biallelic knockout/inactivation. . In one aspect of the provided methods for GM-CSF gene inactivation/gene knockout, the targeted genome editing comprises a zinc finger (ZnF) protein. In a further aspect, targeted genome editing comprises a transcriptional activator-like effector (TALENS). In one embodiment, the targeted genome editing comprises a homing endonuclease, wherein the homing endonuclease is an ARC nuclease (ARCUS) or a meganuclease. In certain embodiments of the methods provided herein, the cell is a CAR T cell. In another embodiment, the CAR T cell is a CD19 CAR-T cell. In one embodiment, the GM-CSF gene silencing is selected from the group consisting of RNA interference (RNAi), short interfering RNS (siRNA) and DNA-induced RNA interference (ddRNAi).

Methods of Prevention or Treatment of Immunotherapy-Related Toxicity

In some embodiments, the method comprises administering a CAR-T cell modified to express a lower level of GM-CSF via GM-CSF gene silencing or GM-CSF gene knockout. Gene silencing and gene knockout methods are well known to those skilled in the art and may include, without limitation, RNAi, CRISPR, siRNA, ddRNAi, TALEN, zinc-finger, homing endonucleases and meganucleases or other suitable techniques.

In some embodiments, administration of CAR-T cells in which GM-CSF is silenced or gene knocked out prevents or significantly reduces the incidence and/or severity of CRS and NT. In some embodiments, administration of CAR-T cells in which GM-CSF is silenced or gene knocked out prevents or significantly reduces BBB destruction. In some embodiments, administration of CAR-T cells in which GM-CSF is silenced or gene knocked out prevents or significantly reduces activation and trafficking of CD14+ myeloid cells to the CNS. In some embodiments, administration of CAR-T cells in which GM-CSF is silenced or gene knockout is administered with the systemic cytokines IL-3, IL-5, IP10, KC, MCP-1, MIP-1a, MIP-1b, M- CSF, MIP-2, MIG, VEGF, IL-1ra, IL-1b, IL-6, IL-12p40, IL12p70, IL-RA, M-CSF, and G-CSF observed upon administration of wild-type CAR-T cells to a lower level than what has been

In some embodiments, administration of CAR-T cells in which GM-CSF is silenced or gene knocked out occurs in combination with a recombinant GM-CSF antagonist, further reducing the incidence or severity of CRS, NT and further preventing or reducing BBB destruction and , further prevents or reduces activation of CD14+ myeloid cells and trafficking to the CNS, and IL-3, IL-5, IP10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, Systemic cytokine levels of MIG, VEGF, IL-1ra, IL-1b, IL-6, IL-12p40, IL12p70, IL1-RA, M-CSF, and G-CSF were observed upon administration of wild-type CAR-T cells. to prevent or reduce further.

How to Improve the Efficacy of Adoptive Cell Therapy

In some embodiments, the method comprises administering a CAR-T cell modified to express a lower level of GM-CSF via GM-CSF gene silencing or GM-CSF gene knockout. In some embodiments, CAR-T cells in which the GM-CSF gene is silenced or gene knocked out are less differentiated after expansion and comprise a higher percentage of naive, stem cell memory and central memory characteristics after expansion compared to wild-type CAR-T cells. do. In some embodiments, CAR-T cells in which the GM-CSF gene is silenced or gene knocked out do not express FAS or express lower levels of FAS than wild-type CAR-T cells. In some embodiments CAR-T cells in which the GM-CSF gene is silenced or gene knocked out are more resistant to activation-induced cell death (AICD), more resistant to senescence, and anergy compared to wild-type CAR-T cells. ) is resistant to In some embodiments, CAR-T cells in which GM-CSF is silenced or gene knocked out result in lower levels of MDSC formation and better CAR-T cell expansion and persistence compared to wild-type CAR-T cells. In some embodiments, administration of CAR-T cells in which the GM-CSF gene is silenced or gene knocked out results in greater expansion and persistence than wild-type CAR-T cells. In some embodiments, CAR-T cells in which the GM-CSF gene is silenced or gene knocked out exhibit a higher level of objective response (complete response and partial response) compared to wild-type CAR-T cells. In some embodiments, CAR-T cells in which the GM-CSF gene is silenced or gene knocked out exhibit lower levels of recurrence at 6 months, 12 months and 24 months compared to wild-type CAR-T cells. In some embodiments, CAR-T cells in which the GM-CSF gene is silenced or gene knocked out demonstrate improved levels of progression-free survival and/or overall survival compared to wild-type CAR-T cells.

In some embodiments, administration of GM-CSF silencing or gene knockout CAR-T cells occurs with a recombinant GM-CSF antagonist that further improves expansion, persistence, resistance to aging and anergy resistance. In another embodiment, administration of GM-CSF silencing or gene knockout CAR-T cells in combination with a recombinant GM-CSF antagonist further reduces MDSC formation. In another embodiment, administration of GM-CSF silencing or gene knockout CAR-T cells in combination with a recombinant GM-CSF antagonist further improves the objective response (complete response and partial response) and relapses at 6 months, 12 months and 24 days. It lowers the level and shows an improved level of progression free survival and/or overall survival.

In some embodiments, the CAR-T cell is a CD19 CAR-T cell; In another embodiment, the CAR-T cell is a BMCA CAR-T cell, and in another embodiment the CAR-T cell is a dual CD19/CD22 CAR-T cell. In another embodiment, the CAR-T cell is a dual CD19/CD20 CAR-T cell.

In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing immune activation. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises ameliorating capillary leak syndrome. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises ameliorating cardiac dysfunction. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises ameliorating encephalopathy. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises alleviating colitis. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises suppressing seizures. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises ameliorating CRS. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises ameliorating neurotoxicity. In various embodiments, the CAR-T cell-associated neurotoxicity in the subject is reduced by about 90% as compared to the reduction in neurotoxicity in the subject treated with the CAR-T cells and a control antibody. In certain embodiments, the recombinant GM-CSF antagonist is an antibody, particularly a GM-CSF neutralizing antibody according to the embodiments described herein, including Example 15.

In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing cytokine storm symptoms. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises increasing impaired left ventricular ejection fraction. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises ameliorating diarrhea. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises improving disseminated intravascular coagulation.

In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing edema. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises amelioration of the rash. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing gastrointestinal bleeding. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises treating gastrointestinal perforation. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises treatment of hemophagocytic lymphohistiocytosis (HLH). In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises treating hepatitis. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing hypotension. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises reducing pituitary.

In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises inhibiting an immune related adverse event. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing immunohepatitis. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises reducing immunodeficiency. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises treating ischemia. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing liver toxicity. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises treating macrophage-activated syndrome (MAS). In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing neurotoxic symptoms.

In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing pleural effusion. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pericardial effusion. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pneumonia.

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing polyarthritis. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises treating occipital reversible encephalopathy syndrome (PRES). In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing pulmonary hypertension. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises treating a thromboembolism. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing transaminitis. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the patient's CRES, neurotoxicity (NT) and/or cytokine release syndrome (CRS) grade. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises improving the patient's CARTOX-10 score.

In one aspect, as demonstrated in Examples 6 and 20-21, the present invention further provides a method of treating or preventing an immunotherapy-related toxicity in a subject, said method comprising administering to the subject a chimeric antigen receptor-expressing T -administering a cell (CAR-T cell) and a recombinant hGM-CSF antagonist, wherein the CAR-T cell has a GM-CSF gene knockout (GM-CSF ^k/o CAR-T cell). In some embodiments, the GM-CSF ^k/o CAR-T cell expresses a reduced level of GM-CSF compared to the level of GM-CSF expression by the wild-type CAR-T cell. In certain embodiments, the GM-CSF ^k/o CAR-T cell produces a level of one or more cytokines and/or chemokines that is lower than or equal to the level of one or more cytokines and/or chemokines expressed by the wild-type CAR-T cell. to manifest In certain embodiments, the one or more cytokines are selected from the group consisting of IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a., TNF-a and VEGF. selected human cytokines. In some embodiments the one or more cytokines are IFN-γ, IL-1a, IL-1b, IL-2, IL-4, IL-5, IL-6, IL7, IL-9, IL-10, IL-12p40 , IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF MIP-2, MIG, RANTES, and TNF-a , eotaxin, G-CSF, and combinations thereof. In various embodiments, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In certain embodiments, the anti-GM-CSF antibody binds human GM-CSF. In another embodiment, the anti-GM-CSF antibody binds to primate GM-CSF. In various embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In some embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In certain embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In various embodiments, the anti-hGM-CSF antibody is a human antibody. In some embodiments, the CAR-T cell is a CD19 CAR-T cell. In certain embodiments, the GM-CSF ^k/o CAR-T cells enhance the antitumor activity of the recombinant hGM-CSF antagonist. In certain embodiments, the GM-CSF ^k/o CAR-T cells improve overall survival of a subject as compared to survival in a subject treated by administration of the wild-type CAR-T cells. In certain embodiments, administration of CAR-T cells and recombinant hGM-CSF antagonists with GM-CSF gene knockout (GM-CSF ^k/o CAR-T cells) to the subject results in immunity such as CRS, neurotoxicity and neuroinflammatory Continuous treatment for the prevention or treatment of therapy-related toxicity. In some embodiments, the subject has cancer. In various embodiments, the cancer is acute lymphoblastic leukemia.

How to Remove Human GM-CSF from a Subject

In one aspect, the present invention provides human GM-CSF in a subject in need thereof, comprising administering to the subject a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^{k/o CAR-T cell).} Methods for neutralizing and/or eliminating are provided.

In one embodiment of this method, the method further comprises administering to the subject a recombinant hGM-CSF antagonist. In certain embodiments, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In one embodiment, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In another embodiment, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. In a further embodiment, the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scan It is selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. In another embodiment, the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. In a further embodiment, the GM-CSF is CAR T-derived GM-CSF or non-CAR T-derived GM-CSF. In some embodiments, the subject has the development of immunotherapy-related toxicity.

How to Reduce Blood Brain Barrier Breakdown and Preserve/Maintain BBB Integrity

In one aspect, the invention provides a method of reducing blood-brain barrier disruption in a subject treated with immunotherapy comprising administering to the subject a recombinant GM-CSF antagonist. In some embodiments, the subject has the development of immunotherapy-related toxicity.

In certain embodiments, immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cytokine administration, cancer vaccine administration, T cell engaging therapy, or any combination thereof. In various embodiments, adoptive cell transfer is a chimeric antigen receptor-expressing T-cell (CAR), a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR) ) the modified natural killer cells, or dendritic cells, or a combination thereof. In certain embodiments, the CAR T-cell is a CD19 CAR-T cell.

In a further embodiment, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In some embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In various embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In certain embodiments, the anti-GM-CSF antibody binds to primate GM-CSF. In some embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, foto ( potto), sifaka, indri, aye-aye, ape or human.

In certain embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In various embodiments, the anti-hGM-CSF antibody binds human GM-CSF. In certain embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In a further embodiment, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In a further embodiment, the anti-hGM-CSF antibody is a human antibody. In certain embodiments, the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. In one more specific embodiment, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered before, concurrently with, or after immunotherapy or a combination thereof.

In various embodiments, the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. In certain embodiments, the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is 95% human JH4 (YFD YWGQGTL VTVSS) and at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence, wherein the V-segment comprises at least 90% identity; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. In some embodiments, the J segment comprises YFDYWGQGTLVTVSS. In certain embodiments, CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In a further embodiment, the VH region CDR1 is a human germline VH1 CDR1; VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. In a further embodiment, the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2, as shown in the VH region shown in FIG. 1 . In some embodiments, the V-segment sequence has the VH V segment sequence shown in FIG. 1 . In various embodiments, the VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in FIG. 1 . In certain embodiments, the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR.

In a further embodiment, the anti-hGM-CSF antibody comprises a human germline JK4 region. In certain embodiments, the VL region CDR3 comprises QQFN(K/R)SPL. In some embodiments, the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. In certain embodiments, the VL region comprises CDR1, or CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . In certain embodiments, the VL region comprises a V segment with at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . In various embodiments, the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . In some embodiments, the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. In a further embodiment, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In a further embodiment, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus.

In various embodiments, the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scan. It is selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. In certain embodiments, the immunotherapy-related toxicity is a CAR-T related toxicity. In one more specific embodiment, the CAR-T related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

In another aspect, the invention provides a method of preserving blood-brain barrier integrity in a subject treated with immunotherapy comprising administering to the subject a recombinant hGM-CSF antagonist.

In a further aspect, the invention provides disruption of the blood-brain barrier in a subject treated with immunotherapy comprising administering a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^{k/o CAR-T cell).} It provides a method for preventing or reducing.

In one embodiment of the methods provided, the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. In another embodiment, the anti-GM-CSF antibody binds to mammalian GM-CSF. In another embodiment, the anti-GM-CSF antibody binds to primate GM-CSF. In further embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, foto ( potto), sifaka, indri, aye-aye, ape or human.

In certain embodiments of the provided methods, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In a further specific embodiment, the anti-hGM-CSF antibody binds human GM-CSF. In a further embodiment, the anti-hGM-CSF antibody is a monoclonal antibody. In certain embodiments, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In one embodiment, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In another embodiment, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In certain embodiments, the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. In various embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered before, concurrently with, or after immunotherapy or a combination thereof.

In one embodiment of the provided methods, the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein at least 95% of the J-segment human JH4 (YFD YWGQGTL VTVSS) and identity and the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. In certain embodiments, the J segment comprises YFDYWGQGTLVTVSS. In certain embodiments, CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is a human germline VH1 CDR1; VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. In certain embodiments, the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2, as shown in the VH region shown in FIG. 1 . In one embodiment, the V-segment sequence has the VH V segment sequence shown in FIG. 1 . In some embodiments, VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in FIG. 1 . In various embodiments, the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. In certain embodiments, the anti-hGM-CSF antibody comprises a human germline JK4 region. In one embodiment, the VL region CDR3 comprises QQFN(K/R)SPL. In certain embodiments, the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. In some embodiments, the VL region comprises CDR1, or CDR2, or both CDR1 and CDR2 of the VL region shown in FIG. 1 . In various embodiments, the VL region comprises a V segment having at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . In certain embodiments, the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . In certain embodiments, the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. In a further embodiment, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In some embodiments, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. In certain embodiments, the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analogue, adnectin, lipocalin scaffold antibody mimetic (lipocalin scaffold antibody mimetic), calixarene antibody mimetic, and antibody-like binding peptidomimetic. In certain embodiments of the provided methods, the subject has immunotherapy-related toxicity. In a further embodiment, the immunotherapy-related toxicity is a CAR-T related toxicity. In a further embodiment, the CAR-T related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof.

In a further aspect, the invention provides a method of reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof comprising administering to the subject a recombinant GM-CSF antagonist.

In some embodiments, administration of the recombinant GM-CSF antagonist reduces disruption of the blood brain barrier to maintain its integrity. In certain embodiments, reducing disruption of the blood brain barrier reduces or prevents entry of pro-inflammatory cytokines into the central nervous system. In various embodiments, the pro-inflammatory cytokine is IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF, TNF-a, FGF-2, IFN-γ, IL-12p40, IL-12p70, sCD40L, MDC, MCP-1, MIP-1a, MIP-1b, or a combination thereof. In certain embodiments, the pro-inflammatory cytokine is IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IP-10, KC, It is selected from the group consisting of MCP-1, MIP, or a combination thereof. In certain embodiments, neuroinflammation in an individual is reduced by 75% to 95% compared to an individual treated with CAR-T cell therapy and a control antibody. In various embodiments, a 75% to 95% reduction in neuroinflammation is similar to neuroinflammation in an untreated control subject. In some embodiments, the subject is administered a chimeric antigen receptor-expressing T-cell (CAR T-cell). In certain embodiments, the subject is administered a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR) modified natural killer cell, or a dendritic cell, or any combination thereof. This is administered In certain embodiments, the CAR T-cell is a CD19 CAR-T cell. In various embodiments, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In certain embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In a further embodiment, the anti-GM-CSF antibody binds to primate GM-CSF. In some embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, foto ( potto), sifaka, indri, aye-aye, ape or human. In one embodiment, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In another embodiment, the anti-hGM-CSF antibody binds human GM-CSF. In a further embodiment, the anti-hGM-CSF antibody is a monoclonal antibody. In a further embodiment, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In certain embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody.

In various embodiments, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In certain embodiments, the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. In a further embodiment, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody is administered before, concurrently with, or after immunotherapy or a combination thereof.

In a further embodiment, the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is 95% human JH4 (YFD YWGQGTL VTVSS) or greater identity and the V-segment contains greater than or equal to 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. In various embodiments, the J segment comprises YFDYWGQGTLVTVSS.

In certain embodiments, CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is a human germline VH1 CDR1; VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. In various embodiments, the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2, as shown in the VH region shown in FIG. 1 . In certain embodiments, the V-segment sequence has the VH V segment sequence shown in FIG. 1 . In certain embodiments, VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in FIG. 1 . In a further embodiment, the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. In a further embodiment, the anti-hGM-CSF antibody comprises a human germline JK4 region. In some embodiments, the VL region CDR3 comprises QQFN(K/R)SPL. In certain embodiments, the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. In various embodiments, the VL region comprises CDR1, or CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . In a further embodiment, the VL region comprises a V segment having at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . In a further embodiment, the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 .

In certain embodiments, the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. In some embodiments, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In various embodiments, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus.

In a further embodiment, the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody mimetic (lipocalin scaffold antibody mimetic), calixarene antibody mimetic, and antibody-like binding peptidomimetic. In some embodiments. The subject further has a CAR-T related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof.

How to reduce or prevent recurrence

In one aspect, the present invention provides a method of reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy, comprising administering to the subject a recombinant GM-CSF antagonist. In some embodiments, reducing the recurrence rate or preventing the occurrence of tumor recurrence in an individual occurs without the occurrence of immunotherapy-related toxicity. In certain embodiments, reducing the rate of recurrence or preventing the occurrence of tumor recurrence in an individual occurs in the presence of the occurrence of an immunotherapy-related toxicity. In some embodiments, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In various embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In some embodiments, the anti-GM-CSF antibody binds human GM-CSF. In certain embodiments, the anti-GM-CSF antibody binds to primate GM-CSF. In various embodiments, the primate is a monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, foto ( potto), sifaka, indri, aye-aye, or ape. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF.

In certain embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. As mentioned above, the anti-GM-CSF antibody is a monoclonal antibody. In another embodiment, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In various embodiments, the anti-hGM-CSF antibody is a human antibody. In some embodiments, the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. In certain embodiments, the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. In various embodiments, the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. In some embodiments, the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is 95% with human JH4 (YFD YWGQGTL VTVSS). at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence, wherein the V-segment comprises at least 90% identity; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. In certain embodiments, the J segment comprises YFDYWGQGTLVTVSS. In a further embodiment, CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. In some embodiments, the VH region CDR1 is a human germline VH1 CDR1; VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences.

In certain embodiments, the anti-hGM-CSF antibody comprises VH CDR1 or VH CDR2, or both VH CDR1 and VH CDR2, shown in the VH region shown in FIG. 1 . In various embodiments, the V-segment sequence has the VH V segment sequence shown in FIG. 1 . In certain embodiments, VH has the sequence of VH#1, VH#2, VH#3, VH#4 or VH#5 shown in FIG. 1 . In some embodiments, the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. In some embodiments, the anti-hGM-CSF antibody comprises a human germline JK4 region. In certain embodiments, the VL region CDR3 comprises QQFN(K/R)SPLT. In various embodiments, the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. In some embodiments, the VL region comprises CDR1, or CDR2, or both CDR1 and CDR2 of the VL region shown in FIG. 1 . In certain embodiments, the VL region comprises a V segment with at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . In some embodiments, the VL region has the sequence of VK#1, VK#2, VK#3, or VK#4 shown in FIG. 1 . In certain embodiments, the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. In some embodiments, the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . In another embodiment, the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus.

In some embodiments, the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scan It is selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. In certain embodiments, the CAR-T cell is a CD19 CAR-T cell. In certain embodiments, the immunotherapy-related toxicity is a CAR-T related toxicity. In some embodiments, the CAR-T related toxicity is CRS, NT, or neuroinflammation.

In certain embodiments, the incidence of tumor recurrence is reduced by 50% to 100% in the first quarter following administration of the recombinant GM-CSF antagonist, as compared to the incidence of tumor recurrence in a subject treated with immunotherapy and not administered the recombinant GM-CSF antagonist do. In certain embodiments, the incidence of tumor recurrence is reduced by 50% to 95% in the first half following administration of the recombinant GM-CSF antagonist. In various embodiments, the incidence of tumor recurrence is reduced by 50% to 90% in the first year after administration of the recombinant GM-CSF antagonist. In some embodiments, the occurrence of tumor recurrence is prevented for a long period of time. As used herein, the term “long-term” refers to an extended period of at least one year, ie, 12 months, from the last date of treatment with the recombinant hGM-CSF antagonist. In some embodiments, the recombinant hGM-CSF antagonist is an hGM-CSF neutralizing antibody. In various embodiments, the recombinant hGM-CSF antagonist is an anti-hGM-CSF antibody, eg, Lenzilumab. In certain embodiments, tumor recurrence is prevented by 12-36 months of age. In some embodiments, the occurrence of tumor recurrence is "completely" (100%) prevented, which as used herein means no recurrence of the tumor for at least 12 months from the last date of treatment with the recombinant hGM-CSF antagonist. In certain embodiments, the subject has acute lymphoblastic leukemia.

In various embodiments of the methods provided herein for reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in an individual treated with immunotherapy, the individual has a refractory cancer, which as used herein (a) A malignant tumor for which surgery is ineffective (also referred to herein as "cancer" or "tumor") and (b) is initially unresponsive or resistant to treatment, wherein the treatment is chemotherapy, radiation, or a combination thereof; or (b) a malignancy that has not responded or has become refractory to said treatment. In some embodiments, the individual is suffering from a “recurrent” cancer, which, as used herein, is a cancer that has responded to treatment but has relapsed. In certain embodiments, the refractory cancer or relapsed cancer is non-Hodgkin's lymphoma (NHL). In various embodiments, the refractory cancer or relapsed cancer is non-Hodgkin's lymphoma (NHL). In certain embodiments, the refractory cancer is refractory aggressive B-cell non-Hodgkin's lymphoma. In some embodiments, the refractory cancer or relapsed cancer is chemotherapy refractory B cell lymphoma. In various embodiments, the refractory cancer or relapsed cancer is hormone refractory prostate cancer. In certain embodiments, the refractory cancer or relapsed cancer is childhood cancer. In some embodiments, the refractory or relapsed childhood cancer is neuroblastoma. In certain embodiments, the refractory childhood cancer or relapsed childhood cancer is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) or uncommon childhood leukemia, which is juvenile myeloid monocytic leukemia or chronic myelogenous leukemia is leukemia In certain embodiments, the refractory cancer or relapsed cancer is juvenile bone cancer. In some embodiments, the refractory cancer or relapsed cancer is adrenal cancer. In various embodiments, the refractory cancer or relapsed cancer is breast cancer. In certain embodiments, the refractory cancer or relapsed cancer is colon cancer, rectal cancer, or colorectal cancer. In certain embodiments, the refractory cancer or relapsed cancer is a T cell lymphoma. In some embodiments, the refractory cancer or relapsed cancer is head and neck cancer. In some embodiments, the refractory cancer or relapsed cancer is brain and/or spinal cord cancer, including but not limited to gliomas and glioblastomas. In a further embodiment, the refractory or relapsed cancer is a tumor of bone or soft tissue, including but not limited to chondrosarcoma. In various embodiments, the refractory or relapsed cancer is bone cancer. In some embodiments, the refractory cancer or relapsed cancer is esophageal cancer. In certain embodiments, the refractory cancer or relapsed cancer is gallbladder cancer. In some embodiments, the refractory cancer or relapsed cancer is kidney cancer. In various embodiments, the refractory or relapsed cancer is melanoma. In some embodiments, the refractory cancer or relapsed cancer is ovarian cancer. In certain embodiments, the refractory cancer or relapsed cancer is pancreatic cancer. In some embodiments, the refractory cancer or relapsed cancer is a skin cancer selected from basal cell carcinoma, squamous cell carcinoma or melanoma. In various embodiments, the refractory or relapsed cancer is lung cancer. In some embodiments, the refractory cancer or relapsed cancer is salivary gland cancer. In a further embodiment, the refractory cancer or relapsed cancer is uterine smooth muscle cancer. In some embodiments, the refractory cancer or relapsed cancer is testicular cancer. In various embodiments, the refractory cancer or relapsed cancer is gastric cancer or gastrointestinal cancer. In certain embodiments, the refractory cancer or relapsed cancer is bladder cancer. In a further embodiment, the refractory cancer or relapsed cancer is an adipose tissue neoplasm. In some embodiments, the refractory or relapsed childhood cancer is adenocarcinoma. In certain embodiments, the refractory cancer or relapsed cancer is a thymoma. In various embodiments, the refractory or relapsed cancer is an angiosarcoma, i.e., of the lining of blood vessels that can develop in any part of the body including, but not limited to, skin, breast, liver, spleen, and deep tissue, i.e., deep tumors. It is cancer. In some embodiments, the refractory or relapsed cancer is a metastasis of any of the aforementioned refractory or relapsed cancers.

In some embodiments, the immunotherapy is activating immunotherapy. In some embodiments, immunotherapy is provided as a cancer treatment. In some embodiments, immunotherapy comprises adoptive cell transfer.

In some embodiments, adoptive cell transfer comprises administration of chimeric antigen receptor-expressing T-cells (CAR T-cells). Those skilled in the art will recognize that CAR is a type of antigen-targeting receptor consisting of an intracellular T-cell signaling domain fused to an extracellular tumor-binding moiety, most commonly a single chain variable fragment (scFv) from a monoclonal antibody. . CAR directly recognizes cell surface antigens independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for a specific antigen in all patients. Early CAR fused the antigen recognition domain to the CD3ζ activation chain of the T-cell receptor (TCR) complex. These first-generation CARs induced T cell effector function in vitro but were largely limited by poor antitumor efficacy in vivo. Subsequent CAR repeats included secondary costimulatory signals with CD3ζ, including the intracellular domain of CD28 or various TNF receptor family molecules such as 4-1BB (CD137) and OX40 (CD134). In addition, third-generation receptors contain two costimulatory signals (most commonly from CD28 and 4-1BB) in addition to CD3ζ. Second- and third-generation CARs dramatically improve antitumor efficacy, in some cases leading to complete remission in patients with advanced cancer. In one embodiment, the CAR T-cell is an immune-responsive cell that has been modified to express a CAR, which is activated when the CAR binds to its antigen.

In one embodiment, the CAR T cell is an immune response cell comprising an antigen receptor, which is activated when the receptor binds its antigen. In one embodiment, the CAR T-cells used in the compositions and methods as disclosed herein are first generation CAR T-cells. In another embodiment, the CAR T-cells used in the compositions and methods as disclosed herein are second-generation CAR T-cells. In another embodiment, the CAR T-cells used in the compositions and methods as disclosed herein are third generation CAR T-cells. In another embodiment, the CAR T-cells used in the compositions and methods as disclosed herein are fourth generation CAR T-cells.

In some embodiments, adoptive cell transfer comprises administering a T-cell receptor (TCR) modified T-cell. One of ordinary skill in the art will understand that TCR-modified T-cells are prepared by isolating T-cells from tumor tissue and isolating TCRα and TCRβ chains. These TCRα and TCRβ were later cloned and transfected into T cells isolated from peripheral blood, expressing TCRα and TCRβ in tumor-recognizing T cells.

In some embodiments, adoptive cell transfer comprises administering tumor infiltrating lymphocytes (TILs). In some embodiments, adoptive cell transfer comprises administering chimeric antigen receptor (CAR)-modified NK cells. Those of skill in the art will recognize that CAR-modified NK cells include NK cells isolated from a patient or commercially available NK engineered to express a CAR that recognizes a tumor specific protein.

In some embodiments, adoptive cell transfer comprises administering dendritic cells.

In some embodiments, immunotherapy comprises administering a monoclonal antibody. In some embodiments, the monoclonal antibody attaches to a specific protein on a cancer cell and marks the immune system to find and destroy the cell. In some embodiments, monoclonal antibodies act by inhibiting immune checkpoints, thereby interfering with suppression of the immune system by cancer cells. In some embodiments, the monoclonal antibody improves the availability of CAR-T in synergy with a checkpoint inhibitor.

