EP4662243A2 - Anti-il1rap antibodies and variants thereof - Google Patents

Abstract

Provided herein are, inter alia, Interleukin-1 receptor accessory protein (IL1RAP) antibodies, fragments and variants thereof as well as recombinant proteins including IL1RAP antibody regions are useful for treating IL1RAP-expressing cancers.

Description

ANTI-IL1RAP ANTIBODIES AND VARIANTS THEREOF

RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/484,181 filed on February 9, 2023, which is hereby incorporated herein by reference in its entirety and for all purposes.

BACKGROUND

[0002] Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy that can lead to hematopoiesis failure and death. Despite increasing knowledge of the disease, current treatment options benefit only a minority of AML patients. The limited success of treatments is believed to be at least partially due to the inability of chemotherapy and/or other molecular targeting therapeutics to eliminate so-called leukemia stem cells (LSCs). Thus, there is a need in the art for treatments, which specifically eliminate LSCs while sparing normal hematopoietic stem cells.

[0003] Immunotherapeutic approaches hold promise as an effective means of treating patients suffering from AML. To be successful, however, immunotherapy must allow for the selective targeting and destruction of LSCs with minimal adverse effects. Provided herein are compositions and methods which cure this and other needs in the art.

BRIEF SUMMARY

[0004] In an aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of an anti- interleukin- 1 receptor accessory protein (IL1RAP) antibody, wherein the antibody does not inhibit ILl-receptor signaling relative to a standard control, thereby treating cancer in the subject.

[0005] In another aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of a recombinant protein including: (i) a first antibody region, wherein the first antibody binding region binds an effector cell ligand; and (ii) a second antibody region, wherein the second antibody region binds an interleukin- 1 receptor accessory protein (IL1RAP) and does not inhibit IL 1 -receptor signaling relative to a standard control, thereby treating cancer in the subject.

[0006] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO: 37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

[0007] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NON, a CDR H2 as set forth in SEQ ID NO:5 and a CDR H3 as set forth in SEQ ID NO:6; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:40, a CDR L2 as set forth in SEQ ID NO:41, and a CDR L3 as set forth in SEQ ID NO:42.

[0008] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8 and a CDR H3 as set forth in SEQ ID NON; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:43, a CDR L2 as set forth in SEQ ID NO:44, and a CDR L3 as set forth in SEQ ID NO:45.

[0009] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 10, a CDR H2 as set forth in SEQ ID NO: 11 and a CDR H3 as set forth in SEQ ID NO: 12; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:46, a CDR L2 as set forth in SEQ ID NO:47, and a CDR L3 as set forth in SEQ ID NO:48.

[0010] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14 and a CDR H3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:49, a CDR L2 as set forth in SEQ ID NO:50, and a CDR L3 as set forth in SEQ ID NO:51.

[0011] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 16, a CDR H2 as set forth in SEQ ID NO:17 and a CDR H3 as set forth in SEQ ID NO:18; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO: 52, a CDR L2 as set forth in SEQ ID NO:53, and a CDR L3 as set forth in SEQ ID NO:54.

[0012] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 19, a CDR H2 as set forth in SEQ ID NO:20 and a CDR H3 as set forth in SEQ ID NO:21; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:55, a CDR L2 as set forth in SEQ ID NO:56, and a CDR L3 as set forth in SEQ ID NO:57.

[0013] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:22, a CDR H2 as set forth in SEQ ID NO:23 and a CDR H3 as set forth in SEQ ID NO:24; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO: 58, a CDR L2 as set forth in SEQ ID NO:59, and a CDR L3 as set forth i [0014] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:25, a CDR H2 as set forth in SEQ ID NO:26 and a CDR H3 as set forth in SEQ ID NO:27; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:61, a CDR L2 as set forth in SEQ ID NO: 62, and a CDR L3 as set forth in SEQ ID NO: 63.

[0015] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:28, a CDR H2 as set forth in SEQ ID NO:29 and a CDR H3 as set forth in SEQ ID NO:30; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO: 64, a CDR L2 as set forth in SEQ ID NO:65, and a CDR L3 as set forth in SEQ ID NO:66.

[0016] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:31, a CDR H2 as set forth in SEQ ID NO:32 and a CDR H3 as set forth in SEQ ID NO:33; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:67, a CDR L2 as set forth in SEQ ID NO:68, and a CDR L3 as set forth in SEQ ID NO:69.

[0017] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:34, a CDR H2 as set forth in SEQ ID NO:35 and a CDR H3 as set forth in SEQ ID NO:36; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:70, a CDR L2 as set forth in SEQ ID NO:71, and a CDR L3 as set forth in SEQ ID NO:72.

