Fas antibody and its use in treating cancers

Abstract

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to the Fas receptor. Also provided herein are methods comprising said antibodies or antigen-binding fragments.

Description

[0001] FAS ANTIBODY AND ITS USE IN TREATING CANCERS

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority to US Provisional Patent Application No. 63 / 624,045, filed January 23, 2024, the entire contents of which are incorporated herein by reference in its entirety for all purposes.

[0004] STATEMENT OF FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under Grant number CA233752 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] BACKGROUND

[0007] Ovarian cancer is the most lethal of gynecological diseases and is known for chemoresistance and frequent reoccurrence with no effective treatment available. Clinical data indicate that antibodies that help eliminate tumor cells by activating and harnessing the power of a patient’s immune system are highly effective against blood cancers and melanomas. However, in the case of ovarian cancer, immune system-activating antibody-based and chimeric antigen receptor T-cell (CAR-T)-based approaches have primarily failed. The latter is mainly attributed to the highly limited penetration of immune effector cell types (such as T- cells, NK-cells, etc.) into the solid ovarian tumor bed, making it the most immune-cold. Thus, strategies that debulk ovarian tumors independent of immune effector cells are critical.

[0008] Fas / CD95 and DR5 / TRAIL-R2 are two critical ovarian cancer tumor-enriched deathactivating receptors. In the case of DR5, multiple death agonist antibodies have been tested in phase-II solid cancer trials with limited clinical success. On the contrary, no Fas agonist antibody has been made to even phase-I human clinical trials. The latter is even more earnestly needed considering that, unlike DR5, Fas also has a well-established role in promoting ovarian cancer tumorigenesis, metastasis, and cancer stem signaling pathways, independent of its death-activating signaling. Therefore, there remains a need for Fas-targeted antibody capable of inhibiting tumor growth and activating tumor death. SUMMARY

[0009] In one aspect, an antibody or antigen-binding fragment thereof that specifically binds Fas receptor (e.g., SEQ ID NO: 10) is provided, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

[0010] In certain exemplary embodiments, the Fas receptor is hypoglycosylated relative to a wild-type Fas receptor. In certain exemplary embodiments, the Fas receptor is aglycosylated. In certain exemplary embodiments, the Fas receptor is expressed on an ovarian cancer cell or other solid tumor cancer cells that are optionally selected from the group consisting of triplenegative breast cancer (TNBC), lung cancer, and glioblastoma multiforme (GBM).

[0011] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

[0012] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof binds Fas receptor expressed on a tumor cell with a higher affinity than Fas receptor expressed on a T-cell,

[0013] In another aspect, a method of treating a subject having a solid tumor is provided, the method comprising contacting the subject with an antibody or antigen-binding fragment thereof that specifically binds Fas receptor, wherein the Fas receptor is hypoglycosylated relative to wild-type Fas receptor or is aglycosylated.

[0014] In certain exemplary embodiments, the solid tumor comprises uterine cancer or ovarian cancer or other solid tumor cancer cells that are optionally selected from the group consisting of triple-negative breast cancer (TNBC), lung cancer, and glioblastoma multiforme (GBM), expressing hypoglycosylated or aglycosylated Fas. In certain exemplary embodiments, the solid tumor comprises ovarian cancer.

[0015] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively. In certain exemplary embodiments, the antibody or antigenbinding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8. In certain exemplary embodiments, the method further comprises contacting the subject with an antibody or antigen-binding fragment thereof that specifically binds folate receptor 1 (FOLR1).

[0016] In certain exemplary embodiments, the antibody that specifically binds Fas receptor and the antibody that specifically binds FOLR1 are in a bispecific antibody format. In certain exemplary embodiments, the bispecific antibody specifically binds both of Fas receptor and FOLR1.

[0017] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds Fas receptor and the antibody or antigen-binding fragment thereof that specifically binds FOLR1 are administered together. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds Fas receptor and the antibody or antigen-binding fragment thereof that specifically binds FOLR1 are administered separately.

[0018] In another aspect, a method of treating ovarian cancer in a subject is provided, comprising contacting the subject with a bispecific antibody that specifically binds Fas receptor that is hypoglycosylated relative to wild-type Fas receptor or is aglycosylated.

[0019] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

[0020] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

[0021] In certain exemplary embodiments, the method further comprises contacting the subject with an antibody or antigen-binding fragment thereof that specifically binds folate receptor 1 (FOLR1).

[0022] In certain exemplary embodiments, the antibody that specifically binds Fas receptor and the antibody that specifically binds FOLR1 are in a bispecific antibody format.

[0023] In certain exemplary embodiments, the bispecific antibody specifically binds both of Fas receptor and FOLR1.

[0024] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds Fas receptor and the antibody or antigen-binding fragment thereof that specifically binds FOLR1 are administered together. In certain exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds Fas receptor and the antibody or antigen-binding fragment thereof that specifically binds FOLR1 are administered separately.

[0025] In another aspect, a method of predicting a positive clinical response to treating a solid tumor in a subject is provided, comprising contacting a solid tumor sample from the subject with an antibody or antigen-binding fragment thereof that specifically binds Fas receptor that is hypoglycosylated relative to wild-type Fas receptor or is aglycosylated, wherein the antibody or antigen-binding fragment thereof binds to the solid tumor sample with a higher affinity than to a non-tumor sample.

[0026] In certain exemplary embodiments, the solid tumor comprises ovarian cancer.

[0027] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

[0028] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

[0029] In another aspect, a single chain variable fragment (scFv) that comprises a variable light chain followed by GS linker and a variable heavy chain comprising SEQ ID NO: 9 is provided.

[0030] In certain exemplary embodiments, the target of the scFv is expressed on solid tumor comprises ovarian and other cancers. In certain exemplary embodiments, the scFv target is a bispecific molecule that optionally comprises FOLR1, EGFR and / or MUC16.

[0031] In certain exemplary embodiments, the scFv target is expressed on CAR-T cells to enhance antigen independent bystander anti-tumor function.

[0032] In certain exemplary embodiments, the scFv target is expressed as bispecific that optionally includes FOLR1, CD24, NaPi2b, EGFR, Mucl6 on CAR-T cells to enhance antigen independent bystander anti-tumor function.

[0033] In another aspect, a method of binding a Fas receptor that is hypoglycosylated relative to wild-type Fas receptor (SEQ ID NOs: 10) or is aglycosylated is provided, comprising contacting the Fas receptor with an antibody or antigen-binding fragment thereof that specifically binds to a patch of positively charged residues (PPCR) motif present in the Fas receptor as set forth as SEQ ID NO: 11 or SEQ ID NO: 12).

[0034] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

[0035] In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate exemplary embodiments of the disclosure, and, together with the general description given above and the detailed description given below, serve to explain the features of the disclosure. The file of this patent contains at least one drawing / photograph executed in color. Copies of this patent with color drawing(s) / photograph(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] FIG. 1A - FIG. ID illustrate salt bridges formed between FasL and residue R87 of Fas. FIG. 1A is an alignment of the Fas PPCR region from dog to humans. FIG. IB is a ribbon depiction of the PPCR region in DR5 and Fas. FIG. 1C depicts the FasL-DCR3 interface at the PPCR region. FIG. ID is similar to FIG. 1C, but includes FasR (grey ribbon) instead of DCR3.

[0039] FIG. 2A - FIG. 2H demonstrate that a single point mutation in the PPCR interacting FasL residues abolishes function. FIG. 2A is a schematic of various His-tagged FasL mutants. FIG. 2B is a reducing gel of proteins purified from CHO cells expressing various FasL mutants using HisTrap Ni Sepharose. FIG. 2C illustrates the binding of His-tagged FasL to immobilized Fc-tagged FasR by ELISA. FIG. 2D - FIG. 2G are the results of a cell killing assay using OVCAR-3 ovarian cancer cells, Jukat cells, and human T-cells 36 hours after treatment of indicated FasL mutants.

[0040] FIG. 3A - FIG. 3C are immunoblots illustrating that wild-type FasL blocks T-cell activation but point mutations E163A and E271A do not. FIG. 3A depicts protein samples from T-cells derived from CD3 -enriched human PBMC samples that were treated with either CD3 / CD28 agonist alone or indicated Fas mutants alone or together for 60 min. followed by immunoblotting of indicated proteins. FIG. 3B depicts protein samples from human T cells treated with FasL mutants for 1 hour followed by crosslinker treatment to capture FasR. FIG. 3C is the same as FIG. 3 A, but with non-human primate T-cell samples. FIG. 4A - FIG. 4G are data demonstrating that pre-clinical Fas agonist antibodies do not engage the PPCR region of Fas. FIG. 4A depicts the results of a Jurkat cell killing assay of different Fas antibodies and dabigatran Fc-conjugated FasL. FIG. 4B depicts the results of a cell survival assay using OVCAR-3 cells treated with the indicated Fas agonist + / - cFLIP inhibitor. The DR5 agonist KMTR2 and FasL are positive controls (n=3). FIG. 4C is an immunoblotting analysis of total cFLIP. FIG. 4D and FIG. 4E illustrate the surface model interface of E09 Fas antibody against the Fas PPCR. VH (green) and VL (gold) are shown as space-filling models, while Fas is represented by the ribbon structure. FIG. 4F is a sequence alignment of human Fas PPCR for alanine screen studies. FIG. 4G illustrates the binding of indicated His-tagged FasL mutants to immobilized E09 IgGl by ELISA.

[0041] FIG. 5A - FIG. 5B illustrate the binding kinetics of immobilized his-tagged FasR (wild-type) and FasR with a R87A substitution against E09 IgGl.

[0042] FIG. 6A - FIG. 6F depict known Fas agonist antibodies. These antibodies do not engage Fas PPCR. FIG. 6A is a schematic showing the construction of Fas agonist bispecific antibodies and antibody ligand bispecific combinations. The antibodies of FIG. 6A were expressed and purified. FIG. 6C are the results of a ovarian cancer OVCAR-3 cell killing assay of Fas agonist monospecific antibodies, Fas agonist bispecific antibodies, and antibody ligand bispecific combinations. FIG. 6D - FIG. 6F depict ovarian tumor efficacy data of the antibodies of FIG. 6A.

[0043] FIG. 7A - FIG. 7J show the generation cells expressing human FasR and anti-FOLRl CAR-T cells. FIG. 7A is a schematic of chimeric FasR with a human extracellular domain and a murine transmembrane domain, intracellular domain, and signal peptide. FIG. 7B is immunoblotting confirmation of murine ovarian ID8 cell clones expressing the human Fas extracellular domain. FIG. 7C illustrates E09 IgGl flow cytometry confirmation of murine ID8 cells expressing the human Fas extracellular domain. FIG. 7D is confirmation of cytotoxic sensitivity of chimeric human Fas stable murine ID8 cells against Fas agonist reagents. FIG. 7E is a schematic of anti-FOLRl (farletuzumab)-expressing CAR-T cell. FIG. 7F is confirmation by immunoblotting of the anti-FOLRl CAR expression in T-cells using a CD3C antibody. FIG. 7G is flow cytometry data using a goat anti-human Fab-specific antibody after two rounds of lentiviral transfections to express the anti-FOLRl CAR. FIG. 71 - FIG. 7J depict survival and granzyme B data of FOLR1-CAR T-cells co-cultured either alone or with OCCAR-3 cells or ID8 cells, or with ID8 human Fas-stable cells at an effector / target (E / T) ration of 5 : 1 for 4 hours. FIG. 8A - FIG. 8F presents data that demonstrates that residue R87 of the PPCR region is critical for CAR-T bystander killing. FIG. 8A depicts the cells (ID8 cells not expressing human Fas) and OVCAR-3 ovarian cancer cells used in the experiments with anti-FOLRl CAR-T cells. FIG. 8B depicts immunoblotting analysis of the total cell lysates, probing for cleaved caspase-8 levels with or without FasR expression. FIG. 8C depicts the results of a granzyme assay. FIG. 8D illustrates a similar experiment seen in FIG. 8A but the ID8 cells stably-expressed human FasR. FIG. 8E depicts immunoblotting of cells for cleaved and uncleaved caspase-8 while FIG. 8F depicts green fluorescent protein (GFP) signal analyzed by FACS. Recombinant wild-type FasR or R87A FasR (5 ug / mL) controls were used prior to co-culturing.

[0044] FIG. 9A - FIG. 9B demonstrates that avidity is critical for CAR-T bystander killing. FIG. 9A is immunoblotting analysis of caspase-8 following co-culturing of a 50:50 mix of ID8 cells that stably expressed human Fas and OVCAR-3 cells along with anti-FOLRl -CAR-T cells in plates either precoated with wild-type FasR or FasR87A. FIG. 9B depicts the results of a caspase activity assay kit in the co-cultures indicated in FIG. 9A.

[0045] FIG. 10A - FIG. 10C illustrates differential N-linked glycosylation patterns of FasR in tumors vs. T-cells. FIG. 10A is a ribbon structure of FasR showing a patch of positively charged residues (PPCR). FIG. 10B is a schematic of FIG. 10A. FIG. 10C shows total cell lysates of T-cells enriched from human PBMCs of 2 different donors. Immunoblotting for FasR shows that ovarian cancer cells predominantly express aglycosylated FasR.

[0046] FIG. 11 A - FIG. 11 E illustrates the rational engineering design and tumor-specific working mechanism of TFTab. FIG.11 A is a surface view and FIG. 1 IB is a schematic view of FasR. The PPCR epitope in the middle of FasR is shielded with hyperglycosylated sugars. FIG. 11C-11E show FasR either partially glycosylated (FIG. 11C) or aglycosylated (FIG. HD), allowing full accessibility to the TFTab (shown in FIG.11 E).