In some embodiments, the antibody is 5AC, 5T4, activin receptor-like kinase 1, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, B7-H3, BAFF, BCMA, C242 antigen , CA-125, carbonic anhydrase 9, CCR4, CD125, CD152, CD184, CD19, CD2, CD20, CD200, CD22, CD221, CD23, CD25, CD27, CD274, CD276, CD28, CD3, CD30, CD33, CD37, CD38, CD4, CD40, CD41, CD44 v6, CD49b, CD5, CD51, CD52, CD54, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CFD, CGRP, ch4D5, CLDN18.2, aggregation factor A, CSF1R , CSF2, CTGF, CTLA-4, DLL3, DLL4, DPP4, DR5, EGFL7, EGFR, endoglin, EpCAM, ephrin receptor A3, episialin, ERBB3 (HER3), FAP, FGF 23, fibrin II, beta chain , Fibronectin extra domain-B, folate hydrolase, folate receptor, Frizzled receptor, GCGR, GD2 ganglioside, GD3 ganglioside, GDF-8, glypican 3, GM-CSF, GM-CSF receptor α-chain, GPNMB, GUCY2C, HER1, HER2/neu, HGF, HHGFR, histone complex, human scatter factor receptor kinase, human TNF, ICOSL, IFN-α, IGF1, IGF2, I GHE, IL-17A, IL-13, IL1A, IL-2, IL -6, IL-6 receptor, IL-8, IL-9, ILGF2, integrin α4, integrin α5β1, integrin α7 β7, integrin αvβ3, IP10, KIR2D, KLRC1, Lewis-Y antigen, MAGE-A, MCP-1, Mesothelin, MIF, MIG, MIP1β, MS4A1, MSLN, MUC1, Mucin CanAg, N-glycolylneuraminic acid, NOGO-A, Notch 1, Notch receptor, NRP1, OX-40, PD-1, PDCD1, PDGF-Rα, phosphate-sodium cotransporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostate carcinoma cells, RHD, RON, RTN4, SDC1 , sIL2Rα, SLAMF7, SOST, Sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGF beta 1, TGF beta 2, TGF-β, TNFR superfamily member 4, TNF-α, TRAIL-R1, TRAIL-R2, TRP-1, TRP-2, TSLP, tumor antigen CTAA16.88, tumor-specific glycosylation of MUC1, tumor-associated It targets a protein selected from the group comprising tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR-1, VEGFR2, vimentin and VWF.

In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a bispecific T-cell participant (BiTE) antibody. In some embodiments, the antibody is a group comprising ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, rituximab, TGN1412, alemtuzumab, OKT3, or any combination thereof is selected from

In some embodiments, immunotherapy comprises administration of cytokines. One of ordinary skill in the art will recognize that cytokines can be administered to enhance the immune system attacking tumors by increasing recognition and killing by immune cytotoxic cells. In some embodiments, the cytokine is IFNα, IFNβ, IFNγ, IFNλ, IL-1, IL-2, IL-6, IL-7, IL-15, IL-21, IL-11, IL-12, IL- 18, GM-CSF, TNFα, or a combination thereof.

In some embodiments, immunotherapy comprises administering an immune checkpoint inhibitor. One of ordinary skill in the art will recognize that immune checkpoints are membrane proteins that prevent T cells from attacking cells expressing them. Immune checkpoints are often expressed by cancer cells and thus prevent T cells from attacking the cancer cells. In some embodiments, the checkpoint protein comprises PD-1/PD-L1 and CTLA-4/B7-1/B7-2. Blocking the checkpoint protein has been shown to uninhibit T cells that attack and kill cancer cells. In some embodiments, the checkpoint inhibitor is selected from the group comprising a molecule that blocks CTLA-4, PD-1, or PD-L1. In some embodiments, the checkpoint inhibitor is an antibody or portion thereof.

In some embodiments, immunotherapy comprises administering a polysaccharide. Those skilled in the art will recognize that certain polysaccharides found in mushrooms enhance the immune system and anti-cancer properties. In some embodiments, the polysaccharide is beta-glucan or lentinan.

In some embodiments, immunotherapy comprises administration or a cancer vaccine. Those skilled in the art will recognize that cancer vaccines expose the immune system to cancer specific antigens and adjuvants. In some embodiments, the cancer vaccine is sipuleucel-T, GVAX, ADXS11-001, ADXS31-001, ADXS31-164, ALVAC-CEA vaccine, AC vaccine, talimogene laherparepvec , BiovaxID, Prostvac, CDX110, CDX1307, CDX1401, CimaVax-EGF, CV9104, DNDN, NeuVax, Ae-37, GRNVAC, tarmogens, GI-4000, GI-6207, GI-6301, ImPACT Therapy, IMA901, hepcortespenlisimut-L, Stimuvax, DCVax-L, DCVax-Direct, DCVax Prostate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328 and PVX-410.

Methods of reducing the level of cytokines or chemokines other than GM-CSF

In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of one or more inflammation-related factors in the body fluid. In some embodiments, inhibiting or reducing the incidence or severity of immunotherapy-related toxicity comprises reducing the concentration of one or more inflammation-related factors in the serum. In some embodiments, inhibiting or reducing the occurrence or severity of immunotherapy-related toxicity comprises reducing the concentration of one or more inflammation-related factors in the cerebrospinal fluid (CSF). In some embodiments, disclosed herein is a method of reducing the concentration of one or more inflammation-related factors in serum. In some embodiments, disclosed herein is a method of reducing the concentration of one or more inflammation-related factors in a tissue fluid. In some embodiments, disclosed herein is a method of reducing the concentration of one or more inflammation-related factors in CSF. In some embodiments, the concentration of one or more inflammation-related factors in the serum is decreased. In some embodiments, the concentration of one or more inflammation-related factors in the tissue fluid is reduced. In some embodiments, the concentration of one or more inflammation-related factors in the CSF is decreased. Those skilled in the art recognize that reducing the concentration of an inflammation-related factor includes reducing or inhibiting the production of the inflammation-related factor in a subject, or inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity in a subject. something to do. In another embodiment, reducing or inhibiting the production of an inflammation-related factor comprises treating an immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of an inflammation-related factor comprises preventing immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of inflammation-related factor levels comprises alleviating immunotherapy-related toxicity. In another embodiment, reducing or inhibiting the production of inflammation-related factors comprises ameliorating immunotherapy-related toxicity.

In some embodiments, the inflammation-associated factor is a cytokine. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises reducing the concentration of one or more cytokines in the serum. In some embodiments, inhibiting or reducing the occurrence or severity of an immunotherapy-related toxicity comprises reducing the concentration of one or more cytokines in the CSF.

In some embodiments, the cytokine is hGM-CSF. In some embodiments, the cytokine is interleukin (IL)-1β. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is sIL2R. In some embodiments, the cytokine is IL-5. In some embodiments, the cytokine is IL-6. In some embodiments, the cytokine is IL-8.

In some embodiments, the cytokine is IP10. In some embodiments, the cytokine is IL-13. In some embodiments, the cytokine is IL-15. In some embodiments, the cytokine is tumor necrosis factor α (TNFα). In some embodiments, the cytokine is interferon γ (IFNγ). In some embodiments, the cytokine is a monokine induced by gamma interferon (MIG). In some embodiments, the cytokine is macrophage inflammatory protein (MIP) 1β. In some embodiments, the cytokine is a C-reactive protein. In some embodiments, reducing or inhibiting the production of a cytokine level comprises reducing or inhibiting the production of one cytokine. In some embodiments, reducing or inhibiting the production of a cytokine level comprises reducing or inhibiting the production of one or more cytokines. In some embodiments, reducing or inhibiting the production of a cytokine level comprises reducing or inhibiting the production of a plurality of cytokines.

In one aspect, the invention provides a method of reducing the level of a cytokine or chemokine other than GM-CSF in an individual having the development of an immunotherapy-related toxicity comprising administering to the individual a recombinant hGM-CSF antagonist and the level of the cytokine or chemokine is reduced relative to the level of the subject administered the isotype control antibody during the development of immunotherapy-related toxicity. In some embodiments, immunotherapy comprises adoptive cell transfer, administration of a monoclonal antibody, administration of a cancer vaccine, T cell engaging therapy, or any combination thereof. In certain embodiments, adoptive cell transfer is a chimeric antigen receptor-expressing T-cell (CAR), a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR) ) the modified natural killer cells, or dendritic cells, or a combination thereof. In some embodiments, the CAR-T cell is a CD19 CAR-T cell. In certain embodiments, the recombinant GM-CSF antagonist is a hGM-CSF antagonist. In various embodiments, the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. In certain embodiments, the anti-GM-CSF antibody binds human GM-CSF. In another embodiment, the anti-GM-CSF antibody binds to primate GM-CSF as described above. In some embodiments, the anti-GM-CSF antibody binds to mammalian GM-CSF. In certain embodiments, the anti-GM-CSF antibody is an anti-hGM-CSF antibody. In some embodiments, the anti-hGM-CSF antibody is a monoclonal antibody. In various embodiments, the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. In some embodiments, the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. In certain embodiments, the anti-hGM-CSF antibody is a recombinant or chimeric antibody. In some embodiments, the anti-hGM-CSF antibody is a human antibody. In certain embodiments, as demonstrated in Example 22, the cytokine or chemokine is IP-10, IL-2, IL-3, IL-5, IL-1Ra, VEGF, TNF-a, FGF-2, IFN -γ, IL-12p40, IL-12p70, sCD40L, MDC, MCP-1, MIP-1a, MIP-1b, or a combination thereof. In some embodiments, the cytokine or chemokine is IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-9, IL-10, IP-10, KC, MCP-1, MIP or and combinations thereof (see Example 22). In certain embodiments, the subject has acute lymphoblastic leukemia.

In one embodiment, the methods disclosed herein do not affect the efficacy of immunotherapy. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 5%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 10%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 15%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 20%. In another embodiment, the methods disclosed herein reduce the efficacy of immunotherapy by less than about 50%.

In one embodiment, the methods described herein increase the efficacy of immunotherapy. In one embodiment, the increase in efficacy allows for improvement of clinical management of immunotherapy, patient outcome and therapeutic index. In other embodiments, the increased efficacy allows administration of higher immunotherapy doses. In another embodiment, the increased efficacy reduces hospital stay and further treatment and monitoring. In one embodiment, the immunotherapy comprises CAR-T.

Appropriate methods of quantifying cytotoxicity can be used to determine whether immunotherapy efficacy is substantially unchanged. For example, cytotoxicity can be quantified using a cell culture-based assay, such as the cytotoxicity assay described in the Examples. Cytotoxicity assays may use dyes that preferentially stain the DNA of dead cells. In other cases, fluorescence and luminescence assays that measure the relative numbers of living and dead cells in a cell population can be used. In this assay, protease activity serves as a marker for cell viability and cytotoxicity, and the labeled cell-penetrating peptide produces a fluorescence signal proportional to the number of viable cells in the sample. In other embodiments, the measurement of cytotoxicity may be qualitative. In other embodiments, the measurement of cytotoxicity can be quantitative.

In one embodiment, the increased efficacy is increased CAR-T cell expansion, decreased number and/or activity of myeloid-derived suppressor cells (MDSCs) that inhibit T-cell function, synergy with checkpoint inhibitors, or any combination thereof. In another embodiment, said increased CAR-T cell expansion comprises at least a 50% increase as compared to a control. In another embodiment, said increased CAR-T cell expansion comprises at least one-quarter log expansion compared to a control. In another embodiment, said increased cell expansion comprises at least 1/2 log expansion compared to a control. In another embodiment, said increased cell expansion comprises at least one log expansion compared to a control. In another embodiment, said increased cell expansion comprises more than 1 log expansion as compared to a control.

In one embodiment, immunotherapy-related toxicity occurs between 2 days and 4 weeks after administration of the immunotherapy. In one embodiment, immunotherapy-related toxicity occurs between 0 and 2 days after administration of the immunotherapy. In some embodiments, the hGM-CSF antagonist is administered to the subject concurrently with immunotherapy as prophylaxis. In another embodiment, the hGM-CSF antagonist is administered to the subject 0-2 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 days after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 days after administration of the immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 hours after administration of the immunotherapy. In another embodiment, the GM-CSF antagonist is administered to the subject 14 hours after administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 hours after administration of the immunotherapy.

In an alternative embodiment, the hGM-CSF antagonist is administered to the subject prior to immunotherapy as prophylaxis. In another embodiment, the hGM-CSF antagonist is administered to the subject one day prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 days prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 days prior to administration of the immunotherapy.

In another embodiment, the hGM-CSF antagonist is administered to the subject 2-3 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 7 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 10 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 14 hours prior to administration of the immunotherapy. In another embodiment, the hGM-CSF antagonist is administered to the subject 2-14 hours prior to administration of the immunotherapy.

In another embodiment, the hGM-CSF antagonist may be administered therapeutically once immunotherapy-related toxicity occurs. In one embodiment, the hGM-CSF antagonist may be administered upon detection of a pathophysiological process leading to or demonstrating the onset of immunotherapy-related toxicity. In one embodiment, the hGM-CSF antagonist can terminate the pathophysiological process and avoid its sequelae. In some embodiments, the pathophysiological process comprises at least one of the following: increased serum cytokine concentration, CSF increased cytokine concentration, serum C-reactive protein (CRP) increased, serum ferritin increased, serum IL-6 increase, endothelial activation, disseminated intravascular coagulation (DIC), increased ANG2 serum concentration, increased serum ANG2:ANG1 ratio, CAR T cell presence in CSF, increased Von Willebrand factor (VWF) serum concentration, blood brain barrier ( BBB) leakage or a combination thereof.

In another embodiment, the hGM-CSF antagonist may be administered therapeutically at multiple time points. In another embodiment, the administration of the hGM-CSF antagonist is at least two time points. In another embodiment, the administration of the hGM-CSF antagonist is at least three time points.

In one embodiment, the hGM-CSF antagonist is administered once. In another embodiment, the hGM-CSF antagonist is administered twice. In another embodiment, the hGM-CSF antagonist is administered three times. In another embodiment, the hGM-CSF antagonist is administered 4 times. In another embodiment, the hGM-CSF antagonist is administered at least 4 times. In another embodiment, the hGM-CSF antagonist is administered four or more times.

Those skilled in the art will recognize that immunotherapy-related toxicities are managed by other therapies. In some embodiments, the hGM-CSF antagonist is co-administered with other treatments. In some embodiments, the other treatment is cytokine directed therapy, anti-IL-6 therapy, corticosteroids, tocilizumab, siltuximab, low dose vasopressor, diuretic, supplemental oxygen, diuretic, thoracic puncture, antiepileptic, benzodiazepine, levi tiracetam, phenobarbital, hyperventilation, hyperosmolarity and standard therapy for certain organ toxicity.

In some embodiments, the immunotherapy-related toxicity comprises a brain disease, injury or dysfunction. In some embodiments, the immunotherapy-related toxicity comprises CAR T cell associated NT. In some embodiments, the immunotherapy-related toxicity comprises CAR T cell associated encephalopathy syndrome (CRES). In some embodiments, the methods provided herein are methods of inhibiting or reducing the occurrence of a brain disease, injury, or dysfunction.

In some embodiments, inhibiting or reducing the occurrence of CRES comprises ameliorating headache. In some embodiments, inhibiting or reducing CRES generation comprises alleviating delirium. In some embodiments, inhibiting or reducing CRES generation comprises reducing anxiety. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing tremor. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing seizure activity. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing confounding. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing changes in arousal.

In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing hallucinations. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing aphasia. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing ataxia. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing ataxia. In some embodiments, inhibiting or reducing the occurrence of CRES comprises ameliorating facial nerve palsy. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing motor weakness. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing seizures. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing non-convulsive EEG attacks. In some embodiments, inhibiting or reducing the occurrence or severity of CRES comprises improving coma recovery.

In some embodiments, inhibiting or reducing the occurrence or severity of CRES comprises reducing endothelial activation. Those skilled in the art will recognize that endothelial activation is an inflammatory and procoagulant state of endothelial cells characterized by increased interaction with leukocytes.

In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing vascular leakage. The term “vascular leak” may be used interchangeably with the terms “vascular leak syndrome” and “capillary leak syndrome”, both of which have the same characteristics and meanings. Those skilled in the art will recognize that vascular leakage associates with endothelial cells and dissociates, resulting in transendothelial migration of plasma leakage and inflammatory cells into body tissues resulting in tissue and organ damage. In addition, neutrophils can cause microcirculation occlusion, reducing tissue perfusion. In some embodiments reducing the occurrence of CRES comprises reducing coagulation in blood vessels.

In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing the concentration of one or more circulating cytokines. In some embodiments, the cytokine is selected from the group comprising hGM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10 and IL-2. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing the serum concentration of ANG2. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing the ANG2:ANG1 ratio in serum.

In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing CRES grade. In some embodiments, inhibiting or reducing CRES generation comprises improving CARTOX-10 score. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing elevated intracranial pressure. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing seizures. In some embodiments, inhibiting or reducing the occurrence of CRES comprises reducing motor weakness.

In some embodiments, the immunotherapy-related toxicity comprises CAR T-cell associated CRS. In some embodiments, provided herein are methods of inhibiting or reducing the incidence or severity of CRS and/or NT.

In some embodiments, inhibiting or reducing the occurrence of CRS or NT is ameliorating fever (stiffness, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia , shock, cardiovascular tachycardia, enlarged pulse pressure, hypotension, capillary leakage, early cardiac output increase, late cardiac output decrease, D-dimer elevation, hypofibrinogenemia with or without bleeding, azotemia, trans transaminitis, hyperbilirubinemia, altered mental status, confusion, delirium, frank aphasia, hallucinations, tremors, dysmetria, gait changes, seizures, organ failure, or combinations thereof , or any other symptoms or characteristics known in the art to be associated with CRS). In some embodiments, inhibiting or reducing the occurrence of CRS comprises reducing the concentration of one or more circulating cytokines. In some embodiments, the cytokine is selected from the group comprising GM-CSF, IFNγ, IL-1, IL-15, IL-6, IL-8, IL-10 and IL-2.

In some embodiments, inhibiting or reducing the occurrence of CRS comprises reducing CRS grade. In some embodiments, inhibiting or reducing the occurrence of NT comprises reducing NT grade. In some embodiments, inhibiting or reducing the occurrence of CRS comprises improving CARTOX-10 score. In some embodiments, inhibiting or reducing the occurrence of NT comprises improving CARTOX-10 score. In some embodiments, inhibiting or reducing the occurrence of CRS comprises reducing elevated intracranial pressure. In some embodiments, inhibiting or reducing the occurrence of CRS comprises reducing seizures. In some embodiments, inhibiting or reducing the occurrence of CRS comprises reducing motor weakness. In some embodiments, inhibiting or reducing the occurrence of NT or CRS comprises inhibiting or reducing the occurrence by less than 60%. In some embodiments, inhibiting or reducing the occurrence of NT or CRS comprises inhibiting or reducing the occurrence by less than 50%. In some embodiments, inhibiting or reducing the occurrence of NT or CRS comprises inhibiting or reducing the occurrence by less than 40%. In some embodiments, inhibiting or reducing the occurrence of NT or CRS comprises inhibiting or reducing the occurrence by less than 30%. In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises inhibiting or reducing the incidence in less than 20% of patients. In some embodiments, inhibiting or reducing the occurrence of NT or CRS comprises eliminating NT or CRS.

In some embodiments, the subject has grade 1 CRS and/or NT. In some embodiments, the individual has grade 2 CRS and/or NT. In some embodiments, the individual has grade 3 CRS and/or NT. In some embodiments, the individual has grade 4 CRS and/or NT. In some embodiments, the individual has any combination of the above.

In some embodiments, inhibiting or reducing the incidence of NT or CRS comprises reducing CRS grade, NT grade, or both. In some embodiments, the grade is reduced to < 3 NT and/or CRS in 95% of patients.

In some embodiments, the subject has a body temperature of 37° C. or higher following administration of the immunotherapy. In some embodiments, the subject has a body temperature of 38° C. or higher following administration of the immunotherapy. In some embodiments, the subject has a body temperature greater than or equal to 39° C. following administration of the immunotherapy. In some embodiments, the subject has a body temperature of 40° C. or higher following administration of the immunotherapy. In some embodiments, the subject has a body temperature of 41° C. or higher following administration of the immunotherapy. In some embodiments, the subject has a body temperature of 42° C. or higher following administration of the immunotherapy.

In some embodiments, the subject has an IL-6 serum concentration greater than 10 pg/ml following administration of immunotherapy. In some embodiments, the subject has an IL-6 serum concentration greater than 12 pg/ml following administration of immunotherapy. In some embodiments, the subject has an IL-6 serum concentration of at least 14 pg/ml following administration of the immunotherapy. In some embodiments, the subject has an IL-6 serum concentration of at least 16 pg/ml following administration of immunotherapy. In some embodiments, the subject has an IL-6 serum concentration of at least 18 pg/ml following administration of the immunotherapy. In some embodiments, the subject has an IL-6 serum concentration of at least 20 pg/ml following administration of immunotherapy. In some embodiments, the subject has an IL-6 serum concentration of at least 22 pg/ml following administration of the immunotherapy.

In some embodiments, the subject has an MCP-1 serum concentration greater than 200 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration greater than 400 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration of at least 600 pg/ml following administration of the immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration greater than 800 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration greater than 1000 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration greater than 1200 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration of at least 1400 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration of at least 1600 pg/ml following administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration of 1800 pg/ml or greater after administration of immunotherapy. In some embodiments, the subject has an MCP-1 serum concentration greater than 2000 pg/ml following administration of immunotherapy.

In some embodiments, the subject has a grade 1 CRES. In some embodiments, the individual has a grade 2 CRES. In some embodiments, the individual has a grade 3 CRES. In some embodiments, the individual has a grade 4 CRES.

In some embodiments, the subject is predisposed to a brain disease, injury or dysfunction prior to immunotherapy. In some embodiments, the predisposition is hereditary. In some embodiments, the trend is obtained. In some embodiments, the tendency is associated with a pre-existing medical condition. In some embodiments, the predisposition is diagnosed prior to immunotherapy. In some embodiments, the tendency is not diagnosed. In some embodiments, the subject undergoes a medical evaluation prior to immunotherapy to determine a predisposition to acquiring an immunotherapy-related brain disease, injury or dysfunction.

In some embodiments, the medical evaluation comprises determining the ANG1 concentration in the body fluid. In some embodiments, the medical evaluation comprises determining the concentration of ANG1 in the serum. In some embodiments, the medical evaluation comprises determining the ANG2 concentration in the body fluid. In some embodiments, the medical evaluation comprises determining the concentration of ANG2 in the serum. In some embodiments, the medical evaluation comprises calculating the ANG2:ANG1 ratio in the serum. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 0.5 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 0.7 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 0.9 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 1 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 1.1 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 1.3 prior to immunotherapy is susceptible to CRES. In some embodiments, an individual with a serum ANG2:ANG1 ratio greater than 1.5 prior to immunotherapy is susceptible to CRES.

In some embodiments, the immunotherapy-related toxicity comprises hemophagocytic lymphohistiocytosis (HLH). In some embodiments, the immunotherapy-related toxicity comprises macrophage-activation syndrome (MAS). In some embodiments, provided herein are methods of inhibiting or reducing the occurrence of HLH. In some embodiments, provided herein are methods of inhibiting or reducing MAS development.

In some embodiments, inhibiting or reducing the occurrence of HLH comprises increasing the survival of the individual. In some embodiments, inhibiting the decrease in the incidence of HLH comprises increasing the time to relapse. In some embodiments, inhibiting or reducing the occurrence of MAS comprises increasing the survival of the individual. In some embodiments, inhibiting the decrease in the incidence of MAS comprises increasing the time to relapse.

In some embodiments, inhibiting or reducing the development of HLH or MAS comprises inhibiting macrophage activation and/or proliferation. In some embodiments, inhibiting or reducing the occurrence of HLH or MAS comprises inhibiting T lymphocyte activation and/or proliferation. In some embodiments, inhibiting or reducing the occurrence of HLH or MAS comprises reducing the concentration of circulating IFNγ. In some embodiments, inhibiting or reducing the occurrence of HLH or MAS comprises reducing the circulating concentration of GM-CSF.

In some embodiments the subject develops fever after immunotherapy. In some embodiments, the subject exhibits an enlarged spleen after immunotherapy. In some embodiments, the subject exhibits cytopenia after immunotherapy. In some embodiments, the subject exhibits cytopenia in two or more cell lines following immunotherapy. In some embodiments, the subject exhibits hypertriglyceridemia following immunotherapy. In some embodiments, the subject exhibits hypofibrinogenemia following immunotherapy. In some embodiments, the subject exhibits hemophagocytosis following immunotherapy. In some embodiments the hematocytosis is observed in bone marrow. In some embodiments the subject exhibits low NK-cell activity following immunotherapy. In some embodiments the subject exhibits absent NK activity following immunotherapy.

In some embodiments, the subject exhibits a serum concentration of ferritin greater than 100 U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin greater than 300 U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin greater than 500 U/ml following immunotherapy. In some embodiments, the subject exhibits a serum concentration of ferritin greater than 700 U/ml following immunotherapy. In some embodiments, the subject exhibits a ferritin serum concentration greater than 900 U/ml after immunotherapy.

In some embodiments, the subject exhibits a soluble CD25 serum concentration greater than 1200 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration greater than 1500 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration of at least 1800 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration of at least 2000 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration of at least 2200 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration of at least 2400 U/ml following immunotherapy. In some embodiments the subject exhibits a soluble CD25 serum concentration greater than 2700 U/ml following immunotherapy. In some embodiments, the subject exhibits a soluble CD25 serum concentration greater than 3000 U/ml following immunotherapy.

In some embodiments, the individual tends to have HLH. In some embodiments, the predisposition is hereditary. In some embodiments, the tendency is associated with a pre-existing medical condition. Those of skill in the art will recognize that sporadic HLH is associated with a number of genetic mutations. In some embodiments, the individual carries a mutation in a gene selected from PRF1, UNC13D, STX11, STXBP2, or RAB27A, or any combination thereof. In some embodiments, the subject has reduced or absent expression of Perforin.

hGM-CSF antagonist

Suitable hGM-CSF antagonists for use selectively interfere with the induction of signal transduction by the hGM-CSF receptor by reducing the binding of hGM-CSF to the receptor. These antagonists are antibodies that bind to the hGM-CSF receptor, antibodies that bind hGM-CSF, GM-CSF analogs such as E21R, and hGM-CSF compete for binding to the receptor, or usually arise from ligand binding to the receptor. It may include other proteins or small molecules that inhibit a signal to

In many embodiments, the hGM-CSF antagonist used in the present invention binds to a polypeptide, e.g., an anti-hGM-CSF antibody, an anti-hGM-CSF receptor antibody, a soluble hGM-CSF receptor, or hGM-CSF to a receptor. It is a modified GM-CSF polypeptide that competes for but is inactive. Such proteins are often produced using recombinant expression techniques. Such methods are well known in the art. General molecular biology methods, including expression methods, are described, for example, in Sambrook and Russell (2001) Molecular Cloning: A laboratory manual 3rd ed. Cold Spring Harbor Laboratory Press; Current Protocols in Molecular Biology (2006) John Wiley and Sons ISBN: 0-471-50338-X.

A variety of prokaryotic and/or eukaryotic based protein expression systems can be used to produce hGM-CSF antagonist proteins. Many of these systems are widely available from commercial vendors.

hGM-CSF antibody

The hGM-CSF antibody of the present invention is an antibody that binds to hGM-CSF with high affinity and is an antagonist of hGM-CSF. Antibodies comprise variable regions with a high degree of identity to human germline VH and VL sequences. In a preferred embodiment, the BSD sequence in CDRH3 of the antibody of the invention comprises the amino acid sequence RQRFPY or RDRFPY. The BSD of CDRL3 includes FNK or FNR.

BSD forms part of CDR3, resulting in a complete V region where additional sequences are used to complete CDR3 and add FR4 sequences. Typically, a portion of CDR3, excluding BSD, and complete FR4 consist of human germline sequences. In some embodiments, the CDR3-FR4 sequence, excluding BSD, differs from the human germline sequence by no more than 2 amino acids in each chain. In some embodiments, the J-segment comprises a human germline J-segment. Human germline sequences can be determined, for example, via the publicly available international ImMunoGeneTics (IMGT) database and V-Base (at the website vbase.mrc-cpe.cam.ac.uk).

The human germline V-segment repertoire consists of 51 heavy chain V-regions, 40 κ V-segments, and 31 λ light chain V-segments, making a total of 3,621 germline V-region pairs, and also stable alleles for these V-segments. Although genetic variants exist, their contribution to the structural diversity of the germline repertoire is limited. The sequences of all human germline V-segment genes are known and the V-base database provided by the MRC Center for Protein Engineering (Cambridge, UK) (see also Chothia et al., 1992, JMol Biol 227: 776-798; Tomlinson) et al., 1995, EMBO J 14: 4628-4638; Williams et al., 1996, see J Mol Biol 264: 220-232).