[0018] In another aspect is provided a recombinant protein including: (i) a first antibody region; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0019] In another aspect is provided a recombinant protein including: (i) a first antibody region, including: (a) a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO: 37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0020] In another aspect is provided a recombinant protein including a first antigen binding domain and a second antigen binding domain independently including a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain includes a CDR Hl sequence, a CDR H2 sequence and a CDR H3 sequence of any one of the antibody clones set forth by Table 1; and wherein said light chain variable domain includes a CDR LI sequence, a CDR L2 sequence and a CDR L3 sequence of any one of the antibody clones set forth by Table 2.

[0021] In another aspect is provided a recombinant protein including: (i) a first antibody region, including: (a) a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO:4, a first CDR H2 as set forth in SEQ ID NO:5 and a first CDR H3 as set forth in SEQ ID NO:6; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:40, a first CDR L2 as set forth in SEQ ID NO:41, and a first CDR L3 as set forth in SEQ ID NO:42; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0022] In another aspect is provided a recombinant protein including: (i) a first antibody region, including: (a) a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO: 13, a first CDR H2 as set forth in SEQ ID NO: 14 and a first CDR H3 as set forth in SEQ ID NO: 15; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:49, a first CDR L2 as set forth in SEQ ID NO:50, and a first CDR L3 as set forth in SEQ ID NO:51; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO: 37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0023] In another aspect is provided a recombinant protein including: (i) a first antibody region, including: (a) a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO:28, a first CDR H2 as set forth in SEQ ID NO:29 and a first CDR H3 as set forth in SEQ ID NO: 30; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:64, a first CDR L2 as set forth in SEQ ID NO:65, and a first CDR L3 as set forth in SEQ ID NO: 66; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO: 37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0024] In another aspect is provided a multivalent recombinant protein including: (i) a first Fc dimerizing domain bound to a first antibody region through a first chemical linker; (ii) a second Fc dimerizing domain bound to a second antibody region through a second chemical linker; wherein the first Fc dimerizing domain is covalently attached to the second Fc dimerizing domain thereby forming a Fc region binding the first antibody region to the second antibody region; and (iii) a third antibody region bound through a third chemical linker to the first antibody region, the second antibody region or the Fc region.

[0025] In another aspect is provided an isolated nucleic acid encoding an antibody as provided herein including embodiments thereof or a recombinant protein as provided herein including embodiments thereof.

[0026] In another aspect is provided a pharmaceutical composition including (i) a therapeutically effective amount of an antibody as provided herein including embodiments thereof and a pharmaceutically acceptable excipient or (ii) a therapeutically effective amount of a recombinant protein as provided herein including embodiments thereof and a pharmaceutically acceptable excipient.

[0027] In another aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of (i) an antibody as provided herein including embodiments thereof or (ii) a recombinant protein as provided herein including embodiments thereof, thereby treating cancer in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGS. 1A-1L show new anti-Interleukin 1 Receptor Accessory Protein antibodies (anti- IL1RAP Abs) production. FIG. 1A shows the ILlRAP-2Ab at SEC200. FIG. IB shows IL1RAP- lOAb at SEC200. FIG. 1C shows ILlRAP-17Ab at SEC200. FIG. ID shows ILlRAP-22Ab at SEC200. FIG. IE shows ILlRAP-24Ab at SEC200. FIG. IF shows ILlRAP-25Ab at SEC200. FIG. 1G shows ILlRAP-33Ab at SEC200. FIG. 1H shows ILlRAP-46Ab at SEC200. FIG. II shows IL1RAP-49 Ab at SEC200. FIG. 1J shows ILlRAP-53Ab at SEC200. FIG. IK shows ILlRAP-58Ab at SEC200. FIG. IL shows ILlRAP-61Ab at SEC200. [0029] FIGS. 2A-2C show new anti-ILlRAP Abs production. FIG. 2A shows the SDS page output in both non-reduced and reduced form of ILlRAP-2Ab, ILlRAP-lOAb, ILlRAP-17Ab, IL1RAP- 22Ab, ILlRAP-24Ab, ILlRAP-25Ab and ILlRAP-33Ab. FIG. 2B shows the SDS page output in both non-reduced and reduced form of ILlRAP-46Ab, ILlRAP-49Ab, ILlRAP-53Ab, ILlRAP-58Ab and ILlRAP-61Ab. FIG. 2C shows the table of the proteins with its yield in mg/L.