[0047] FIG. 12A - FIG. 12B is data demonstrating that the newly engineered TFTab specifically engages residue R87 in FasR. FIG. 12A is the ribbon structure of FasR showing wild-type (left) and PPCR mutant R86A and R87A FasR. FIG. 12B depicts the binding of Fc-tagged E09 IgG and TFTab IgG against His-tagged wild-type FasR or FasR87Aby ELISA.

[0048] FIG. 13A - FIG. 13C is data demonstrating that the TFTab antibody is selectively cytotoxic to tumor cells but not to T-cells. FIG. 13A is a schematic showing the constructs of a FOLRl-FasR targeting bispecific antibody (Farle-TFT bispecific; top) and a FOLRl-Fas ligand bispecific combination (Farle-FasL; bottom). FIG. 13B are the results of an OVCAR-3 cell killing assay of Farle-FasL and Farle-TFT along with controls against a panel of patient- derived xenograft cell lines. FIG. 13C are the results of a cell killing assay of 0VCAR3 tumor cell and human T-cells (CD3 -enriched from two donors) using Farle-FasL and Farle-TFT antibodies.

[0049] FIG. 14A - FIG. 14D depict data confirming N-linked glycosylation in TFTab. The two left lanes of FIG. 14A and FIG. 14B show E09 and TFTab IgGl with intact N297 glycosylation sites in the CH2 domain. Lanes 4, 5 and 6 show TFTab generated with the N297A mutation. Lane 4 is wild-type TFTab with the NYS motif near CRD3. Lane 5 and 6 is TFTab with NYS-NYG and NYS-NYI mutations. The gel of FIG. 14A was stained with a glyco-specific stain and the same gel (FIG. 14B) was stained with Coomassie blue. FIG. 14C and FIG. 14D are overlaid ion chromatograms of the N-glycans in sample TFT-IgGl NYS- NYI mutant (FIG. 14C) and a sample of N297A TFT-IgGl (FIG. 14D).

[0050] FIG. 15A - FIG. 15C demonstrates that the TFT antibody displays limited binding to T-cells. FIG. 15A is an immunoblot detecting FasR. T-cell samples demonstrated hyperglycosylated FasR. FIG. 15B is fly cytometry data of the TFT antibody against 0VCAR3 and T-cells. FIG. 15C confirmed N-linked glycosylation of recombinant FAs-IgG-Fc by asparagine (N) mutation at 102 and 120 (lanes 1-4) plus also via treatment of PNGaseF (lanes 5 and 7).

[0051] FIG. 16A - FIG. 16C is data that demonstrates that removal of N-linked glycans sensitized T-cells to TFT antibody cytotoxicity. FIG. 16A is a schematic of the experiment described in FIG. 16B. FIG. 16B is immunoblotting for FasR in CD3-enriched T-cells cultured in the presence of tunicamycin showing increased non-glycosylated FasR compared to glycosylated Fas. FIG. 16C are the results of a cell killing assay of human T-cells (CD8 enriched) + / - tunicamycin-treated in the presence of Farle-FasL and Farle-TFT antibodies. T- cell cytotoxicity is evident with Farle-TFT antibody only in presence of tunicamycin.

[0052] FIG. 17A - FIG. 17C demonstrate the N102 glycan of FasR forms multiple hydrogen bonds at PPCR interface FasR. FIG. 17A shows that the alpha-fold molecular dynamic (MD) simulation studies confirm hydrogen bonding of glycan atoms at and near R87 residue of FasR. FIG. 17B illustrates a magnified part of FIG. 17A showing R87-glycan hydrogen bonding. FIG. 17C illustrates a representation of MD simulation data summarizing total H-bonds due to N-linked glycosylation against full length protein (red) and total H-bonds due to N-linked glycosylation near PPCR region only.

[0053] FIG. 18A - FIG. 18C illustrate MD simulation studies of glycosylated and nonglycosylated FasR. FIG. 18A are schematics showing pairwise RMSD values. FIG. 18B are heat maps of dynamic cross-correlation matrix analysis. FIG. 18C depicts principal component trajectory analysis of glycosylated versus non-glycosylated FasR.

[0054] FIG. 19A - FIG. 19B depicts data demonstrating the caspase-9 and anti-tumor related activities of the bispecific antibodies. FIG. 19A shows caspase-8 activity after FasL treatment in 0VCAR3 and T-cells done side by side after the indicated time. FIG. 19B shows the antitumor activity of indicated molecules (right) against OVCAR-3 tumor grafts. All antibodies were generated with ADCC mutation in CH2 residues 234 and 235 positions.

[0055] FIG. 20A - FIG. 20B illustrate TFTab’s PPCR interface confirming the role saltbridges to achieve the optimal Fas cytotoxic activity. FIG. 20A is a schematic showing the beta strand and the CDR3 loop of TFT VH and FasR residues near the PPCR. FIG. 20B is data from a cell killing assay of OVCAR-3 cells treated with either wild-type Farle-TFT or Farle-OTFT with E-A mutations in the CDR3 loop. Farle-FasL is for comparison is shown in the left. IC50 values are on the top.

[0056] FIG. 21A - FIG. 21C illustrate that low expression of ALG12 is associated with a worse survival outcome. FIG. 21A is a graph showing the % cases affected with various ALG family gene loss and gain in copy number variations in ovarian and uterine cancers. FIG. 21B- 21C shows ALG expression-dependent comparison of disease-free survival of ovarian and uterine cancer patients vs. glioma, breast cancer and lung cancer patients. With ALG12 loss, only ovarian and uterine cancer patients showed a highly significant difference.

[0057] FIG. 22 depicts immunoblots showing that over 75% of tested ovarian cancer patient tissue showed ALG 12 downregulation regardless of cisplatin resistance.

[0058] FIG. 23A - FIG. 23F illustrate the specificity of TFTab to tumor cells via aglycosylated FasR. FIG. 23A and FIG. 23B are immunoblots of 0VCAR3 or Hey-A8 cells cultured in either complete media (CM) or serum-free media (SF) and probed for FasR glycosylation status. FIG. 23C illustrates that the addition of tunicamycin decreases the amount of glycosylated FasR in Hey-A8 cells. FIG. 23D is a graph illustrating a survival assay of Hey-A8 cells in the presence of E09 anti-FasR antibody, TFTab, or FasL. FIG. 23E demonstrates that in the presence of a bispecific TFT molecule, or a bispecific molecule comprising the binding site of E09, the TFT-bispecific was significantly more successful at killing Hey-A8 cells. FIG. 23F shows that TFTab is only effective in Hey-A8 cells in the presence of Tunicamycin, which results in aglycosylated FasR.

[0059] FIG. 24A - FIG. 24C illustrate the angles achieved by the extracellular and transmembrane domains of glycosylated and non-glycosylated Fas relative to the plasma membrane. FIG. 24A is an illustration of tilt angle. FIG. 24B is a graph illustrating the measured tilt angles of domain 1. FIG. 24C is a graph illustrating the measured tilt angles of domain 2.

[0060] FIG. 25 Top and Bottom illustrate two extreme orientations of non-glycosylated Fas. FIG 25 Top is an illustration of the lying down orientation of non-glycosylated Fas. FIG. 25 Bottom is an illustration of the standing orientation of non-glycosylated Fas.

[0061] FIG. 26 Top and Bottom illustrate two extreme orientations of glycosylated Fas. FIG 26 Top is an illustration of the lying down orientation of glycosylated Fas. FIG. 26 Bottom is an illustration of the standing orientation of glycosylated Fas.

[0062] FIG. 27A - FIG. 27C illustrate the RMSD value of glycosylated Fas vs. non- glycosylated Fas. FIG. 27A is a PDB ribbon structure of Fas showing PPCR (red) and glycosylation sites (teal). FIG. 27B is a graph showing the RMSD value of glycosylated (blue) Fas vs non-glycosylated (yellow) Fas over time (n=3). FIG. 27C is a graph of the flexible distance of non-glycosylated (blue) Fas from receptor molecules center of mass (COM) compared to glycosylated (green) Fas.

[0063] FIG. 28 is a heat map illustrating the contact frequency analysis between the groups of NTD residues with the TFT antibody PPCR residues. The contact frequency horizontal bar explains the color coding in the contact map. A value of 1 indicates that the specific pair of residues is in contact in every frame of the trajectory, while a value of 0 means the residues are never in contact. Values between 0 and 1 represent the fraction of frames in which the residues are in contact. As evident in the bottom heatmap, glycosylated Fas values are close to 1 almost all the time, while non-glycosylated values are not consistent.

[0064] FIG. 29A - FIG. 29B are graphs of the contact occurrence frequency between NTD and the PPCR epitope. FIG. 29A is a graph showing the high frequency contacts that occur in at least 20% of frames for glycosylated and non-glycosylated FAS. FIG. 29B is a graph showing the high frequency contacts that occur in at least 60% of frames for glycosylated FAS.

[0065] FIG. 30A - FIG. 30C illustrate the steric hinderance of NTD against PPCR epitope. FIG. 30A illustrates that no salt bridges are evident between Asp39 (D39) of the NTD (blue) and Arg86 (R86) of the PPCR (red) in non-glycosylated FAS. FIG. 30B illustrates that salt bridges (<5 Armstrong) are evident between Asp39 (D39) of the NTD (blue) and Arg86 (R86) of the PPCR (red) in glycosylated FAS. FIG. 30C illustrates 50 different trajectories that demonstrate the close interaction of NTD domain (blue), which sterically interferes with R86 and R87 (PPCR epitope) accessibility with a large size 150Kda antibody vs small size 60Kda FasL trimer. FIG. 31A - FIG. 31E illustrate the amenable system of Hey-A8 ovarian cells for confirming TFT killing specificity toward non-glycosylated Fas. FIG. 31A - FIG. 31B are immunoblots showing that trypsinized or scraped HeyA8 cells express a glycosylated form of Fas in complete media (CM), HeyA8 cells in serum-free media (SF) express a non-glycosylated form of Fas, and 0VCAR3 cells express a non-glycosylated form of Fas in both conditions. FIG. 31B is similar to FIG. 31A except an increasing concentration of lysates was used. FIG. 31C is an immunoblot showing that Tunicamycin drug also results in a higher non-glycosylated form of Fas in HeyA8 cells. FIG. 31D Top shows color-coded antibodies used in in vitro experiment in FIG. 31D Bottom. FIG. 31D Bottom is a graph showing that preclinical Fas IgGl E09 does not activate cell death of HeyA8 cells in SF conditions, while TFT IgGl does. Other FasL controls and CM media controls are included. FIG. 3 IE Left shows a bispecific engager confirmation of TFT scFv or E09 scFv with DB scFv (a random control antibody targeting Paradexa drugs). FIG. 31E Right is a graph showing that the bispecific engager confirmation of TFT scFv only eliminates HeyA8 cells in SF conditions but not in CM conditions.

[0066] FIG. 32A - FIG. 32C illustrate anti-tumor efficacy and animal survival data for HeyA8 xenografts. FIG. 32A illustrates antibodies used in in vivo experiment in FIG. 32B and FIG. 32 C. FIG. 32B shows each antibody’s effect on HeyA8-induced tumor size in the presence and absence of Tunicamycin drug. FIG. 32C is a graph of Kaplan Meier survival analysis data: the probability of animals survival over time grafted with Hey-A8 tumors is significantly higher (average 80 days) when treated with Farle-TFT + tunicamycin (which makes Fas epitope available for antibody) compared to Farle-TFT- tunicamycin (which keep Fas epitope hidden due to glycosylation).

[0067] FIGs. 33A-33E illustrate anti-tumor efficacy and animal survival data for chemoresistant xenografts. FIG. 33A are immunoblots of HeyA8 and chemoresistant cancer cell lines. FIGs. 33B - FIG. 33C illustrate mono- and bi-specific antibodies used in the in vivo experiment in FIG. 33D and FIG. 33E. FIG. 33D shows each antibody’s effect on PDX1031-induced tumor size. FIG. 32 E is a graph of Kaplan Meier survival analysis data. Farle-TFT treated mice survived an average of approximately 95 days, similar to Farle-FasL treated animals, which survived approximately 85 days. Farle-E09 treated animals only survived an average of approximately 50 days.

[0068] FIG. 34 depicts the extracellular domain sequence of FasR: R86, R87, shown in blue, which is epitope targeting by the anti-PPCR antibody. N102 and N120, shown in red, are N- glycosylation sites. DETAILED DESCRIPTION

[0069] Provided herein are antibodies or antigen-binding fragments thereof that specifically bind the Fas receptor, and methods comprising said antibodies or antigen-binding fragments for treating a subject having a solid tumor.

[0070] I. Definitions

[0071] That the disclosure may be more readily understood, select terms are defined below.

[0072] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article.

[0073] “About” or “approximately” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, ±5%, ±1%, or ±0.1% of a given value or range, as such variations are appropriate to perform the disclosed methods.