The antibodies or antibody fragments described herein can be expressed in prokaryotic or eukaryotic microbial systems or in cells of higher eukaryotes, such as mammalian cells.

Antibodies that may be used in the present invention may be in any format. For example, in some embodiments, the antibody may be a complete antibody comprising a constant region, such as a human constant region, or a fragment of an intact antibody, such as _{Fab, Fab', F(ab') 2 , scFv, Fv, or} It may be a derivative or single domain antibody, such as a nanobody or camelid antibody. Such antibodies may additionally be recombinantly engineered by methods well known to those skilled in the art. As noted above, such antibodies can be produced using known techniques.

In some embodiments, the hGM-CSF antagonist is an antibody that binds hGM-CSF or an antibody that binds hGM-CSF receptor α or β subunits. The antibody may be raised against the hGM-CSF (or hGM-CSF receptor) protein or fragment or it may be recombinantly produced. Antibodies to GM-CSF for use in the present invention may be neutralizing or non-neutralizing antibodies that bind to GM-CSF and increase the in vivo clearance of hGM-CSF such that hGM-CSF levels in circulation are reduced. . Often the hGM-CSF antibody is a neutralizing antibody.

Methods for preparing polyclonal antibodies are known to those skilled in the art (eg, Harlow & Lane, Antibodies, A Laboratory Manual (1988); Methods in Immunology). Polyclonal antibodies can be raised in mammals by one or more injections of an immunizing agent and, if desired, an adjuvant. The immunizing agent includes a GM-CSF or GM-CSF receptor protein, such as a human GM-CSF or GM-CSF receptor protein, or a fragment thereof.

In some embodiments, the GM-CSF antibody for use in the present invention is purified from human plasma. In one such embodiment, the GM-CSF antibody is a polyclonal antibody isolated from other antibodies generally present in human plasma. Such isolation procedures can be performed using known techniques such as, for example, affinity chromatography.

In some embodiments, the GM-CSF antagonist is a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods such as those described by Kohler & Milstein, Nature 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is generally immunized with an immunizing agent, such as human GM-CSF, to induce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Human monoclonal antibodies can be prepared using a variety of techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. MoI. Biol. 227:381 (1991); Marks et al, J. MoI. Biol. 222:581 (1991)). Cole et al. and Boerner et al. can also be used for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, p.77 (1985) and Boerner et al., J.Immunol. 147(l):86-95 (1991) )). Similarly, human antibodies can be made by introducing a human immunoglobulin locus into a transgenic animal, eg, a mouse in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which is very similar to that seen in humans in all respects, including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and in the following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al, Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

In some embodiments the anti-GM-CSF antibody is a chimeric or humanized monoclonal antibody. As mentioned above, humanized antibody forms are chimeric immunity in which residues from the complementarity determining regions (CDRs) of a human antibody are replaced with residues from the CDRs of a non-human species such as mouse, rat or rabbit having the desired specificity, affinity and ability. It is globulin.

In some embodiments of the invention, the antibody is further engineered to reduce immunogenicity, eg, such that the antibody is suitable for repeated administration. Methods for generating antibodies with reduced immunogenicity include humanization/humanization procedures and modification techniques such as deimmunization, wherein the antibody is further engineered, for example, in one or more framework regions to remove T cell epitopes. do.

In some embodiments, the antibody is a humaneered antibody. Hugh Money adjuster antibody connecting the DNA sequence encoding a binding specificity determinant (BSD) of the heavy chain of the CDR3 region derived from the reference antibody to the human V _H segment sequences and connected to a light chain CDR3 BSD reference antibody derived from a human V _L segment sequence It is an engineered human antibody having the binding specificity of a reference antibody, obtained by Methods for Humaneering are provided in US Patent Application Publication No. 20050255552 and US Patent Application Publication No. 20060134098. A method for signal-free secretion of antibody fragments from E. coli is described in US Patent Application No. 20070020685.

The antibody may be further de-immunized to remove one or more predicted T-cell epitopes from the V-region of the antibody. Such a procedure is described, for example, in WO 00/34317.

The heavy chain constant region is often a gamma chain constant region, eg, a gamma-1, gamma-2, gamma-3 or gamma-4 constant region. In some embodiments, for example, when the antibody is a fragment, the antibody may be conjugated with another molecule, such as polyethylene glycol (PEGylated) or serum albumin, to provide an extended half-life in vivo. Examples of PEGylation of antibody fragments are (for abciximab) Knight et al (2004) Platelets 15: 409; (for anti-CEA antibodies) Pedley et al (1994) Br.J. Cancer 70: 1126; Chapman et al (1999) Nature Biotech. 17:780.

Antibodies for use in the present invention bind to hGM-CSF or hGM-CSF receptor. Several techniques can be used to determine antibody binding specificity. For a description of immunoassay formats and conditions that can be used to determine the specific immunoreactivity of an antibody, see, for example, Harlow & Lane, Antibodies, A Laboratory Manual (1988).

An exemplary antibody suitable for use in the present invention is cl9/2 (mouse/human chimeric anti-hGM-CSF antibody). In some embodiments, monoclonal antibodies that compete for binding to the same epitope as cl9/2 or bind to the same epitope as cl9/2 are used. The ability of a particular antibody to recognize the same epitope as another antibody is typically determined by the ability of a first antibody to competitively inhibit binding of a second antibody to an antigen. A number of competitive binding assays can be used to measure competition between two antibodies for the same antigen. For example, a sandwich ELISA assay can be used for this purpose. This is done by coating the surface of the wells with a capture antibody. An unsaturated concentration of the tagged-antigen is then added to the capture surface. This protein binds to the antibody through specific antibody-epitope interactions. A secondary antibody covalently linked to a detectable moiety (eg HRP, a labeled antibody defined as a detection antibody) is washed and added to the ELISA. When this antibody recognizes the same epitope as the capture antibody, the specific epitope is no longer available for binding and cannot bind to the target protein. However, if this secondary antibody recognizes a different epitope on the target protein, it can bind and this binding can be detected by quantifying the level of activity (and thus antibody binding) using the relevant substrate. A background is defined using a single antibody as capture and detection antibody, whereas a maximal signal can be established by capture with an antigen-specific antibody and detection with an antibody to the antigen's tag. Using background and maximal signal as reference, antibodies can be evaluated in pairs to determine epitope specificity.

Binding of the second antibody to the antigen is reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%, and often at least about 90% in the presence of the first antibody using any of the assays described above. It is considered that the first antibody competitively inhibits the binding of the second antibody.

In some embodiments of the invention, known antibodies are used, eg, antibodies that compete with or bind to binding to the same epitope as cl9/2. Methods of mapping epitopes are well known in the art. For example, one approach to the localization of the functionally active region of human granulocyte-macrophage colony stimulating factor (hGM-CSF) is to map the recognized epitope by neutralizing the anti-hGM-CSF monoclonal antibody. For example, the epitope to which cl9/2 (having the same variable region as the neutralizing antibody LMM 102) binds is defined using a protease fragment obtained by enzymatic digestion of bacterially synthesized hGM-CSF (Dempsey). , et al, Hybridoma 9:545-558, 1990). RP-HPLC fractionation of the trypsin lysate identified an immunoreactive “tryptic core” peptide comprising 66 amino acids (52% of protein). Further digestion of this "triptic core" with S. aureus V8 protease results in a unique immunoreactive hGM-CSF product comprising two peptides, residues 86-93 and 112-127, linked by a disulfide bond between residues 88 and 121. This was created. Individual peptides were not recognized by the antibody.

In some embodiments, antibodies suitable for use in the present invention have high affinity binding to the human GM-CSF or hGM-CSF receptor. There is high affinity binding between the antibody and antigen when the dissociation constant (KD) of the antibody is < about 10 nM, typically <1 nM, preferably <100 pM. In some embodiments, the antibody has a dissociation rate of at least ^{about 10 -4 per second.}

Various methods can be used to determine the binding affinity of an antibody for a target antigen, such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA, as is well known to those skilled in the art. Exemplary methods for determining binding affinity are described in Krinner et al, (2007) MoI. Immunol. Feb;44(5):916-25. (Epub 2006 May H)) by surface plasmon resonance analysis on a BIAcore ™ 2000 instrument (Biacore AB, Freiburg, Germany) using a CM5 sensor chip. In some embodiments, the hGM-CSF antagonist is a neutralizing antibody to hGM-CSF, its receptor, or its receptor subunit, that binds in a manner that interferes with binding of hGM-CSF to the receptor or receptor subunit of hGM-CSF. In some embodiments, an anti-hGM-CSF antibody for use in the present invention inhibits binding to the alpha subunit of the hGM-CSF receptor. Such an antibody may bind to and inhibit binding of hGM-CSF, for example in a region where hGM-CSF binds to a receptor. In another embodiment, the anti-hGM-CSF antibody inhibits hGM-CSF function without blocking its binding to the alpha subunit of the hGM-CSF receptor.

II. heavy chain

The heavy chain of the anti-hGM-CSF antibody of the invention comprises a heavy chain V-region comprising the following elements:

1) human heavy chain V-segment sequence comprising FR1-CDR1-FR2-CDR2-FR3

2) a CDRH3 region comprising the amino acid sequence R(Q/D)RFPY

3) FR4 contributed by the human germline J-gene segment.

An example of a V-segment sequence that aids binding to hGM-CSF in combination with the CDR3-FR4 segment described above with a complementary VL region is shown in FIG. 1 . The V-segment may be derived from, for example, the human VH1 subclass. In some embodiments, the V-segment is a human VH1 subclass segment having a high level of amino acid sequence identity, e.g., at least 80%, 85% or 90% or greater identity, to germline segments VH1 1-02 or VH1 1-03. to be. In some embodiments, the V-segment differs from VH1 1-02 or VH1 1-03 by no more than 15 residues, preferably no more than 7 residues.

The FR4 sequence of the antibody of the invention is provided by a sequence having a high degree of amino acid sequence identity to a human JH1, JH3, JH4, JH5 or JH6 gene germline segment, or a human germline JH segment. In some embodiments, the J segment is a human germline JH4 sequence.

CDRH3 also includes sequences derived from the human J-segment. In general, the CDRH3-FR4 sequence, except for BSD, differs from the human germline J-segment by no more than 2 amino acids. In an exemplary embodiment, the J-segment sequence of CDRH3 is derived from the same J-segment used in the FR4 sequence. Thus, in some embodiments, the CDRH3-FR4 region comprises a BSD and fully human JH4 germline gene segment. Exemplary combinations of CDRH3 and FR4 sequences are shown below, where BSD is bold and human germline J-segment JH4 residues are underlined:

In some embodiments, an antibody of the invention comprises germline segments VH 1-02 or VH 1-03; _{or at least 90% identity or at least 91% to the V segment portion of one of the V segments of the V H} region shown in Figure 1, such as VH#1, VH#2, VH#3, VH#4 or VH#5, V segments with 92% 93%, 94%, 95%, 965, 97%, 98%, 99% or 100% identity.

In some embodiments, the V-segment of the VH region has CDR1 and/or CDR2 as shown in FIG. 1 . For example, an antibody of the invention may have a CDR1 having the sequence GYYMH or NYYIH; or a CDR2 having the sequence WINPNSGGTNYAQKFQG or WINAGNGNTKYSQKFQG.

In certain embodiments, the antibody has both CDR1 and CDR2 from one of the VH region V-segments depicted in FIG. 1 , and a CDR3 comprising R(Q/D)RFPY, eg, RDRFPYYFDY or RQRFPYYFDY. Thus, in some embodiments, an anti-GM-CSF antibody of the invention may have, for example, CDR3-FR4 having the sequence R(Q/D)RFPYYFDYWGQGTLVTVSS and CDR1 and/or CDR2 as shown in FIG. 1 . In some embodiments, the VH region of an antibody of the invention has a CDR3 with the binding specificity determinant R(Q/D)RFPY, a CDR2 from a human germline VH1 segment, or a CDR1 from a human germline VH1. In some embodiments, both CDR1 and CDR2 are from a human germline VH1 segment.

III. light chain

The light chain of the anti-hGM-CSF antibody of the invention comprises a light chain V-region comprising the following elements:

1) human light chain V-segment sequence comprising FR1-CDR1-FR2-CDR2-FR3

2) a CDRL3 region comprising the sequence FNK or FNR, for example QQFNRSPLT or QQFNKSPLT.

3) FR4 contributed by the human germline J-gene segment.

The V _L region consists of the V lambda or V kappa V min segment. An example of a V kappa sequence that aids binding in combination with a complementary V _{H region is set forth in FIG. 1 .}

The VL region CDR3 sequence comprises a J-segment derived sequence. In an exemplary embodiment, the J-segment sequence of CDRL3 is derived from the same J-segment used in FR4. Thus, in some embodiments the sequence may differ from human kappa germline V-segment and J-segment sequences by no more than 2 amino acids. In some embodiments, the CDRL3-FR4 region comprises a BSD and fully human JK4 germline gene segment. Exemplary CDRL3-FR4 combinations for the kappa chain are shown below, where the minimum essential binding specificity determinant is bold and the JK4 sequence is underlined.

The Vkappa segment is generally a VKIII subclass. In some embodiments, the segment has at least 80% sequence identity to the human germline VKIII subclass, eg, at least 80% identity to the human germline VKIIIA27 sequence. In some embodiments, a Vkappa segment may differ from VKIIIA27 by no more than 18 residues. In another embodiment, the V _L region V segment of _{an antibody of the invention comprises a human kappa V segment sequence of the V L} region shown in Figure 1, eg, VK#1, VK#2, VK#3 or VK#4. has at least 85% identity or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the V segment sequence of

In some embodiments, the variable region consists of a human V-gene sequence. For example, the variable region sequence may have at least 80% identity, or at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity to a human germline V-gene sequence. % identity or at least 99% identity or greater.

In some embodiments, the V-segment of the VL region has CDR1 and/or CDR2 as shown in FIG. 1 . For example, an antibody of the invention comprises the CDR1 sequence of RASQSVGTNVA or RASQSIGSNLA; or the CDR2 sequence STSSRAT.

In a specific embodiment, an anti-GM-CSF antibody of the invention comprises a CDR3 sequence comprising FNK or CDR1 and CDR2 in combination with a CDR3 comprising FNR as shown in one of the V-segments of the VL region shown in FIG. 1 . can have For example, CDR3 may be QQFNKSPLT or QQFNRSPLT. In some embodiments, such GM-CSF antibody may comprise a FR4 region that is FGGGTKVEIK. Thus, an anti-GM-CSF antibody of the invention may comprise, for example, CDR1 and CDR2 from one of the VL regions depicted in FIG. 1 and a CDR3-FR4 region that is FGGGTKVEIK.

IV. Preparation of hGM-CSF antibody

The antibody of the present invention may comprise any of the VH regions VH#1, VH#2, VH#3, VH#4 or VH#5 as shown in FIG. 1 . In some embodiments, an antibody of the invention may comprise any of the VL regions VK#1, VK#2, VK#3 or VK#4 as shown in FIG. 1 . In some embodiments, the antibody comprises a VH region VH#1, VH#2, VH#3, VH#4, or VH#5; and VL regions VK#1, VK#2, VK#3 as shown in FIG. 1, as described, for example, in US Pat. Nos. 8,168,183 and 9,017, 674, each of which is incorporated herein by reference in its entirety. or VK#4.

Antibodies can be tested to see if they retain activity that antagonizes hGM-CSF activity. Antagonist activity can be measured using several endpoints, including proliferation assays. Neutralizing antibodies and other hGM-CSF antagonists can be identified or assessed using any number of assays that assess hGM-CSF function. Cell-based assays for GM-CSF receptor signaling are also conveniently used, such as, for example, assays that measure the proliferation rate of a GM-CSF dependent cell line in response to a limited amount of GM-CSF. The human TF-1 cell line is suitable for use in such assays. Krinner et al, (2007) Mol. See Immunol. In some embodiments, a neutralizing antibody of the invention inhibits hGM-CSF stimulated TF-I cell proliferation by at least 50% when a hGM-CSF concentration that stimulates 90% maximal TF-I cell proliferation is used. Thus, typically, neutralizing antibodies or other hGM-CSF antagonists for use in the present invention have an EC50 of less than 10 nM (eg, Table 2). Additional assays suitable for use in identifying neutralizing antibodies suitable for use in the present invention will be well known to those skilled in the art. In another embodiment, the neutralizing antibody comprises at least about 75%, 80%, 90%, 95% or 100 of the antagonist activity of the mouse monoclonal antibody LMM102 and the antibody chimeric cl9/2 having the variable regions of the CDRs, for example WO03/068920. Inhibit hGM-CSF stimulated proliferation by %.

An exemplary chimeric antibody suitable for use as an hGM-CSF antagonist is cl9/2. The cl9/2 antibody binds to hGM-CSF with a monovalent binding affinity of about 10 pM as determined by surface plasmon resonance analysis. The heavy and light chain variable region sequences of cl9/2 antibodies are known (eg WO03/068920). The CDRs defined according to Kabat are:

CDRH1 DYNIH

CDRH2 YIAPYSGGTGYNQEFKN

CDRH3 RDRFPYYFDY

CDRL1 KASQNVGSNVA

CDRL2 SASYRSG

CDRL3 QQFNRSPLT.

CDRs can also be determined using other definitions well known in the art, such as Chothia, International ImMunoGeneTics Database (IMGT) and AbM.

In some embodiments, the antibody used in the present invention competes or binds for binding to the same epitope as cl9/2. The GM-CSF epitope recognized by cl9/2 was identified as a product with two peptides linked by a disulfide bond between residues 88 and 121, residues 86-93 and residues 112-127. The cl9/2 antibody inhibits GM-CSF dependent proliferation of a human TF-1 leukemia cell line with an EC50 of 30 pM when cells are stimulated with 0.5 ng/ml GM-CSF. In some embodiments, the antibody used in the present invention binds to the same epitope as cl9/2.

Antibodies for administration, such as cl9/2, can be further humanized. For example, the cl9/2 antibody can be further engineered to include a human V gene segment.

High affinity antibodies can be identified using well-known assays to determine binding activity and affinity. These techniques include ELISA assays as well as binding determinations using surface plasmon resonance or interferometry. For example, affinity can be determined by biolayer interferometry using a ForteBio (Mountain View, CA) Octet biosensor. Antibodies of the invention generally bind with similar affinity to both glycosylated and non-glycosylated forms of hGM-CSF.

Antibodies of the invention compete with c19/2 for binding to GM-CSF. The ability of an antibody described herein to block or compete for binding to GM-CSF is dependent on the ability of the antibody to bind to the same epitope c19/2 or to an epitope that is close to, eg, overlaps with, the epitope bound by c19/2. indicates that it binds to In another embodiment, an antibody described herein as a reference antibody to assess whether other antibodies compete for binding to GM-CSF, e.g., a combination of _{V H} and V _{L regions shown in the table set forth in FIG. 1 .} antibodies can be used. If the binding of the reference antibody to the antigen is reduced by at least 30%, usually at least about 40%, 50%, 60% or 75%, and often at least about 90%, in the presence of the test antibody, the test antibody is are thought to be competitively suppressed. Many assays are available to assess binding, including ELISA, and other assays such as immunoblots. In some embodiments, an antibody of the invention has a dissociation rate that is at least 2-3 fold slower, but at least 6-10 fold potency, than a reference chimeric cl9/2 monoclonal antibody assayed under the same conditions. It is greater than the reference antibody in neutralizing hGM-CSF activity in a cell-based assay measuring hGM-CSF activity.

Methods for isolating antibodies with V-region sequences close to human germline sequences have been previously described (US Patent Application Publication Nos. 20050255552 and 20060134098). Antibody libraries can be expressed in suitable host cells, including mammalian cells, yeast cells, or prokaryotic cells. For expression in some cellular systems, a signal peptide can be introduced at the N-terminus to direct secretion into the extracellular medium. Antibodies can be secreted from bacterial cells, such as E. coli, with or without a signal peptide. A method for signal-free secretion of antibody fragments from E. coli is described in US Patent Application 20070020685.

In some embodiments, the hGM-CSF-binding antibody of the invention comprises an antibody having a CDR from one of the VH-regions of the invention shown in Figure 1 and an antibody having a CDR from one of the VL-regions shown in Figure 1; When combined, they are produced and expressed in several formats in an appropriate expression system. Thus, antibodies can be either scFv, Fab, Fab' (including immunoglobulin hinge sequences), F(ab*?*) ₂ , (formed by disulfide bond formation between the hinge sequences of two Fab molecules), whole immunoglobulin or truncated It can be expressed as an immunoglobulin, or as a fusion protein in a prokaryotic or eukaryotic host cell either inside the host cell or by secretion. A methionine residue may optionally be present at the N-terminus, for example in a polypeptide produced in a signalless expression system. Each of the VH-regions described herein can be paired with a respective VL region to generate an anti-hGM-CSF antibody. In one embodiment, the fusion protein is an anti-hGM-CSF-binding antibody or fragment thereof of the invention (in a non-limiting example, the anti-hGM-CSF antibody fragment is Fab, a Fab', a F(ab')2 , a scFv, or a dAB), and human transferrin, which is disclosed in Shin, SU., et al. Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 2820-2824, 1995, fused to the antibody after the terminus, after hinge or after CH3 of heavy chain constant region 1 (CH1).

Exemplary combinations of heavy and light chains are shown in the table provided in FIG. 1 . In some embodiments, an antibody VL region, eg, VK#1, VK#2, VK#3 or VK#4 of FIG. 1 , is combined with a human kappa constant region to form a complete light chain. Also, in some embodiments, the VH region is associated with a human gamma-1 constant region. An appropriate gamma-1 allotype may be selected, such as the f-allotype. Thus, in some embodiments, the antibody comprises a VH selected from VH#1, VH#2, VH#3, VH#4 or VH#5, and a VK#1, VK#2, VK#3 or VK#4 selected from IgG with VL , eg with f-allotype ( FIG. 1 ).

The antibody of the present invention inhibits hGM-CSF receptor activation by, for example, inhibits binding of hGM-CSF to the receptor, and exhibits high affinity binding to hGM-CSF, for example, 500 pM. In some embodiments, the antibody has ^{a dissociation constant of about 10 -4} /sec or less. Without wishing to be bound by theory, antibodies with slower dissociation constants offer improved therapeutic benefits. For example, an antibody of the invention has an off-rate 3-fold slower than c19/2, and for example, 10-fold more potent GM-CSF neutralization in cell-based assays such as IL-8 production. generate activity (see, eg, Example 2).

Any number of expression systems can be used to generate antibodies, including both prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is mammalian cell expression, such as a CHO cell expression system. Many of these systems are widely available from commercial suppliers. In embodiments wherein the antibody comprises both a V _H region and a V _L region, the V _H region and V _L region are expressed using a single vector, for example in a bicistronic expression unit or under the control of different promoters. can be In another embodiment, the VH and VL regions may be expressed using separate vectors. The VH or VL regions described herein may optionally express methionine at the N-terminus.

Antibodies of the invention can be produced in any number of formats, including Fab, Fab', F(ab') _{2 , scFv or dAB.} Antibodies of the invention may also comprise human constant regions. The constant region of the light chain may be a human kappa or lambda constant region. The heavy chain constant region is usually a gamma chain constant region, for example a gamma-1, gamma-2, gamma-3 or gamma-4 constant region. In another embodiment, the antibody may be IgA.

In some embodiments of the invention, the antibody VL region, e.g., VK#1, VK#2, VK#3, or VK#4 of Figure 1, is a human kappa constant region (e.g., SEQ ID NO: 10) complete form a light chain

In some embodiments of the invention, the V _H region is associated with a human gamma-1 constant region. Any suitable gamma-1 f-allotype can be selected, such as the f-allotype. _{Thus, in some embodiments, the antibody is an f-allotype having a V H} ( FIG. 1 ) selected from VH#1, VH#2, VH#3, VH#4 or VH#5, e.g., SEQ ID NO: 11 IgG with _{In some embodiments, the antibody has a V L} selected from VK#1, VK#2, VK#3 or VK#4 ( FIG. 1 ). In certain embodiments, the antibody has a kappa constant region as set forth in SEQ ID NO: 10, and a heavy chain constant region as set forth in SEQ ID NO: 11, wherein the heavy and light chain variable regions are the following combinations from the sequence set forth in Figure 1 : ( a) VH#2, VK#3; (b) VH#1, VK#3; (c) VH#3, VK#1; (d) VH#3, VL#3; (e) VH#4, VK#4; (f) VH#4, VK#2; (g) VH#5, VK#1; (h) VH#5, VK#2; (i) VH#3, VK#4; or (j) VH#3, VL#3.

In some embodiments, for example, when the antibody is a fragment, the antibody may be conjugated with another molecule, such as polyethylene glycol (PEGylated) or serum albumin, to provide an extended half-life in vivo. Examples of PEGylation of antibody fragments are (for abciximab) Knight et al (2004) Platelets 15: 409; (for anti-CEA antibodies) Pedley et al (1994) Br.J. Cancer 70: 1126; Chapman et al (1999) Nature Biotech. 17:780.

In some embodiments, an antibody of the invention is in the form of a Fab' fragment. The full-length light chain is _{generated by fusion of the V L} region to a human kappa or lambda constant region. Although either constant region can be used in any light chain, in a typical embodiment, a kappa constant region is used in combination with a Vkappa variable region, and a lambda constant region is used with a Vlambda variable region.

The heavy chain of the Fab' is an Fd' fragment produced by the fusion of the human heavy chain constant region sequence, the first constant (CH1) domain and the V _{H region of the invention to the hinge region.} The heavy chain constant region sequence may be from any of the immunoglobulin classes, but is usually from IgG, and may be from IgG1, IgG2, IgG3 or IgG4. In addition, the Fab' antibody of the invention may be a heterologous sequence, for example the hinge sequence may be from one immunoglobulin subclass and the CH1 domain may be from a different subclass.

Ⅴ. Administration of an anti-GM-CSF antibody for the treatment of a disease in which GM-CSF is a target.

The present invention also provides a method of treating a patient suffering from a disease associated with GM-CSF for which it is desirable to inhibit GM-CSF activity, ie, for which GM-CSF is a therapeutic target. In some embodiments, the patient is suffering from chronic inflammatory diseases such as arthritis, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, systemic-onset Still's disease and other inflammatory diseases of the joint; inflammatory bowel diseases such as ulcerative colitis, Crohn's disease, Barrett's syndrome, ileitis, enteritis, eosinophilic esophagitis and gluten intolerance; inflammatory diseases of the respiratory system such as asthma, eosinophilic asthma, adult respiratory distress, allergic rhinitis, silicosis, chronic obstructive pulmonary disease, irritable lung disease, interstitial lung disease, diffuse parenchymal disease, bronchiectasis; inflammatory diseases of the skin such as psoriasis, scleroderma, and inflammatory dermatosis such as eczema, atopic dermatitis, urticaria and pruritus; Diseases involving inflammation of the central nervous system and peripheral nervous system such as multiple sclerosis, idiopathic demyelinating polyneuropathy, Galen Valle's syndrome, chronic inflammatory demyelinating polyneuropathy, neurofibromatosis and neurodegenerative diseases such as Alzheimer's disease. A variety of other inflammatory diseases can be treated using the methods of the present invention. These include systemic lupus erythematosus, immune-mediated renal diseases such as glomerulonephritis and spondyloarthropathies; and diseases with undesirable chronic inflammatory components such as systemic sclerosis, idiopathic inflammatory myopathy, Sjogren's syndrome, vasculitis, sarcoidosis, thyroiditis, gout, otitis, conjunctivitis, sinusitis, sarcoidosis, Behcet's syndrome, autoimmune lymphoproliferative syndrome (or ALPS, Also known as Canale-Smith syndrome), Ras-associated autoimmune leukoproliferative disorder (or RALD), Noonan syndrome, hepatobiliary diseases such as hepatitis, primary cholangiosclerosis, granulomatous hepatitis and sclerosing cholangitis. In some embodiments, the patient is free from inflammation after damage to the cardiovascular system. Various other inflammatory diseases include Kawasaki disease, multiple Castleman's disease, tuberculosis and chronic cholecystitis. Additional chronic inflammatory diseases include, for example, Harrison's Principles of Internal Medicine, 12th Edition, Wilson, et al., eds., McGraw-Hill, Inc. is described. In some embodiments, the patient treated with the antibody is a tumor or cancer in which GM-CSF contributes to cancer cell growth, such as acute myeloid leukemia, plexiform neurofibromatosis, autoimmune lymphoproliferative syndrome (or ALPS, also Canale). -Smith syndrome), Ras-associated autoimmune leukoproliferative disorder (or RALD), Noonan syndrome, chronic myelogenous monocytic leukemia, juvenile myelogenous monocytic leukemia, and acute myeloid leukemia . In some embodiments, the patient treated with an antibody of the invention due to ischemic damage to the cardiovascular system such as ischemic heart disease, stroke and atherosclerosis does not have or is at risk of heart failure. In some embodiments, the patient treated with an antibody of the invention suffers from asthma. In some embodiments, the patient treated with an antibody of the invention is suffering from Alzheimer's disease. In some embodiments, the patient treated with an antibody of the invention has osteopenia, eg, osteoporosis. In some embodiments, the patient treated with an antibody of the invention suffers from thrombocytopenic purpura. In some embodiments, the patient has type I or type II diabetes. In some embodiments, the patient may not have one or more diseases for which GM-CSF is a therapeutic target, for example, the patient may suffer from rheumatoid arthritis and heart failure, or osteoporosis and rheumatoid arthritis, and the like.