[0030] FIGS. 3A-3L show Ab binding to IL 1 RAP on SPR. Binding on SPR, immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics anti-ILlRAP Abs, pos Ctrl (ILlRAP-scFv BiTe) and negative control (IgG) in HBS-EP+ running buffer at different concentrations (nM) and 25’C. FIG. 3A shows a plot of ILlRAP-2Ab binding to IL1RAP on SPR. FIG. 3B shows a plot of ILlRAP-lOAb binding to IL1RAP on SPR. FIG. 3C shows a plot of ILlRAP-17Ab binding to IL1RAP on SPR. FIG. 3D shows a plot of ILlRAP-22Ab binding to IL1RAP on SPR. FIG. 3E shows a plot of ILlRAP-24Ab binding to IL1RAP on SPR. FIG. 3F shows a plot of ILlRAP-25Ab binding to IL1RAP on SPR. FIG. 3G shows a plot of ILlRAP-33Ab binding to IL1RAP on SPR. FIG. 3H shows a plot of ILlRAP-46Ab binding to IL1RAP on SPR. FIG. 31 shows a plot of ILlRAP-49Ab binding to IL1RAP on SPR. FIG. 3J shows a plot of IL1RAP- 53Ab binding to IL1RAP on SPR. FIG. 3K shows a plot of ILlRAP-58Ab binding to IL1RAP on SPR. FIG. 3L shows a plot of IL 1 RAP-61 Ab binding to IL 1 RAP on SPR.

[0031] FIG. 4 shows Ab binding to IL1RAP on cells of MV411.

[0032] FIG. 5 shows Ab binding to IL 1 RAP on cells of THP1.

[0033] FIG. 6 shows Ab binding to IL1RAP on cells of K562 (IL1RAP neg).

[0034] FIG. 7A shows ADCC with PBMC (10: 1) of MV411 at 24 hours. FIG. 7B shows ADCC with PBMC (10: 1) of KASUMI at 24 hours.

[0035] FIGS. 8A-8B shows IFN-Gamma Release (10: 1) using PBMCS and MV411 at 24 hours (FIG. 8A) and at 48 hours (FIG. 8B). [0036] FIG. 9 shows a summary of in-vitro characterization for novel anti-human IL1RAP antibodies.

[0037] FIGS. 10A-10F show Anti-ILIRAP Fab production. FIG. 10A shows the ILlRAP-2Ab at SEC200. FIG. 10B shows ILlRAP-22Ab at SEC200. FIG. 10C shows ILlRAP-24Ab at SEC200. FIG. 10D shows ILlRAP-46Ab at SEC200. FIG. 10E shows ILlRAP-53Ab at SEC200. FIG. 10F shows ILlRAP-58Ab at SEC200.

[0038] FIGS. 11A-11B show Anti-ILIRAP Fab production. FIG. 11A shows the non-reduced state of Anti-ILIRAP Fab production. FIG. 11B shows the reduced state of Anti-ILIRAP Fab production.

[0039] FIGS. 12A-12G show Anti-ILIRAP Fab binding to IL 1 RAP on SPR. Binding on SPR, immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics anti- IL1RAP Fabs, pos Ctrl (ILlRAP-2Ab) and negative control (Fab) in HBS-EP+ running buffer at different concentrations (nM) and 25’C. FIG. 12A shows a plot of IL1RAP-2 Fab binding to Anti- ILIRAP Fab binding to IL1RAP on SPR. FIG. 12B shows a plot of IL1RAP-22 Fab binding to Anti- ILIRAP Fab binding to IL1RAP on SPR. FIG. 12C shows a plot of IL1RAP-24 Fab binding to Anti- ILIRAP Fab binding to IL1RAP on SPR. FIG. 12D shows a plot of IL1RAP-46 Fab binding to Anti- ILIRAP Fab binding to IL1RAP on SPR. FIG. 12E shows a plot of IL1RAP-53 Fab binding to Anti- ILIRAP Fab binding to IL 1 RAP on SPR. FIG. 12F shows a plot of IL 1 RAP-58 Fab binding to Anti- ILIRAP Fab binding to IL1RAP on SPR. FIG. 12G shows a table with binding properties of antibodies provided herein including embodiments thereof.

[0040] FIGS. 13A-13B show Protein Thermal Shift of IL1RAP 2Fab, IL1RAP 22Fab, IL1RAP 24Fab, IL1RAP 46Fab, IL1RAP 53Fab and IL1RAP 58Fab.