[0074] As used herein, the terms “Fas receptor” or “FasR” refer to a transmembrane protein, also known as “CD95” or “tumor necrosis factor receptor super family member 6” or “TNFRSF6”. FasR is a death receptor on the surface of cells that leads to programmed cell death (apoptosis) if it binds its ligand, Fas ligand (FasL). The extracellular domain of FasR comprises some or all of the amino acid sequence of SEQ ID NO: 10. In some embodiments, the extracellular domain of FasR comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0075] As used herein, the term “PPCR” refers to a “patch of positively charged residues.” This motif (amino acids Cys-Arg-Arg-Cys-Arg; CRRCR) is highly conserved and can be found in the cysteine-rich domain 2 (CRD2) of FasR (Mondal, et al. Cell Death Differ. 2023. 30(11):2408-2431. doi: 10.1038 / s41418-023-01229-7, incorporated herein in its entirety). FasL interacts with the FasR by multiple low-affinity interactions upstream, downstream, and within the PPCR epitope throughout the receptor extracellular domain.

[0076] As used herein, the term “glycosylation” refers to the process by which a carbohydrate is covalently attached to a target molecule. This modification to a protein residue may be a co- translational and / or a post-translational modification. The carbohydrate may be covalently attached to the nitrogen of an asparagine (Asn, N) or an arginine (Arg, R) in the event of N- linked glycosylation, or to the hydroxyl oxygen of a serine (Ser, S), threonine (Thr, T), tyrosine (Tyr, Y), hydroxylysine (Hyl), or hydroxyproline (Hyp) in the event of (9-linked glycosylation. Glycosylation may serve many purposes, e.g., allows for the proper folding of a protein, provides stability, and cell-to-cell adhesion. In some cases, glycosylation plays an important role in the optimization of antibodies. In some embodiments, N102 and / or N120 of FasR are glycosylated on T-cells. In some embodiments, glycosylation of N102 and / or N120 of FasR partly shield hydrogen bonds with the FasR PPCR region. In some embodiments, the shielding prevents access to the PPCR region by anti-FasR PPCR antibodies. In some embodiments, N102 and / or N120 of FasR are non-glycosylated or hypoglycosylated (e.g., limited glycosylation) in tumor cells. In some embodiments, non-glycosylation or hypoglycosylation ofN102 and / or N120 of FasR allows for anti-FasR PPCR binding to FasR. Standard laboratory assays such as immunohistochemistry (IHC) studies of tumor tissues + / - PNGase F or flow cytometry studies or Mass-Spec analysis could be used to determine Fas N-glycosylation status in patient tumors. In certain exemplary embodiments, the patients expressing non- or hypoglycosylated Fas would be targeted by the TFT antibody.

[0077] As used herein, the term “Chimeric antigen receptor T-Cell” or a “CAR-T cell” is a T- cell that expresses a “chimeric antigen receptor” or a “CAR”. Typically, a CAR comprises a tumor associated-antigen recognition or binding moiety, a hinge, a transmembrane domain, one or more intracellular signaling domain, and a CD3 signaling domain. In some embodiments, the tumor associated-antigen recognition or binding moiety is an antibody or an antigen-binding fragment thereof.

[0078] As used herein, the term “bystander killing” refers to the phenomenon where targeted tumor cells are destroyed along with bystander tumor cells, regardless of whether they express and antigen.

[0079] As used herein, the term “bispecific binding protein” refers to a binding protein that specifically binds to two different antigen targets (e.g., FasR and a tumor-associated antigen) through at least two distinct antigen-binding domains.

[0080] As used herein, the terms “specifically binds to” or “binds specifically to” refers to the ability of an antigen-binding domain to bind to an antigen (e.g. FasR and / or a tumor-associated antigen) containing an epitope with an Kd of at least about 1 x 10-6 M, 1 x 10-7 M, 1 x 10-8 M, 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M, 1 x 10-12 M, or more, and / or to bind to an epitope with an affinity that is at least twofold greater than its affinity for a nonspecific antigen.

[0081] Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art. Expressions such as “specifically binds to”, or “with specificity for” are used interchangeably. Those terms are not construed to refer exclusively to those antibodies, polypeptides and / or multichain polypeptides which actually bind to the recited target / binding partner, but also to those which, although provided in a nonbound form, retain the specificity to the recited target. Binding specificity can be quantitatively determined by an affinity constant KA (or KA) and a dissociation constant KD (or KD).

[0082] As used herein, the term “affinity”, concentration (EC50) or the equilibrium dissociation constant (KD) means the strength of the binding of an antibody or polypeptide to an epitope. The affinity of an antibody is given by a specific type of equilibrium constant, which is the dissociation constant KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant KA is defined by 1 / KD. Exemplary methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One exemplary and standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analytical device). In a non-limitative manner, a KD of less than 50 nM as determined by SPR, and under physiological conditions (e.g. at a pH ranging from 6 to 8 under normal buffer conditions), may generally be considered as indicative of specificity of binding for antigen-antigen binding domain (ABD) interactions.

[0083] As used herein, the term “and / or” is a grammatical conjunction that is to be interpreted as encompassing that one or more of the cases it connects may occur. For example, the wording "such native sequence proteins can be made using standard recombinant and / or synthetic methods" indicates that native sequence proteins can be made using standard recombinant and synthetic methods or native sequence proteins can be made using standard recombinant methods or native sequence proteins can be made using synthetic methods.

[0084] As used herein, the terms “therapeutically effective amount” of the multifunctional binding protein or pharmaceutical composition thereof is meant a sufficient amount of the antibody-like multifunctional binding protein to treat said cancer disease, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the polypeptides and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific polypeptide employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific polypeptide employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

[0085] As used herein, the term “subject” or “individual” or “patient” are used interchangeably and may encompass a human or a non-human mammal, rodent or non-rodent. The term includes, but is not limited to, mammals, e.g., humans including man, woman and child, other primates (monkey), pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.

[0086] As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a pharmaceutically acceptable carrier” encompasses a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

[0087] As used herein, “a plurality of’ may thus include two or two or more.

[0088] As used herein, “antibody” or “immunoglobulin” may refer to a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain generally includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). In particular, classes IgG, IgA, and IgD have three heavy chain constant region domains, which are designated CHI CH2, and CH3; and the IgM and IgE classes have four heavy chain constant region domains, CHI, CH2, CH3, and CH4. The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the antigen-binding fragment (Fab) of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.

[0089] As used herein, the term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences as described, for example, by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0090] The human antibody can have at least one or more amino acids replaced with an amino acid residue, e.g. , an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. Typically, the human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human germline immunoglobulin sequence. In a particular embodiment, these replacements are within the CDR regions as described in detail below.

[0091] An “antigen” is an entity (e.g., a proteinaceous entity or peptide) to which an antibody or antigen-binding portion thereof binds. In various embodiments of the present invention, an antigen is Fas receptor (FasR), e.g., as presented on a cell (e.g., a FasR-positive cancer cell).

[0092] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). As used herein, when referring to “IgG” or “Immunoglobulin G” in general, IgGl, IgG2, IgG3 and IgG4 are included, unless defined otherwise. In particular, IgG is IgGl.

[0093] As used herein, the term “antibody-like” or “immunoglobulin-like” polypeptide may also refer to non-conventional or synthetic antigen-binding polypeptides or binding protein, including single domain antibodies and fragments thereof, in particular variable heavy chain of single domain antibodies, and chimeric, humanized, bispecific or multimeric antibodies.

[0094] As used herein, the term “domain” may be any region of a protein, generally defined on the basis of sequence homologies or identities, which is related to a specific structural or functional entity. Accordingly, the term “region”, as used in the context of the present disclosure, is broader in that it may comprise additional regions beyond the corresponding domain.

[0095] As used herein, the term “Fc region” or “fragment crystallizable region”, or alternatively “Fc portion”, encompasses all or parts of the “Fc domain”, which may thus include all or parts of an immunoglobulin hinge region (which natively bears a first binding site to FcyRs), a CH2 domain (which natively bears a second binding site to FcyRs), and a CH3 domain of an immunoglobulin (e.g. of an IgG, IgA or IgD immunoglobulin), and / or when applicable of a CH4 domain of an immunoglobulin (e.g. for IgM and IgE). In certain embodiments, the Fc region includes all or parts of, at least, a CH2 domain and a CH3 domain, and optionally all or parts of an immunoglobulin hinge region. The term may thus refer to a molecule comprising the sequence of a non-antigen-binding fragment resulting from digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and can contain the hinge region. The original immunoglobulin source of the native Fc is, in particular, of human origin and can be any of the immunoglobulins, although IgGl are particularly exemplary. Native Fc molecules are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 13 depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgGl, IgG2, lgG3, IgGAl, and IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms. Under that terminology, a “Fc region” may thus comprise or consist of CH2-CH3 (e.g., (CH2-CH3)A or (CH2-CH3)B or a binding pair thereof, and optionally all or part of an immunoglobulin hinge region, comprising a binding site to a human FcyR. Unless specified otherwise, the term “Fc region” may refer to either a native or variant Fc region. The term “Fc variant” as used herein refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term “Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues has be modified, that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0096] The fragment crystallizable (Fc) regions (e.g., native or variant) according to the present disclosure retain a capacity to bind to a human Fc-y receptor polypeptide (Fey) which generally occurs on native Fc regions through binding of the antibody Fc-hinge region. As a reference, overall structures of IgGl, IgG2, and IgG4 are similar with more than 90% sequence homology, the major differences residing in the hinge region and CH2 domain, which form primary binding sites to FcyRs. The hinge region also functions as a flexible linker between the Fab and Fc portion.

[0097] Fc regions having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions, which modifications increase the affinity and avidity of the variant Fc region for an FcyR (including activating and inhibitory FcyRs) are further considered as Fc regions. In some embodiments, said one or more amino acid modifications increase the affinity of the Fc region for FcyRIIIA and / or FcyRIIA. In another embodiment, the variant Fc region further specifically binds FcyRIIB with a lower affinity than does the Fc region of the reference parent antibody (e.g., an antibody having the same amino acid sequence as the antibody except for the one or more amino acid modifications in the Fc region). Hence, native and variant Fc regions considered herein generally comprise a domain (i.e. , a CH2 domain) capable of binding to human CD16, e.g., a human Fc domain comprising N-linked glycosylation at amino acid residue N297 (according to EU numbering).

[0098] As used herein, the term “Fc-competent” thus refers to a binding protein that is capable of binding specifically to a FcyR, in particular of an activating FcyR, in particular to one selected from FcyRI (CD64a), FcyRIIa (CD32a), and FcyRIIIa (CD16a), and more particularly to FcyRIIIa (CD 16a). Alternatively, several modifications are reported to directly affect the binding to FcyRs, including mutation on residues 297 (according to EU numbering), or alternatively on residues 234 and 235 in the lower hinge region (according to the EU numbering system).

[0099] As used herein, the term “Fc-silent” refers to a binding protein with a Fc region, wherein the Fc region lacks a binding site to a FcyR (e.g., a Fc region lacking a CH2 domain with said binding site and hinge region with said binding site); in particular FcyRI, FcyRIIa, and FcyRIIIa, and more particularly to FcyRIIIa (CD16a).

[0100] As used herein, the term “variable”, as in “variable domain”, refers to certain portions of the relevant binding protein which differ extensively in sequence between and among antibodies and are used in the specific recognition and binding of a particular antibody for its particular target. However, the variability is not evenly distributed throughout the entire variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) also known as hypervariable regions, both in the light chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework (FR) regions or sequences.

[0101] As used herein, the term “VH domain,” or “VH domain” can be used interchangeably and refer to the corresponding heavy chain immunoglobulin variable domain.

[0102] As used herein, the term “VL domain”, or “VL domain” can be used interchangeably and refer to the corresponding light chain immunoglobulin variable domain.

[0103] When the VH or VL domains are associated to a first antigen-binding domain (ABD) or to a second antigen-binding domain, they may also be respectively referred herein as “VH1” and “VL1”, or “VH2” and “VL2”.

[0104] The terms “binding pair V (VH / VL)”, “VH / VL pair” or “(VH / VL) pair” or “VL / VH pair” or “(VL / VH) pair” can be used interchangeably. Heavy chain and light chain variable domain can pair in parallel to form the antigen binding domains (ABDs). Each binding pair includes both a VH and a VL region. Unless instructed otherwise, these terms do not specify which immunoglobulin variable regions are VH or VL regions and which ABD will bind specifically the protein expressed on the surface of an immune effector cell or a target cell (e.g., NKp46 and CD 123).

[0105] As used herein, the term “single chain variable fragment” (scFv) refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g. , with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0106] As used herein, the terms “antibody fragment” or “antigen binding fragment” refer to a fragment of an antibody. In some embodiments, the antibody fragment is an antigen binding fragment. In some embodiments, the term antigen binding fragment refers to the fragment of an antibody that binds to a target antigen. In some embodiments, the antigen binding fragment is an scFv. In some embodiments, the antigen binding fragment is a VHH (e.g., a single domain antibody or sdAb; nanobody), a Fab, a F(ab’)2, or a Fab’. In some embodiments, the antigen binding fragment comprises three heavy chain complementarity determining regions, or CDRs. In some embodiments, the antigen binding fragment comprises three heavy chain CDRs and three light chain CDRs. In some embodiments, the antibody fragment is a Fc domain. The Fc domain, or the fragment crystallization region, is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. The Fc domain allows antibodies to activate the immune system, for example, through binding to the Fc receptors. In some embodiments, glycosylation of the Fc region is essential for Fc receptor-mediated activity. In some embodiments, it is desirable to have aglycosylated Fc domains.

[0107] As used herein, the term “hypervariable region’ when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. This term may be substituted by the terms “Complementarity Determining Regions” or “CDRs”.