Two other examples of neutralizing anti-GM-CSF antibodies are the human ElO antibody and the human G9 antibody described in Li et al, (2006) PNAS 103 (10):3557-3562. ElO and G9 are IgG class antibodies. ElO has 870 pM binding affinity for GM-CSF and G9 has 14 pM affinity for GM-CSF. Both antibodies are specific for binding to human GM-CSF and exhibit potent neutralizing activity as assessed by TF1 cell proliferation assay.

A further exemplary neutralizing anti-GM-CSF antibody is the MT203 antibody described by Krinner et al. (MoI Immunol. 44:916-25, 2007; Epub 2006 May 112006). MT203 is an IgG1 class antibody that binds to GM-CSF with picomolar affinity. The antibody shows potent inhibitory activity as assessed by TF-1 cell proliferation assay and the ability to block IL-8 production in U937 cells.

Additional antibodies suitable for use with the present invention will be known to those skilled in the art.

An hGM-CSF antagonist, which is an anti-hGM-CSF receptor antibody, may also be used in conjunction with the methods of the present disclosure. Such hGM-CSF antagonists include antibodies to the hGM-CSF receptor alpha chain or beta chain. The anti-hGM-CSF receptor antibody used in the present invention may be in any antibody format as described above, eg intact, chimeric, monoclonal, polyclonal, antibody fragment, humanized, humaneered, etc. can Examples of suitable anti-hGM-CSF receptor antibodies, eg, neutralizing, high affinity antibodies, suitable for use in the present invention are known (see, eg, US Pat. No. 5,747,032 and Nicola et al., Blood 82:1724, 1993). ).

Non-antibody GM-CSF antagonist

Other proteins that may interfere with the productive interaction of hGM-CSF with its receptor include mutant hGM-CSF protein, and one or both of the hGM-CSF receptor chains that bind hGM-CSF and compete for binding to cell surface receptors. secreted proteins comprising at least a portion of the extracellular portion of For example, a soluble hGM-CSF receptor antagonist can be prepared by fusing the coding region of sGM-CSFR alpha with the CH2-CH3 region of murine IgG2a. Exemplary soluble hGM-CSF receptors are described in Raines et al. (1991) Proc. Natl. Acad. Sci USA 88: 8203. Examples of GM-CSFR alpha-Fc fusion proteins are provided, for example, in Brown et al (1995) Blood 85:1488. In some embodiments, the Fc component of such a fusion can be engineered to modulate binding, for example to increase binding to an Fc receptor.

Other hGM-CSF antagonists include hGM-CSF mutations. See, for example, Hercus et al. Proc. Natl. Acad. hGM-CSF in which amino acid residue 21 of hGM-CSF is mutated to arginine or lysine (E21R or E21K), as described in Sci USA 91:5838, 1994, prevents propagation of hGM-CSF-dependent leukemia cells in a mouse xenograft model It has been shown to have in vivo activity in the blood (Iversen et al. Blood 90:4910, 1997). As will be understood by one of ordinary skill in the art, such antagonists may include conservatively modified variants of hGM-CSF with substitutions such as those mentioned at amino acid residue 21, or hGM-CSF with amino acid analogs, for example to prolong half-life. variants may be included.

In some embodiments, the hGM-CSF antagonist may be a peptide. For example, the hGM-CSF peptide antagonist may be a peptide designed to structurally mimic the location of specific residues in the B and C helices of human GM-CSF associated with receptor binding and biological activity (e.g., Monfardini et al, J. Biol. Chem 271:2966-2971, 1996).

In another embodiment, the hGM-CSF antagonist is an “antibody mimetic” that targets and binds an antigen in a manner similar to an antibody. Some of these "antibody mimetics" use a non-immunoglobulin protein scaffold as an alternative protein framework for the variable region of an antibody. For example, Ku et al. (Proc. Natl. Acad. Sci. USA 92(14):6552-6556 (1995)) reported that two of the loops of cytochrome b562 were randomized and selected for binding to bovine serum albumin in a cytochrome b562-based antibody. alternatives are disclosed. Individual mutants were found to bind BSA selectively, similar to anti-BSA antibodies. U.S. Pat. Nos. 6,818,418 and 7,115,396 disclose antibody mimetics featuring a fibronectin or fibronectin-like protein scaffold and one or more variable loops. This fibronectin-based antibody mimetic, known as Adnectin, exhibits many of the same properties as native or engineered antibodies, including high affinity and specificity for all target ligands. The structure of this fibronectin-based antibody mimetic is similar to that of the variable region of an IgG heavy chain. Thus, these mimetics naturally exhibit similar antigen-binding properties and affinity to native antibodies. In addition, these fibronectin-based antibody mimetics exhibit certain advantages over antibodies and antibody fragments. For example, these antibody mimetics do not rely on disulfide bonds for basal fold stability and are therefore generally stable under conditions that degrade the antibody. In addition, since the structure of this fibronectin-based antibody mimetic is similar to that of an IgG heavy chain, the process for loop randomization and shuffling can be used in vitro, similar to the affinity maturation process of an antibody in vivo.

Beste et al. (Proc. Natl. Acad. Sci. U.S.A. 96(5):1898-1903 (1999)) discloses antibody mimetics based on lipocalin scaffolds (Anticalin®). Lipocalin consists of a β-barrel with four hypervariable loops at the ends of the protein. The loop is subjected to random mutagenesis and selected for binding to eg fluorescein. Three variants showed specific binding to fluorescein, and one variant showed binding similar to the anti-fluorescein antibody. Further analysis showed that all random positions are variable, indicating that Anticalin is suitable for use as an alternative to antibodies. Thus, anticalins are small single-chain peptides, typically 160 to 180 residues, that offer several advantages over antibodies, including reduced production cost, increased storage stability and reduced immune response.

U.S. Pat. No. 5,770,380 discloses synthetic antibody mimetics using a rigid non-peptide organic scaffold of calixarene, to which multiple variable peptide loops are attached as binding sites. The peptide loops all protrude geometrically on the same side in the calisaren with respect to each other. This geometrical identification allows all loops to be used for binding, increasing the binding affinity for the ligand. However, compared to other antibody mimetic, calisaren-based antibody mimetic is less susceptible to attack by protease enzymes because it is not composed only of peptides. Scaffolds do not consist purely of peptides, DNA or RNA. This means that this antibody mimetic is relatively stable and long-lived under extreme environmental conditions. In addition, because calisaren-based antibody mimetics are relatively small, they are less likely to elicit an immunogenic response.

Murali et al. (Cell MoI Biol 49 (2):209-216 (2003)) describes a method for reducing antibodies to smaller peptidomimetics, "antibody-like binding peptidomimetics" which may be useful alternatives to antibodies ( ABiP).

In addition to the non-immunoglobulin protein framework, antibody properties were also mimicked in compounds containing RNA molecules and non-natural oligomers (eg, protease inhibitors, benzodiazepines, purine derivatives and beta-turn mimetics). Accordingly, non-antibody GM-CSF antagonists may also include such compounds.

therapeutic administration

In some embodiments, the methods of the present disclosure comprise administering an hGM-CSF antagonist, (eg, an anti-hGM-CSF antibody) as a pharmaceutical composition to an individual having CRS or a cytokine storm. In some embodiments, the hGM-CSF antagonist is administered in a therapeutically effective amount using a dosing regimen suitable for treating the disease.

In some embodiments, a therapeutically effective amount is an amount that at least partially arrests the condition or symptom thereof. For example, a therapeutically effective amount may inhibit immune activation, decrease circulating cytokine levels, decrease T cell activation, or reduce fever, malaise, fatigue, anorexia, myalgia, arthralgia, nausea, vomiting, Headache, skin rash, diarrhea, tachypnea, hypoxemia, hypoxia, shock, cardiovascular tachycardia, enlarged pulse pressure, hypotension, capillary leakage, increased cardiac output (early), potentially decreased cardiac output (late), elevated D-dimer , hypofibrinogenemia with or without bleeding, azotemia, transaminitis, hyperbilirubinemia, headache, changes in mental state, confusion, delirium, difficulty finding words or overt frank aphasia, hallucinations, tremors, It may relieve dysmetria, gait changes, or seizures.

The methods of the present invention comprise administering to a patient a therapeutically effective amount of an anti-GM-CSF antibody as a pharmaceutical composition using a dosage regimen suitable for the treatment of the disease. Compositions can be formulated for use in a variety of drug delivery systems. In addition, for suitable formulations, one or more physiologically acceptable excipients or carriers may be included in the composition. Suitable formulations for use in the present invention are described in Remington: The Science and Practice of Pharmac y, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005. For a brief review of drug delivery methods see Langer, Science 249:1527-1533 (1990).

Anti-GM-CSF antibodies for use in the methods of the invention are provided in a solution suitable for infusion into a patient, such as sterile isotonic aqueous water for injection. The antibody is dissolved or suspended in an acceptable carrier at an appropriate concentration. In some embodiments the carrier is aqueous, such as water, saline, phosphate buffered saline, and the like. The composition may include auxiliary pharmaceutical substances necessary to approximate the physiological state, such as pH adjusting agents and buffers, isotonicity agents, and the like.

The pharmaceutical composition of the present invention can be administered to a patient, for example osteopenia, rheumatoid arthritis, juvenile idiopathic arthritis, systemic-onset Still's disease, asthma, eosinophilic asthma, eosinophilic esophagitis, multiple sclerosis, psoriasis, atopic dermatitis, plexiform nerve Fibromatosis, autoimmune lymphoproliferative syndrome (or ALPS, also known as Canale-Smith syndrome), Ras-associated autoimmune leukoproliferative disorder (or RALD), Noonan syndrome, chronic myelogenous monocytic leukemia, juvenile myelomonocytic leukemia, acute Treating or at least partially stopping myeloid leukemia, multiple Castleman's disease, chronic obstructive pulmonary disease, interstitial lung disease, diffuse pulmonary parenchymal disease, idiopathic thrombocytopenia purpura, Alzheimer's disease, heart failure, Kawasaki disease, heart damage or diabetes due to an ischemic event It is administered to the patient in an amount sufficient to: or in an amount capable of at least partially arresting the symptoms of the disease and its complications or the disease. An amount appropriate to accomplish this is defined as a “therapeutically effective dose”. A therapeutically effective dose is determined by monitoring the patient's response to therapy. The usual criteria for indicating a therapeutically effective dose include alleviation of symptoms of a disease in a patient. The effective amount will depend on the severity of the disease and the general health of the patient, including other factors such as age, weight, sex, route of administration, and the like. The antibody may be administered singly or in multiple doses depending on the patient's need and tolerability and dose and frequency. In any event, the method provides a sufficient dose of the anti-hGM-CSF antibody to effectively treat the patient.

Antibodies may be administered alone or in combination with other therapies to treat a disease of interest.

The antibody may be administered by injection or infusion via any suitable route including, but not limited to, intravenous, subcutaneous, intramuscular or intraperitoneal routes. In some embodiments, the antibody may be administered by infusion. In an exemplary embodiment, the antibody may be stored at 10 mg/ml in sterile isotonic saline solution for injection at 4° C. and diluted in 100 ml or 200 ml 0.9% sodium chloride for injection prior to administration to a patient. The antibody is administered by intravenous infusion for 1 hour at a dose of 0.2 to 10 mg/Kg. In another embodiment, the antibody is administered by intravenous infusion over a period of, for example, 15 minutes to 2 hours. In another embodiment, the administration procedure is via subcutaneous or intramuscular injection.

In some embodiments, the hGM-CSF antagonist, eg, an anti-hGM-CSF antibody, is administered by the paraspinal route. Paraspinal administration involves anatomical local delivery performed to place the therapeutic molecule directly in the vicinity of the spine upon initial administration. Paraspinal administration is described, for example, in US Pat. No. 7,214,658 and Tobinick & Gross, J. Neuroinflammation 5:2, 2008.

The dose of the hGM-CSF antagonist is selected to provide effective treatment to an individual diagnosed with CRS or a cytokine storm. Dosages typically range from about 0.1 mg/Kg body weight to about 50 mg/Kg body weight or from about 1 mg to about 2 g body weight per patient. Doses often range from about 1 to about 20 mg/Kg or from about 50 mg to about 2000 mg/patient. The dosage is administered at an appropriate frequency, which may range from once daily to once every three months, depending on the pharmacokinetics (eg, half-life of the antibody in circulation) and pharmacokinetic response (eg, duration of therapeutic effect of the antibody) of the antagonist. In some embodiments where the antagonist is an antibody or modified antibody fragment, an in vivo half-life of about 7 to about 25 days and administration of the antibody dose is repeated from once weekly to once every 3 months. In another embodiment, the antibody is administered approximately once a month.

The VH region and/or VL region of the present invention may also be used for diagnostic purposes. For example, VH and/or VL regions can be used in clinical assays such as detection of GM-CSF levels in a patient. VH or VL regions of the invention may also be used, for example, to produce anti-Id antibodies.

Unless defined otherwise herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Also, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

In one embodiment, "treating" includes therapeutic treatment and "preventing" includes prophylactic measures, wherein the object is to prevent or reduce the targeted pathological condition or disorder as described above. Thus, in one embodiment, treatment can directly affect or include curing, suppressing, suppressing, preventing, reducing the severity, delaying the onset of, reducing symptoms associated with a disease, disorder or condition, or a combination thereof. Thus, in one embodiment, “treating”, “ameliorating” and “alleviating” particularly mean delaying progression, promoting remission, inducing remission, increasing remission, accelerating recovery, increasing efficacy or decreasing tolerance of an alternative therapeutic agent, or their refers to a combination. In one embodiment, "preventing" refers to, inter alia, delaying the onset of symptoms, preventing recurrence of disease, decreasing the number or frequency of recurrent episodes, increasing delay between symptomatic episodes, or a combination thereof. In one embodiment, "inhibiting" or "inhibiting" refers to particularly reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing delay in symptoms, ameliorating symptoms, reducing secondary symptoms, 2 reducing primary infection, prolonging patient survival, or a combination thereof.

In this disclosure, singular forms include plural references, and reference to a particular number includes at least that particular value unless the context clearly dictates otherwise. As used herein, the term “plurality” means one or more. When a range of values is expressed, another embodiment includes the particular value and/or the other particular value.

Similarly, when values are expressed as approximations using the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable. In some embodiments, the term “about” refers to a deviation of 0.0001-5% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviation of 1-10% from the indicated number or range of numbers. In some embodiments, the term “about” refers to a deviation of up to 25% from the indicated number or range of numbers. The term "comprises" includes all elements listed, but may also include additional unnamed elements, and is interchangeable with the terms "includes," "includes," or "includes" having all the same characteristics and meanings. can be used interchangeably. The term “consisting of” means consisting of the recited elements or steps and may be used interchangeably with the term “consisting of” having all the same characteristics and meanings.

Example

Example 1- Exemplary Humaneered Antibodies to GM-CSF

A panel of engineered Fab' molecules with a specificity of cl9/2 was generated from an epitope-centric human V-segment library as described in US Patent Application Publication Nos. 20060134098 and 20050255552. The epitope central library was constructed from human V-segment library sequences linked to the CDR3-FR4 region comprising the BSD sequences of CDRH3 and CDRL3 along with human germline J-segment sequences. Human germline JH4 sequence was used for the heavy chain, and human germline JK4 sequence was used for the light chain.

Full-length humanized V-regions were selected from Vh1 restriction libraries that support binding to recombinant human GM-CSF. A "full-length" V-kappa library was used as the basis for the construction of a "cassette" library as described in US Patent Application Publication 20060134098, where only a portion of the murine cl9/2 antibody V-segment was initially human sequence. replaced by the library of Two types of cassettes were constructed. A cassette for the V-kappa chain was made by bridge PCR with overlapping consensus sequences within the framework 2 region. In this way "front-end" and "middle" human cassette libraries were constructed for the human V-kappa III isotype. Human V-kappa III cassettes supporting binding to GM-CSF were identified by colony-lift binding assay and ranked according to affinity in ELISA. V-kappa human "front-end" and "intermediate" cassettes were fused together by bridge PCR to reconstruct the fully human V-kappa region supporting GM-CSF binding activity. Thus, the humanoid Fab consists of humanoid V-heavy and V-kappa regions that support binding to human GM-CSF.

Binding activity was determined by surface plasmon resonance (spr) analysis. Biotinylated GM-CSF was captured on a CM5 biosensor chip coated with streptavidin. Humanized Fab fragments expressed in E. coli were diluted to a starting concentration of 30 nM in 10 mM HEPES, 150 mM NaCl, 0.1 mg/ml BSA and 0.005% P20 at pH 7.4. Each Fab was diluted 4-fold using a 3-fold dilution series and each concentration was tested twice at 37°C to determine binding kinetics with different density antigen surfaces. Data from all three surfaces were globally fit to extract dissociation constants.

Binding kinetics was analyzed by Biacore 3000 surface plasmon resonance (SPR). Recombinant human GM-CSF antigen was biotinylated and immobilized on a streptavidin CM5 sensor chip. Fab samples were diluted to a starting concentration of 3 nM and run in 3-fold dilution series. Assays were run in 10 mM HEPES, 150 mM NaCl, 0.1 mg/ml BSA and 0.005% p20 at pH 7.4 and 37°C. Each concentration was tested in duplicate. Fab' binding assays were run on two antigen density surfaces providing redundant data sets. The mean affinity (KD) for each of the six different humanized anti-GM-CSF Fab clones calculated using the 1:1 Langmuir binding model is shown in Table 2.

Fabs were tested for GM-CSF neutralization using a TF-1 cell proliferation assay. GM-CSF-dependent proliferation of human TF-1 cells was measured after culturing for 4 days with 0.5 ng/ml GM-CSF using MTS assay (Cell titer 96, Promega) to determine viable cells. All Fabs inhibit cell proliferation in this assay, indicating that they are neutralizing antibodies. There is a good correlation between the relative affinity of anti-GM-CSF Fab and EC50 in cell-based assays. Anti-GM-CSF antibodies with monovalent affinities ranging from 18 pM-104 pM demonstrate effective neutralization of GM-CSF in a cell-based assay.

An exemplary engineered anti-GM-CSF V region sequence is shown in Figure 1.

Table 2: Affinity of anti-GM-CSF Fabs determined by surface plasmon resonance assay compared to activity (EC50) in GM-CSF dependent TF-1 cell proliferation assay

Example 2 Evaluation of Humanized GM-CSF Antibodies

This example demonstrates the binding activity of a humanized anti-GM-CSF antibody in a cell-based assay compared to a chimeric IgG1k antibody (Ab2) with variable regions of the mouse antibody LMM102 (Nice et al., Growth Factors 3:159, 1990). and biological efficacy. Ab1 is a human IgG1k antibody against GM-CSF with the same constant region as Ab2.

Surface Plasmon Resonance Analysis of Binding of Human GM-CSF to Ab1 and Ab2

Surface plasmon resonance analysis using a Biacore 3000 instrument was used to compare binding kinetics and monovalent affinity for the interaction of Ab1 and Ab2 with glycosylated human GM-CSF. Ab1 or Ab2 was captured on the surface of the Biacore chip using polyclonal anti-human F(ab')2. Glycosylated recombinant human GM-CSF expressed in human 293 cells was used as an analyte. Kinetic constants were determined in two independent experiments (see FIGS. 2A-2B and Table 3). The results show that GM-CSF bound to Ab2 and Ab1 with similar monovalent affinity in this experiment. However, Ab1 was "on-rate" two times slower than Ab2, but "off-rate" about three times slower.

Table 3: Kinetic constants at 37° C. determined from surface plasmon resonance analysis of FIGS. 2A-2B; The association constant (ka), dissociation constant (kd) and calculated affinity (KD) are indicated.

GM-CSF does not require glycosylation for biological activity, but is naturally glycosylated at both N-linked and O-linked glycosylation sites. To determine whether GM-CSF glycosylation affects binding of Ab1 or Ab2, antibodies were compared in an ELISA using recombinant GM-CSF from two different sources; GM-CSF expressed in E. coli (not glycosylated) and GM-CSF expressed in human 293 cells (glycosylated). The results in Figures 3A-3B and Table 4 showed that both antibodies bind glycosylated and non-glycosylated GM-CSF with equivalent activity. Both antibodies also showed similar EC50 values in this assay.

Table 4. EC50 summary for binding of Ab2 and Ab1 to human GM-CSF from two different sources as determined by ELISA. Binding of human 293 cells (glycosylated) or E. coli (non-glycosylated) to recombinant GM-CSF was determined in two independent experiments. Experiment 1 is shown in Figures 3A-3B.

Ab1 is a humanoid antibody derived from the mouse variable region present in Ab2. Ab1 was tested for overlapping epitope specificity (Ab2) by competition ELISA.

Biotinylated Ab2 was prepared using known techniques. Biotinylation did not affect binding of Ab2 to GM-CSF as determined by ELISA. In the assay, either Ab2 or Ab1 was added at various concentrations along with a fixed amount of biotinylated Ab2. Detection of biotinylated Ab2 was assayed in the presence of unlabeled Ab or Ab1 competitor ( FIGS. 4A-4B ). Both Ab1 and Ab2 compete with biotinylated Ab2 for binding to GM-CSF, indicating binding to the same epitope. Ab1 competed more effectively for binding to GM-CSF than Ab2, consistent with slower dissociation kinetics for Ab1 when compared to Ab2 by surface plasmon resonance analysis.

Neutralization of GM-CSF activity by Ab1 and Ab2

Biological efficacy was assessed using a cell-based assay for neutralization of GM-CSF activity. This assay measures IL-8 secretion in GM-CSF-induced U937 cells. IL-8 secreted into the culture supernatant is determined by ELISA after 16 hours induction with 0.5 ng/ml E. coli-derived GM-CSF.

A comparison of the neutralizing activity of Ab1 and Ab2 in this assay is shown in a representative assay in FIG. 5 . In three independent experiments, Ab1 inhibited GM-CSF activity more effectively than Ab2 when comparing IC50 (Table 5).

Table 5. Comparison of IC50 for inhibition of GM-CSF induced IL-8 expression. Data from three independent experiments shown in Figure 5 and mean IC50 are expressed in ng/ml and nM.

summary

Humanid Ab1 bound to GM-CSF with a calculated equilibrium binding constant (KD) of 25 pM. Ab2 bound to GM-CSF with a KD of 30.5 pM. Ab2 showed a 2-fold higher association constant (ka) than Ab1 for GM-CSF whereas Ab1 showed a 3-fold slower dissociation kinetic (kd) than Ab2. Ab2 and Ab1 showed similar binding activity to glycosylated and non-glycosylated GM-CSF in antigen binding ELISA. Competition ELISA confirmed that both antibodies compete for the same epitope. Ab1 exhibited higher competitive binding activity than Ab2. In addition, Ab1 showed higher GM-CSF neutralizing activity than Ab2 in the GM-CSF-induced IL-8 induction assay.

Example 3 Administration of Neutralizing Anti-GM-CSF Antibodies in a Mouse Model of Immunotherapy-Related Toxicity

A mouse model of immunotherapy-related toxicity can be used to demonstrate the efficacy of anti-GM-CSF antibodies to prevent and treat immunotherapy-related toxicity. In one model of immunotherapy-related toxicity, mice are injected with CAR T cells at a dose that induces toxicity. See, eg, van der Stegen et al. (J. Immunol 191:4589-4598 (2013)) describes a CRS model induced by a single dose ip injection of ^{30x10 6 cells termed T4+ T cells.} T4+ T cells are engineered T cells expressing the chimeric Ag receptor (CAR) T1E28z. T cells engineered to express T1E28z are activated by cells expressing ErbB1- and ErbB4-based dimers and ErbB2/3 heterodimers.

To evaluate the efficacy of anti-GM-CSF antibodies for preventing and treating CRS, mice were divided into groups (n=10) and each group received one of the following: a) a single ip saline injection; b) ^{ip injection of 30x10 6} T4+ T cells; c) intravenous (iv) injection of 0.25 mg anti-GM-CSF monoclonal antibody 22E9 (recombinant rat anti-mouse-GM-CSF antibody) by ip injection of ^{30x10 6 T4+ T cells and co-administration with T4+ T cells;} d) Intranasal (in) injection of 0.25 mg anti-GM-CSF monoclonal antibody 22E9 by ip injection of ^{30x10 6 T4+ T cells and co-administration with T4+ T cells;} e) ^{ip injection of 30x10 6} T4+ T cells and iv injection of 0.25 mg GM-CSF monoclonal antibody 22E9 6 hours before T4+ T cell administration; f) ^{ip injection of 30x10 6} T4+ T cells and 0.25 mg GM-CSF monoclonal antibody 22E9 in injection 6 hours before T4+ T cell administration; g) ^{ip injection of 30x10 6} T4+ T cells and iv injection of 0.25 mg GM-CSF monoclonal antibody 22E9 2 hours after T4+ T cell administration; Ehsms h) ^{Injection of 0.25 mg GM-CSF monoclonal antibody 22E9 in 30x10 6} ip injection of T4+ T cells and 2 hours after T4+ T cell administration. Additional doses, time of administration, and route of administration are evaluated.

To evaluate the effectiveness of anti-GM-CSF antibody 22E9, organs are harvested from mice, formalin fixed, and subjected to histopathological analysis. Blood was collected and human IFNγ, human IL-2 and mouse IL-6, IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ and TNFα were performed by methods described in the literature such as ELISA analysis. is assessed by wells. Mice weight, behavior and clinical symptoms are observed.

Example 4 Effect of Anti-GM-CSF Antibodies on Immunotherapy

A mouse model can be used to show that GM-CSF antagonists do not adversely affect the efficacy of cancer immunotherapy. SCID beige mice can be inoculated with a cancer cell line and treated with an immunotherapeutic agent known to induce CRS as T4+ T cells with or without anti-GM-CSF antibody.

To evaluate whether anti-GM-CSF antibodies affect the efficacy of immunotherapy, mice were divided into groups (n=10), and each group received one of the following: a) 30x10 ⁶ SKOV3 cells subcutaneously ( sc) injection; b) a sc injection of ^{30x10 6 SKOV3 cells and ip injection of 30x10 6} T4+ T cells; or c) a sc transplantation of ^{30x10 6 SKOV3 cells, ip injection of 30x10 6} T4+ T cells and iv injection of 0.25 mg of anti-GM-CSF antibody 22E9.

To evaluate the effect of anti-GM-CSF antibody 22E9 on T4+ T cell efficacy, tumor size is measured every 4 days with calipers, and tumor volume is calculated with the following formula: 0.5 × (large diameter) × (small diameter) ² . Mice weight, behavior and clinical symptoms are observed. At the end of the experiment, the animals are sacrificed and the tumor tissue is harvested and weighted.

Example 5 - Mouse model of human CRS

A mouse model for CRS was developed to investigate the CRS therapeutic or preventive effect of humanized anti-GM-CSF monoclonal antibody (FIGS. 17a.-17b.).

Method: The model used is the primary AML model. Immunocompromised NSG-S mice further transfected with human SCF, IL-3 and GM-CSF were transplanted with AML blasts derived from CD123-positive AML patients. After 2-4 weeks, bleeding was done to confirm engraftment and high disease burden achievement. Mice were then ^{treated with a high dose of CAR-T123 at 1 x 10 6} cells, a 10-fold higher dose than previously studied.

RESULTS: Within 1-2 weeks after CAR-T cell injection, these mice were observed to develop a disease characterized by weakness, leanness, crooked body, withdrawal and poor motor response. Mice eventually died of disease within 7-10 days. Symptoms are associated with massive T cell expansion in mice and elevations of several human cytokines, such as IL-6, MIP 1α, IFN-γ, TNFα, GM-CSF, MIP1β and IL-2, after CAR-T cell treatment. It appeared in a pattern similar to that seen in human CRS. The GM-CSF fold change was significantly greater than that of other cytokines ( FIGS. 17 a-b ).

Example 6 - Generation of GM-CSF knockout CAR-T

GM-CSF CRISPR knockout T cells were generated and exhibited reduced expression of GM-CSF, but similar levels of other cytokines and degranulation, demonstrating immune cell functionality (see FIGS. 15A-15G ).