[0041] FIGS. 14A-14F show Anti-ILIRAP Fab dual binding to IL1RAP on SPR. Immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics anti-ILlRAP-46Fab and followed by other Fabs at 300nM and 25’C. FIG. 14A shows ILlRAP-46Fab - ILlRAP-46Fab. FIG. 14B shows ILlRAP-46Fab - ILlRAP-2Fab. FIG. 14C shows ILlRAP-46Fab - ILlRAP-24Fab. FIG. 14D shows ILlRAP-46Fab - ILlRAP-22Fab. FIG. 14E shows ILlRAP-46Fab - IL1RAP- 53Fab. FIG. 14F shows ILlRAP-46Fab - ILlRAP-58Fab.

[0042] FIGS. 15A-15F show Anti-ILIRAP Fab dual binding to IL1RAP on SPR. Immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics anti-ILlRAP-24Fab and followed by other Fabs at 300nM and 25’C. FIG. 15A shows ILlRAP-24Fab - ILlRAP-2Fab. FIG. 15B shows ILlRAP-24Fab - ILlRAP-22Fab. FIG. 15C shows ILlRAP-24Fab - ILlRAP-24Fab. FIG. 15D shows ILlRAP-46Fab - ILlRAP-22Fab. FIG. 15E shows ILlRAP-46Fab - IL1RAP- 53Fab. FIG. 15F shows ILlRAP-46Fab - ILlRAP-58Fab.

[0043] FIGS. 16A-16E show Humanized IL1RAP-24 Fab Production. FIG. 16A shows 5k59 at SEC200. FIG. 16B shows 5k59 K41T at SEC200. FIG. 16C shows 4nzr at SEC200. FIG. 16D shows the SDS page output of non-reduced and reduced Humanized IL1RAP-24 Fab Production. FIG. 16E shows the table representing yield in mg/L of 5k59, 5k59 K41T and 4nzr.

[0044] FIGS. 17A-17E show Humanized IL1RAP-24 Fab binding to IL1RAP on SPR. Binding on SPR, immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics Humanized IL1RAP-24 Fabs and IL1RAP-24 Fab in HBS-EP+ running buffer at different concentration (nM) and 25’C. FIG. 17A shows a plot of IL1RAP-24 5k59 Fab binding to IL1RAP on SPR. FIG. 17B shows a plot of IL1RAP-24 5k59 K41T Fab binding to IL1RAP on SPR. FIG. 17C shows a plot of IL1RAP-24 4nzr Fab binding to IL1RAP on SPR. FIG. 14D shows a plot of IL1RAP-24 Fab binding to IL1RAP on SPR. FIG. 17E shows a table with binding properties of antibodies provided herein including embodiments thereof

[0045] FIGS. 18A-18B show Protein Thermal Shift of IL1RAP-24 5k59 Fab, IL1RAP-24 5k59 K41T Fab, IL1RAP-24 4nzr Fab and IL1RAP-24 Fab.

[0046] FIGS. 19A-19E show Humanized IL1RAP-24 Fab binding to IL1RAP on SPR. Binding on SPR, immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics Humanized IL1RAP-24 Fabs and IL1RAP-24 Fab in HBS-EP+ running a buffer at a different concentration (nM) and 25’C. FIG. 19A shows a plot of IL1RAP-24 5k59 Fabs binding to IL1RAP on SPR. FIG. 19B shows a plot of IL1RAP-24 5k59 K41T Fabs binding to IL1RAP on SPR. FIG. 19C shows a plot of IL1RAP-24 4NZR Fabs binding to IL1RAP on SPR. FIG. 19D shows a plot of IL1RAP-24 Fabs binding to IL1RAP on SPR. FIG. 19E shows the thermal stability of all the abovementioned proteins.

[0047] FIGS. 20A-20I show Anti-ILIRAP Fabs binding to Cyno-ILIRAP on SPR. Binding on SPR, immobilization Cyno-ILIRAP on CM5 chip (600RU) through EDC/NHS coupling and kinetics anti-ILlRAP Fabs in HBS-EP+ running a buffer at a different concentration (nM) and 25’C. FIG. 20A shows a plot of ILlRAP-2Fab binding to IL1RAP on SPR. FIG. 20B shows a plot of IL1RAP- 22Fab binding to Cyno-ILIRAP on SPR. FIG. 20C shows a plot of ILlRAP-24Fab binding to Cyno- ILIRAP on SPR. FIG. 20D shows a plot of ILlRAP-46Fab binding to Cyno-ILIRAP on SPR. FIG. 20E shows a plot of ILlRAP-53Fab binding to Cyno-ILIRAP on SPR. FIG. 20F shows a plot of humanized ILlRAP-24Fab binding to Cyno-ILIRAP on SPR. FIG. 20G shows a plot of IL1RAP- 58Fab binding to Cyno-ILIRAP on SPR. FIG. 20H shows a plot of negative control. FIG. 201 shows a table with the binding properties of antibodies provided herein including embodiments thereof.