[0108] Thus, as used herein “Complementarity Determining Regions” or “CDRs” refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR-L1, CDR-L2, CDR-L3 and CDR - Hl, CDR-H2, CDR-H3, respectively. A conventional antibody antigen-binding domain, therefore, includes six CDRs, comprising the CDR set from each of a heavy and a light chain variable region. Also, as used herein, “Framework Regions” (FRs) refer to amino acid sequences interposed between CDRs, i.e., to those portions of immunoglobulin light and heavy chain variable regions that are relatively conserved among different immunoglobulins in a single species. The light and heavy chains of an immunoglobulin each have four FRs, designated FR-L1, FR-L2, FR-L3, FR-L4, and FR-H1, FR-H2, FR-H3, FR-H4, respectively. Accordingly, the light chain variable domain may thus be designated as (FR-Ll)-(CDR-Ll)- (FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4) and the heavy chain variable domain may thus be designated as (FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H)-(FR4-H3).

[0109] The hypervariable region generally comprises amino acid residues from a “complementarity-determining region” or “CDR” (e.g. residues 24-34 (LI ), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991) and / or those residues from a "hypervariable loop" (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (Hl ), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). The numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Accordingly, phrases such as “Kabat position”, “variable domain residue numbering as in Kabat” and “according to Kabat” herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0110] Optionally, CDRs are as defined by EU, Kabat, Chotia or IMGT numbering. Correspondences between those classifications are known in the Art, by reference to the IMGT®, or international ImMunoGeneTics information system® (CNRS and Montpellier University), and as further detailed in Lefranc (Biomolecules; 2014; 4, 1102-1139) and Dondelinger (Frontiers in Immunology; 2018; 9, 2278). CDRs may also be defined according to the Honegger-Pluckthun (“Honegger”) numbering scheme described in Honnegger and Pluckthun (2001), J. Mol. Biol., vol. 309(3):657-670.

[0111] Unless instructed otherwise, the numbering of residues will be considered herein by reference to the EU, Kabat, Chothia, IMGT, or Honegger-Pluckthun numbering convention. In case of conflict regarding the exact position of hypervariable regions within a reference sequence, the Kabat numbering convention will prevail. In case of conflict regarding the exact position of constant regions within a reference sequence, the EU numbering convention will prevail. In some embodiments, the term “linker” refers to 1 - 100 contiguous amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or between two polypeptide domains. A linker may be inserted between VH, VL, CH and / or CL domains to provide sufficient flexibility and mobility for the domains of the light and heavy chains depending on the format of the molecule. A linker is typically inserted at the transition between variable domains between variable and knockout domain, or between variable and constant domains, respectively, at the amino sequence level. The transition between domains can be identified because the approximate sizes of the immunoglobulin domains are well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements such as beta-sheets or alpha-helices as demonstrated by experimental data or as can be determined by techniques of modeling or secondary structure prediction.

[0112] As used herein, the term “cytotoxicity” refers to the quality of a compound, such as the multifunctional binding protein according to the present disclosure, to be toxic to tumoral cells as measured by target cell lysis. Cytotoxicity may be induced by different mechanisms of action and can thus be divided into cell-mediated cytotoxicity, apoptosis, antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC).

[0113] As used herein, the term “antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane-surface antigens have been bound by specific antibodies or the multifunctional binding protein of the present disclosure.

[0114] As used herein, a “pharmaceutically acceptable carrier” is intended to include any and all carrier (such as any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like) which is compatible with pharmaceutical administration, in particular parenteral administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the present disclosure. For example, preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In a non-exhaustive manner, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM (e.g., 0.05M) phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will in an embodiment be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In certain embodiments, isotonic agents are included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0115] As used herein, and unless instructed otherwise, the term “at least one” may encompass “one or more,” or even “two or more” (or “a plurality”). For instance, it may encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

[0116] 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

[0117] 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

[0118] 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than

[0119] 100.

[0120] As used herein, and unless instructed otherwise, the term “less than” may encompass all values from 0 to the corresponding threshold, For instance, it may encompass less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or less than 100, when applicable.

[0121] As used herein, the terms “treat”, “treating”, and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject (e.g., a subject in need thereof), an anti-FasR antibody or antigen binding portion as described herein. In certain embodiments the subject is a subject diagnosed with and / or under treatment for a cancer in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0122] As used herein, “preventing” means a prophylactic use (i.e. , on a subject susceptible of developing a given disease and encompasses the treatment of a cancer patient).

[0123] II. Fas Receptor Antibodies

[0124] Provided herein are antibodies or antigen-binding fragments thereof that bind to the Fas receptor (FasR). In some embodiments, the anti-FasR antibodies are agonistic antibodies. In some embodiments, the antibody or antigen-binding fragments thereof comprises three heavy chain complementary determining region (CDR) sequences and three light chain CDR sequences. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2, and 3, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 13, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 14, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 15, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 16, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 17, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 18, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 19, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 20, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 21, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 22, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 23, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 24, respectively. In some embodiments, the three heavy chain CDR sequences comprise SEQ ID NO: 1, 2 and 25, respectively. In some embodiments, the three light chain CDR sequences comprise SEQ ID NO: 4, 5, and 6, respectively.

[0125] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDR sequences and three light chain CDR sequences comprising: SEQ ID NOs: 1, 2 and 3 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 13 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2, and 14 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2, and 15 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2, and 16 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 17 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 18 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 19 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 20 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 21 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 22 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 23 and SEQ ID NOs: 4, 5 and 6, respectively; SEQ ID NOs: 1, 2 and 24 and SEQ ID NOs: 4, 5 and 6, respectively; or SEQ ID NOs: 1, 2 and 25 and SEQ ID NOs: 4, 5 and 6, respectively.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain CDR sequences and three light chain CDR sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the CDR sequences are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 1, 2, 3, 4, 5 and / or 6.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence and a light chain variable region (LCVR) sequence. In some embodiments, the HCVR sequence is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least

[0128] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0129] 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the HCVR sequence comprises an amino acid sequence of SEQ ID NO: 7. In some embodiments, the

[0130] LCVR sequence is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least

[0131] 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0132] 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to

[0133] SEQ ID NO: 8. In some embodiments, the LCVR sequence comprises an amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequences of the CDRs, the HCVR, and / or the LCVR may comprise substitutions. In some embodiments, the substitutions may be conservative amino acid substitutions. In some embodiments, the amino acid sequence may comprise at least one conservative amino acid substitution. For example, the HCVR and / or the LCVR can differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid substitutions. In some embodiments, the conservative amino acid substitution(s) can occur in the CDR1, CDR2, and / or CDR3 regions. As used herein, the term “conservative amino acid substitution” refers to an amino acid substitution that functionally performs similar to the original amino acid that the conservative amino acid substitution is similar to. With regard to amino acids, a similar amino acid may refer to the structure and / or the charge of the amino acid. An example of conservative substitution is the exchange of an amino acid in one of the following groups for another amino acid of the same group ( U.S. Patent No. 5,767,063 ; Kyte and Doolittle, J. Mol. Biol. 157: 105-132 (1982)):

[0134] (1) Hydrophobic: Norleucine, Ile, Vai, Leu, Phe, Cys, Met;

[0135] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0136] (3) Acidic: Asp; Glu;

[0137] (4) Basic: Asn, Gin, His, Lys, Arg;

[0138] (5) Residues that influence chain orientation: Gly, Pro;

[0139] (6) Aromatic: Trp; Tyr; Phe;

[0140] (7) Small amino acids: Gly, Ala, Ser.

[0141] For example, substitutions can be made by changing, e.g., Vai to Leu; Ser to Thr; or Asp to Glu. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, when it is desired to alter the pKa of an amino acid side chain while retaining the size and structure of the side chain, Glu may be substituted by Gin, and Asp may be substituted by Asn

[0142] In some embodiments, the antibodies bind to a non-glycosylated (or aglycosylated) or hypoglycosylated FasR. In some embodiments, tumor or cancer cells comprise the aglycosylated or hypoglycosylated FasR. In some embodiments, the antibody or antigenbinding fragment thereof binds Fas receptor expressed on a tumor cell with a higher affinity than Fas receptor expressed on a T cell. In some embodiments, the anti-FasR antibody recognizes tumor or cancer cells. In some embodiments, the anti-FasR antibody does not recognize immune cells (e.g., T-cells). In some embodiments, the anti-FasR antibody or antigen-binding fragments thereof specifically binds to a FasR comprising the amino acid sequence of SEQ ID NO: 10.

[0143] In some embodiments, the Fas receptor is expressed on an ovarian cancer cell or other solid tumor cancer cells. In some embodiments, the solid tumor cancer cell includes, but is not limited to, triple-negative breast cancer, lung cancer, or brain cancer (e.g., glioblastoma multiforme).

[0144] Also provided herein is a single chain variable fragment (scFv) that consists of a variable light chain followed by a GS linker and a variable heavy chain. In some embodiments, the target of the scFv is expressed on solid tumor comprising ovarian and other cancers.

[0145] In some embodiments, the antibodies of the disclosure can be provided in various isotypes and with different constant regions. The Fc region of the antibodies primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity, and antibody-dependent cell phagocytosis (ADCP) activity. These “cellular effector functions”, as distinct from effector T cell function, involve the recruitment of cells bearing Fc receptors to the site of the target cells, resulting in killing of the antibody-bound cell.

[0146] An antibody as disclosed herein may be one that exhibits reduced effector function. In some embodiments, the one or more mutations reduces one or more of antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), or complement dependent cytotoxicity (CDC). In certain embodiments, an antibody according to the present invention may lack ADCC, ADCP and / or CDC activity. In either case, an antibody according to the present invention may comprise, or may optionally lack, an Fc region that binds to one or more types of Fc receptor. Use of different antibody formats, and the presence or absence of FcR binding and cellular effector functions, allow the antibody to be tailored for use in particular therapeutic purposes as discussed elsewhere herein.

[0147] In some embodiments, the first and the second Fc domain comprise one or more mutations that reduces Fc effector function. In some embodiments, the first Fc domain and the second Fc domain each comprise a L234A and L235A mutation. These IgGl mutations are also known as the “LALA” mutations and are described in further detail in Xu et al. (Cell Immunol. 2000; 200:16-26). In some embodiments the first Fc domain and the second Fc domain each comprise a L234A, L235A, G237A, and / or P329G mutation. The Fc domain amino acid positions referred to herein are based on EU antibody numbering. Alternatively, an antibody may have a constant region which is effector null. An antibody may have a heavy chain constant region that does not bind Fey receptors, for example the constant region may comprise a L235E mutation. Another optional mutation for a heavy chain constant region is S228P, which increases stability. A heavy chain constant region may be an IgG4 comprising both the L235E mutation and the S228P mutation. This "IgG4-PE" heavy chain constant region is effector null. A disabled IgGl heavy chain constant region is also effector null. A disabled IgGl heavy chain constant region may contain alanine at position 234, 235 and / or 237 (EU index numbering), e.g., it may be an IgGl sequence comprising the L234A, L235A and / or G237A mutations ("LALAGA").

[0148] Human IgGl constant regions containing specific mutations or altered glycosylation on residue Asn297 (e.g., N297Q, N297D, and N297K, EU index numbering) have been shown to reduce binding to Fc receptors.

[0149] In other embodiments, it may be desirable to enhance the binding of the Fc region of a multispecific antibody to human Fc gamma receptor IIIA (FcgRIIIA) relative to that of the Fc region of a corresponding naturally occurring antibody. In certain embodiments, a constant region may be engineered for enhanced ADCC and / or CDC and / or ADCP. The potency of Fc- mediated effects may be enhanced by engineering the Fc domain by various established techniques. Such methods increase the affinity for certain Fc-receptors, thus creating potential diverse profiles of activation enhancement. This can be achieved by modification of one or several amino acid residues. Example mutations are one or more of the residues selected from 239, 332 and 330 for human IgGl constant regions (or the equivalent positions in other IgG isotypes). An antibody may thus comprise a human IgGl constant region having one or more mutations independently selected from S239D, I332E and A330L (EU index numbering).

[0150] Increased affinity for Fc receptors can also be achieved by altering the natural glycosylation profile of the Fc domain by, for example, generating under fucosylated or de- fucosylated variants. Non-fucosylated antibodies harbor a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcyRIIIA binding capacity. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to FcyRIIIA. Thus, an antibody may comprise a human IgG heavy chain constant region that is a variant of a wildtype human IgG heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region binds to human Fey receptors selected from the group consisting of FcyRIIB and FcyRIIA with higher affinity than the wild type human IgG heavy chain constant region binds to the human FcyRIIIA. The antibody may comprise a human IgG heavy chain constant region that is a variant of a wild type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human FcyRIIB with higher affinity than the wild type human IgG heavy chain constant region binds to human FcyRIIB. The variant human IgG heavy chain constant region can be a variant human IgGl, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system). In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (EU index numbering system).

[0151] The enhancement of CDC may be achieved by amino acid changes that increase affinity for C 1 q, the first component of the classic complement activation cascade. Another approach is to create a chimeric Fc domain created from human IgGl and human IgG3 segments that exploit the higher affinity of IgG 3 for Cl q. Antibodies of the present invention may comprise mutated amino acids at residues 329, 331 and / or 322 to alter the Clq binding and / or reduced or abolished CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain Fc regions with modifications at residues 231 and 239, whereby the amino acids are replaced to alter the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has a constant region comprising one or more mutations selected from E345K, E430G, R344D and D356R, in particular a double mutation comprising R344D and D356R (EU index numbering system).