Example 7-Anti-GM-CSF Neutralizing Antibodies Do Not Inhibit CAR-T Mediated Death, Proliferation or Cytokine Production, but Neutralize GM-CSF

Anti-GM-CSF neutralizing antibodies do not inhibit CAR-T mediated death, proliferation or cytokine production, but successfully neutralize GM-CSF. (See FIGS. 16A-16I ).

Example 8-Anti-GM-CSF Neutralizing Antibodies Do Not Inhibit CAR-T Efficacy In Vivo

Humanized anti-GM-CSF monoclonal antibody, a neutralizing hGM-CSF antibody, does not inhibit CAR-T efficacy in vivo ( FIGS. 18A-18C ). CAR-T efficacy in a xenograft model in combination with an anti-GM-CSF neutralizing antibody according to embodiments described herein. As shown in FIG. 18A , NALM-6-GFP/luciferase cells (human, peripheral blood leukemia pre-B cells) were injected into NSG mice and bioluminescence imaging (BLI0) was performed to confirm tumor growth. Mice were treated with (1) anti-GM-CSF antibody (10 mg/Kg daily for 10 days) and (a) CART19 or (b) untransduced human T cells (UTD) 1×10 ⁶ cells or (2) IgG control antibody ( 10 mg/Kg daily for 10 days) and (a) CART19 or (b) untransduced human T cells (UTD) 1×10 ⁶ cells. 18B and 18C demonstrate that anti-GM-CSF neutralizing antibodies did not inhibit CAR-T efficacy in vivo.

Example 9-Anti-GM-CSF Neutralizing Antibodies Do Not Impair CAR-T Effect on Survival

In vitro and in vivo preclinical data show that anti-GM-CSF neutralizing antibody (humanized anti-GM-CSF monoclonal antibody) does not impair the effect of CAR-T on survival in mouse models. (Fig. 19).

Anti-GM-CSF neutralizing antibody does not interfere with CAR-T cell function in vivo in the absence of PBMCs. Survival rates were similar for CAR-T + control and CAR-T + anti-GM-CSF neutralizing antibodies.

Example 10-Anti-GM-CSF Neutralizing Antibodies Can Increase CAR-T Expansion

In vitro and in vivo preclinical data show that anti-GM-CSF neutralizing antibody (humanized anti-GM-CSF monoclonal antibody) can increase CAR-T expansion ( FIG. 20 ). Anti-GM-CSF neutralizing antibodies can increase CAR-T cancer cell death in vitro. Antibodies may increase proliferation of CAR-T cells and enhance efficacy. CAR-T proliferation was increased by GM-CSF neutralizing antibody in the presence of PBMCs. (unaffected without PBMC). The antibody did not inhibit degranulation, intracellular GM-CSF production or IL-2 production.

Example 11 - CAR-T Extensions Associated with Improved Overall Response Rate

CAR-T extensions associated with improved overall response rates. (Fig. 21). CAR AUC (area under the curve) defined as the cumulative level of CAR+ cells/μL of blood during the first 28 days after CAR-T administration. P values calculated by Wilcoxon rank sum test (Neelapu, et al ICML 2017 Abstract 8).

Example 12 - Study protocol for anti-GM-CSF neutralizing antibodies according to embodiments described herein

A study protocol for an anti-GM-CSF neutralizing antibody (humanized anti-GM-CSF monoclonal antibody) according to embodiments described herein. (See Fig. 22). CRS and NT are assessed daily during hospitalization and at clinic visits during the first 30 days. Eligible subjects to receive GM-CSF neutralizing antibody on days -1, 1 and 3 of CAR-T treatment. Tumor assessments to be performed at baseline and at 1, 3, 6, 9, 12, 18 and 24 months. Blood samples (PBMC and serum) on days -5, -1, 0, 1, 3, 5, 7, 9, 11, 13, 21, 28, 90, 180, 270 and 360.

Example 13 - GM-CSF depletion increases CAR-T cell expansion

GM-CSF depletion increases CAR-T cell expansion. ( FIGS. 23A-23B ) FIG. 23A shows increased ex-vivo expansion of ^{GM-CSF k/o} CAR-T cells compared to control CAR-T cells.

23B demonstrates stronger proliferation following in vivo treatment with an anti-GM-CSF neutralizing antibody (human anti-GM-CSF monoclonal antibody) according to embodiments described herein.

Examples 14-100 Safety Profile of Anti-GM-CSF Neutralizing Abs in Over 100 Human Patients*

Stage 1: Single dose in healthy adult volunteers, dose escalation.

The goals were to analyze safety/tolerability, PK and immunogenicity.

Enrollment/dose:

(n=12)

3 / 1 mg/kg

3 / 3 mg/kg

3 / 10 mg/kg

3 / placebo

Stability Results:

Clean Safety Profile:

No drug-related serious side effects (SAEs)

non-immunogenic

Step 2 :1) Dosage at 0, 2, 4, 8, 12 weeks in rheumatoid arthritis patients.

The objective is to analyze efficacy, safety/tolerability, PK and immunogenicity.

Enrollment/dose:

(n=9)

7 / 600 mg

2 / placebo

Safety Results:

Clean Safety Profile:

No drug-related serious side effects (SAEs)

non-immunogenic

2) Doses at 0, 2, 4, 8, 12, 16 and 20 weeks in patients with severe asthma.

The objectives were to analyze efficacy, safety/tolerability, PK and immunogenicity.

Registration/Capacity:

(n=160)

78/400 mg

82 / placebo

Safety Results:

Clean safety profile:

No drug-related serious side effects (SAEs)

non-immunogenic

* 94 patients in the study described above and 12 patients in the ongoing CMML Clinical I trial where the drug was well tolerated; An additional 76 patients received a chimeric version of the GM-CSF neutralizing Ab (KB002) and had a similar safety profile.

All studies were randomized, double-blind, placebo-controlled, IV dosing. (See Figure 24.)

Example 15- Effect of anti-GM-CSF antibodies on CART activity and toxicity

This study will investigate the effect of GMCSF blockade with anti-GM-CSF antibody on chimeric antigen receptor T cell (CART) activity and toxicity.

This can be done through two AIMSs:

AIM # 1: Investigation of the effect of GMCSF blockade with anti-GM-CSF antibody on CART cell effector function

AIM # 2: Study of GMCSF Blocking Effect Using Anti-GM-CSF Antibody on Reduction of Cytokine Release Syndrome after CART Cell Therapy Study Strategies.

The following experiment is proposed:

Anti-GM-CSF antibody and CART cells (cytokine production (GM-CSF, IL-2, INFg, IL-6, IL-8, MCP- An in vitro study of the combination of four different doses of GMCSF blockade using 30 plex Lumiex containing 1) antigen-specific killing, degranulation, proliferation and depletion).

An in vitro study of the combination of anti-GM-CSF antibody and four different doses of GMCSF blockade with CART cells using two models:

CD19 positive cell line (NALM6) engrafted xenografts treated with CART19 with or without anti-GM-CSF antibody; and

Xenografts from patients with primary ALL followed by treatment with CART19 with or without anti-GM-CSF antibody.

Mice were administered i.p. 10 mg/Kg of anti-GM-CSF antibody immediately before CART cell transplantation. and 10 mg/Kg/day i.p. for 10 days. Mice are followed for tumor response and survival. Retroorbital bleeding is obtained weekly from 1 week after CART cell treatment. Disease burden, T cell expansion kinetics, expression of exhaustion markers and cytokine levels (30 Plex) are analyzed. Upon completion of the experiment, the spleen and bone marrow are collected and analyzed for tumor characteristics and CAR-T cell count.

Using the following model, in the presence of PBMCs, anti-GM-CSF antibodies (with and without murine GM-CSF blockade) and CART cells in the CRS model (in which high doses of CART cells were used to induce CRS) In vivo study of the combination of GMCSF blockade using:

Xenografts from patients with primary ALL followed by treatment with CART19 with or without anti-GM-CSF antibody.

Mice were treated with anti-GM-CSF antibody at 10 mg/Kg immediately before CART cell transplantation and 10 mg/Kg/day for 10 days i.p. is injected Mice are followed for tumor response, CRS toxicity symptoms, and survival. Analyze disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30 Plex). After the experiment, the spleen and bone marrow are collected and analyzed for tumor characteristics and CAR-T cell count.

In vivo neurotoxicity assay

Using the model discussed in #3 above, MRI images of mice when they are sick to assess the development of neurotoxicity following CART cell treatment. Images show CART cells and anti-GM-CSF antibody vs. Mice receiving control antibody are compared. Repeat experiments are performed. Mice will be euthanized after 14 days of CART cells in these replicates. Brain tissue is analyzed for the presence of cytokines, monocytes, human T cells via multiplex analysis and integrity of the blood brain barrier by IHC, flow cytometry and microscopy.

Example 16

Anti-hGM-CSF Neutralizing Antibodies Reduce Neuroinflammation in CAR-T Cell Associated Neurotoxicity (NT)

There is extensive scientific evidence to suggest that GM-CSF is essential for cytokine release syndrome (CRS), neurotoxicity (NT) and the initiation of the inflammatory cascade that appears after initiation of CAR-T cell therapy. The hypothesis studied is that blocking soluble GM-CSF with a neutralizing antibody (lenzilumab) could eliminate or prevent the incidence and severity of both CRS and NT observed with CAR-T cell therapy. Importantly, CAR-T cell activity should be preserved or improved whenever possible. The experimental design included the effect of GM-CSF blockade with an anti-GM-CSF antibody (lenzilumab) on CAR-T cell effector function, CAR-T efficacy in a tumor xenograft model, CRS development in a CRS xenograft model, and MRI. We test the development of NT using imaging and volumetric analysis to quantify neuroinflammation seen with CAR-T cell therapy. In vitro and in vivo experiments with CAR-T +/- lenzilumab in both the presence and absence of human PBMCs were studied. (See Examples 9 and 10, FIGS. 19 and 20A-20B).

Way

An in vitro study was performed to evaluate the combination of the GM-CSF neutralizing antibody lenzilumab with human CD19 CAR-T cells for antigen-specific killing, degranulation, proliferation and depletion in the presence or absence of human PBMCs.

To evaluate the effect of anti-GM-CSF antibody (lenzilumab) on CAR-T cell proliferation and efficacy, an in vivo study was performed using a follow-up model (with and without murine GM-CSF blockade).

Effector/target control experiments: CD19 positive cell line (NALM6) engrafted xenografts treated with CART19 with or without anti-GM-CSF antibody (lenzilumab) in the absence of human PBMCs.

NSG mice were administered 10 mg/Kg of anti-GM-CSF antibody (lenzilumab) i.p. immediately before CAR-T cell transplantation. Tumor response and survival were evaluated after injection and daily administration at the same dose for 10 days. Post-orbital bleeding was obtained starting weekly starting 1 week after CAR-T cell treatment. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30 Plex) were also analyzed. After the experiment, spleen and bone marrow were collected and analyzed for tumor characteristics and CAR-T cell count.

CRS/NT trial: patient-derived xenografts with primary ALL followed by treatment with CART19 with or without lenzilumab in the presence of human PBMCs:

To evaluate the effect of lenzilumab in abrogating or preventing the onset and severity of CAR-T-induced CRS and NT, using xenografts from primary ALL patients treated with CART19 with or without lenzilumab, An in vivo study was performed with human CAR-T cells (with or without murine GM-CSF blockade) in a CRS model in the presence of high doses of CAR-T cells to induce CRS. NSG mice were administered i.p. with lenzilumab 10 mg/Kg daily immediately before and after CAR-T cell transplantation for 10 days. administered. Mice were followed for tumor response, survival, CRS and NT symptoms. Brain MRI scans were taken at baseline during and at the end of CAR-T cell treatment, and volumetric analysis was performed to evaluate and quantify neuroinflammation and MRI T1 high intensity signals across treatment groups. Body weight and post-orbital bleeding were obtained at baseline, 2 days later, 1 week after CAR-T cell treatment and weekly thereafter. Disease burden, T cell expansion kinetics, expression of depletion markers and cytokine levels (30 Plex) were analyzed. At the end of the experiment, the spleen and bone marrow were collected and the tumor characteristics and the number of CAR-T cells were analyzed.

result

in vitro model

In this experiment, the effect of GM-CSF neutralization with lenzilumab on CAR-T cell effector function was investigated. GM-CSF is secreted by CAR-T cells at very high levels (above 1,500 pg/ml) and the use of lenzilumab completely neutralized GM-CSF but inhibited CAR-T degranulation, intracellular GM-CSF production or IL2 production. did not interfere Moreover, lenzilumab did not inhibit CAR-T antigen specific proliferation or CAR-T killing. The effector-to-target ratio (E:T) was CAR-T + lenzilumab vs. Similar to CAR-T control antibody, p = ns ( FIGS. 16A-16D and 16J ).

In vivo model:

Effector/target control experiments:

To study the effect of lenzilumab on CART19 cell function in vivo, we engrafted immune-compromised NOD-SCID-g-/- with the CD19+ ALL cell line NALM6 in the absence of human PBMCs. Treatment with CART19 in combination with lenzilumab resulted in potent antitumor activity and improved overall survival, similar to CART19 with control antibody, despite complete neutralization of GM-CSF levels in these mice, indicating that GM-CSF It does not impair CAR-T cell activity in vivo in the absence of PBMCs ( FIGS. 16F and 16G ).

CRS and NT experiments:

Using human ALL blasts, human CD19 CAR-T, and human PBMC, lenzilumab in combination with CAR-T cell therapy showed neuroinflammation compared to CAR-T alone as assessed by quantitative MRI T1 high intensity signal. was found to decrease by ~90%. This is a groundbreaking discovery and demonstrated for the first time in vivo that neuroinflammation caused by CAR-T cell therapy can be effectively eliminated. MRI images after treatment with lenzilumab + CAR-T cells were similar to baseline pre-treatment scans, in sharp contrast to MRI images after treatment with control antibody + CAR-T cells, which showed a significant increase in inflammation. Moreover, a decrease in myeloid cells was observed in the brains of mice treated with lenzilumab + CAR-T compared to mice treated with CAR-T and control antibody. This finding is consistent with data reported in clinical trials using CD19 CAR-T cell therapy in which an increase in myeloid cells was observed in the CSF of patients with severe grade >3 neurotoxicity. In addition, lenzilumab in combination with CAR-T cell therapy has been shown to reduce the incidence and severity of CRS compared to CAR-T + control antibody. This finding is supported by the statistically significant reduction in body weight observed in mice treated with CAR-T and controls, the most objective markers and characteristic symptoms of CRS seen in vivo. Body weights in mice treated with lenzilumab + CAR-T were maintained at baseline levels compared to CAR-T + controls ( p<0.05 ). Moreover, mice treated with CAR-T + control antibody showed somatic symptoms consistent with CRS including hunched posture, withdrawal and weakness, whereas mice treated with CAR-T + lenzilumab appeared healthy. Importantly, lenzilumab + CAR-T also showed a significant 5-fold increase in proliferation of CAR-T cells compared to CAR-T + control in these CRS/NT experiments involving PBMCs. It has been shown in previous clinical trials with various CD19 CAR-T cell therapies that improving CAR-T proliferation or expansion is associated with improved efficacy (including ORR, CR), suggesting that lenzilumab is potentially anti-inflammatory. suggest that it can improve tumor response. This finding may be partly explained by the reduction in MDSC expansion and trafficking known to be presented by GM-CSF. Finally, the combination of lenzilumab and CAR-T results in a much better leukemia control as quantified by flow cytometry compared to CAR-T and control antibody. Compared to untreated mice (including 500,000 to 1.5M leukemia cells) and CAR-T + control antibody (containing 15,000 to 100,000 leukemia cells), CAR-T + lenzilumab treatment significantly reduced the number of leukemia cells (500 to 5,000 cells). cells) improved overall disease control (see FIGS. 25A-25D ).

The MRI image of FIG. 25A shows a clear improvement in neurotoxicity (NT) (neuroinflammation) in the brain of mice receiving CAR-T cells and an anti-GM-CSF neutralizing antibody according to embodiments described herein. In contrast, the brains of mice administered CAR-T cells and control antibody showed signs of neurotoxicity on MRI images. 25B graphically depicts a 90% reduction in NT in mice in group 1 compared to increased NT in group 2 mice.

The extent of quantitative improvement (90% reduction in NT) after administration of CAR-T cells and an anti-GM-CSF neutralizing antibody according to embodiments described herein was an unexpected finding.

conclusion

Combining an anti-GM-CSF antibody (lenzilumab) with CAR-T cell therapy prevents the onset and severity of CRS and NT while ameliorating CAR-T expansion/proliferation in human ALL blasts, human CD19 CAR-T and human The use of PBMCs shows the potential to improve overall leukemia control in vivo. This is the first time that it has been demonstrated that CAR-T-induced neurotoxicity can be eliminated in vivo. An important clinical trial using lenzilumab in combination with CAR-T cell therapy is planned to validate these findings for improved safety and efficacy.

Example 17

GM-CSF Blockade During Chimeric Antigen Receptor T Cell Therapy May Reduce Cytokine Release Syndrome and Neurotoxicity and Improve Effector Function

Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). CRS is associated with extreme elevation of cytokines and massive T cell expansion, but the exact mechanism for NT remains unknown. Preliminary studies suggest that NT may be mediated by myeloid cells that cross the blood-brain barrier. This is supported by a correlation analysis of the CART19 pivotal trial with increased CD14 cell counts in the cerebrospinal fluid of patients with severe NT (Locke et al, ASH 2017). Therefore, the purpose of this study was to investigate the role of GM-CSF neutralization in the prevention of CRS and NT after CART cell treatment through monocyte regulation.

First, the effect of GM-CSF blockade on CART cell effector function was investigated. Here, a human GM-CSF neutralizing antibody (lenzilumab, Humanigen, California, Burlingame) that has been proven to be safe in a phase 2 clinical trial was used. When CART19 cells are stimulated with CD19+ luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, lenzilumab (10 ug/kg) neutralizes GM-CSF but does not impair CART cell function in vitro. It has been found that macrophage-associated malignancies reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were transplanted with a high disease burden of NALM6 and administered with low doses of CART19 or control T cells (inducing tumor recurrence) in combination with lenzilumab or an isotype control antibody. processed. The combination of CART19 and lenzilumab resulted in significant antitumor activity and overall survival advantage compared to control T cells ( FIG. 26A ), indicating that GM-CSF neutralization did not impair CART cell activity in vivo. This antitumor activity was validated in an ALL patient-derived xenograft model.

Next, we investigated the effect of GM-CSF neutralization on CART cell-related toxicity in a novel patient-derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemia blasts (1-3x10 ⁶ cells) derived from patients at high risk of ALL recurrence. Mice were then treated with high doses of CART19 cells (2-5x10 ^{6 intravenously).} Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched body, and weight loss, which correlated with a massive elevation of circulating human cytokine levels. Magnetic resonance imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema associated with central nervous system (CNS) infiltration. This is similar to that reported in the CART19 clinical trial in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CS) and a murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) prevented weight loss (Figure 26B), reduced significant bone marrow cytokines (Figures 26C-26D), It resulted in reduced cerebral edema ( FIG. 26E ), enhanced leukemia control in the brain ( FIG. 26F ), and decreased brain macrophages ( FIG. 26G ).

Finally, we hypothesized that disruption of GM-CSF via CRISPR/Cas9 gene editing during CART cell manufacturing would result in functional CART cells with reduced GM-CSF secretion. A guide RNA targeting exon 3 of the GM-CSF gene was designed and GM-CSF ^k/o CART19 cells were generated. Preliminary data suggest that this CART produces significantly less GM-CSF upon activation, but continues to produce similar production of other cytokines and exhibits normal effector function in vitro ( FIG. 26H ). Using the NALM6 high tumor burden recurrent xenograft model as described above, GM-CSF ^k/o CART19 cells resulted in slightly improved disease control compared to CART19 cells ( FIG. 26I ).

Therefore, modulating myeloid cell behavior through GM-CSF blockade could help control CART-mediated toxicity and improve leukemia control by reducing their immunosuppressive function. These studies shed light on a novel approach to abrogate NT and CRS via GM-CSF neutralization, potentially enhancing CART cell function. Based on these results, a phase 2 clinical trial was designed using lenzilumab as a modality to prevent CART-related toxicity in patients with diffuse large B-cell lymphoma.

Example 18

In vitro GM-CSF neutralization enhances CAR-T cell proliferation in the presence of monocytes, but does not impair CAR-T cell effector function.

Cell lines and primary cells

The NALM6 cell line was purchased from ATCC, Manassas, Virginia, and the MOLM13 cell line was provided as a gift from the Mayo Clinic's Jelinek laboratory (purchased from DSMZ, Braunschweig, Germany). These cell lines were transduced using luciferase-ZsGreen lentivirus (Addgene, Cambridge, MA, USA) and sorted with 100% purity. Cell line R10 (RPMI 1640 (Gibco, Gaithersburg, MD, US), 10% fetal bovine serum (FBS, Millipore Sigma, Ontaria, Canada) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, USA))) cultured in Primary cells were obtained from the Mayo Clinic Biobank for acute leukemia patients according to Mayo Clinic Institutional Review Board (IRB) approved protocols. The use of recombinant DNA in the laboratory was approved by the Mayo Clinic Institutional Biosafety Committee (IBC).

Primary cells and CAR-T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from non-confirmed normal donor blood collection cones obtained according to Mayo Clinic IRB approved protocols using SepMate tubes (STEMCELL Technologies, Vancouver, Canada). T cells were isolated with negative selection magnetic beads using EasySepTM Human T Cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada). Monocytes were isolated using the Human Monocyte Isolation Kit of Miltenyi Biotec, Bergisch Gladbach, Germany, which isolates CD14+ monocytes. Primary cells were treated with X-Vivo 15 (Lonza, Walkersville, MD, USA) supplemented with 10% human serum albumin (Corning, NY, USA) and 1% penicillin-streptomycin-glutamine (Gibco, Gaithersburg, MD, USA). It was cultured in the prepared T cell medium. MD, USA). CART19 cells were generated via lentiviral transduction of normal donor T cells as described herein below. The second generation CART19 construct was de novo synthesized (IDT) and cloned into a third generation lentivirus under the control of the EF-1α promoter. The CD19 inducing single chain variable region fragment was derived from clone FMC63. A second generation 4-1BB costimulatory (FMC63-41BBz) CAR construct was synthesized and used in these experiments. Lentiviral particles were plasmids in the presence of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), VSV-G and packaging plasmids (Addgene, Cambridge, MA, USA) to 293T virus-producing cells (Ikeda lab, received as a gift from Mayo Clinic). was generated through transient transfection. T cells isolated from normal donors were stimulated at a 1:3 ratio using Cell Therapy Systems Dynabeads CD3/CD28 (Life Technologies, Oslo, Norway), followed by lentiviral particles 24 h after stimulation at a multiplicity of infection (MOI) 3.0. was transduced with To determine titers and subsequent MOIs, titers were determined by transducing ^{1x10 5} primary T cells in 100 ul of T cell medium with 50 ul of lentivirus after lentiviral particles were concentrated. First, T cells were stimulated with CD3/CD28 beads and then transduced with lentiviral particles 24 hours later. Transduction was performed in triplicate and serial dilutions. Fresh T cell medium was added one day later.

Titers were determined based on the percentage of CAR positive cells (percentage of CAR+ cells x number of T cells at transduction x specific dilution/volume) and expressed in units of transduction/ml (TU/ml). Magnetic bead removal was performed on day 6 and CAR-T cells were harvested and cryopreserved on day 8 for future experiments. CAR-T cells were thawed 12 hours before use in experiments and placed in T cell medium.

GM-CSF neutralizing antibody and isotype control

Renzilumab (Humanigen, Burlingame, CA), an hGM-CSF neutralizing antibody according to embodiments described herein and as described in US Pat. Nos. 8,168,183 and 9,017, 674, each of which is incorporated herein by reference in its entirety, comprises: It is a novel, first-in-class Humaneered® monoclonal antibody that neutralizes human GM-CSF. For in vitro experiments, lenzilumab or nVivo MAb human IgG1 isotype control (BioXCell, West Lebanon, NH, USA), 10 ug/ml was used. For the in vivo experiments, 10 mg/Kg of lenzilumab or isotype control was intraperitoneally injected daily for 10 days starting from the day of CART19 injection. In some experiments, anti-mouse GM-CSF neutralizing antibody (InVivoMAb anti-mouse GM-CSF, BioXCel, West Lebanon, NH, USA) or the corresponding isotype control (InVivoMAb rat IgG2a isotype control BioXCel, West Lebanon, NH, USA) ) were used as indicated in the experimental outline.

T cell function experiment

Cytokine assays were performed 24 or 72 h after co-culture of CAR-T cells with the target in a 1:1 ratio as indicated. Human High Sensitivity T Cell Magnetic Bead Panel (Millipore Sigma, Ontario, Canada), Milliplex Human Cytokine/Chemokine MAGNETIC BEAD Premixed 38 Plex Kit (Millipore Sigma, Ontario, Canada), or Milliplex Mouse Cytokine/Chemokine MAGNETIC BEAD Premixed 32 Plex Kit Millipore Sigma, Ontario, Canada) performed on supernatants collected from these experiments or sera as indicated. It was analyzed using a Luminex (Millipore Sigma, Ontario, Canada). Intracellular cytokine assay and T cell degranulation assay were performed using monensin (BioLegend, San Diego, CA, USA), hCD49d (BD Biosciences, San Diego, CA, USA), and hCD28 (BD Biosciences, San Diego, CA, USA). CAT-T cells were incubated for 4 hours with the target in the presence of 1:5 ratio, followed by incubation. After 4 hours, cells were harvested, surface staining, intracellular staining, and fixation and permeabilization were performed with fixation media A and B (Life Technologies, Oslo, Norway). For proliferation assays, CFSE (Life Technologies, Oslo, Norway) labeled effector cells (CART19) and irradiated target cells were co-cultured in a 1:1 ratio. In some experiments, CD14 monocytes were added to the co-culture in a 1:1:1 ratio as indicated. Cells were co-cultured for 3-5 days as indicated in specific experiments, then cells were harvested and surface stained with anti-hCD3 (eBioscience, San Diego, CA, USA) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA). PMA/ionomycin (Millipore Sigma, Ontario, Canada) was used as a positive non-specific stimulator of T cells at different concentrations as indicated in specific experiments. For apoptosis assays, CD19 ⁺ Luciferase ⁺ ALL cell lines NALM6 or CD19 ^- Luciferase ⁺ control MOLM13 cells were incubated with effector T cells at the indicated rates for 24, 48 or 72 hours as listed in the specific experiments. Killing was calculated by bioluminescence imaging on a Xenogen IVIS-200 spectral camera (PerkinElmer, Hopkinton, MA, USA) as a measure of remaining viable cells. Samples were treated with 1ul D-luciferin (30ug/ml) per 100ul sample volume for 10 minutes prior to imaging (Gold Biotechnology, St. Louis, MO, USA).

Multi-parameter flow cytometry

Anti-human and anti-mouse antibodies were purchased from Biolegend, eBioscience or BD Biosciences (San Diego, CA, USA). Cells were isolated from in vitro culture or from animal peripheral blood. BD FACS lysis (BD Biosciences, San Diego, CA, USA) followed by phosphate buffered saline supplemented with 2% FBS (Millipore Sigma, Ontario, Canada) and 1% sodium azide (Ricca Chemical, Arlington, TX, USA). Washed once and dyed at 4°C. For cell number quantification, Countbright beads (Invitrogen, Carlsbad, CA, USA) were used according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). In all assays, populations of interest were gated based on forward-to-side scatter properties followed by singlet gating, and live cells were stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) prior to sampling. Gated. Surface expression of CAR was detected by staining with goat anti-mouse F(ab')2 antibody (Invitrogen, Carlsbad, CA, USA). Flow cytometry was performed on 3-laser cytometers, Canto II (BD Biosciences, San Diego, CA, USA) and CytoFLEX (Beckman Coulter, Chaska, MN, USA). Analysis was performed using FlowJo X10.0.7r2 software (Ashland, OR, USA) and Kaluza 2.0 software (Beckman Coulter, Chaska, MN, USA).

result

If GM-CSF neutralization after CAR-T cell treatment should be utilized as a strategy to prevent CRS and neurotoxicity, it should not inhibit CAR-T cell efficacy. Therefore, initial experiments aimed to investigate the effect of GM-CSF neutralization on CAR-T cell effector function. CART19 cells were co-cultured with or without the CD19 ALL cell line NALM6 in the presence of lenzilumab (hGM-CSF neutralizing antibody) or an isotype control (IgG). It was demonstrated that lenzilumab, but not the IgG control antibody, was indeed able to completely neutralize hGM-CSF ( FIG. 27A ) but did not inhibit CAR-T cell antigen specific proliferation ( FIG. 27B ). When CART19 cells were ^{co-cultured with the CD19 +} cell line NALM6 in the presence of monocytes, lenzilumab in combination with CART19 demonstrated an exponential increase in antigen-specific CART19 proliferation compared to CART19 + isotype control IgG (P<0.0001) , Fig. 27C). To investigate CAR-T specific cytotoxicity, CART19 or control UTD T cells were ^{incubated with luciferase + CD19 +} NALM6 cell lines and treated with isotype control antibody or GM-CSF neutralizing antibody ( FIG. 27D ). GM-CSF neutralizing antibody treatment did not inhibit the ability of CAR-T cells to kill NALM6 target cells ( FIG. 27D ). Overall, these results indicate that lenzilumab does not inhibit CAR-T cell function in vitro and enhances CART19 cell proliferation in the presence of monocytes, suggesting that GM-CSF neutralization may improve CAR-T cell-mediated efficacy. suggests

Example 19

GM-CSF neutralization enhances CAR-T cell antitumor activity in vivo in xenograft models.