[0048] FIG. 21A shows generation of Bi-specific Ab using Fab Arm Exchange. FIG. 21B shows the table of targets.

[0049] FIG. 22A-22E show Bispecific Ab: ILR24X3.1 (anti-ILlRAP and anti-CD3) on CEX. FIG. 22A shows a plot of IL1RAP24 K409R. FIG. 22B shows a plot of H26H8 F405L. FIG. 22C shows a plot of BiFOOl . FIG. 22D shows the combined plot of FIGS. 22A-22C. FIG. 22E shows the SDS page output of non-reduced and reduced BiFOOl.

[0050] FIGS. 23A-23E show Bi-specific Ab: ILR24X3.1 binding to IL1RAP and CD3. FIG. 23A shows a plot of BiFOOl to IL1RAP. FIG. 23B shows a plot of BiFOOl to human CD3E6-FC. FIG. 23C shows a plot of ILlRAP-21Fab to IL1RAP. FIG. 23D shows a plot of H26H8 Fab to human CD3e6-Fc. FIG. 23E shows a table with binding properties of antibodies provided herein including embodiments thereof.

[0051] FIGS. 24A-24D show Bi-specific Ab: ILR24X3.1FcEN on CEX. FIG. 24A shows a plot of IL1RAP24 K409R FcEN. FIG. 24B shows a plot of H26H8 F405L FcEN. FIG. 23C shows a plot of ILR24X3. IFcEN. FIG. 24D shows the combined plot of FIGS. 24A-24C.

[0052] FIGS. 25A-25F show Bi-specific Ab: ILR24X3.1FcEN binding to human Fc gamma receptor (FcyRIIIA, CD16a). FIG. 25A shows a plot of ILR24X3.1FcEN. FIG. 25B shows a plot of H26H8 F405L FcEN. FIG. 25C shows a plot of IL1RAP24 K409R FcEN. FIG. 25D shows a plot of ILR24X3.1. FIG. 25E shows a plot of H26H8 F405L. FIG. 25F shows a plot of IL1RAP24 K409R.

[0053] FIGS. 26A-26J show IL 1 RAP silent mutations on IL 1 RAP-24 Ab. FIG. 26A shows IL1RAP-24 Fab BOS in SEC200. FIG. 26B shows IL1RAP-24 Fab Y54S in SEC200. FIG. 26C shows IL 1 RAP-24 Fab Q99E in SEC200. FIG. 26D shows IL1RAP-24 Fab T101E in SEC200. FIG. 26E shows IL1RAP-24 Fab I30SY54S in SEC200. FIG. 26F shows IL1RAP-24 Fab Q99ET101E in SEC200. FIG. 26G shows the SDS page output of the non-reduced and reduced form of IL1RAP silent mutant. FIG. 26H shows the table of the abovementioned proteins with yield in mg/L. FIG. 261 shows IL1RAP-24 Fab I30SQ99E in SEC200. FIG. 26J shows SDS page output of non-reduced and reduced form of IL1RAP-24 Fab I30SQ99E.

[0054] FIGS. 27A-27I show ILIRAP silent mutant binding to IL1RAP on SPR. Binding on SPR, immobilization ILIRAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics ILIRAP - 24 Fabs in HBS-EP+ running a buffer at a different concentration (nM) and 25’C. FIG. 27A shows ILIRAP -24 Fab binding to ILIRAP on SPR. FIG. 27B shows IL1RAP-24 Fab BOS binding to ILIRAP on SPR. FIG. 27C shows IL1RAP-24 Fab Y54S binding to ILIRAP on SPR. FIG. 27D shows IL1RAP-24 Fab Q99E binding to ILIRAP on SPR. FIG. 27E shows IL1RAP-24 Fab T101E binding to ILIRAP on SPR. FIG. 27E shows IL1RAP-24 Fab 130 SQ99E binding to ILIRAP on SPR. FIG. 27G shows IL1RAP-24 Fab 130 SY54S binding to IL1RAP on SPR. FIG. 27H shows IL1RAP-24 Fab Q99ET101E binding to IL1RAP on SPR. FIG. 271 shows a table with binding properties of antibodies provided herein including embodiments thereof.