[0152] The functional properties of the antibody may be further tuned by combining amino acid substitutions that alter Fc binding affinity with amino acid substitutions that affect binding to FcRn. Binding proteins with amino acid substitutions that affect binding to FcRn (also referred to herein as “FcRn variants”) may in certain situations also increase serum half-life in vivo as compared to an unmodified binding protein. As will be appreciated, any combination of Fc and FcRn variants may be used to tune clearance of the antigen-antibody complex. Suitable FcRn variants that may be combined with any of the Fc variants described herein that include without limitation N434A, N434S, M428L, V308F, V259I, M428L / N434S, V259I / V308F, Y436I / M428L, Y436I / N434S, Y436V / N434S, Y436V / M428L, M252Y, M252Y / S254T / T256E, and V259I / V308F / M428L. In some embodiments, the antibody or antigen-binding fragments as described herein can be engineered as a bispecific molecule. In some embodiments, the bispecific molecule can be bivalent (two binding domains), trivalent, or multivalent. In some embodiments, the bispecific molecule may comprise a first binding domain of a scFv, VHH, or a Fab. In some embodiments, the bispecific molecule may comprise a second binding domain of a scFv, VHH, or a Fab. In some embodiments, one or more of the binding domains may be a ligand or part of a ligand or protein known to bind to the target. In some embodiments, the bispecific molecule may comprise a Fc domain. In some embodiments, one of the binding domains specifically binds to FasR on a tumor cell. In some embodiments, the bispecific molecule binds to the PPCR epitope of FasR. In some embodiments, the other binding domain specifically binds to a tumor-associated antigen. In some embodiments, the tumor-associated antigen is associated with a solid tumor. In some embodiments, the solid tumor may be, but not limited to, ovarian cancer, uterine cancer, triple negative breast cancer, lung cancer, or brain cancer (e.g., glioblastoma multiforme). In some embodiments, the tumor-associated antigen maybe be selected from, but not limited to, folate receptor 1 (FOLR1), CD24, NaPi2b, epidermal growth factor receptor (EGFR), or MUC 16.

[0153] In some embodiments, the anti -FasR antibody as described herein is used in a method of treating a cancer in a subject. In some embodiments, the method comprises an anti-FasR antibody that specifically binds Fas receptor that is hypogylcosylated relative to wild-type Fas receptor or is aglycosylated. In some embodiments, the glycosylation status of FasR is used as a diagnostic. Glycosylation status of FasR may be used to select patients who may respond to a method of treating a cancer in a subject comprising the anti-FasR antibody as described herein. For example, a patient who exhibits a hypoglycosylated or aglycosylated FasR may be more responsive to therapy comprising the anti-FasR antibody as described herein compared to a patient who exhibits wild-type or hyperglycosylated FasR.

[0154] III. Production of Antibodies or Binding Fragments Thereof

[0155] In some embodiments, polypeptides described herein (e.g., antibodies and its binding fragments) are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and can be produced by recombinant expression techniques.

[0156] In some instances, an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which can involve the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

[0157] Alternatively, a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.

[0158] In certain aspects, provided herein are nucleic acid sequences encoding the anti-FasR antibodies disclosed herein. In some embodiments, the nucleic acid sequences encode the binding domain of the anti-FasR antibody. In some embodiments, the binding domain of the anti-FasR antibody comprises a HCVR and a LCVR. In some embodiments, the binding domain of the anti-FasR antibody comprises a scFv. In some embodiments, the binding domain of the anti-FasR antibody comprises a Fab. In some embodiments, the binding domain of the anti-FasR antibody comprises a F(ab’)2. In some embodiments, the binding domain of the anti-FasR antibody comprises a Fab’.

[0159] It is understood that due to the degeneracy of the genetic code, most amino acids are specified by more than one codon in the transcribed messenger RNA (mRNA). As such, any codon in the transcribed mRNA encoding any of the anti-FasR antibodies as disclosed herein may be used. In some embodiments, the nucleic acid encoding the amino acid sequence of an anti-FasR antibody as described herein comprises at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% identity to a reference nucleic acid sequence provided herein.

[0160] In some instances, an antibody or binding fragment thereof is optionally generated by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246:1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937). In some embodiments, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

[0161] In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0162] In some embodiments, an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody. In specific embodiments, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

[0163] In some embodiments, a variety of host-expression vector systems is utilized to express an antibody or its binding fragment described herein. Such host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0164] For long-term, high-yield production of recombinant proteins, stable expression is exemplary. In some instances, cell lines that stably express an antibody are optionally engineered. Rather than using expression vectors that contain viral origins of replication, host cells are transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells are then allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn are cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the antibody or its binding fragments.

[0165] In some instances, a number of selection systems are used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes are employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance are used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87- 95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11 (5): 155-215) and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds., 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY ; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1). In some instances, the expression levels of an antibody are increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing an antibody is amplifiable, an increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of the antibody, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell Biol. 3:257).

[0166] In some instances, an antibody or its binding fragment thereof is generated by chemical peptide synthesis techniques. Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the exemplary method for the chemical synthesis of the antibody binding fragments. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc, 85: 2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111. (1984).

[0167] In some instances, any method known in the art for purification of an antibody can be used. Illustrative methods include, but are not limited to, chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0168] In certain embodiments the binding affinity (avidity) of the antibody (e.g., anti-FasR antibody) is determined and can be optimized and / or increased (e.g., by mutation and successive rounds of panning a library, etc.). Methods of determining binding affinity are well known to those of skill in the art. Briefly, for example, the KD of the antibody is determined from the kinetics of binding to, e.g. the target cell in a BIAcore, a biosensor based on surface plasmon resonance. For this technique, the antigen or cell is coupled to a derivatized sensor chip capable of detecting changes in mass. When antibody is passed over the sensor chip, antibody binds to the antigen resulting in an increase in mass that is quantifiable. Measurement of the rate of association as a function of antibody concentration can be used to calculate the association rate constant (kon). After the association phase, buffer is passed over the chip and the rate of dissociation of antibody (koff) determined. Kon is typically measured in the range l.OxlO2to 5.0xl06and koff in the range 1.0 x ] ()1to I .Ox 10fi. The equilibrium constant KD is often calculated as koffZkon and thus is typically measured in the range IO5to 1012. Affinities measured in this manner correlate well with affinities measured in solution by fluorescence quench titration.

[0169] IV. Combination Therapies

[0170] As used herein, the term “combination therapy” refers to a method of prevention or treatment in which a subject in need thereof, e.g., a human subject, is administered a combination of two or more therapeutic agents. These “combinations” can comprise an anti- FasR agonist antibody and one or more additional therapeutic agents. The combinations may be co-formulated for administration or may be provided separately, for example as separate compositions, for administration to a subject or patient in need thereof. In some embodiments, the combinations have additive activity. In other embodiments, the combinations have synergistic activity.

[0171] Illustrative therapeutic agents include, but are not limited to, anti-cancer antibodies (e.g., HERCEPTIN®), antimetabolites, alkylating agents, topoisomerase inhibitors, microtubule targeting agents, kinase inhibitors, protein synthesis inhibitors, somatostatin analogs, glucocorticoids, aromatose inhibitors, mTOR inhibitors, protein Kinase B (PKB) inhibitors, phosphatidylinositol, 3-Kinase (PI3K) Inhibitors, cyclin dependent kinase inhibitors, anti-TRAIL molecules, MEK inhibitors, and the like. In certain embodiments the anti-cancer compounds include, but are not limited to flourouracil (5-FU), capecitabine / XELODA, 5-Trifluoromethyl-2'-deoxyuridine, methotrexate sodium, raltitrexed / Tomudex, pemetrexed / Alimta®, cytosine Arabinoside (Cytarabine, Ara- C) / Thioguanine, 6-mercaptopurine (Mercaptopurine, 6-MP), azathioprine / Azasan, 6- thioguanine (6-TG) / Purinethol (TEVA), pentostatin / Nipent, fludarabine phosphate / Fludara®, cladribine (2-CdA, 2-chlorodeoxyadenosine) / Leustatin, floxuridine (5-fluoro-2) / FUDR (Hospira, Inc.), ribonucleotide Reductase Inhibitor (RNR), cyclophosphamide / Cytoxan (BMS), neosar, ifosfamide / Mitoxana, thiotepa, BCNU-l,3-bis(2-chloroethyl)-l-nitosourea, 1,- (2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl CCNU, hexamethylmelamine, busulfan / Myleran, procarbazine HCL / Matulane, dacarbazine (DTIC), chlorambucil / Leukaran®, melphalan / Alkeran, cisplatin (Cisplatinum, CDDP) / Platinol, carboplatin / Paraplatin, oxaliplatin / Eloxitan, bendamustine, carmustine, chloromethine, dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin, nimustine, prednimustine, ranimustine, satraplatin, semustine, streptozocin, temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine, doxorubicin HCL / Doxil, daunorubicin citrate / Daunoxome®, mitoxantrone HCL / Novantrone, actinomycin D, etoposide / Vepesid, topotecan HCL / Hycamtin, teniposide (VM-26), irinotecan HCL(CPT-1 1), Camptosar®, camptothecin, Belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbine tartrate, vindesine sulphate, paclitaxel / Taxol, docetaxel / Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, vinfiunine, and the like. In certain embodiments the anti-cancer drug(s) comprise one or more drugs selected from the group consisting of carboplatin (e.g., PARAPLATIN®), Cisplatin (e.g., PLATINOL®, PLATINOL-AQ®), Cyclophosphamide (e.g., CYTOXAN®, NEOSAPv®), Docetaxel (e.g., TAXOTERE®), Doxorubicin (e.g., ADRIAMYCIN®), Erlotinib (e.g., TARCEVA®), Etoposide (e.g., VEPESID®), Fluorouracil (e.g., 5-FU®), Gemcitabine (e.g., GEMZAR®), imatinib mesylate (e.g., GLEEVEC®), Irinotecan (e.g., CAMPTOSAR®), Methotrexate (e.g., FOLEX®, MEXATE®, AMETHOPTERIN®), Paclitaxel (e.g., TAXOL®, ABRAXANE®), Sorafmib (e.g., NEXAVAR®), Sunitinib (e.g., SUTENT®), Topotecan (e.g., HYCAMTIN®), Vinblastine (e.g., VELBAN®), Vincristine (e.g., ONCOVIN®, VINCASAR PFS®). In certain embodiments the anti-cancer drug comprises one or more drugs selected from the group consisting of retinoic acid, a retinoic acid derivative, doxorubicin, vinblastine, vincristine, cyclophosphamide, ifosfamide, cisplatin, 5 -fluorouracil, a camptothecin derivative, interferon, tamoxifen, and taxol. In certain embodiments the anti-cancer compound is selected from the group consisting of abraxane, doxorubicin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab, megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid, vinblastine, etc.), an antisense molecule, an SiRNA, and the like.

[0172] In some embodiments, the additional therapeutic agent comprises an encapsulation system, such as a viral capsid, a liposome, or micelle that contains a therapeutic composition such as a drug, a nucleic acid (e.g. an antisense nucleic acid or another nucleic acid to be delivered to the cell), or another therapeutic moiety that is shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. No. 4,957,735, Connor et al. (1985) Pharm. Ther., 28: 341-365, and the like).

[0173] In some embodiments, the one or more additional therapeutic agents comprises a CAR-T cell.

[0174] In some embodiments, the anti-FasR agonist antibody is administered to the subject intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the anti-FasR agonist antibody is administered to the subject intravenously. In some embodiments, the one or more therapeutic agents is administered to the subject intravenously, subcutaneously, intraperitoneally, or intramuscularly. In some embodiments, the anti-FasR agonist antibody and the one or more therapeutic agents have the same route of administration. In some embodiments, the anti-FasR agonist antibody and the one or more therapeutic agents have different routes of administration.

[0175] V. Pharmaceutical Compositions

[0176] In some embodiments, pharmaceutical compositions comprising an anti-FasR agonist antibody are contemplated. In some embodiments, a pharmaceutical composition formulated for administration in a variety of unit dosage forms depending upon the route of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recognized that the antibodies described herein or pharmaceutical compositions comprising antibodies described herein, when administered orally, are protected from digestion. This can be accomplished by a number of means known to those of skill in the art, e.g., by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are well known in the art.

[0177] In some embodiments, a composition, e.g., a pharmaceutical composition, containing an anti-FasR agonist antibody or antigen-binding portions thereof, formulated together with a pharmaceutically acceptable carrier are provided.

[0178] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In an exemplary embodiment, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody or antigen-binding fragment thereof, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0179] In certain embodiments the antibody and / or antigen-binding fragment thereof can be administered in the “native” form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, and the like, provided the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the present method(s). Salts, esters, amides, prodrugs and other derivatives of the active agents can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley- Interscience, and as described above.

[0180] By way of illustration, a pharmaceutically acceptable salt can be prepared for any of the antibodies and / or antigen-binding fragments thereof described herein having a functionality capable of forming a salt. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered.

[0181] In various embodiments pharmaceutically acceptable salts may be derived from organic or inorganic bases. The salt may be a mono or polyvalent ion. Of particular interest are the inorganic ions, lithium, sodium, potassium, calcium, and magnesium. Organic salts may be made with amines, particularly ammonium salts such as mono-, di- and trialkyl amines or ethanol amines. Salts may also be formed with caffeine, tromethamine and similar molecules.