Xenograft mouse model

Male and female 8-12 week old NOD-SCID-IL2rγ ^−/- (NSG) mice were housed and managed within the Department of Comparative Medicine at the Mayo Clinic according to breeding protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC). did. Mice were maintained in an animal barrier space approved by the IBC for BSL2+ level experiments.

NALM6 cell line xenograft

CD19 ⁺ , luciferase ⁺ ALL NALM6 cell lines were used to establish ALL xenografts according to IACUC approved protocols. Here, 1x10 ⁶ cells were injected intravenously (IV) via tail vein injection. 4-6 days after injection, mice were subjected to bioluminescence imaging using a Xenogen IVIS-200 Spectrum camera (PerkinElmer, Hopkinton, MA, USA) to confirm engraftment. Imaging was performed 10 minutes after intraperitoneal (IP) administration of 10ul/g D-luciferin (15mg/ml, Gold Biotechnology, St. Louis, MO, USA). Mice were then randomized based on bioluminescence imaging and received different treatments as described in specific experiments. In general, 1-1.5x10 ⁶ CAR-T cells (and corresponding to the total number of untransduced (UTD) T cells) were injected IV per mouse. The transduction efficiency of CAR-T cells was generally about 50%. ^{For example, mice that received 1.5×10 6} CAR-T cells with 50% transduction efficiency of CAR-T cells received a total of 3 million T cells, and those UTD mice ^{received 3×10 6} UTD. Imaging was performed weekly to assess and follow disease burden. Bioluminescence images were acquired using a Xenogen IVIS-200 Spectrum camera (PerkinElmer, Hopkinton, MA, USA) and analyzed using Living Image version 4.4 (Caliper LifeSciences, PerkinElmer). Tail vein bleeding was performed 7-8 days after CAR-T cell injection to evaluate T cell expansion and cytokines and chemokines, and then as needed. Mouse peripheral blood was subjected to red blood cell lysis using BD FACS Lyse (BD Biosciences, San Diego, CA, USA) and then used for flow cytometry studies. Antibody treated mice were started IP with antibody treatment (10 mg/Kg lenzilumab or isotype control) daily for a total of 10 days on the day of CART cell treatment.

RNA-seq of mouse brain tissue

RNA was isolated using miRNeasy Micro kit (Qiagen, Gaithersburg, MD, USA) and treated with RNase-Free DNase Set (Qiagen, Gaithersburg, MD, USA). RNA-seq was performed on an Illumina HTSeq 4000 (Illumina, San Diego, CA, USA) by the Genome Analysis Core of the Mayo Clinic. Binary base call data were converted to fastq using Illumina bcl2fastq software. Bolger, AM, et al., Bioinformatics , which is incorporated herein by reference in its entirety. The adapter sequence was removed using Trimmomatic as described by 2014;30(15):2114-2120.10.1093/bioinformatics/btu170, Leggett RM, et al., Front Genet , incorporated herein by reference in its entirety. 2013;4:288. FastQC was used for quality check as described by Prepublished on 2014/01/02 as DOI 10.3389/fgene.2013.00288. The latest human (GRCh38) and mouse (GRCm38) reference genomes were downloaded from NCBI. Dobin A, et al., Bioinformatics , incorporated herein by reference in its entirety. 2013;29(1):15-21. A genomic index file was generated using STAR as described in 10.1093/bioinformatics/bts635, and paired-end reads were mapped to the genome for each condition. Anders S, et al., Bioinformatics , incorporated herein by reference in its entirety. 2015;31(2):166-169. HTSeq was used to generate expression counts for each gene as described in Prepublished on 2014/09/28 as DOI 10.1093/bioinformatics/btu638, Love MI, et al., Genome Biol, which is incorporated herein by reference in its entirety. . 2014;15(12):550. ^{DeSeq2 38} was used to calculate the differential expression as described in Prepublished on 2014/12/18 as DOI 10.1186/s13059-014-0550-8. Kuleshov MV et al., Nucleic Acids Research 2016;44(W1):W90-W97, incorporated herein by reference in its entirety. As described in 10.1093/nar/gkw377, gene ontology was evaluated using Enrichr. 35 summarizes the steps described above. RNA sequencing data is available from Gene Expression Omnibus under registration number GSE121591.

statistics

Prism Graph Pad (La Jolla, CA, USA) and Microsoft Excel (Microsoft, Redmond, WA, USA) were used for data analysis. In the heat map, high cytokine concentrations were normalized to “1” and low concentrations were normalized to “0” through Prism. Statistical tests are described in the figure legend.

result

To confirm that GM-CSF depletion did not inhibit CART19 effector function, the role of GM-CSF neutralization with lenzilumab on CART19 antitumor activity in a xenograft model was investigated. First, a recurrence model was used to actively investigate whether the antitumor activity of CART19 cells was affected by GM-CSF neutralization. NOD/SCID/interleukin-2 receptor gamma null (NSG) mice were ^{injected with 1x10 6} luciferase ⁺ NALM6 cells and then imaged 6 days later to allow sufficient time for the mice to achieve a high tumor burden. Mice were randomized to receive a single injection of CART19 or UTD cells, followed by administration of an isotype control antibody or lenzilumab on day 10 ( FIG. 28A ). GM-CSF analysis of sera collected 8 days after CART19 injection showed that lenzilumab successfully neutralized GM-CSF in the context of CART19 therapy ( FIG. 28B ). Bioluminescence imaging one week after CART19 injection showed that CART19 in combination with lenzilumab effectively controlled leukemia in a high tumor burden recurrence model and was significantly better than control UTD cells ( FIGS. 28C-28D ). Treatment with CART19 in combination with lenzilumab resulted in potent anti-tumor activity and improved overall survival, similar to CART19 with control antibody, despite neutralization of GM-CSF levels, indicating that GM-CSF had It does not impair cell activity ( FIG. 36 ). Second, these experiments were performed in xenograft models derived from patients with primary ALL in the presence of human PBMCs, as they represent a more relevant xenograft model. After chemotherapy was controlled with busulfan, blast cells derived from a patient with recurrent ALL were injected into mice. Monitor mice for engraftment for several weeks via continuous tail vein bleeding and ^{, when CD19 +} blast cells in the blood are approximately 1/uL, randomize mice to lenzilumab + anti-mouse GM-CSF neutralizing antibody or isotype control IgG The antibody was combined with PBMC to receive CART19 treatment for 10 days from the day of CART 19 injection ( FIG. 28E ). In this primary ALL xenograft model, GM-CSF neutralization in combination with CART19 therapy resulted in significant improvement in leukemia disease control that persisted over time for at least 35 days after CART19 administration compared to CART19 + isotype controls ( Figure 28F). This suggests that GM-CSF neutralization may play a role in reducing relapse and increasing sustained complete response after CART19 cell treatment.

Example 20

GM-CSF ^k/o Generation of CART19

Sanjana NE et al., Improved vectors and genome-wide libraries for CRISPR screening, incorporated herein by reference in its entirety. 2014;11(8):783-784. Prepublished on 2014/07/31 as described in DOI 10.1038/nmeth.3047, targeting exon 3 of human GM-CSF through gRNA screening previously reported to have high efficiency against human GM-CSF. Guide RNA (gRNA) was selected. This gRNA was ordered with a CAS9 third generation lentiviral construct (lentiCRISPRv2) controlled by the U6 promoter (GenScript, Township, NJ, USA). Lentiviral particles encoding this construct were generated as described above. T cells were double transduced with CAR19 and GM-CSFgRNA-lentiCRISPRv2 lentivirus 24 hours after stimulation with CD3/CD28 beads. CAR-T cell expansion was then continued as described above. To analyze the effectiveness of GM-CSF targeting, genomic DNA was extracted from ^{GM-CSF k/o} CART19 cells using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). The DNA of interest was PCR amplified using Choice Taq Blue Mastermix (Thomas Scientific, Minneapolis, MN, USA) and the gel was extracted using QIAquick Gel Extraction Kit (Qiagen, Germantown, MD, USA) to determine editing. PCR amplicons were transferred for Eurofins sequencing (Louisville, KY, USA) and allele modification frequencies were calculated using Tracking of Indels by Decomposition (TIDE). This method requires two parallel PCR reactions and a pair of standard capillary sequencing assays; Then, Brinkman EK, et al., Easy quantitative assessment of genome editing by sequence trace decomposition, which is incorporated herein by reference in its entirety. Nucleic Acids Research . 2014;42(22):e168. Prepublished on 2014/10/11 as described in DOI 10.1093/nar/gku936, two resulting sequencing traces were created using a simple web tool and specially designed software delivered as R code available at tide.nki.nl. Analyze. Figure 34B describes the gRNA sequence and primer sequence, and Figures 34A(i)-34A (iii) describes the schema for generating the ^{GM-CSF k/o CART19 outline.}

Example 21

GM-CSF CRISPR knockout CAR-T cells show reduced expression of GM-CSF, similar levels of key cytokines and chemokines, and enhanced antitumor activity.

To definitively rule out a role for important GM-CSF in CAR-T cell function, Sanjana NE, et al., Nature Methods . 2014;11(8):783-784. As described in Prepublished on 2014/07/31 as DOI 10.1038/nmeth.3047 (incorporated herein by reference in its entirety), the GM-CSF gene produces a high-efficiency knockout and CAR using gRNA that replicates into the CRISPR lentiviral backbone. -T cells were destroyed during preparation. About 60% knockout efficiency was achieved in CART19 cells using this gRNA ( FIG. 37 ). When CAR-T cells were stimulated with the CD19 cell line NALM6, GM-CSF ^k/o CAR-T cells produced statistically significantly less GM-CSF compared to CART19 with the wild-type GM-CSF locus (“wild-type CART19 cells”). produced. GM-CSF knockout in CAR-T cells did not impair the production of IFN-γ, IL-2 or other key T cell cytokines, including CAR-T cell antigen specific degranulation (CD107a) (Figure 29B), although GM - showed reduced expression of CSF. To confirm that GM-CSF ^k/o CAR-T cells continue to exhibit normal function, their in vivo efficacy was tested in a high tumor burden recurrent xenograft model of ALL (described in Figure 28A). ^{The use of GM-CSF k/o} CART19 instead of wild-type CART19 in this xenograft model significantly reduced the serum levels of human GM-CSF 7 days after CART19 treatment ( FIG. 29B ). Bioluminescence imaging data suggest that GM-CSF ^k/o CART19 cells display enhanced leukemia control compared to CART19 in this model ( FIG. 38 ). Importantly, GM-CSF ^k/o CART19 cells showed a significant improvement in overall survival compared to wild-type CART19 cells ( FIG. 29C ). Human GM-CSF was statistically significantly decreased through the t test in ^{GM-CSF k/o} CART19 cells compared to wild-type CART19 ( FIG. 29D ). Mouse GM-CSF appeared to be visually increased, although not statistically significant, via the t test (P = 0.472367) (Fig. 29E). This lack of mouse GM-CSF reduction ^{is not necessarily surprising, as GM-CSF k/o} CART19 cells (human) are the only cells in mice harboring knockouts and therefore mouse GMCSF is unlikely to be directly affected. On visual inspection, the chemokine mouse IP-10, which attracts numerous cell types, including T cells and monocytes, ^{appears to be paradoxically increased in GM-CSF k/o} CART19 compared to CART19, although this is not statistically significant. P = 0.4877 by t test (Fig. 29E). By visual inspection, mouse MIP1α (inflammatory cytokine important for neutrophil attraction) and mouse M-CSF (cytokine important for macrophage differentiation) appear to be reduced but not statistically significant with P = 0.2437 and P = 0.3619 (Fig. 29E). Mouse IL-1b, an important inflammatory cytokine produced by macrophages, and mouse IL-15, a cytokine produced by macrophages aiding NK cell proliferation, had P values of P = 0.0741 and P = 0.0900, respectively, for CART19 ( It appears to be reduced in GM-CSF ^k/o CART19 compared to FIG. 29E ) ( FIG. 29E ). Important human T cell cytokines ^{were not inhibited by GM-CSF k/o} ( FIG. 29D ). These xenografts were produced with a high burden of the NALM6 cell line, highlighting that our CRS/NI model (Figures 30A-30D, 31, 32A-32D and 33A-33D) requires the use of the resulting primary ALL cells. Should be. Therefore, it is not surprising that the cytokine profiles differ between the two models as NALM6 xenografts ( FIGS. 29A-29E ) do not develop CRS or NI. Taken together, in the context of the NALM6 high tumor burden model without CRS, the results in Figures 29A-29E are the results of Figures 27A-27D and Figures 27A-27D and Figs. Check 28A-28F. In addition, the results in Figures 29A-29E indicate that GM-CSF ^k/o CART may represent a treatment option for "built in" GM-CSF control as a modification during CAR-T cell preparation.

Example 22

Patient-derived xenograft model for neuroinflammation (NI) and cytokine release syndrome/in vivo GM-CSF neutralization ameliorates cytokine release syndrome and neuroinflammation after CART19 treatment in xenograft model.

Primary patient-derived ALL xenografts

To establish a primary ALL xenograft, NSG mice were first administered IP (Selleckchem, Houston, TX, USA) with 30 mg/Kg busulfan. The next day, mice were injected with ^{2.5x10 6} primary blast cells derived from peripheral blood of patients with relapsed or refractory ALL. Mice were monitored for ˜10-13 weeks for engraftment. When CD19 ⁺ cells were consistently observed in the blood (approximately 1 cell/uL), use antibody therapy (10 mg/Kg lenzilumab or isotype control IP for a total of 10 days from the day of CAR-T cell therapy) or Randomized to receive different treatments ^{with CART19 (2.5x10 6} cells IV) and PBMCs (1x10 ⁵ cells IV) derived from the same donor without use. Mice were periodically monitored for leukemia burden via tail vein bleeding.

Primary patient-derived ALL xenografts for CRS/NI

Similar to the above experiment, mice were IP-injected with 30 mg/Kg busulfan (Selleckchem, Houston, TX, USA). The next day, mice received ^{1-3x10 6} primary blast cells derived from peripheral blood from relapsed ALL patients. Mice were monitored for engraftment for ~10-13 weeks via tail vein bleeding. ^{Mice received CART19 (2-5×10 6} cells IV) as indicated when serum CD19 cells ≧10 cells/ul and started antibody treatment for a total of 10 days. To measure the health of the mice, body weight was measured daily. Mouse brain MRI was performed 5-6 days after CART19 injection, tail vein bleeding for cytokines/chemokines was performed, and T cell analysis was performed 4-11 days after CART19 injection.

MRI acquisition

A Bruker Avance II 7 Tesla vertical bore small animal MRI system (Bruker Biospin) was used for image acquisition to evaluate central nervous system (CNS) vascular permeability. Inhalation anesthesia was induced and maintained via 3-4% isoflurane. Respiratory rates were monitored during the acquisition session using an MRI compatible vital sign monitoring system (Model 1030; SA Instruments, Stony Brook, NY). Mice were given gadolinium IP injections using a weight-based dosing of 100 mg/Kg, after a standard delay of 15 min, using a volume acquisition T1-weighted spin echo sequence (repeat time = 150 ms, echo time = 8 ms, field of view) :32 mm × 19.2 mm × 19.2 mm, matrix: 160 × 96 × 96; average number = 1) T1-weighted images were obtained. Gadolinium-enhanced MRI changes indicate blood-brain-barrier disruption. Volume analysis was performed using the Analyze Software package developed by Mayo Clinic's Biomedical Imaging Resource.

result

In this model, conditioned NSG mice were engrafted with primary ALL blasts and engraftment was monitored for several weeks until a high disease burden occurred ( FIG. 30A ). ^{When the level of CD19 +} blast cells in peripheral blood was >10/uL, mice were randomly assigned to receive different treatments as indicated (Fig. 30A). CART19 treatment (using control IgG antibody or GM-CSF neutralizing antibody) successfully eradicated the disease ( FIG. 30B ). Within 4-6 days after treatment with CART19, mice began to develop symptoms consistent with CRS and NI: ataxia, crooked body, and gradual weight loss. This was associated with elevations of major serum cytokines and chemokines 4-11 days after CART19 injection, similar to that seen in human CRS following CAR-T cell therapy (human GM-CSF, TNF-α, IFN-γ, IL-10). , IL-12, IL-13, IL-2, IL-3, IP-10, MDC, MCP-1, MIP-1α, MIP-1β, and mouse IL-6, GM-CSF, IL-4, IL -9, including IP-10, MCP-1, and MIG). These mice treated with CART19 also developed NI showing abnormal T1 enhancement in brain MRI analysis, suggesting blood-brain barrier disruption and possibly brain edema (Figure 30C), and flow cytometry analysis of harvested brains showed human CART19 reveals cell infiltration ( FIG. 30D ). In addition, RNA-seq analysis of brain sections from mice that developed these signs of NI revealed the expression of genes regulating T cell receptors, cytokine receptors, T cell immune activation, T cell trafficking, and T cell and myeloid cell differentiation. showed significant upregulation. (Fig. 31, Table 6).

Table 6: Table of standard pathways altered in brain from patient-derived xenografts after treatment with CART19 cells, tabulated.

The effect of neutralizing GM-CSF on CART19 toxicity was investigated using a xenograft patient-derived model for NI and CRS shown in Figure 30A. To rule out the co-establishment effect of mouse GM-CSF, mice were treated with CART19 cells in combination with 10 days of GM-CSF antibody therapy (10 mg/Kg lenzilumab and 10 mg/Kg anti-mouse GM-CSF neutralizing antibody) or an isotype control antibody. received GM-CSF neutralizing antibody therapy statistically significantly reduced CRS-induced weight loss after CART19 therapy ( FIG. 32A ). Cytokine and chemokine analysis after 11 days of CART19 cell treatment showed that human GM-CSF was neutralized by the antibody ( FIG. 32B ). In addition, GM-CSF neutralization was performed in several human (IP-10, IL-3, IL-2, IL-1Ra, IL-12p40, VEGF, GM-CSF) (Figure 32C) and mouse (MIG, MCP-1, KC) , IP-10) ( FIG. 32C ) resulted in a significant decrease in cytokines and chemokines. Interferon gamma-inducing proteins (IP-10, CXCL10) are produced by monocytes, among other cell types, and serve as chemoattractants for numerous cell types, including monocytes, macrophages, and T cells. IL-3 plays an important role in myeloid progenitor cell differentiation. IL-2 is a key T cell cytokine. Interleukin-1 receptor antagonists (IL-1Ra) inhibit IL-1. (IL-1 is produced by macrophages and is an important family of inflammatory cytokines.) IL-12p40 is a subunit of IL-12 produced by macrophages, among other cell types, and capable of promoting Th1 differentiation. Vascular endothelial growth factor (VEGF) promotes angiogenesis. Monokines induced by gamma interferon (MIG, CXCL9) are T cell chemoattractants. Monocyte chemoattractant protein 1 (MCP-1, CCL2) attracts monocytes, T cells and dendritic cells. KC (CXCL1) is produced by macrophages among other cell types and attracts myeloid cells such as neutrophils. There was also a non-statistically significant decrease in several other human and mouse cytokines and chemokines after GM-CSF neutralization. This suggests that GMCSF plays a role in the downstream activity of several cytokines and chemokines that are tools in the cascade leading to CRS and NI.

Brain MRI 5 days post CAR19 treatment showed that GM-CSF neutralization reduced T1 enhancement as a measure of brain inflammation, blood-brain barrier disruption and edema compared to CART19 + control antibody. MRI images after GM-CSF neutralization (using lenzilumab and anti-mouse GM-CSF antibody) were similar to baseline pretreatment scans, suggesting that GM-CSF neutralization helped effectively abrogate NI associated with CART19 therapy ( 33A and 33B). Using human ALL blasts and human CART19 in this patient-derived xenograft model, GM-CSF neutralization after CART19 reduced neuroinflammation by 75% compared to CART19 and isotype controls ( FIG. 33B ). This is an important finding and demonstrated for the first time in vivo that NI-induced CART19 can be effectively discarded. Human CD3 T cells were present in the brain after CART19 treatment analyzed by flow cytometry, with differences in the raw mean with a decrease in brain CD3 T cells due to GM-CSF neutralization, but did not meet statistical significance (Fig. 33C, 30D and 39). Finally, the difference in raw means (which did not reach statistical significance), along with a decrease in CD11b+ bright macrophages, was found in mice receiving GM-CSF neutralization during CAR-T cell treatment compared to isotype controls during CAR-T treatment. observed in the brain ( FIG. 33D ), suggesting that possibly GM-CSF neutralization helps to reduce macrophages in the brain.

The results of Examples 18-19 and 22 demonstrate that neutralization of GM-CSF can eliminate toxicity and enhance their therapeutic activity after CAR-T cell treatment. In particular, GM-CSF neutralization in combination with CART19 therapy has been shown to prevent the development of CRS and significantly reduce the severity of NI in xenograft models using human ALL blasts and human CART19. GM-CSF neutralization resulted in a decrease in chemokines associated with myeloid trafficking, such as IP-10, MCP-1, KC and other inflammatory cytokines and chemokines, and a raw mean decrease in T cell infiltration and myeloid cell activation in the brain (statistically not noticed) related to Interestingly, our experiments suggest that GM-CSF inhibition enhances CART19 proliferation, antitumor activity and overall survival in vivo. Based on these results, GM-CSF neutralization can be viewed as a potential next-generation strategy to enable conventional CAR-T cell immunotherapy.

In the studies described herein, GM-CSF neutralization with lenzilumab did not impair CART19 effector function in vitro. In two different xenograft models (NALM6 xenograft and patient-derived xenograft), CART19 combined with lenzilumab effectively eradicated tumors despite GM-CSF neutralization and significantly improved leukemia disease control for 35 days after treatment, whereas CART19 and isotype control was unable to maintain disease control after 35 days. Finally, in Examples 21-22, GM-CSF ^k/o CART19 cells displayed potent effector function in vitro and demonstrated significantly improved overall survival compared to CART19 in vivo.

The CRS and NI models described herein are unique and relevant ALL patient-derived xenograft models for the development of therapies for toxicity following human CAR-T cell therapy. In the model described here, the time interval between CAR-T cell infusion to symptom onset, brain MRI changes, cytokine and chemokine elevation, and effector cell infiltration into the CNS are all similar to those reported in patients developing toxicity after CART19 therapy. do. Mice developed CRS and NI symptoms (weight loss, impaired motor function, crouching). Changes in brain MRI were detected 4-6 days after CART19 cell injection. Brain MRI T1 uptake suggests blood-brain barrier disruption and possibly brain edema, and Gust et al. 2017 Cancer Discovery . 2017;7(12):1404-1419. Prepublished on 2017/10/14 as DOI 10.1158/2159-8290.CD-17-0698 (incorporated herein by reference in its entirety), similar to changes seen in human brain MRI in cases of severe neurotoxicity.

Interestingly, Gust et al. 2017 explain that blood-brain barrier permeability prevented protection of CSF from systemic cytokines that induce perivascular stress and secretion of endothelial activating cytokines, and that patients showed evidence of endothelial activation. In the CRS/NI model described herein, GM-CSF was found to be neutralized in the sera of mice receiving CART19 therapy with a GM-CSF neutralizing antibody compared to CART19 and an isotype control antibody. Thus, T cells in the mouse brain themselves could provide GM-CSF production, and serum GM-CSF, among other cytokines and chemokines, could possibly reach CSF. Endothelial cells can also produce GM-CSF, which can lead to exacerbation cycles. NI was associated with T cell infiltration and myeloid cell activation in the CNS, which was similar to CSF changes in CAR-T-induced neurotoxicity patients and non-human primate models. The model described herein is similar to a previously reported patient-derived xenograft model in which CRS was developed following CAR-T cell therapy. A recent report suggested that IL-1 blockade prevents NI through depletion of myeloid cells. However, the development of NI in this model is delayed and is associated with meningeal thickening, unlike the model described here and observed in patients receiving CART19 treatment. Thus, the model described here serves as a reliable way to investigate novel interventions for the prevention and treatment of CRS and neurotoxicity following CART19 cell treatment. The results described herein show that GM-CSF neutralization results in reduction of major myeloid and several inflammatory cytokines and chemokines, suggesting that GM-CSF is an important cytokine in the downstream activation of several cytokines and chemokines; Blocking contributes to a raw mean reduction in myeloid and T cell infiltration of the brain/CNS (not reaching statistical significance); Blocking helps reduce neuroinflammation of obvious neurotoxicity.

Interestingly, an exponential increase in CART19 cell proliferation was observed, antitumor activity was enhanced, and overall survival was improved with GM-CSF blockade. For example, CART19 antigen specific proliferation in the presence of monocytes was increased in vitro after GM-CSF neutralization. Moreover, in all patient-derived xenografts, CART19 cells resulted in longer-lasting disease control when combined with lenzilumab. In addition, ^{it was demonstrated that GM-CSF k/o} CAR-T cells were more effective in controlling leukemia in NALM6 xenografts and improved overall survival. Although the mechanism for enhanced CART effector function after GM-CSF depletion is currently unclear, the results provided herein indicate that monocytes impair T cell expansion ex vivo and that M2-polarized macrophages inhibit CART19 antigen-specific proliferation. matches with This is an important finding as improved CAR-T cell proliferation in CAR-T clinical trials is consistently associated with improved efficacy and response (ie, overall and complete response rate).

Activated T cells are known to produce GM-CSF. Since T cells do not possess all subunits for the GM-CSF receptor, under normal circumstances GM-CSF usually does not provide direct feedback to T cells, but in some situations it is possible at very high levels. Instead, this GM-CSF affects the behavior of numerous other cell types, including macrophages and dendritic cells. Subsequent activation of these cells results in T cell stimulating actions such as cytokine production and antigen presentation. T cell stimulation can further induce the production of GM-CSF and other cytokines to act on other cell types such as macrophages and dendritic cells to induce cycles. The large number of activated T cells produced in a very short time in CAR-T cell therapy is likely to push this cycle to extremes. The results described here suggest that blocking GM-CSF helps prevent and actually enhance this immune overstimulation without compromising T cell function. The exact mechanism for enhanced CAR-T cell effector function after GM-CSF blockade is unclear.

Finally, the results presented here further suggest that ^{the development of GM-CSF k/o} CART19 cells may represent a novel way to partially control GM-CSF production, which can now be included in CAR-T cell production. These results indicate that these cells function normally and may represent an independent therapeutic approach to improve the therapeutic window after CAR-T cell treatment. Anti-GM-CSF antibodies, such as lenzilumab, are clinical-stage therapeutic solutions that neutralize GM-CSF and eliminate both overt neurotoxic CRS and neuroinflammation and potentially improve CAR-T cell function.

The studies described herein represent significant advances in understanding and preventing toxicity following CAR-T cell treatment. These results strongly suggest that modulating myeloid cell behavior through GM-CSF blockade helps improve leukemia control by controlling CAR-T cell-mediated toxicity and reducing immunosuppressive function. These studies shed light on novel approaches to clear neuroinflammation of CRS and overt neurotoxicity through GM-CSF neutralization, potentially enhancing CAR-T cell function.

Example 23

Administration of anti-GM-CSF monoclonal antibody (lenzilumab) significantly reduced neuroinflammation induced by CAR-T therapy and maintained blood-brain-barrier integrity in xenograft models

This preclinical study is designed to closely replicate the results observed in the CAR-T clinical trial and utilizes human acute lymphoblastic leukemia (ALL), human CD19-targeted CAR-T (CART19) and human peripheral blood mononuclear cells (PBMC) and performed in mice.

Primary patient-derived ALL xenografts

ALL xenografts were established in mice essentially as described in Example 22.

MRI acquisition

The integrity of the BBB can be monitored non-invasively by magnetic resonance imaging (MRI). Conventional MR contrast agents (CA) containing gadolinium are used in conjunction with MRI to detect and quantify BBB leakage. Under normal circumstances, the CA does not cross the intact BBB.