[0055] FIGS. 28A-28B show the Protein Thermal Shift of IL1RAP-24 Fab I30S, IL1RAP-24 Fab Y54S, IL1RAP-24 Fab Q99E, IL1RAP-24 Fab T101E, IL1RAP-24 Fab I30SY54S, IL1RAP-24 Fab Q99ET101E, IL1RAP-24 Fab I30SQ99E and IL1RAP-24 Fab.

[0056] FIG. 29A-29C show Meditope-enabled humanized IL1RAP-24 (meILlRAP-24) antibody production. FIG. 29A shows meILlRAP-24 Fab V2. FIG. 29B shows SDS page output of nonreduced and reduced forms of meILlRAP-24 Fab V2. FIG. 29C shows a table of meILlRAP-24 Fab V2 with yield in mg/L.

[0057] FIG. 30A-30C show meILlRAP-24 Fab binding to IL1RAP on SPR. Binding on SPR, immobilization IL1RAP on CM5 chip (500RU) through EDC/NHS coupling and kinetics IL1RAP- 24 Fabs in HBS-EP+ running buffer at different concentration (nM) and 25’C. FIG. 30A shows IL1RAP-24 Fab binding to IL1RAP on SPR. FIG. 30B shows meILlRAP-24 Fab binding to ZL1RAP on SPR. FIG. 30C shows a table with binding properties of antibodies provided herein including embodiments thereof.

[0058] FIGS. 31A-31C show meILlRAP-24 Fab binding to meditope on SPR. Binding on SPR and immobilization diphenyl-meditope on CM5 chip (200RU) through EDC/NHS coupling and kinetics meditope-enabled Fabs in HBS-EP+ running buffer at different concentrations (nM) and 25’C. FIG. 31 A shows meILlRAP-24 Fab-V2 binding to meditope on SPR. FIG. 3 IB shows meTras I83E Fab binding to meditope on SPR. FIG. 31C shows a table with binding properties of antibodies provided herein including embodiments thereof.

[0059] FIGS. 32A-32B show Protein Thermal Shift of meILlRAP-24 Fab-V2 and IL1RAP-24 Fab. [0060] FIG. 33A shows the fabrack activation. FIG. 33B shows a plot representing MV411. FIG. 33C shows secondary Ab: AF647 Goat Anti-Human IgG, F(ab’)2 fragment specific, and meditope- 647 in cell line MV411.

[0061] FIG. 34A shows the fabrack activation. FIG. 34B shows a plot representing Molml3. FIG. 34C shows secondary Ab: AF647 Goat Anti-Human IgG, F(ab’)2 fragment specific, and meditope-647 in cell line Molml3.

[0062] FIG. 35 shows secondary Ab: AbF488 Gt F(ab’)2 aHulgG in Fabrack Jurkat nfat-luc CD28 CD19t cell line.

[0063] FIG. 36 shows ILlRAP/meditope simultaneous binding of meditope-enabled anti-ILlRAP antibody ofMV411 cell line.

[0064] FIG. 37 shows ILlRAP/meditope simultaneous binding of meditope-enabled anti-ILlRAP antibody of THP-1 cell line.

[0065] FIG. 38 shows ILlRAP/meditope simultaneous binding of meditope-enabled anti-ILlRAP antibody of Molm3 cell line.

[0066] FIGS. 39A-39C show Biparatopic antibody ILR24XILR53, ILR24XILR2, ILR24XILR22 clones provided herein including embodiments thereof.

[0067] FIGS. 40A-40C show Biparatopic (BPAb) /Thpl. In comparison to the other two BP Abs (#24*2 and #24*22), BP Abs(anti-IL1RAP # 24*53) has a higher percentage of binding. In comparison to all Biparatopic (BP Abs), monoclonal anti-ILlRAP #24 has the best binding.

[0068] FIGS. 41A-41C show Biparatopic (BPAb) binding to MV411 cells.

[0069] FIG. 42A shows binding of Biparatopic antibodies provided herein including embodiments thereof to THP1 cells. FIG. 42B shows binding of Biparatopic antibodies provided herein including embodiments thereof to MV411 cells. In compared to anti-ILlRAP monoclonal #24 Ab, the MFI of Biparatopic (BPAb) (#24*2; #24*22, and #24*53) is lower. BiFOOl "(anti- IL1RAP #24 & anti CD-3) no FC mutation" has a greater MFI than BIF002 "(anti-ILlRAP #24 & anti CD-3) with FC mutation.