[0182] Methods of formulating pharmaceutically active agents as salts, esters, amide, prodrugs, and the like are well known to those of skill in the art. For example, salts can be prepared from the free base using conventional methodology that typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or can be brought out of solution by addition of a less polar solvent.

[0183] Suitable acids for preparing acid addition salts include, but are not limited to both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt can be reconverted to the free base by treatment with a suitable base. Certain exemplary acid addition salts of the active agents herein include halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of the active agents of this invention are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Particularly basic salts include, but are not limited to, alkali metal salts, e.g., the sodium salt, and copper salts.

[0184] For the preparation of salt forms of basic drugs, the pKa of the counterion is at least about 2 pH units lower than the pKa of the drug. Similarly, for the preparation of salt forms of acidic drugs, the pKa of the counterion is at least about 2 pH units higher than the pKa of the drug. This permits the counterion to bring the solution's pH to a level lower than the pHmax to reach the salt plateau, at which the solubility of salt prevails over the solubility of free acid or base. The generalized rule of difference in pKa units of the ionizable group in the active pharmaceutical ingredient (API) and in the acid or base is meant to make the proton transfer energetically favorable. When the pKa of the API and counterion are not significantly different, a solid complex may form but may rapidly disproportionate (i.e. , break down into the individual entities of drug and counterion) in an aqueous environment. The counterion can be a pharmaceutically acceptable counterion.

[0185] Suitable anionic salt forms include, but are not limited to acetate, benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate (embonate), phosphate and diphosphate, salicylate and disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide, valerate, and the like, while suitable cationic salt forms include, but are not limited to aluminum, benzathine, calcium, ethylene diamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, zinc, and the like.

[0186] Preparation of esters typically involves functionalization of hydroxyl and / or carboxyl groups that are present within the molecular structure of the antibody and / or antigen-binding fragment thereof. In certain embodiments, the esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH where R is alky, and can be lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

[0187] Amides can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine.

[0188] Pharmaceutical compositions comprising the antibodies described herein can be administered alone or in combination therapy, i.e., combined with other agents. For example, the combination therapy can include an antibody with at least one or more additional therapeutic agents, such as the anti-cancer agents described infra. The pharmaceutical compositions can also be administered in conjunction with radiation therapy and / or surgery.

[0189] A composition comprising the antibodies and / or antigen-binding fragment thereof described herein can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

[0190] In certain embodiments, administration of an anti-FasR antibody or antigen-binding fragment thereof may be facilitated by coating the antibody or antigen-binding fragment composition, or co-administering the antibody or antigen-binding fragment, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include, but are not limited to, saline and aqueous buffer solutions. Liposomes include, but are not limited to, water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol, 7: 27).

[0191] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0192] In various embodiments the therapeutic compositions are typically sterile and stable under the conditions of manufacture and storage. The composition(s) can be formulated as a solution, a microemulsion, in a lipid or liposome, or other ordered structure suitable to contain high drug concentration(s). In certain embodiments the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be exemplary to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., antibodies and / or antigen-binding fragments thereof described herein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, illustrative methods of preparation include vacuum drying, and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0193] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, in certain embodiments, the antibodies and / or antigen-binding fragments thereof described herein may be administered once or twice daily, or once or twice weekly, or once or twice monthly by subcutaneous injection.

[0194] In certain embodiments it is advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated. Each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0195] In certain embodiments the formulation comprises a pharmaceutically antioxidant. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. For the therapeutic compositions, formulations of the antibody and / or antigen-binding fragments thereof as described herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.001 percent to about ninety percent of active ingredient, from about 0.005 percent to about 70 percent, or from about 0.01 percent to about 30 percent.

[0196] Formulations of the anti-FasR antibody or antigen-binding fragments thereof as described herein that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of antibodies and / or antigen-binding fragments thereof described herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In certain embodiments the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0197] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection, and infusion.

[0198] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions comprising antibodies and / or antigen-binding fragments thereof described herein include, but are not limited to water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate, and the like. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In various embodiments these compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Particular examples of adjuvants that are well-known in the art include, for example, inorganic adjuvants (such as aluminum salts, e.g., aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g., squalene), oil-based adjuvants, virosomes {e.g., virosomes that contain a membrane-bound hemagglutinin and neuraminidase derived from the influenza virus).

[0199] Prevention of presence of microorganisms in formulations may be ensured both by sterilization procedures, and / or by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0200] In certain embodiments, when the antibodies and / or antigen-binding fragments thereof described herein are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (or from 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0201] Regardless of the route of administration selected, the antibodies and / or antigenbinding fragments thereof described herein, that may be used in a suitable hydrated form, and / or the pharmaceutical compositions, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0202] Actual dosage levels of the active ingredients (e.g., antibodies and / or antigen-binding fragments thereof described herein) in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of antibodies and / or antigen-binding fragments thereof described herein will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. In certain embodiments, administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, optionally administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered a single dosage, or as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for antibodies and / or antigen-binding fragments thereof described herein to be administered alone, it is typically common to administer the compound(s) as a pharmaceutical formulation.

[0203] In certain embodiments the therapeutic compositions can be administered with medical devices known in the art. For example, in an illustrative embodiment, antibodies and / or antigen-binding fragments thereof described herein can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of useful well- known implants and modules are described for example in U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate, in U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medications through the skin, in U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate, in U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery, in U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments, and in U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0204] In certain embodiments, the anti-FasR antibody and / or antigen-binding fragments thereof as described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., Ranade (1989) J. Clin. Pharmacol. 29: 685). Illustrative targeting moieties include, but are not limited to folate or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (Bloeman et al. (1995) FEB'S Lett. 357: 140; Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134).

[0205] VI. Kits / Articles of Manufacture

[0206] In certain embodiments kits and articles of manufacture for use with one or more methods described herein are provided. In certain embodiments such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[0207] In various embodiments the articles of manufacture provided herein can contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[0208] For example, in certain embodiments, kits provide container(s) containing the anti- FasR antibody and / or reagents for use with the anti-FasR antibody as disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[0209] A kit typically includes labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[0210] In certain embodiments, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein. SEQUENCE LISTING

[0211] In the protein sequence notation used herein, the left-hand direction is the amino terminal direction (the “N-terminus” or “N-term”) and the right-hand direction is the carboxyl- terminal direction (the “C-terminus” or “C-term”), in accordance with standard usage and convention.

[0212] EXAMPLES

[0213] Example 1. Characterization of the Fas PPCR epitope for its crucial optimal death signaling function.

[0214] The amino acid sequences of the extracellular domains (ECD) of DR5 and Fas were aligned. A highly conserved cysteine stabilized arginine-rich patch (similar to DR5 PPCR; Shivange G„ et al. Cell Rep. 2021; 37(5): 109953. doi: 10.1016 / j.celrep.2021.109953) was identified in human, monkey, mouse and dog Fas receptors (FIG. 1A). Because a crystal structure study of Fas ligand (FasL)-Fas receptor does not exist, a decoy receptor known as DCR3 and a preclinical anti-Fas antibody (E09) binding interface studies with FasL to extract FasL and Fas structures (FIG. IB). Similar to DCR3 R89+, the corresponding R86 of Fas formed hydrogen bonds with the Q220 and D221 of FasL (FIG. 1C). However, R87 of Fas formed salt bridges with E271 (GH loop) and E163 of FasL, while Y90 of DCR3 forms hydrophobic interactions with Y166 of FasL (FIG. ID). The DCR3 Y90 and FasL Y166 interface sterically blocked the accessibility of FasL negatively charged residues E271 and El 63 to engage R87 of FasR in a lock and key manner, explaining the saturating function of DCR3 (FIG. 1C and FIG. ID). This confirmed the importance of FasL salt bridges with Fas PPCR for its activation.

[0215] AnN-terminal His-tagged FasL with E163A, D164A, E270A, E271A, ED163-164AA, EE270-271AA mutations was generated (FIG. 2A and FIG. 2B). When tested, a FasL with D165A and E270A mutations bound to FasR similar to wildtype FasL (FIG. 2C) and demonstrated cytotoxic activity against tumor cells, Jurkat cells, and T-cells similar to wildtype FasL (FIG. 2D-2G). PPCR engaging FasL residues such as E163A, E271A, ED163-164AA, and EE270-271AA mutants lost both binding, apoptotic cytotoxicity, and FasR clustering activity against tumor and T-cells (FIG. 2H). This confirmed the importance of salt bridges with Fas PPCR for its activation.

[0216] Fas is known to have a key role in activation induced cell death (AICD) and T-cell homeostasis. During ACID, membrane or soluble FasL inhibits the activation of various scaffolds and kinases downstream of the TCR-CD3 complex. When human peripheral blood mononuclear cells (PBMC)-derived pan-T cells were stimulated with anti-CD3 / CD28 agonist antibodies in the presence of wild-type IgGl-Fc-Fas, it interfered with TCR signaling by inhibiting tyrosine phosphorylation of ^-chain-associated protein of 70 kD (ZAP70), phospholipase-y (PLC-y), and protein kinase C (FIG. 3A). However, E163A and E271A FasL mutants were ineffective. At the same time, non-PPCR engaging mutations (D164A, E270A) sit right next to El 63 and E271 effectively blocked the TCR activation cascade and Fas clustering (FIG. 3A and FIG. 3B). Similar results were obtained when a cross-species specific anti-CD3 antibody and CD28 agonists were used to stimulate monkey PBMCs, followed by treatment with wild-type and various FasL mutants (FIG. 3C). These results confirm the importance of FasL salt bridges with the Fas PPCR for AICD function.

[0217] Example 2. Fas PPCR-targeting is crucial for optimal death signaling by Fas antagonist antibodies.

[0218] The activity of previously described preclinical Fas agonist antibodies E09 and EP6- 1B (Chodorge M., et al. Cell Death Differ. 2012;19(7):l 187-95. doi: 10.1038 / cdd.2011.208) in the context of Fas PPCR. Both antibodies showed cytotoxicity to Jurkat cells, however significantly lower than FasL (FIG. 4A). These antibodies remained untested against tumor cell lines unless co-treated with cycloheximide (FIG. 4B). The latter decreased overall levels of cFLIP (FLICE-like inhibitory protein; also known as CASP8 and FADD-like apoptosis regulator), suggesting differential cFLIP expression and regulation in tumors vs. Jurkat cells (FIG. 4C).

[0219] Published studies suggest potential interface closeness between the CDR3 of the variable heavy chain region of E09 with R86 (corresponding to R89 of DCR3; FIG. 4D and FIG. 4E), however this has not been tested experimentally. It was hypothesized that the reason E09 is not effective against tumor cells (and only limitedly effective against Jurkat cells, FIG. 4A) is due to its inability to engage the critical PPCR residue R87, whose guanidinium side chain interface is in opposite orientation to the variable domain of VH (CDR3) (FIG. 4E). Although R86 of Fas forms hydrogen bonds with tyrosine (Y114) of E09, analysis demonstrated that R87 is critical. As the side chain of R86 is oriented perpendicular to R87, R86 may not simultaneously engage R87 and PPCR (FIG. ID). It was found that when R86A, R87A and R89A mutants were tested for binding, none interfered with E09 binding (FIG. 4F- 4G and FIG. 5A-5B).

[0220] FasL directly engages R87 (FIG. 1) and activates cytotoxic death in both tumor and Jurkat cells (FIG. 2), while E09 was partly cytotoxic only to Jurkat cells (FIG. 2A). To further confirm R87 engagement is critical for effective cytotoxicity of tumor cells, E09 and EP6 were engineered with a FasL to make a bispecific antibody (E09-FasL and EP6-FasL; FIG. 6A and FIG. 6B). As a control, E09 and EP6 antibodies were engineered as bispecific antibodies (FIG. 6A and FIG. 6B). Both EP6-FasL and E09-FasL were significantly more effective in OVCAR-3 and HEY -A8 ovarian cancer cells. Further, it was found that a single point mutation in FasL, R163A or E271A, rendered both E09-FasL and EP6-FasL ineffective in tumor cells and tumor xenografts (FIG. 6C). Similar results were seen in Hey-A8 ovarian tumor efficacy studies (FIG. 6D-6F).

[0221] Example 3. PPCR epitope targeting is critical for CAR-T bystander off-targeting tumor cell death function.

[0222] Mutations in target antigen or antigen loss contribute to acquired resistance against all tumor types to most targeted therapies, including chimeric antigen receptor T cell therapy (CAR-T). In heterogeneous tumors, where antigen-negative tumor cells do not bind the scFv of a designated CAR-T, Fas-FasL mediated off-target (bystander) killing of Fas-enriched tumor cells has been shown to be critical in human clinical trials as well as in murine studies (Upaadhyay R., et al. Cancer Discov. 2021 ; 11 (3):599-613. doi: 10.1158 / 2159-8290.CD-20- 0756). Therefore, to test if PPCR is critical for CAR-T-mediated bystander killing, human FasR-expressing GFP-stable murine ID8 cells were generated (FIG. 7A). A chimeric Fas construct was engineered to have a human Fas extracellular domain (ECD) fused with mouse Fas transmembrane and intracellular domains. The chimeric receptor-expressing cell lines bound and was activated by human Fas agonists (FIG. 7B-7D).

[0223] A folate receptor 1 (FOLRl)-targeting CAR-T cells were generated (FIG. 7E-7G). The antigen binding domain of the FOLR1 CAR was the scFv of farletuzumab. When tested in various ratios with high and medium FOLR1+ OVCAR3 and colo205 cells, the CAR-T cells were highly effective in complete cell lysis and granzyme activation even in an 8: 1 ratio (FIG. 7I-7J).