However, even when the BBB is partially damaged due to the small size of CA, it leaks from the blood into the brain tissue.

MRI was obtained essentially as described in Example 22. Ku, MC et al., Methods Mol Biol. 2018;1718:395-408. A gadolinium-enhanced MRI method based on T1-weighted images taken before and after CA injection, as described by doi: 10.1007/978-1-4939-7531-0_23, is consistent with that used in the current preclinical study of lenzilumab and CART19. do. This gadolinium-enhanced MRI method is useful for investigating BBB permeability in an in vivo mouse model and can be readily applied to several experimental disease states, including neuroinflammatory disorders, or to evaluate undesired drug effects.

confocal microscope

Confocal microscopy was used to assess damage/breakdown of the blood brain barrier (also referred to herein as the BBB). This microscopy technique uses spatial filtering to remove out-of-focus light or flares from specimens thicker than the focal plane. Confocal microscopy offers several advantages over conventional light microscopy, including controllable depth of field, image removal that degrades out-of-focus information, and the ability to collect serial optical sections from thick specimens.

result

BBB integrity is preserved and neuroinflammation is significantly reduced after CAR-T and lenzilumab treatment.

In the present study, MRI images taken on day 5 qualitatively revealed diffuse neuroinflammation with CAR-T therapy, and MRI images taken after the combination of lenzilumab + CAR-T showed significantly less neuroinflammation, similar to untreated controls. showed that (see Fig. 33A). Quantification of MRI via gadolinium-enhanced T1 high-intensity signal showed that lenzilumab + CAR-T vs. CAR-T + control antibody showed a significant 75% reduction in neuroinflammation and BBB injury ( FIG. 33A ). In addition, confocal microscopy clearly shows in the high-resolution image that the BBB was significantly damaged after CAR-T treatment (Fig. 40A), which is consistent with the MRI image qualitatively showing diffuse neuroinflammation with CAR-T treatment (see Fig. 33A). ). In contrast, confocal microscopy shows the maintenance of BBB integrity using lenzilumab in combination with CAR-T ( FIG. 40A ), which shows a significant reduction in neuroinflammation compared to CAR-T + isotype control. Consistent with qualitative and quantitative MRI images taken after the combination of lumab + CAR-T. 40A shows confocal microscopy BBB data. 33A is an exemplary quantitative MRI using gadolinium-enhanced T1-high intensity, three treatment groups: untreated vs. untreated. CART19 + lenzilumab vs. CART19 + isotype control is shown. Confocal microscopy results are important as they help explain the pathology of CAR-T-induced neuroinflammation. These data suggest that the BBB is damaged after CAR-T administration, allowing for a large influx of pro-inflammatory cytokines into the CNS, which is thought to propagate neuroinflammation. These data are consistent with data reported in the CAR-T clinical trial. Patients who developed grade 3 neurotoxicity in a ZUMA-1 study using chimeric antigen receptor (CAR) transduced autologous T cells administered intravenously at a target dose of 2 x 10 ^{6 anti-CD19 CAR T cells/kg (Yescarta)} A significant increase in pro-inflammatory cytokines was identified in the CNS of Current confocal microscopy data help to explain why and how the addition of lenzilumab significantly reduces CAR-T-induced neuroinflammation. MRI images qualitatively showed diffuse neuroinflammation and damage to the BBB on day 5 after CAR-T therapy (CART19) administration. Co-administration of lenzilumab with CAR-T preserved/maintained the integrity of the BBB, and MRI images taken 5 days after combination of lenzilumab CAR-T showed a significant reduction in neuroinflammation, similar to untreated controls. (See Figure 33A). Moreover, confocal microscopy shows that these results are in full agreement with MRI imaging data showing a 75% reduction in neuroinflammation and BBB damage following lenzilumab and CAR-T compared to CAR-T and control antibodies (Y in this analysis The axis is the gadolinium-enhanced T1 high-intensity signal).

CART19 cell proliferation increased exponentially and leukemia management was significantly improved after CART and lenzilumab treatment.

Lenzilumab administration following CART19 therapy also resulted in an exponential increase in CART19 cell proliferation and a significant improvement in leukemia control lasting at least 35 days after CART19 infusion as described in Example 22. These results suggest that GM-CSF neutralization with an anti-GM-CSF monoclonal antibody (lenzilumab) may play a role in reducing relapse and increasing sustained complete response after CART19 treatment. This is an important finding, given that more than 50% of adult lymphoma patients who first respond to CART19 therapy relapse within the first year of follow-up.

Figure 40B (Santomasso, BD, et al., published OnlineFirst on June 7, 2018; modified from DOI: 10.1158/2159-8290.CD-17-1319; incorporated by reference in its entirety) shows high levels in CSF shows that the amount of protein in α (shown in the data of Santomasso) is indicative of BBB disruption and protein leakage into the CNS (increased blood-cerebrospinal fluid (CSF) barrier permeability during neurotoxicity). This shows that BBB disruption is central to the pathophysiology of NI and links the xenograft model results presented here with the clinical results of Santomasso. In embodiments of the methods provided herein, the method further comprises (by the clinician) performing a lumbar puncture and measuring the CSF level of a protein/albumen that is capable of predicting subsequent clinical expectations and preemptive measures of NT grade. do.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art, in light of the teachings of the present invention, will appreciate the specific details without departing from the spirit or scope of the appended claims. It will be clear that changes and modifications are possible.

Example 24

Gene editing technology to knock out the GM-CSF gene in T cells

Several strategies are being pursued by different groups to integrate gene editing into the development of next-generation chimeric antigen receptor (CAR) T cells for various cancer treatments. Severe toxicity (cytokine release syndrome and neutral toxicity) is associated with CAR T cell therapy and the patient's prognosis may be poor. The major initiator of the toxic process appears to be GM-CSF derived from CART cells.

Gene editing (eg, using engineered nucleases) can be used for KO GM-CSF genes and/or genes encoding proteins essential for GM-CSF gene expression in T cells.

Nucleases useful for such genome editing include, but are not limited to, CRISPR-associated (Cas) nucleases, zinc-finger nucleases (ZFNs), transcriptional activator-like effector (TALE) nucleases, and meganucleases. known homing endonucleases (HE).

Use of zinc-finger nucleases against GM-CSF

DNA sequence-specific nucleases cleave the GM-CSF gene and DNA double-strand break repair results in inactivation of the gene. A sequence-specific nuclease is produced by joining a Fok1 endonuclease domain with a sequence-specific DNA binding domain (zinc finger). Targeted nucleases act as dimers and provide site-specific cleavage using two different DNA recognition domains. Engineering of the Fok 1 endonuclease allows heterodimers to form rather than homodimers. Thus, the absolute heterodimeric Fok1-EL variant provides a higher level of specificity.

Clinical experience to date on genetic KO approaches using ZFN technology is limited. However, in a small safety study in which the CCR5 receptor was knocked out and T cells reintroduced into HIV patients using ZFN technology, modified T cells had a significant survival advantage over unmodified ones upon discontinuation of antiretroviral drug therapy.

The best effect was observed when double allele disruption was achieved. This suggests that the KO technique with the highest rate of gene disruption is likely to be the most effective (Singh 2017, Tebas 2014). Biallelic targeting efficiency in some human cell types is increased by RAD51 compared to expression and valproic acid treatment (Takayama 2017).

Exons 1-4 of the human GM-CSF gene can be targeted with ZFNs that form pairs within a selected target region. A potential advantage of targeting close to the translation initiation codon within the DNA sequence is that the gene knockout still does not result in large fragments of the protein being synthesized. Such protein fragments may have unwanted biological activity.

A variety of tools are available to identify potential zinc finger nuclease (ZFN) sites at specific target sequences. For example, such a tool can be found at http://bindr.gdcb.iastate.edu/ZiFiT/. A vector for the expression of ZFN pairs identified in this way (used in GM-CSF gene KO) was tested in human cells expressing GM-CSF and the effect of gene disruption on each pair was determined based on the GM-CSF production inside the cell pool. measured by change. ZFN pairs showing the highest reduction in GM-CSF levels are selected for testing in human CART cells.

For example, autologous T-cells can be transduced ex vivo with a replication-deficient recombinant Ad5 viral vector encoding a GM-CSF-specific ZFN pair resulting in modification of the GM-CSF gene. A vector supports only transient expression of the gene encoded by the vector. The two ZFNs bind to a complex bp sequence found specifically in the region (within exons 1,2, 3 or 4) selected for mutagenesis of the GM-CSF gene. Expression of GM-CSF-specific ZFNs induces double-strand breaks in cellular DNA, which are repaired by the cellular machinery, leading to random sequence insertions or deletions in transduced cells. These insertions and deletions disrupt the GM-CSF coding sequence, leading to frame shift mutations and termination of protein expression.

T cell preparation/patient-specific samples

Study subjects underwent 10 liters of leukocyte collection to collect ^{>10 9 leukocytes.} The leukocyte collection product is enriched for CD4+ cells by depleting monocytes via countercurrent centrifugal elution and magnetically depleting CD8+ T-cells, both using a disposable closed system disposable set. The resulting enriched CD4+ T-cells are activated with anti-CD3/anti-CD28 mAb coated paramagnetic beads and transduced with a vector encoding CAR T and a vector encoding ZFN. The cells are then expanded and cultured in a closed system. T-cell expansion continues after transfer to the WAVE Bioreactor for further expansion in perfusion conditions. At the end of the incubation period, cells are depleted of magnetic beads, washed, concentrated and cryopreserved.

Primary T cells can also be treated with other agents, such as valproic acid to increase the bi-allelic targeting efficiency of ZFNs.

putative target sequence

exon 1

ATG TGG CTG CAG AGC CTG CTG CTC TCG GGC

TAC ACC GAC GTC TCG GAC GAC GAG AGC CCG

CTC GCC CAG CCC CAG CAC GCA GCC

GAG CGG GTC GGG GTC GTG CGT CGG

exon 2

AAT GAA ACA GTA GAA GTC ATC TCA GAA ATG

TTA CTT TGT CAT CTT CAG TAG AGT CTT TAC

GAA GTC ATC TCA GAA ATG TTT GAC

CTT CAG TAG AGT CTT TAC AAA CTG

exon 3 design

GAG CCG A CC TGC CTA CAG ACC CGC CTG GAG

CTC GGC TGG ACG GAT GTC TGG GCG GAC CTC

GCC TAC AGA CCCGCCT GGA GCT GTA

CGG ATG TCT GGCGGA CCT CGA CAT

exon 4

GAA ACT TCC TGT GCA ACC CAG ATT ATC ACC

CTT TGA AGG ACA CGT TGG GTC TAA TAG TGG

TGC AAC CCA GAT TATC ACC TTT GAA

ACG TTG GGT CTA ATAG TGG AAA CTT

Talen

The GM-CSF gene/s of T cells can also be inactivated using activator-like effector nucleases (TALENS). TALENS is similar to ZFNs in that it contains a Fok1 nuclease domain fused to a sequence specific DNA binding domain. The targeted nuclease then creates a double-stranded break in the DNA and error-prone repair produces a mutated target gene. TALENS can be easily designed using a simple protein-DNA code that uses DNA binding TALE (transcriptional activator-like effector) repeat domains for individual bases of the binding site. The robustness of TALEN means that genome editing is a reliable and facile process (Reyon D., et al., 2012 Nat Biotechnol. 2012 May;30(5):460-5. doi: 10.1038/nbt.2170, in its entirety. is incorporated herein by reference)

For example, some TALE target sequences within exon 1 of the human GM-CSF gene are:

One.

TGGCTGCAGAGCCTGCTG CTCTTGGGCACTGTGG CCTGCAGCATCTCTGCA

2.

TTGGGCACTGTGGCCCTGC AGCATCTCTGCACCCG CCCGCTCGCCCAGCCCCA

Example of TALE target sequence in exon 4 of human GM-CSF gene:

One.

TGTGCAACCCAGATTATC ACCTTTGAAAGTTTCA AAGAGAACCTGAAGGA

2.

TCCTGTGCAACCCAGATT ATCACCTTTGAAAGTT TCAAAGAGAACCTGAA

3.

TTATCACCTTTGAAAG TTTCAAAGAGAACCTGA AGGACTTTCTGCTTGTCA

CRISPR Cas-9 mediated GM-CSF gene KO in primary T cells.

Clustered regularly interspaced short palindromic repeats (CRISPR), the Cas-9 system, consists of Cas9, RNA-guided nucleases, and short guide RNAs (gRNAs) that facilitate the generation of site-specific DNA breaks that are repaired by cellular endogenous mechanisms. . Cas9/gRNA RNP delivery to primary human T cells results in highly efficient target genetic modification. A CRISPR/Cas9-mediated method to knockout the GM-CSF gene is described by a detailed protocol. Oh, SA, Seki, A., & Rutz, S. (2018) Current Protocols in Immunology , 124, e69. doi: 10.1002/cpim.69, and Seki and Rutz, J Exp. Med . 2018 Vol. 215 No. 3 985-997 (incorporated herein by reference in its entirety).

It has been reported that GM-CSF inactivation by gene KO reduces cytokine release syndrome and neurotoxicity and improves antitumor activity in CAR T-treated mice bearing tumor xenografts (Sterner RM et al., 2018 Blood 2018: blood-2018-10-881722; doi: https://doi.org/10.1182/blood-2018-10-881722, incorporated herein by reference in its entirety).

Inactivation of GM-CSF gene by CRISPR approach targeting exon 1 or 2 or 3 or 4

Multiple, eg, 3 Cas9 constructs targeting three different sequences within the GM-CSF gene can be used to ensure efficient gene inactivation in all samples. This is easily accomplished using CRISPR compared to other gene editing methods.

A high frequency of bi-allelic KO has been reported using Cas9 (Zhang, Y., et al. Methods . 2014 September; 69(2): 171-178. doi:10.1016/j.ymeth. 2014.05.003, incorporated herein by reference in its entirety). This high frequency of double allele KO offers a possible advantage.

Other gene silencing techniques for GM-CSF KO in CAR T cells

Other methods that can be used for gene silencing are well known to those of skill in the art, and are not limited to homing endonuclease (HE), also known as meganuclease, RNA interference (RNAi), short interfering RNS (siRNA). ), DNA-induced RNA interference (ddRNAi).

Combination of neutralizing antibodies against GM-CSF gene KO and non-CAR T-derived GM-CSF in CAR T cells.

Removal/neutralization of all GM-CSF in patients requires anti-GM-CSF antibody or anti-receptor antibody or soluble receptor-Fc fusion, used in conjunction with GM-CSF gene KO in CART cells (for these combinations claim required).

All publications, accession numbers, patents and patent applications cited herein are hereby incorporated by reference as if each were specifically and individually indicated to be incorporated by reference.

Exemplary V of anti-GM-CSF antibodies of the present invention _H Region sequence:

SEQ ID NO: 1 (VH#1, Figure 1)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRRDRFPYYFDYWGQGTLVTVSS

SEQ ID NO: 2 (VH#2, Figure 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRDRFPYYFDYWGQGTLVTVSS

SEQ ID NO: 3 (VH#3, Figure 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCARRQRFPYYFDYWGQGTLVTVSS

SEQ ID NO: 4 (VH#4, Figure 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVAITRDTSASTAYMELSSLRSEDTAVYYCVRRQRFPYYFDYWGQGTLVTVSS

SEQ ID NO: 5 (VH#5, Figure 1)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVRRQRFPYYFDYWGQGTLVTVSS

Exemplary V of anti-GM-CSF antibodies of the present invention _L Region sequence:

SEQ ID NO: 6 (VK#1, FIG. 1)

EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGGGTKVEIK

SEQ ID NO: 7 (VK#2, Fig. 1)

EIVLTQSPATLSVSPGERATLSCRASQSVGTNVAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGGGTKVEIK

SEQ ID NO: 8 (VK#3, Fig. 1)

EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNRSPLTFGGGTKVEIK

SEQ ID NO: 9 (VK#4, Fig. 1)

EIVLTQSPATLSVSPGERATLSCRASQSIGSNLAWYQQKPGQAPRVLIYSTSSRATGITDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFNKSPLTFGGGTKVEIK

SEQ ID NO: 10 Exemplary kappa constant region

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 11 Exemplary heavy chain constant region, f-allotype:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQUENCE LISTING <110> HUMANIGEN, INC. <120> METHODS OF TREATING IMMUNOTHERAPY-RELATED TOXICITY USING A GM-CSF ANTAGONIST <130> P-566450-PC15 <140> PCT/US19/050494 <141> 2019-09-10 <150> US 16/283,694 <151> 2019-02-22 <150> US 16/248,762 <151> 2019-01-15 <150> US 16/204,220 <151> 2018-11-29 <150> PCT/US2018/053933 <151> 2018-10-02 <150> US 16/149,346 <151> 2018-10-02 <150> US 62/729,043 <151> 2018-09-10 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody heavy chain variable region (VH) VH#1 <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 2 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody heavy chain variable region (VH) VH#2 <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 3 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody heavy chain variable region (VH) VH#3 <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 4 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody heavy chain variable region (VH) VH#4 <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Ala Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 5 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody heavy chain variable region (VH) VH#5 <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody kappa light chain variable region (VL) VK#1 <400> 6 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Arg Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody kappa light chain variable region (VL) VK#2 <400> 7 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Thr Asn 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Lys Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody kappa light chain variable region (VL) VK#3 <400> 8 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Arg Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibody kappa light chain variable region (VL) VK#4 <400> 9 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Val Leu Ile 35 40 45 Tyr Ser Thr Ser Ser Arg Ala Thr Gly Ile Thr Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asn Lys Ser Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> synthetic kappa constant region <400> 10 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 11 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> synthetic f-allotype heavy chain constant region <400> 11 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 3 (CDR3) binding specificity determinant (BSD) <400> 12 Arg Gln Arg Phe Pro Tyr 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 3 (CDR3) binding specificity determinant (BSD) <400> 13 Arg Asp Arg Phe Pro Tyr 1 5 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) human J-segment JH4 <400> 14 Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 15 <210> 15 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 3 (CDR3) <400> 15 Arg Gln Arg Phe Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 3 (CDR3) <400> 16 Arg Asp Arg Phe Pro Tyr Tyr Phe Asp Tyr 1 5 10 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 3 (CDR3) <220> <221> VARIANT <222> (5)..(5) <223> Xaa = Lys or Arg <400> 17 Gln Gln Phe Asn Xaa Ser Pro Leu Thr 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 3 (CDR3) <400> 18 Gln Gln Phe Asn Lys Ser Pro Leu Thr 1 5 <210> 19 <211> 98 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> germline heavy chain variable region VH1 1-02 <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 20 <211> 98 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> germline heavy chain variable region VH1 1-03 <400> 20 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 21 <211> 96 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <223> germline kappa light chain variable region VKIII A27 <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 3 (CDR3) <220> <221> VARIANT <222> (2)..(2) <223> Xaa = Gln or Asp <400> 22 Arg Xaa Arg Phe Pro Tyr 1 5 <210> 23 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic combination of heavy chain variable region (VH) complementarity-determining region 3 (CDR3) binding specificity determinant (BSD) and human germline J-segment JH4 (CDRH3 and FR4) <220> <221> VARIANT <222> (2)..(2) <223> Xaa = Gln or Asp <400> 23 Arg Xaa Arg Phe Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 1 5 10 15 Val Thr Val Ser Ser 20 <210> 24 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 1 (CDR1) <400> 24 Gly Tyr Tyr Met His 1 5 <210> 25 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 1 (CDR1) <400> 25 Asn Tyr Tyr Ile His 1 5 <210> 26 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 2 (CDR2) <400> 26 Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 27 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 2 (CDR2) <400> 27 Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 3 (CDR3) <400> 28 Gln Gln Phe Asn Arg Ser Pro Leu Thr 1 5 <210> 29 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic combination of light chain variable region (VL) complementarity-determining region 3 (CDR3) binding specificity determinant (BSD) and human germline J-segment JK4 (CDRL3 and FR4) <400> 29 Gln Gln Phe Asn Arg Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 1 5 10 15 Glu Ile Lys <210> 30 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> synthetic combination of light chain variable region (VL) complementarity-determining region 3 (CDR3) binding specificity determinant (BSD) and human germline J-segment JK4 (CDRL3 and FR4) <400> 30 Gln Gln Phe Asn Lys Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 1 5 10 15 Glu Ile Lys <210> 31 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 1 (CDR1) <400> 31 Arg Ala Ser Gln Ser Val Gly Thr Asn Val Ala 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 1 (CDR1) <400> 32 Arg Ala Ser Gln Ser Ile Gly Ser Asn Leu Ala 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 2 (CDR2) <400> 33 Ser Thr Ser Ser Arg Ala Thr 1 5 <210> 34 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) FR4 region <400> 34 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 1 (CDRH1) <400> 35 Asp Tyr Asn Ile His 1 5 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic heavy chain variable region (VH) complementarity-determining region 2 (CDRH2) <400> 36 Tyr Ile Ala Pro Tyr Ser Gly Gly Thr Gly Tyr Asn Gln Glu Phe Lys 1 5 10 15 Asn <210> 37 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 1 (CDRL1) <400> 37 Lys Ala Ser Gln Asn Val Gly Ser Asn Val Ala 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 2 (CDRL2) <400> 38 Ser Ala Ser Tyr Arg Ser Gly 1 5 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> synthetic light chain variable region (VL) complementarity-determining region 1 (CDR1) <220> <221> VARIANT <222> (6)..(6) <223> Xaa = Val or Ile <220> <221> VARIANT <222> (8)..(8) <223> Xaa = Thr or Ser <220> <221> VARIANT <222> (10)..(10) <223> Xaa = Val or Leu <400> 39 Arg Ala Ser Gln Ser Xaa Gly Xaa Asn Xaa Ala 1 5 10 <210> 40 <211> 54 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF EXON 1 putative ZNF targeting sequence <400> 40 atgtggctgc agagcctgct gctctcgggc ctcgcccagc cccagcacgc agcc 54 <210> 41 <211> 54 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF EXON 1 complementary strand of putative ZNF targeting sequence <400> 41 tacaccgacg tctcggacga cgagagcccg gagcgggtcg gggtcgtgcg tcgg 54 <210> 42 <211> 54 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF EXON 2 putative ZNF targeting sequence <400> 42 aatgaaacag tagaagtcat ctcagaaatg gaagtcatct cagaaatgtt tgac 54 <210> 43 <211> 54 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF EXON 2 complementary strand of putative ZNF targeting sequence <400> 43 ttactttgtc atcttcagta gagtctttac cttcagtaga gtctttacaa actg 54 <210> 44 <211> 55 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> design Exon 3 GM-CSF <400> 44 gagccgacct gcctacagac ccgcctggag gcctacagac ccgcctggag ctgta 55 <210> 45 <211> 55 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 3 complementary strand <400> 45 ctcggctgga cggatgtctg ggcggacctc cggatgtctg ggcggacctc gacat 55 <210> 46 <211> 55 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 4 <400> 46 gaaacttcct gtgcaaccca gattatcacc tgcaacccag attatcacct ttgaa 55 <210> 47 <211> 55 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Exon 4 complementary strand GM-CSF <400> 47 ctttgaagga cacgttgggt ctaatagtgg acgttgggtc taatagtgga aactt 55 <210> 48 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 1 <400> 48 tggctgcaga gcctgctgct cttgggcact gtggcctgca gcatctctgc a 51 <210> 49 <211> 52 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 1 <400> 49 ttgggcactg tggcctgcag catctctgca cccgcccgct cgcccagccc ca 52 <210> 50 <211> 50 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 4 <400> 50 tgtgcaaccc agattatcac ctttgaaagt ttcaaagaga acctgaagga 50 <210> 51 <211> 50 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 4 <400> 51 tcctgtgcaa cccagattat cacctttgaa agtttcaaag agaacctgaa 50 <210> 52 <211> 51 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> GM-CSF Exon 4 <400> 52 ttatcacctt tgaaagtttc aaagagaacc tgaaggactt tctgcttgtc a 51

Claims (342)