[0070] FIG. 43 shows ADCC, 10: 1 RATIO; PBMCs as effector cells and MV411 as Target cells. However, the MFI and percentage of binding of BiParatopic Ab #24*2 were lower than that of monoclonal Ab #24, 24*2 BP Ab showing better cytotoxic activity in ADCC experiments using MV411 as a target cell in comparison to monoclonal #24 Ab.

[0071] FIG. 44A shows IL1RAP-24 K409R-scFvH26H8 (CD3 scFv to C-term of Fc of IL1RAP- 24Ab K409R, linker: S(G4S)3. FIG. 44B shows the SDS page output of a non-reduced and reduced form of IL1RAP-24 K409R-scFvH26H8. FIGS. 44C-44D show preliminary production for biparatropic-bite antibody development. FIG. 44E shows theyield of antibodies provided herein including embodiments thereof in mg/L.

[0072] FIG. 45A shows CD3 binding. FIG. 45B shows IL 1RAP binding. Immobilization CD3s8- Fc (1000RU) or IL1RAP (monomer, 500RU) on CM5 chip through EDC/NHS coupling. Sample preparation: IL1RAP-24 K409R-scFvH26H8, H26H8 Fab or IL1RAP-24 K409R in HBS-EP+ running a buffer at a different concentration and 25’C. FIG. 45C shows binding properties of antibodies provided herein including embodiments thereof.

[0073] FIGS. 46A-46D show Biparatropic-BiTE: ILR24XILR2X3.1 (2 * anti -IL 1 RAP and anti- CD3) on CEX. FIG. 46A shows a plot of IL1RAP2 F405L. FIG. 46B shows a plot of IL1RAP24 K409R scFvH26H8. FIG. 46C shows a plot of ILR24XILR2X3.1. FIG. 46D shows a combined plot of binding activity.

[0074] FIG. 47A shows SCFVW26H8-003-IL1RAP24 (tumor-activated CD3 scFv linked to N-term of LC of IL1RAP 24 Ab, linker: SSGAGGG). FIG. 47B shows the SDS page output of a non-reduced and reduced form of SCFVW26H8-003-IL1RAP24. FIG. 47C shows binding of CD3 with IL1RAP- 24. FIG. 47D shows binding of CD3 with IL1RAP-24 and IL1RAP-53 or CD123. [0075] FIG. 48A shows cleavable scFvW26H8-003-TEVFc (tumor-activated CD3 scFv, linker: GGSAGPAENLYFQGGGGSGGGSG). FIG. 48B shows non- cleavables cFvW26H8-003-TEVFc (tumor-activated CD3 scFv, linker: GGSAGPAENLYFQGGGGSGGGSG). FIG. 48C shows the SDS page output of non-reduced and reduced form of scFvW26H8-003-TEVFc. FIG. 48D shows binding of CD3 and FIG. 48E shows binding of scFv CD3. FIG. 48F shows the abovementioned proteins with yield I mg/L.

[0076] FIG. 49A shows SCFVH26H8-IL1RAP-24 K409R (CD3 scFv to C-term of LC of IL1RAP- 24Ab K409R, linker: S(G4S)3. .FIG. 49B shows the SDS page output of a non-reduced and reduced form of scFvH26H8-ILlRAP-24 K409R. FIG. 49C shows binding of CD3 with IL1RAP- 24. FIG. 49D shows binding of CD3 with IL1RAP-24 and IL1RAP-53 or CD123. FIG. 49E shows the abovementioned proteins with yield in mg/L.

[0077] FIG. 50A shows scFvW26H8-003-ILlRAP24-2 (tumor-activated CD3 scFv linked to N- term of HC of IL1RAP 24Ab, linker: SSGAGGG). .FIG. 50B shows the SDS page output of nonreduced and reduced form of SCFVW26H8-003-IL1RAP24-2. FIG. 50C shows binding of CD3 with IL1RAP-24. FIG. 50D shows binding of CD3 with IL1RAP-24 and IL1RAP-53 or CD123. FIG. 50E shows the scFvW26H8-003-ILlRAP24-2 with yield in mg/L.

[0078] FIG. 51A shows SCFVH26H8-IL1RAP24 K409R-2 (CD3 scFv linked to N-term of HC of IL1RAP 24Ab, linker: GGSAGGG). FIG. 51B shows the SDS page output of a non-reduced and reduced form of scFvH26H8-ILlRAP24 K409R-2. FIG. 51C shows binding of CD3 with IL1RAP- 24. FIG. 51D shows binding of CD3 with IL1RAP-24 and IL1RAP-53 or CD123. FIG. 51E shows the scFvH26H8-ILlRAP24 K409R-2 with yield in mg/L.