[0224] The co-culture of parental ID8 cells with OVCAR3 and CAR-T cells demonstrated high granzyme levels but no sign of caspase-8 cleavage (FIG. 8A-8C). However, when huFas- expressing ID 8 cells were used along with OVCAR3 and CAR-T cells, a significantly high caspase-8 cleavage and GFP loss were evident (FIG. 8D-8F). The only difference in these two scenarios was the presence of huFOLRl antigen-negative and huFas-positive ID8 cells. Since farletuzumab CAR-T cells have no direct capability to engage murine FOLR1 in GFP-stable HuFas ID8 cells, the decrease in GFP signal was contributed by huFas activation and caspase- 8 bystander activation in murine ID8 cells. Altogether, it suggested the antigen-independent and Fas-dependent bystander activation of ID8HuFas cells by CAR-T. Further, pre-coating the culture plates with recombinant rFasWT completely inhibited caspase-8 cleavage (FIG. 9A). On the contrary, a single R87A PPCR mutation in rFAS was ineffective (FIG. 9A). Notably, the Fas signaling, and caspase-8 activation was significantly enhanced in the presence of FOLR1 antigen expressing OVCAR-3, due to high-affinity CAR-T binding to FOLR1 antigen enhancing the avidity of CAR-T membrane FasL binding to huFas expressing ID cells (FIG. 9B).

[0225] Example 4. Generation of an anti-PPCR and tumor-specific Fas targeting antibody (TFT) via rationally exploiting differential Fas glycosylation profiling in T-cells and tumor cells.

[0226] As discussed in Example 1, Fas has a highly conserved PPCR motif similar to DR5. However, unlike DR5, Fas has two N-linked glycosylation sites N102 and N120 (FIG. 10A- 10B). Notably, Fas is hyperglycosylated at N102 and N 120 in T cells but not in tested ovarian tumor cells (FIG. 10C). The sugar moieties on FasR not only form the partly shielded hydrogen bonds with the Fas PPCR epitope region but also contribute to its differential conformational ensembles as compared to non-glycosylated FasR (FIG. 11A-11B). The latter limits FasR’s solvent-accessible surface area (SASA) against a tumor-specific Fas targeting antibody (TFTab; FIG. 11B). However, Fas is either hypoglycosylated or aglyosylated in ovarian tumor cells (FIG. 10C; FIG. 11C-11D), which allows higher SASA of Fas. The latter results in TFTab interactions with high affinity (FIG. 11C-11E). Therefore, TFTab selectively activates apoptotic cytotoxicity of tumor cells but not T-cells.

[0227] Using the well-characterized Fas R87 as being crucial for its agonist signaling in the context of the consensus N102 and N 120 N-linked glycosylation site, the goal of this aim is to engineer, characterize, and mechanistically elucidate the anti-tumor efficacy and T -cell safety of a highly novel Fas PPCR epitope targeting tumor-specific antibody.

[0228] By random mutagenesis, >400 clones were screened to find a lead antibody which engaged Fas PPCR, specifically the R87 side chain. The antibody named “Tumor-specific Fas- Targeting” (TFTab) has already been humanized and lost binding entirely by a single R87A mutation (FIG. 12A-12B). The size and sequence were confirmed by mass spectrometry. Surprisingly, the TFT antibody was found to activate apoptotic cytotoxicity of various tumor cells tested but not of T-cells. To enhance the specificity toward ovarian cancer but not T- cells, the TFT antibody was engineered into a bispecific antibody that targeted anti-FOLRl. This antibody was known as FOLR1-TFT (FIG. 13A). The bispecific antibody was highly superior in killing FOLR1 enriched ovarian tumor cells (FIG. 13B). As a positive control, an anti-FOLRl was engineered to FasL instead of TFTab. Both bispecific combinations were almost equally effective against tumor cells when tested. Only FOLRl-FasL was cytotoxic to T-cells, while FOLR1-TFT was not (FIG. 13C). These results strongly support the high specificity of the FOLR1-TFT approach against FOLR1+ ovarian tumors.

[0229] Example 5. In vitro studies to characterize tumor-specific cytotoxicity of FORL1-TFT.

[0230] In order to characterize the tumor-specific cytotoxicity of FORL1-TFT, a panel of patient-derived ovarian cancer cell lines (both chemo-sensitive and chemo-resistant) having variable degrees of FOLR1 and Fas expression will be exposed to FORL1-TFT. FORL1-TFT will be neutralized with recombinant FOLR1 to confirm that co-engagement of the two targets drives avidity-based killing. The FOLR1 IgG will be replaced with a control IgGl to generate a control molecule. Both molecules will be tested side by side. Human T-cells derived from over 20 different PBMC donors will be tested using FORL1-TFT, the control TFT, and FOLRl-FasL antibodies along with IgGl controls to assess T cell safety. Specific T cell subtypes (e.g., CD3+, CD4+, CD8+) will be enriched from over 20 donors and separately tested for the extrinsic apoptotic signaling pathway and cell survival assays using either FORL1-TFT or FOLRl-FasL. Activation of T-cell signaling kinases will also be analyzed, as well as Fas clustering, caspase-8 activation, and other signaling components.

[0231] Example 6. Characterization of the selective tumor specificity apoptotic mechanism of a novel TFT antibody.

[0232] Analysis of the TFT antibody elucidated a single consensus N-linked glycosylation consensus “NYS” in the heavy chain variable domain (VH) right next the cysteine-rich domain CRD3. TFTab glycosylation was confirmed at only this particular NYS site in addition to Fc glycosylation, common to all IgGl antibodies (FIG. 14A-14D). This led to the hypothesis that similar to the glycosylating sugars on the HIV- 1 -envelope (Env) trimer and G120 protein, which help to sterically avoid the essential antibody-mediated neutralization of the virus, the molecular crowding of N-linked sugars near CRD3 of TFT could potentially sterically interfere with the antibody accessibility against Fas PPCR in T-cells versus tumor cells. To test the latter, the total Fas profiles of whole cell lysates from CD3 -enriched human T-cells and ovarian tumor PDX cells and OVCAR-3 cells were compared. The hyperglycosylated form of Fas was evident in T-cells compared to that of OVCAR-3 and ovarian PDX cells (FIG. 15A). TFTab binding as measured by FACS was significantly lower (<80%) compared to tumor cells at the same time (FIG. 15B). Furthermore, it was confirmed that the recombinant Fas sequence underwent N 102 or N120 N-linked glycosylation (FIG. 15C). Notably, removing N-glycans enhanced the cytotoxic sensitivity of TFTab against T-cells (FIG. 16A-16C). Altogether, these results suggested hyperglycosylation mediated interference of TFT antibody function.

[0233] To further investigate the role of N-linked sugars, simulation studies of glycosylated and non-glycosylated Fas ectodomain were performed. Some key points from the studies were: 1) N102 glycan sugars form numerous hydrogen bonds near the PPCR region but specifically only with R87 in PPCR (FIG. 17A-17C); 2) pairwise root mean square distance (RMSD) value changes were significantly lesser in glycosylated Fas compared to non-glycosylated Fas (FIG. 18A); 3) dynamic cross-correlation matrix (DCCM) analysis indicated that various residues (and atoms) moved in a similar fashion and together in glycosylated Fas compared to non- glycosylated Fas (FIG. 18B); and 4) the principal component analysis showed the differential motion trajectory in glycosylated or non-glycosylated Fas. Point 4 indicated that the preferred binding of glycosylated Fas was with its natural substrate (FasL), and higher caspase-8 activity of FasL against T-cells was consistently found to be higher compared to that found in tumor cells (FIG. 19A). Considering the new PPCR-targeting TFTab had limited engagement with glycosylated Fas on the T-cell surface, it is highly likely that conformational ensembles of glycosylated (and molecular crowding) near PPCR interfere with TFTab binding similar to previously described epitopes of glycosylated GP120 (HIV).

[0234] Finally, alanine scanning of the TFTab heavy chain’s CRD3 was carried out. This confirmed that the antibody salt bridges with Fas PPCR, similar to FasL, are essential for its activity (FIG. 20A-20B). These studies confirm that a combination of factors such as saltbridges between TFT and Fas-PPCR, TFTab’s N-linked glycosylation near its CRD3 potentially resulting in steric crowding, and a PPCR epitope backbone selective binding of TFTab contributes to the specificity and optimal activity of TFTab against limitedly or non- glycosylated Fas against tumor cells.

[0235] Example 7. In vitro study to determine tumor specificity of TFT antibody.

[0236] Hey-A8 Fas knockout lines will be used to determine tumor specificity. Similar to the ID8 stable Fas lines, stable cells lines that express wild-type Fas, FasN102, or FasN 120 will be generated. Multiple single cell-confirmed clones will be tested side by side for TFT-binding, activity, and function. In addition, the Hey-A8 Fas wild-type clones will be treated with Tunicamycin alongside FasN102, FasN120, and FasN102A / N120Afollowed by Fas activation, clustering, and cytotoxic function using Farle-TFT, Farle-FasL, TFTab IgGl, or FasL.

[0237] To understand the importance of glycosylation versus non-glycosylation on Fas conformation, two different lipid membranes: a single-component, liquid-disordered (Ld) fluid bilayer made of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; referred to here as the Ld bilayer) and a bilayer with a ternary lipid mixture (DOPC, sphingomyelin (SM), and cholesterol (Choi)), which is more ordered (liquid-ordered (Lo)) and less fluid and is often taken as a physical model of putative membrane “rafts” (referred to here as the Lo bilayer) will be used to insert Fas for simulation studies. These 500 ns of each molecular dynamics (MD) simulation study will provide comprehensive data in four systems (Ld-NG, Ld-G, Lo-NG, Lo-G), and every simulation will be repeated three times to improve sampling. In addition, average values of the tilt angles will be determined in all four systems as described previously for CD2. More ever, an alanine scanning study of TFTab will be conducted (similar to FIG. 20) to confirm the role of its N-glycan-mediated (near its CDR3) steric hindrance in regulating safety toward T- cells. To this end, the consensus sequence around NXS / T will be mutated, followed by tumor cells and T-cell survival studies, Tumor cell apoptotic pathway signaling studies, and T-cell activation studies. For rigor and reproducibility results will be confirmed with >20 different PBMC sources.

[0238] Example 8. In vivo determination of the anti-tumor efficacy and T-cell safety of the TFT antibody.

[0239] The in vivo anti-tumor efficacy of the bispecific FOLR1-TFT antibody was examined alongside Farle-FasL, and Farle-E09 in a pre-clinical murine (Athymic nude Foxnlnu) ovarian cancer xenograft model. It has been hypothesized that the Farle-TFT antibody: 1) will work in vivo independent of ADCC activating NK cells and (2) will have higher overall anti-tumor cytotoxicity with added ADCC function. To test the first hypothesis, Farle-TFT and Farle- FasL with ADCC inactivating mutations (L234A, L235A called LALA-Fc) in the IgGl-Fc hinge region, as the mouse model has functional NK cells. The LALA-Fc mutations mediated loss of binding function to FcyRIIIA. The antibodies Farle-TFT, Farle-FasL, Farle-E09, and RIgGl-FasL, were intraperitoneally (IP) injected at a 25 pg dose. Both Farle-TFT and Farle- FasL completely reduced tumors, while Farle-E09 and RIgGl-FasL only stabilized tumors (FIG. 19B). Similar results were obtained with xenografts generated with another OvCa cell line, Hey-A8 cells. The tumor regression efficacy data in Fig. 20b is ADCC-independent, which confirmed hypothesis 1. Hypothesis 1 was also supported by the fact that a LALA-Fc mutant Farle-E09 has deficient anti-tumor activity (FIG. 19B).

[0240] Example 9. ALG gene status is a determinant of ovarian cancer outcome.

[0241] A family of glycosyltransferases, known as asparagine-linked glycosylation (ALG) catalyzes the transfer of each monosaccharide onto a dolichyldisphosphate carrier. As glycosylation status of FasR is important for TFT binding, glycosylation status in a number of cancers was interrogated.

[0242] ALG gene copy number variations were measured in ovarian and uterine cancer samples (FIG. 21A). The blue bars in FIG. 21A represent the fold loss in copy number and the red bars represent the fold gain. ALG 12 in particular shows a significant % of cases of both uterine and ovarian cancer samples exhibiting a fold loss. When comparing ovarian and uterine cancer samples based on ALG12 expression, it was discovered that those cancers with low ALG12 expression correlated with lower survival (FIG. 21B). The correlation between ALG 12 expression and survival in other cancers (from left to right: Glioma, breast cancer, and lung adenocarcinoma) was also investigated (FIG. 21C). The same correlation as seen in ovarian and uterine cancers was not apparent in these other types of cancers. Next, the question as to whether ovarian cancer patient tissue that was previously treated with an intervention, in this case cisplatin, had differential expression of ALG 12 was investigated. Total lysates of either pan CD3+ or CD8+ enriched T-cells expressing hyperglycosylated FasR were run next to ovarian cancer patient tissue lysates (FIG. 22). Immunoblotting for ALG12 found that over 75% of tested ovarian cancer patient tissue exhibited ALG 12 downregulation regardless of cisplatin status. This illustrates that ALG 12 and resulting glycosylation status of FasR is correlates to disease outcome.

[0243] Example 10. TFTab specifically targets cells comprising aglycosylated FasR.