A method comprising administering to a subject a CAR-T cell (GM-CSF ^{k/o CAR-T cell) having GM-CSF gene inactivation or gene knockout.}
A method of neutralizing and/or eliminating human GM-CSF (hGM-CSF) in a subject treated with CAR-T cells. The method of claim 1 , further comprising administering to the subject a recombinant hGM-CSF antagonist. The method of claim 2 , wherein the recombinant hGM-CSF antagonist is an anti-hGM-CSF antibody. 4. The method of claim 3, wherein the anti-hGM-CSF antibody is a recombinant antibody fragment that is Fab, Fab', F(ab')2, scFv, Fv or dAB. The method of claim 4 , wherein the anti-hGM-CSF antibody has the VH region sequence shown in FIG. 1 and the VL region sequence shown in FIG. 1 . 5. The method of claim 4, wherein the VH region or the VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 3. The method of claim 2, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 8. The method of claim 7, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. The method of claim 1 , wherein the GM-CSF is CAR T derived GM-CSF and/or non-CAR T derived GM-CSF. The method of claim 1 , wherein the individual has the development of an immunotherapy-related toxicity. The method of claim 1 , further comprising prophylactically administering to the subject (a) GM-CSF ^k/o CAR-T cells and/or (b) an anti-hGM-CSF antagonist prior to the subject being treated with immunotherapy. wherein the subject is prevented from developing an immunotherapy-related toxicity following prophylactic administration of (a) and/or (b). The method of claim 1 , wherein the individual is treated with immunotherapy, wherein the immunotherapy comprises administering GM-CSF ^k/o CAR-T cells. A method of GM-CSF gene inactivation or GM-CSF knockout (KO) in cells, comprising targeted genome editing or GM-CSF gene silencing. 14. The method of claim 13, further comprising an endonuclease as a nucleic acid cutting enzyme. 15. The method of claim 14, wherein the endonuclease is a Fok1 restriction enzyme or flap endonuclease 1 (FEN-1). The method according to claim 14, wherein the endonuclease is Cas9 (CRISPR associated protein 9). 14. The method of claim 13, wherein the GM-CSF gene inactivation by CRISPR/Cas9 targets and edits the GM-CSF gene in exon 1, exon 2, exon 3 or exon 4. The method of claim 13 , wherein the GM-CSF gene inactivation comprising CRISPR/Cas9 targets and edits the GM-CSF gene in exon 3. 14. The method of claim 13, wherein the GM-CSF gene inactivation comprising CRISPR/Cas9 targets and edits the GM-CSF gene in exon 1. 15. The method of claim 14, wherein the GM-CSF gene inactivation comprises multiple CRISPR/Cas9 enzymes, each Cas9 enzyme targeting and editing a different sequence of the GM-CSF gene in exon 1, exon 2, exon 3 or exon 4 How to. The method of claim 14 , wherein the GM-CSF gene inactivation comprises bi-allele CRISPR/Cas9 targeting and knockout/inactivation of the GM-CSF gene. 22. The method of claim 21, further comprising treating the primary T cells with valproic acid to enhance bi-allelic gene knockout/inactivation. The method of claim 13 , wherein the targeted genome editing comprises a zinc finger (ZnF) protein. The method of claim 13 , wherein the targeted genome editing comprises transcription activator-like effector nucleases (TALENS). The method of claim 13 , wherein the targeted genome editing comprises a homing endonuclease and the homing endonuclease is an ARC nuclease (ARCUS) or a meganuclease. The method of claim 13 , wherein the targeted genome editing comprises flap endonuclease (FEN-1). The method of claim 13 , wherein the cell is a CAR T cell. 28. The method of claim 27, wherein the CAR T cells are CD19 CAR-T cells. The method of claim 27 , wherein the CAR T cell is a BCMA CAR-T cell. The method of claim 13 , wherein the GM-CSF gene silencing is selected from the group consisting of RNA interference (RNAi), short interfering RNA (siRNA) and DNA-induced RNA interference (ddRNAi). A method for preventing or reducing immunotherapy-related toxicity, comprising administering to a subject a CAR-T cell (GM-CSF ^k/o CAR-T cell) with GM-CSF gene inactivation or GM-CSF knockout As,
The GM-CSF gene is inactivated or knocked out by the method of any one of claims 13 to 26, the method. The method of claim 31 , wherein the immunotherapy-related toxicity is a CAR-T related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 32. The method of claim 31, ^{further comprising administering the GM-CSF k/o} CAR-T cells in combination with an hGM-CSF antagonist. 34. The method of claim 33, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 32. The method of claim 31, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv or dAB. 36. The method of claim 35, wherein the anti-hGM-CSF antibody has a VH region sequence shown in Figure 1 and a VL region sequence shown in Figure 1 . 37. The method of claim 36, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 38. The method of claim 37, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 39. The method of claim 38, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. comprising administering to the subject a recombinant GM-CSF antagonist,
A method of reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy. 41. The method of claim 40, wherein the immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cytokine administration, cancer vaccine administration, T cell engaging therapies, or any combination thereof. , Way. 41. The method of claim 40, wherein the subject comprises diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, and follicular having DLBCL arising from sexual lymphoma. 42. The method of claim 41, wherein the adoptive cell transfer is CAR chimeric antigen receptor-expressing T-cell, T-cell receptor (TCR) modified T-cell, tumor-infiltrating lymphocyte (TIL), chimeric antigen (CAR) receptor) modified natural killer cells, or dendritic cells, or a combination thereof. 41. The method of claim 40, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 41. The method of claim 40, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 46. The method of claim 45, wherein the anti-GM-CSF antibody binds human GM-CSF. 46. The method of claim 45, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 48. The method of claim 47, wherein the primate is monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, photo. (potto), sifaka (sifaka), indri (indri), aye-eye (aye-aye) or ape (ape). 46. The method of claim 45, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 46. The method of claim 45, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv or dAB. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is a recombinant antibody, a humanized antibody, a CDR grafted antibody, or a chimeric antibody. 51. The method of claim 50, wherein the anti-hGM-CSF antibody is a human antibody. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody binds to the same epitope as chimeric 19/2. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises VH region CDR3 and VL region CDR3 of chimeric 19/2. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of chimeric 19/2. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is human. comprises at least 95% identity to JH4 (YFD YWGQGTL VTVSS) and wherein the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. 60. The method of claim 59, wherein the J segment comprises YFDYWGQGTLVTVSS. 61. The method of claim 59 or 60, wherein CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 61. The method of claim 59 or 60, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. 61. The method of claim 59 or 60, wherein the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2 as shown in the VH region shown in Figure 1. 61. The method of claim 59 or 60, wherein the V-segment sequence has the VH V segment sequence shown in FIG. 61. The method of claim 59 or 60, wherein VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 . 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 67. The method of claim 66, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 68. The method of claim 66 or 67, wherein the VL region CDR3 comprises QQFN(K/R)SPLT. 69. The method of claim 68, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 67. The method of claim 66, wherein the VL region comprises CDR1, CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . 67. The method of claim 66, wherein the VL region comprises a V segment having at least 95% identity to a VKIII A27 V-segment sequence as shown in FIG. 1 . 67. The method of claim 66, wherein the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. 56. The method of any one of claims 50-55, wherein the anti-hGM-CSF antibody has a VH region sequence shown in Figure 1 and a VL region sequence shown in Figure 1 . 56. The method of any one of claims 50-55, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 45. The method of claim 44, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 77. The method of claim 76, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 44. The method of claim 43, wherein the CAR-T cell is a CD19 CAR-T cell. 41. The method of claim 40, wherein reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in the subject occurs without the occurrence of immunotherapy-related toxicity. 41. The method of claim 40, wherein reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in the subject occurs in the presence of the occurrence of an immunotherapy-related toxicity. 81. The method of claim 80, wherein the immunotherapy-related toxicity is a CAR-T related toxicity. 82. The method of claim 81, wherein the CAR-T related toxicity is cytokine release syndrome, neurotoxicity or neuroinflammation. 41. The method of claim 40, wherein the incidence of tumor recurrence is from 50% to 100% in the first quarter after administration of the recombinant GM-CSF antagonist compared to the incidence of tumor recurrence in an individual treated with immunotherapy and not administered the recombinant GM-CSF antagonist reduced way. 41. The method of claim 40, wherein the incidence of tumor recurrence is reduced by 50% to 95% in the first half after administration of the recombinant GM-CSF antagonist. 41. The method of claim 40, wherein the occurrence of tumor recurrence is prevented by 12-36 months. 41. The method of claim 40, wherein the occurrence of tumor recurrence is completely (100%) prevented for about 10 years. 41. The method of claim 40, wherein the individual has acute lymphoblastic leukemia. 41. The method of claim 40, ^{further comprising administering the GM-CSF k/o} CAR-T cells in combination with an hGM-CSF antagonist. A cytokine or chemokine other than GM-CSF in an individual treated with immunotherapy (and having the development of immunotherapy-related toxicity) comprising administering to the individual a recombinant hGM-CSF antagonist prior to or during immunotherapy. As a method of reducing the level of
wherein the level of the cytokine or chemokine is reduced as compared to the level of the subject during the development of an immunotherapy-related toxicity. 91. The method of claim 89, wherein the immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cancer vaccine administration, T cell engaging therapy, or any combination thereof. 91. The method of claim 90, wherein said adoptive cell transfer is CAR chimeric antigen receptor-expressing T-cell, T-cell receptor (TCR) modified T-cell, tumor-infiltrating lymphocyte (TIL), chimeric (CAR) antigen receptor) modified natural killer cells, or dendritic cells, or any combination thereof. 92. The method of claim 91, wherein the CAR-T cells are CD19 CAR-T cells or BCMA CAR-T cells. 91. The method of claim 89, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 91. The method of claim 90, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 91. The method of claim 90, wherein the anti-GM-CSF antibody binds human GM-CSF. 91. The method of claim 90, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 91. The method of claim 90, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 91. The method of claim 90, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 95. The method of claim 94, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 95. The method of claim 94, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, Fv, scFv or dAB. 95. The method of claim 94, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 95. The method of claim 94, wherein the anti-hGM-CSF antibody is a recombinant antibody, a humanized antibody, a CDR grafted antibody, or a chimeric antibody. 95. The method of claim 94, wherein the anti-hGM-CSF antibody is a human antibody. 91. The method of claim 89, wherein the cytokine or chemokine is IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a., TNF-a, IL-1Ra, A human cytokine or chemokine selected from the group consisting of FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a, MIP-1b, and combinations thereof, a method. 91. The method of claim 89, wherein the cytokine or chemokine is IFN-γ, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-9, IL. -10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG , RANTES, TNF-a, eotaxin, G-CSF, IL-1Ra, FGF-2, sCD40L, and combinations thereof. 91. The method of claim 89, wherein the individual has DLBCL arising from acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma. 91. The method of claim 89, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 108. The method of claim 107, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. A method of treating or preventing immunotherapy-related toxicity in a subject comprising administering to the subject a chimeric antigen receptor-expressing T-cell (CAR-T cell), comprising:
The method, wherein the CAR-T is a cell with GM-CSF gene inactivation or gene knockout (GM-CSF ^{k / o} CAR-T cell). 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells express a reduced level of GM-CSF compared to the level of GM-CSF expression by wild-type CAR-T cells. 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cell has a level of one or more cytokines and/or equal to or lower than the level of one or more cytokines and/or chemokines expressed by the wild-type CAR-T cell. A method of expressing a chemokine. 110. The method of claim 109, wherein the one or more cytokines are IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a., TNF-a, IL-1Ra, A human cytokine and/or chemokine selected from the group consisting of FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a, MIP-1b, and combinations thereof, a method. 110. The method of claim 109, wherein the one or more cytokines are IFN-γ, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-9, IL -10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2, MIG , RANTES, TNF-a, eotaxin, G-CSF, IL-1Ra, FGF-2, sCD40, and combinations thereof. 110. The method of claim 109, wherein the CAR-T cells are CD19 CAR-T cells or BCMA CAR-T cells. 110. The method of claim 109, wherein the GM-CSFK/O CAR-T cells improve the relapse rate compared to the relapse rate of an individual treated for wild-type CAR-T cells. 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve objective response rates (complete response and partial response) compared to a subject treated by administration of the wild-type CAR-T cells. In, way. 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve progression free survival in an individual as compared to progression free survival in an individual treated by administration of the wild-type CAR-T cells. , Way. 110. The method of claim 109, wherein the individual has DLBCL arising from acute lymphoblastic leukemia, diffuse large B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma, high grade B cell lymphoma, or follicular lymphoma. 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells enhance the antitumor activity of the recombinant hGM-CSF antagonist. 110. The method of claim 109, wherein the GM-CSF ^k/o CAR-T cells improve overall survival of the subject as compared to survival in the subject treated by administration of the wild-type CAR-T cells. 110. The method of claim 109, wherein the individual has acute lymphoblastic leukemia. 110. The method of claim 109, further comprising administering a recombinant hGM-CSF antagonist. 123. The method of claim 122, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 123. The method of claim 122, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 125. The method of claim 124, wherein the anti-GM-CSF antibody binds human GM-CSF. 125. The method of claim 124, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 125. The method of claim 124, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 125. The method of claim 124, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 129. The method of claim 128, wherein the anti-hGM-CSF antibody is a human antibody. 124. The method of claim 123, wherein the anti-hGM-CSF receptor antibody or soluble hGM-CSF receptor or receptor subunit, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody mimetic a method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic 135. The method of claim 134, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. administering to the subject a recombinant hGM-CSF antagonist;
A method of reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in a subject treated with immunotherapy. 137. The method of claim 136, wherein the individual has DLBCL arising from acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma. 137. The method of claim 136, wherein the individual has refractory/relapsed cancer, wherein the individual is non-Hodgkin's lymphoma (NHL) or chemotherapy refractory B cell lymphoma. 137. The method of claim 136, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 137. The method of claim 136, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 137. The method of claim 136, wherein the anti-GM-CSF antibody binds human GM-CSF. 137. The method of claim 136, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 137. The method of claim 136, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 137. The method of claim 136, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 145. The method of claim 144, wherein the anti-hGM-CSF antibody is a human antibody. 140. The method of claim 139, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 150. The method of claim 150, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 137. The method of claim 136, further comprising administering the GM-CSF K/O CAR T cells in combination with an hGM-CSF antagonist. 140. The method of claim 139, wherein the GM-CSF ^k/o CAR T cell has a GM-CSF gene inactivation or a GM-CSF knockout (GM-CSF ^k/o CAR-T cell), and wherein the GM-CSF gene is 26. A method which has been inactivated or knocked out by the method of any one of claims 25 to 26. comprising administering to the subject a recombinant GM-CSF antagonist,
A method of reducing disruption of the blood-brain barrier in a subject treated with immunotherapy. 155. The method of claim 154, wherein the individual has the development of an immunotherapy-related toxicity. 155. The method of claim 154, wherein the immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cytokine administration, cancer vaccine administration, T cell engaging therapy, or any combination thereof. 157. The method of claim 156, wherein the adoptive cell transfer comprises a CAR chimeric antigen receptor-expressing T-cell, a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen (CAR). receptor) modified natural killer cells, or dendritic cells, or a combination thereof. 158. The method of claim 157, wherein the CAR T-cell is a CD19 CAR-T cell or a BCMA CAR-T cell. 155. The method of claim 154, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 155. The method of claim 154, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 160. The method of claim 160, wherein the anti-GM-CSF antibody binds mammalian GM-CSF or binds primate GM-CSF. 162. The primate of claim 161, wherein the primate is monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, photo. (potto), sifaka, indri, aye-aye, ape or human. 160. The method of claim 159, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is administered prior to, concurrently with, following, or in combination with, immunotherapy. 164. The method of claim 163, wherein the anti-hGM-CSF antibody binds human GM-CSF. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 164. The method of claim 163, wherein the anti-hGM-CSF antibody is a human antibody. 170. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. 170. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. 170. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2, and CDR3 of a chimeric 19/2 antibody. 170. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment human JH4 (YFD YWGQGTL VTVSS) contains at least 95% identity and the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. 175. The method of claim 174, wherein the J segment comprises YFDYWGQGTLVTVSS. 178. The method of claim 174 or 175, wherein CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 175. The method of claim 174 or 175, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. 175. The method of claim 174 or 175, wherein the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2 as shown in the VH region shown in FIG. 1 . 175. The method of claim 174 or 175, wherein the V-segment sequence has the VH V segment sequence shown in FIG. 1 . 175. The method of claim 174 or 175, wherein VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 . 164. The method of claim 163, wherein the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 182. The method of claim 181, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 183. The method of claim 181 or 182, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 184. The method of claim 183, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 182. The method of claim 181, wherein the VL region comprises CDR1, CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . 182. The method of claim 181, wherein the VL region comprises a V segment having at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . 182. The method of claim 181, wherein the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . 170. The method of any one of claims 163-170, wherein the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. 190. The method of claim 188, wherein the anti-hGM-CSF antibody has a VH region sequence shown in Figure 1 and a VL region sequence shown in Figure 1 . 190. The method of claim 188, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 160. The method of claim 159, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, a cytochrome b562 antibody mimetic, a hGM-CSF peptide analog, adnectin, a lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 156. The method of claim 155, wherein the immunotherapy-related toxicity is a CAR-T related toxicity. 156. The method of claim 155, wherein the CAR-T related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof. 160. The method of claim 159, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, a cytochrome b562 antibody mimetic, a hGM-CSF peptide analog, adnectin, a lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 195. The method of claim 194, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. administering to the subject a recombinant hGM-CSF antagonist;
A method of preserving blood-brain barrier integrity in subjects treated with immunotherapy. 197. The method of claim 196, wherein the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. 201. The method of claim 197, wherein the anti-GM-CSF antibody binds mammalian GM-CSF or binds primate GM-CSF. 199. The primate of claim 198, wherein the primate is monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, photo. (potto), sifaka, indri, aye-aye, ape or human. 201. The method of claim 197, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is administered prior to, concurrently with, following immunotherapy, or a combination thereof. 201. The method of claim 200, wherein the anti-hGM-CSF antibody binds to human GM-CSF. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 201. The method of claim 200, wherein the anti-hGM-CSF antibody is a human antibody. 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is human. contains at least 95% identity to JH4 (YFD YWGQGTL VTVSS) and the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. 212. The method of claim 211, wherein the J segment comprises YFDYWGQGTLVTVSS. 223. The method of claim 211 or 212, wherein CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 223. The method of claim 211 or 212, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. 223. The method of claim 211 or 212, wherein the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2 as shown in the VH region shown in FIG. 1 . 223. The method of claim 211 or 212, wherein the V-segment sequence has the VH V segment sequence shown in FIG. 223. The method of claim 211 or 212, wherein the VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 . 201. The method of claim 200, wherein the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 229. The method of claim 218, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 221. The method of claim 218 or 219, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 223. The method of claim 220, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 223. The method of claim 220, wherein the VL region comprises CDR1, CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . 218. The method of claim 218, wherein the VL region comprises a V segment having at least 95% identity to the VKIII A27 V-segment sequence as shown in FIG. 1 . 218. The method of claim 218, wherein the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . 208. The method of any one of claims 200-207, wherein the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. 225. The method of claim 225, wherein the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . 225. The method of claim 225, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 197. The method of claim 196, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 197. The method of claim 196, wherein the individual has immunotherapy-related toxicity. 229. The method of claim 229, wherein the immunotherapy-related toxicity is a CAR-T related toxicity. 234. The method of claim 230, wherein the CAR-T related toxicity is cytokine release syndrome, neurotoxicity, neuroinflammation, or a combination thereof. 197. The method of claim 196, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A room selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 232. The method of claim 232, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. administering to the subject a recombinant hGM-CSF antagonist;
A method of reducing or preventing CAR-T cell therapy-induced neuroinflammation in a subject in need thereof. 234. The method of claim 234, wherein administration of the recombinant hGM-CSF antagonist reduces disruption of the blood-brain barrier and thereby maintains its integrity. 234. The method of claim 235, wherein reducing disruption of the blood-brain barrier reduces or prevents entry of pro-inflammatory cytokines into the central nervous system. 237. The method of claim 236, wherein the pro-inflammatory cytokine is IFN-γ, GRO, MDC, IL-2, IL-3, IL-5, IL-7, IP-10, CD107a., TNF- a, a human cytokine selected from the group consisting of IL-1Ra, FGF-2, IL-12p40, IL-12p70, sCD40L, VEGF, MCP-1, MIP-1a, MIP-1b, and combinations thereof, the method. 237. The method of claim 236, wherein the pro-inflammatory cytokine is IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-9. , IL-10, IL-12p40, IL-12p70, ILF, IL-13, LIX, IL-15, IP-10, KC, MCP-1, MIP-1a, MIP-1b, M-CSF, MIP-2 , MIG, RANTES, TNF-a, eotaxin, G-CSF, IL-1Ra, FGF-2, sCD40L, and combinations thereof. 234. The method of claim 234, wherein the neuroinflammation in the individual is reduced by 75% to 95% as compared to an individual treated with the CAR-T cell therapy and a control antibody. 246. The method of claim 239, wherein a 75% to 95% reduction in neuroinflammation is similar to neuroinflammation in an untreated control subject. 234. The method of claim 234, wherein the subject is administered a chimeric antigen receptor-expressing T-cell. 234. The method of claim 234, wherein the subject is a T-cell receptor (TCR) modified T-cell, a tumor-infiltrating lymphocyte (TIL), a chimeric antigen receptor (CAR) modified natural killer cell, or a dendritic cell, or any thereof. A method, wherein the combination is administered. The method of claim 241 , wherein the CAR T-cell is a CD19 CAR-T cell or a BCMA CAR-T cell. 234. The method of claim 234, wherein the recombinant hGM-CSF antagonist is a hGM-CSF antagonist. 234. The method of claim 234, wherein the recombinant hGM-CSF antagonist is an anti-GM-CSF antibody. 245. The method of claim 245, wherein the anti-GM-CSF antibody binds mammalian GM-CSF or binds primate GM-CSF. 245. The primate of claim 245, wherein the primate is monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, photo. (potto), sifaka, indri, aye-aye, ape or human. 246. The method of claim 246, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is administered prior to, concurrently with, following immunotherapy, or a combination thereof. 249. The method of claim 248, wherein the anti-hGM-CSF antibody binds human GM-CSF. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 249. The method of claim 248, wherein the anti-hGM-CSF antibody is a human antibody. 256. The method of any one of claims 248-255, wherein the anti-hGM-CSF antibody binds to the same epitope as the chimeric 19/2 antibody. 256. The method of any one of claims 248-255, wherein the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of a chimeric 19/2 antibody. 256. The method of any one of claims 248-255, wherein the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of a chimeric 19/2 antibody. 256. The anti-hGM-CSF antibody of any one of claims 248-255, wherein the anti-hGM-CSF antibody comprises a VH region comprising a CDR3 binding specificity determinant RQRFPY or RDRFPY, a J segment and a V-segment, wherein the J-segment human JH4 (YFD YWGQGTL VTVSS) contains at least 95% identity and the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. 262. The method of claim 259, wherein the J segment comprises YFDYWGQGTLVTVSS. 267. The method of claim 259 or 260, wherein CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 260. The method of claim 259 or 260, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. 260. The method of claim 259 or 260, wherein the anti-hGM-CSF antibody comprises VH CDR1, or VH CDR2, or both VH CDR1 and VH CDR2 as shown in the VH region shown in FIG. 1 . 260. The method of claim 259 or 260, wherein the V-segment sequence has the VH V segment sequence shown in FIG. 1 . 267. The method of claim 259 or 260, wherein VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 . 249. The method of claim 248, wherein the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 249. The method of claim 248, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 267. The method of claim 266 or 267, wherein the VL region CDR3 comprises QQFN(K/R)SPL. 269. The method of claim 268, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 268. The method of claim 268, wherein the VL region comprises CDR1, CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . 268. The method of claim 268, wherein the VL region comprises a V segment having at least 95% identity to a VKIII A27 V-segment sequence as shown in FIG. 1 . 268. The method of claim 268, wherein the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . 256. The method of any one of claims 248-255, wherein the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. 274. The method of claim 273, wherein the anti-hGM-CSF antibody has a VH region sequence shown in Figure 1 and a VL region sequence shown in Figure 1 . 268. The method of claim 268, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 234. The method of claim 234, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 234. The method of claim 234, wherein the individual further has a CAR-T related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 234. The method of claim 234, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. a method selected from the group comprising lipocalin scaffold antibody mimetic, calixarene antibody mimetic, and antibody-like binding peptidomimetic 289. The method of claim 278, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. A method comprising administering to a subject a CAR-T cell having a GM-CSF gene knockout (GM-CSF ^k/o CAR-T cell),
A method of preventing or reducing blood-brain barrier disruption in a subject treated with immunotherapy. 280. The method of claim 280, further comprising administering to the individual a recombinant hGM-CSF antagonist. The method of claim 281 , wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 289. The method of claim 282, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 289. The method of claim 283, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 283. The method of claim 283, wherein the anti-hGM-CSF antibody has a VH region sequence shown in Figure 1 and a VL region sequence shown in Figure 1 . 283. The method of claim 283, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 282. The method of claim 282, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 280. The method of claim 280, wherein the individual further has a CAR-T related toxicity selected from cytokine release syndrome, neurotoxicity, or a combination thereof. 282. The method of claim 282, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 289. The method of claim 289, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 280. The method of claim 280, ^{further comprising administering a GM-CSF k/o} CAR T cell in combination with an hGM-CSF antagonist, wherein the GM-CSF gene is in the method of any one of claims 12-25. Inactivated or knocked out by the method. comprising administering to the subject a recombinant GM-CSF antagonist,
A method of blocking or reducing hGM-CFS expression in a subject treated with immunotherapy to prevent or treat immunotherapy-related toxicity. 292. The method of claim 292, wherein the immunotherapy comprises adoptive cell transfer, monoclonal antibody administration, cytokine administration, cancer vaccine administration, T cell engaging therapy, or any combination thereof. 302. The method of claim 293, wherein the adoptive cell transfer is CAR chimeric antigen receptor-expressing T-cell, T-cell receptor (TCR) modified T-cell, tumor-infiltrating lymphocyte (TIL), chimeric antigen (CAR). receptor) modified natural killer cells, or dendritic cells, or a combination thereof. 292. The method of claim 292, wherein the recombinant GM-CSF antagonist is a hGM-CSF antagonist. 292. The method of claim 292, wherein the recombinant GM-CSF antagonist is an anti-GM-CSF antibody. 295. The method of any one of claims 292-294, wherein the anti-GM-CSF antibody binds human GM-CSF. 297. The method of claim 296, wherein the anti-GM-CSF antibody binds to primate GM-CSF. 298. The primate of claim 298, wherein the primate is monkey, baboon, macaque, chimpanzee, gorilla, lemur, lorise, tarsier, galago, photo. (potto), sifaka (sifaka), indri (indri), aye-eye (aye-aye) or ape (ape). 297. The method of claim 296, wherein the anti-GM-CSF antibody binds to mammalian GM-CSF. 297. The method of claim 296, wherein the anti-GM-CSF antibody is an anti-hGM-CSF antibody. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is a monoclonal antibody. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is an antibody fragment that is Fab, Fab', F(ab')2, scFv or dAB. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is a human GM-CSF neutralizing antibody. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is a recombinant or chimeric antibody. 302. The method of claim 301, wherein the anti-hGM-CSF antibody is a human antibody. 303. The method of claim 301 or 302, wherein the anti-hGM-CSF antibody binds to the same epitope as chimeric 19/2. 303. The method of claim 301 or 302, wherein the anti-hGM-CSF antibody comprises the VH region CDR3 and the VL region CDR3 of chimeric 19/2. 303. The method of claim 301 or 302, wherein the anti-hGM-CSF antibody comprises the VH and VL regions CDR1, CDR2 and CDR3 of chimeric 19/2. 303. The method of claim 301 or 302, wherein the anti-hGM-CSF antibody comprises a CDR3 binding specificity determinant RQRFPY or RDRFPY, a VH region comprising a J segment and a V-segment, wherein the J-segment is human JH4 (YFD YWGQGTL). VTVSS) and the V-segment comprises at least 90% identity to the human germline VH1 1-02 or VH1 1-03 sequence; or a VH region comprising the CDR3 binding specificity determinant RQRFPY. 310. The method of claim 310, wherein the J segment comprises YFDYWGQGTLVTVSS. 312. The method of claim 310 or 311, wherein CDR3 comprises RQRFPYYFDY or RDRFPYYFDY. 332. The method of claim 310 or 311, wherein the VH region CDR1 is a human germline VH1 CDR1; the VH region CDR2 is human germline VH1 CDR2; or both CDR1 and CDR2 are from human germline VH1 sequences. 312. The method of claim 310 or 311, wherein the anti-hGM-CSF antibody comprises a VH CDR1, or a VH CDR2, or both a VH CDR1 and a VH CDR2 shown in the VH region shown in FIG. 1 . 312. The method of claim 310 or 311, wherein the V-segment sequence has the VH V segment sequence shown in FIG. 1 . 312. The method of claim 310 or 311, wherein VH has the sequence of VH#1, VH#2, VH#3, VH#4, or VH#5 shown in FIG. 1 . 307. The method of any one of claims 301-306, wherein the anti-hGM-CSF antibody comprises a VL-region comprising a CDR3 comprising the amino acid sequence FNK or FNR. 315. The method of any one of claims 313-315, wherein the anti-hGM-CSF antibody comprises a human germline JK4 region. 318. The method of claim 317 or 318, wherein the VL region CDR3 comprises QQFN(K/R)SPLT. 319. The method of claim 319, wherein the anti-hGM-CSF antibody comprises a VL region comprising a CDR3 comprising QQFNKSPLT. 318. The method of claim 317, wherein the VL region comprises CDR1, CDR2, or both CDR1 and CDR2 of the VL region depicted in FIG. 1 . 318. The method of claim 317, wherein the VL region comprises a V segment having at least 95% identity to the VKIII A27 V-segment sequence depicted in FIG. 1 . 318. The method of claim 317, wherein the VL region has the sequence of VK#1, VK#2, VK#3 or VK#4 shown in FIG. 1 . 307. The method of any one of claims 301-306, wherein the anti-hGM-CSF antibody has a VL region having a CDR3 comprising the VH region CDR3 binding specificity determinants RQRFPY or RDRFPY, and QQFNKSPLT. 307. The method of any one of claims 301-306, wherein the anti-hGM-CSF antibody has a VH region sequence shown in FIG. 1 and a VL region sequence shown in FIG. 1 . 307. The method of any one of claims 301-306, wherein the VH region or VL region, or both the VH and VL region amino acid sequences, comprise a methionine at the N-terminus. 296. The method of claim 295, wherein the hGM-CSF antagonist is an anti-hGM-CSF receptor antibody or a soluble hGM-CSF receptor, cytochrome b562 antibody mimetic, hGM-CSF peptide analog, adnectin, lipocalin scaffold antibody. A method selected from the group comprising a lipocalin scaffold antibody mimetic, a calixarene antibody mimetic, and an antibody-like binding peptidomimetic. 327. The method of claim 327, wherein the soluble hGM-CSF receptor comprises a soluble hGM-CSF receptor-Fc fusion protein. 292. The method of claim 292, wherein the CAR-T cell is a CD19 CAR-T cell. 292. The immune system of claim 292, wherein blocking or reducing hGM-CFS expression further reduces the rate of tumor recurrence or further prevents the occurrence of tumor recurrence in the subject, and reduces the rate of tumor recurrence or prevents the occurrence of tumor recurrence in the subject. and occurs without the occurrence of therapy-related toxicity. 329. The method of claim 329, wherein reducing the rate of tumor recurrence or preventing the occurrence of tumor recurrence in the subject occurs in the presence of the occurrence of an immunotherapy-related toxicity. 335. The method of claim 330, wherein the immunotherapy-related toxicity is a CAR-T related toxicity. 332. The method of claim 331, wherein the CAR-T related toxicity is cytokine release syndrome, neurotoxicity or neuroinflammation. 335. The method of claim 330, wherein the incidence of tumor recurrence is from 50% to 100% in the first quarter following administration of the recombinant GM-CSF antagonist compared to the incidence of tumor recurrence in an individual treated with the immunotherapy and not administered the recombinant GM-CSF antagonist. reduced way. 330. The method of claim 330, wherein the incidence of tumor recurrence is reduced by 50% to 95% in the first half after administration of the recombinant GM-CSF antagonist. 330. The method of claim 330, wherein the incidence of tumor recurrence is reduced by 50% to 90% in the first year after administration of the recombinant GM-CSF antagonist. 330. The method of claim 330, wherein the occurrence of tumor recurrence is prevented for a long period of time. 335. The method of claim 330, wherein the occurrence of tumor recurrence is prevented by 12-36 months. 292. The method of claim 292, wherein the occurrence of tumor recurrence is completely (100%) prevented. 292. The method of claim 292, wherein the individual has DLBCL arising from acute lymphoblastic leukemia, diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma, high-grade B-cell lymphoma, or follicular lymphoma. 292. The method of claim 292, wherein the individual has acute lymphoblastic leukemia. 302. The method of claim 292, further comprising administering a ^{GM-CSF k/o} CAR T cell in combination with an hGM-CSF antagonist, wherein the GM-CSF gene is selected from the group consisting of targeted genome editing or GM-CSF gene silencing. ), which is inactivated or knocked out by a GM-CSF gene inactivation or GM-CSF knockout (KO) method in a cell, comprising a method.

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