[0244] In order to test the specificity of tumor targeting and killing of TFT ab, Hey-A8 ovarian cancer cells were cultured and then trypsinized. Hey-A8 cells that expressed a glycosylated form of FasR were cultured in complete media, while Hey-A8 cells that expressed an aglycosylated form of FasR were cultured in serum-free media. OVCAR3 ovarian cancer cells express an aglycosylated form of FasR under both conditions (FIG. 23A and FIG. 23B). Cells were also exposed to tunicamycin which resulted in higher expression of aglycosylated FasR (FIG. 23C). TFTab was compared to preclinical FasR antibody E09. It was found that E09 does not activate cell death of Hey-A8 cells in serum- free media while TFTab does (FIG. 23D). Next, a bispecific engager comprising the TFT scFv with a control antibody scFv targeting Paradexa drugs was tested. In this construct, TFT only eliminated Hey-A8 cells in serum-free conditions (FIG. 23E). Tumor efficacy of TFTab was also tested, showing that TFTab is only effective in the presence of tunicamycin (FIG. 23F).

[0245] Example 11: Simulations to determine the stability and accessibility of glycosylated and non-glycosylated Fas

[0246] A key feature of Fas, unlike other death receptors such as DR5, is the presence of two conserved glycosylation sites, one at the end of CRD2 domain (N112) and the other in the CRD3 domain (N120). As these glycosylation sites are close to PPCR epitope, it was proposed that they could stabilize the orientation of Fas at the cell surface in a membrane lipid-dependent manner.

[0247] Heretofore published crystal structures of the Fas receptor were missing the 29 amino acid sequence QVTDINSKGLELRKTVTTVETQNLEGLHH (SEQ ID NO. 26) located adjacent to the signal peptide, MLGIWTLLPLVLTSVARLSSKSVNA (SEQ ID NO. 27). To determine their structural role, molecular dynamics simulation studies were carried out using the full Fas sequence including the omitted 29 residues, herein identified as the N-terminal domain (NTD).

[0248] Simulations using a liquid-disordered (Ld) fluid bilayer made of 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) were performed. Each simulation as carried out for 500 ns (n=4). The angle between the bilayer normal (z-axis) and the normalized vector connecting the centers of mass (COMs) of FasR transmembrane residues and extracellular residues was measured.

[0249] For 0i, glycosylated Fas exhibited a dominant peak at approximately 30-35°, indicating a prevalent orientation (FIG. 24A-B). A secondary peak at about 75-80° indicated occasional deviations from this main orientation. Non-glycosylated Fas showed a broader distribution of 0i angles with the primary peak at around 75-80°, indicating a more horizontal alignment with the lipid bilayer. Additional peaks at lower angles highlight greater variability in orientation (FIG. 24A-B).

[0250] For O2, both glycosylated and non-glycosylated Fas showed peaks at lower angles (10- 20°, FIG. 24A, 24C). Non-glycosylated Fas had a sharper, higher peak at a slightly lower angle compared to the glycosylated state. Glycosylated Fas exhibited a broader distribution extending to slightly higher angles (FIG. 24A, 24C). Glycosylation significantly affected the orientation of Domain 1 , causing it to adopt a variety of positions relative to the bilayer. For Domain 2, the effect of glycosylation was less dramatic, but it still introduced more flexibility in orientation. The two domains behaved differently, indicating that glycosylation had domain-specific effects on the Fas receptor's structure.

[0251] Based on these tilt angle distributions, the data demonstrated two primary orientations for both glycosylated and non-glycosylated models of the Fas receptor: a “lying down” orientation and a “standing” orientation. In the “lying down” orientation, the ECD appeared to be aligned more horizontally with the lipid bilayer plane (larger 9i). This was more prevalent in the non-glycosylated FAS, as evidenced by the higher tilt angles and broader distribution in Domain 1. In the “standing” orientation, the ECD was more perpendicular to the bilayer plane (smaller 0i). This was more common in the glycosylated FAS, as shown by the lower tilt angles, particularly for Domain 1. Notably, this distinction between lying down and standing orientations appeared specific to the monomeric form of FAS, as trimerization tended to stabilize the receptor in a standing position.

[0252] The orientation of Fas indicated that non-glycosylated Fas had a higher probability of membrane contact as compared to glycosylated (FIGs. 24-26). These results also indicated that glycosylated Fas monomers could bind more effectively to antibodies due to greater accessibility, which can be attributed to lower interaction with the DOPC bilayer and a predominantly standing orientation. Conversely, experimental data indicated that non- glycosylated Fas might exhibit enhanced binding capabilities, indicating that factors other than orientation and membrane interactions are also at play.

[0253] As the accessibility of the Fas PPCR epitope is a key feature of the TFT antibody design, further investigations were necessary to elucidate the experimental observations. Accessibility is primarily influenced by two factors: the tendency of the PPCR epitope to interact with the DOPC lipid bilayer, and the interaction between the N-terminal domain (NTD) of the FAS receptor and the PPCR, which might sterically hinder antibody binding. Thus, the interaction between the NTD flexibility and PPCR epitope interaction with the antibody were investigated.

[0254] The root mean square deviation (RMSD) of the N-terminal domain (NTD) of the FAS receptor in its glycosylated versus non-glycosylated states were analyzed. The RMSD of the NTD is shown as a function of simulation time with respect to the initial configuration in FIG. 27A-B. The non-glycosylated NTD (orange) exhibited higher RMSD values compared to the glycosylated NTD (blue), indicating that glycosylation reduced the flexibility of the NTD. A contact frequency map analysis was performed to investigate specific interactions between the NTD and the PPCR epitope of the FAS receptor. FIG. 28 presents the contact map analysis between two groups of residues: Group 1 representing the NTD, and Group 2 corresponding to the antibody epitope region. The contact frequency bar on the right explains the color coding in the contact map. A value of 1 indicated that the specific pair of residues was in contact in every frame of the trajectory, while a value of 0 meant that the residues were never in contact. Values between 0 and 1 represented the fraction of frames in which the residues were in contact, Several bright yellow regions were shown in the contact map for the glycosylated Fas (FIG. 28), indicating interaction frequencies approaching 100% between specific residue pairs. Tn contrast, the non-glycosylated Fas exhibited a more diffuse pattern of interactions, with lower overall frequencies. Thus, the interactions between the NTD and the PPCR epitope in the non-glycosylated FAS were more transient throughout the simulations (FIG. 28 top), while the glycosylated FAS demonstrated interactions that were more specific and persistent throughout the simulation (FIG. 28 Bottom). When quantified (FIG. 29), the distinct differences in interaction patterns between glycosylated and non-glycosylated FAS were recapitulated. Glycosylated FAS exhibited interactions that were maintained for over 60% of the simulation time, while the non-glycosylated form showed no such persistent interactions.

[0255] The contact frequency was also evident with the formation of salt bridges between NTD residues with PPCR residues. As shown in FIG. 30B, the salt bridges formed between ASP39 (D39) of the NTD (blue) and ARG86 (R86) of the PPCR (red) in glycosylated FAS, but not in non-glycosylated FAS (FIG. 30A). A series of trajectories further confirmed the steric hinderance of NTD (blue) domain with PPCR (red, FIG. 300). The interaction of NTD close to PPCR epitopes effectively neutralized ARG87's ability to bind with TFT antibodies. In contrast, in non-glycosylated FAS, ARG86 appeared more available for interrnolecular interactions, potentially facilitating antibody binding.

[0256] This glycosylation-dependent difference in ARG86 and ARG97 availability can explain the selective binding observed with the described novel TFT antibody, which engaged non-glycosylated FAS but not the glycosylated counterpart These findings indicate a potential mechanism by which glycosylation modulated F AS receptor interactions, with implications for antibody design and therapeutic strategies targeting the FAS pathway as described herein.

Claims

CLAIMSWhat is claimed is:

1. An antibody or antigen-binding fragment thereof that specifically binds a Fas receptor, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

2. The antibody or the antigen-binding fragment thereof of claim 1, wherein the Fas receptor is hypoglycosylated relative to a wild-type Fas receptor.

3. The antibody or the antigen-binding fragment thereof of claim 1, wherein the Fas receptor is aglycosylated.

4. The antibody or the antigen-binding fragment thereof of claim 1, wherein the Fas receptor is expressed on an ovarian cancer cell or another solid tumor cancer cell that is optionally selected from the group consisting of a triple-negative breast cancer (TNBC) cell, a lung cancer cell, and a glioblastoma multiforme (GBM) cell.

5. The antibody or the antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

6. The antibody or the antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof binds a Fas receptor expressed on a tumor cell with a higher affinity than a Fas receptor expressed on a T-cell.

7. A method of treating a subject having a solid tumor, the method comprising contacting the subject with an antibody or antigen-binding fragment thereof that specifically binds a Fas receptor, wherein the Fas receptor is hypoglycosylated relative to a wild-type Fas receptor or is aglycosylated.

8. The method of claim 7, wherein the solid tumor comprises a uterine cancer or an ovarian cancer or another solid tumor cancer cell that is optionally selected from the group consisting of a triple-negative breast cancer (TNBC) cell, a lung cancer cell, and a glioblastoma multiforme (GBM) cell, and wherein the cell expresses hypoglycosylated or aglycosylated Fas.

9. The method of claim 8, wherein the solid tumor comprises ovarian cancer.

10. The method of claim 7, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

11. The method of claim 7, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

12. The method of claim 7, wherein the method further comprises contacting the subject with an antibody or antigen-binding fragment thereof that specifically binds folate receptor 1 (FOLR1).

13. The method of claim 12, wherein the antibody that specifically binds the Fas receptor and the antibody that specifically binds the FOLR1 are in a bispecific antibody format.

14. The method of claim 13, wherein the bispecific antibody specifically binds both the Fas receptor and the FOLR1.

15. The method of claim 12, wherein the antibody or the antigen-binding fragment thereof that specifically binds the Fas receptor and the antibody or the antigen-binding fragment thereof that specifically binds the FOLR1 are administered together.

16. The method of claim 12, wherein the antibody or the antigen-binding fragment thereof that specifically binds the Fas receptor and the antibody or the antigen-binding fragment thereof that specifically binds the FOLR1 are administered separately.

17. A method of treating ovarian cancer in a subj ect, comprising contacting the subj ect with a bispecific antibody that specifically binds Fas receptor that is hypoglycosylated relative to wild-type Fas receptor or is aglycosylated.

18. The method of claim 17, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

19. The method of claim 17, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

20. The method of claim 17, wherein the method further comprises contacting the subject with an antibody or the antigen-binding fragment thereof that specifically binds folate receptor 1 (FOLR1).

21. The method of claim 20, wherein the antibody that specifically binds the Fas receptor and the antibody that specifically binds the FOLR1 are in a bispecific antibody format.

22. The method of claim 21 , wherein the bispecific antibody specifically binds both the Fas receptor and the FOLR1.

23. The method of claim 20, wherein the antibody or the antigen-binding fragment thereof that specifically binds the Fas receptor and the antibody or the antigen-binding fragment thereof that specifically binds the FOLR1 are administered together.

24. The method of claim 20, wherein the antibody or the antigen-binding fragment thereof that specifically binds the Fas receptor and the antibody or the antigen-binding fragment thereof that specifically binds the FOLR1 are administered separately.

25. A method of predicting a positive clinical response to treating a solid tumor in a subject comprising contacting a solid tumor sample from the subject with an antibody or an antigenbinding fragment thereof that specifically binds Fas receptor that is hypoglycosylated relativeto wild-type Fas receptor or is aglycosylated, wherein the antibody or the antigen-binding fragment thereof binds to the solid tumor sample with a higher affinity than to a non-tumor sample.

26. The method of claim 25, wherein the solid tumor comprises ovarian cancer.

27. The method of claim 25, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

28. The method of claim 25, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

29. A single chain variable fragment (scFv) that comprises a variable light chain followed by a GS linker and a variable heavy chain comprising SEQ ID NO: 9.

30. The scFv of claim 29, wherein its target is expressed on a solid tumor.

31. The solid tumor of claim 30, wherein the tumor comprises ovarian cancer or any other solid tumor expressing hypo or non-glycosylated Fas.

32. The scFv of claim 29, wherein the scFv target is a bispecific molecule that optionally comprises FOLR1, EGFR and / or MUC16.

33. The scFv of claim 29, wherein the scFv target is expressed on CAR-T cells to enhance antigen independent bystander anti-tumor function.

34. The scFv of claim 29, wherein the scFv target is expressed as bispecific that optionally includes FOLR1, CD24, NaPi2b, EGFR, and / or Mucl6 on CAR-T cells to enhance antigen independent bystander anti-tumor function.

35. A method of binding a Fas receptor that is hypoglycosylated relative to wild-type Fas receptor (SEQ ID NO: 10) or is aglycosylated, comprising contacting the Fas receptor with an antibody or an antigen-binding fragment thereof that specifically binds to a patch of positively charged residues (PPCR) motif present in the Fas receptor as set forth as SEQ ID NO: 11 or SEQ ID NO: 12.

36. The method of claim 35, wherein the antibody or the antigen-binding fragment thereof comprises three heavy chain complementary determining region (CDR) sequences comprising SEQ ID NOs: 1, 2, and 3, respectively, and three light chain CDR sequences comprising SEQ ID NOs: 4, 5, and 6, respectively.

37. The method of claim 35, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) sequence comprising SEQ ID NO: 7 and a light chain variable region (LCVR) sequence comprising SEQ ID NO: 8.

38. The Fas receptor of claim 1, comprising SEQ ID NO: 10.