[0079] FIG. 52A shows different cell lines and their immune targets in AML cells. FIG. 52B shows a bar graph of different cell lines with MFI ratios considering IL1RAP as a target. FIG. 52C shows a bar graph of different cell lines with MFI ratios considering CLL1 as a target. FIG. 52D shows a bar graph of different cell lines with MFI ratios considering CD33 as a target. FIG. 52E shows a bar graph of different cell lines with MFI ratios considering CD 123 as a target. FIG. 52F is a bar graph showing the percentage of cells expressing IL1RAP in different cell lines. FIG. 52G is a bar graph showing the percentage of cells expressing CLL1 in different cell lines. FIG. 52H is a bar graph showing the percentage of cells expressing CD33 in different cell lines. FIG 521 is a bar graph showing the percentage of cells expressing CD 123 in different cell lines

[0080] FIG. 53A shows a bar graph of different blasts with MFI ratios considering IL1RAP as a target. FIG. 53B shows a bar graph of different blasts with MFI ratios considering CLL1 as a target. FIG. 53C shows a bar graph of different blasts with MFI ratios considering CDD33 as a target. FIG. 52D shows a bar graph of different blasts with MFI ratios considering CD 123 as a target. FIG. 53E is a bar graph showing the percentage of cells expressing IL1RAP in different blasts. FIG. 53F is a bar graph showing the percentage of cells expressing CLL1 in different blasts. FIG. 53Gis a bar graph showing the percentage of cells expressing CD33 in different blasts. FIG. 53H is a bar graph showing the percentage of cells expressing CD123 in different blasts. FIG 531 is a bar graph showing the percentage of cells expressing CD34 in different blasts.

[0081] FIGS. 54A-54F show comparison of IL1-RAP expression on normal donor CD34+ Cell vs. blasts.

[0082] FIG. 55A shows IL2-Rap qPCR results which were normalized using b2m as a housekeeping gene. FIG. 55B shows CD 123 qPCR results which were normalized using b2m as a housekeeping gene.

[0083] FIG. 56 shows IL1-RAP Flow results of different AML cell By Basma. Quantification of IL1-RAP molecules on the surface of AML cell lines USING MESF Beads.

[0084] FIG. 57 shows consistent IL1-RAP expression of commercial vs. #24 ILIRAP-Abs.

[0085] FIG. 58 is a binding test depicting AML banding with IL 1 RAP Ab (BIFO14(IL1RAP #24#2+ CD3), IL1RAP Ab (BIF001 (IL1RAP #24+ CD3), IL1RAP Ab (IL1RAP #24), Commercial IL1RAP and blocked unstained samples in MV4-11, THP-1, and human T-cells, respectively. [0086] FIG. 59 is a binding test depicting T cell and AML banding with BIF018 (IL1RAP +CD3 mutated control), BIF00 l(Fc unmutated), commercial IL1RAP/CD2 samples in MV4-11, THP-1, and human T-cells, respectively.

[0087] FIG.60A is a plot depicting the effect on concentration ofBIFOOl and BIF002, after different time intervals (24h and 48h), on a percentage of specific lysis in ThP-1 with E:T of 1 :5. FIG. 60B is a plot depicting the effect on concentration of various samples (BIF014, and control Cetuximab), incubated at different time intervals (24h and 48), on a percentage of specific lysis in Molml3 with E:T of 1 :5. FIG. 60C is a plot depicting the effect on concentration ofBIFOOl, incubated at different time intervals (24ha nd 48h), on a percentage of specific lysis in K562 with E:T of 1 :5. FIG. 60D is a plot depicting the effect on concentration of BIFOOl and control Cetuximab, incubated at different time intervals (24h and 48h), on a percentage of specific lysis in K562 with E:T of 1 :5.

[0088] FIG. 61A depicts apoptotic cell % of MV4-11 with PBMC(M21) at 48h. FIG. 61B depicts apoptotic cell % of THP-1 with PBMC(M21) at 48h.

[0089] FIGS. 62A-62D show Target (T) with T cell (E-M40) and BITE BIF002 with different ET ratio. FIG. 62A shows Molm 13 (T) with T-cell (E-M40) and BITE in 24 hours. FIG. 62B shows Molm 13 (T) with T-cell (E-M40) and BITE in 48 hours. FIG. 62C shows MV4-11 with T-cell (E- M40) and BITE in 24 hours. FIG. 62D shows MV4-11 with T-cell (E-M40) and BITE in 48 hours.

[0090] FIG. 63A is a plot depicting apoptosis induced cell death at various E:T ratios, at various concentrations. Molm 13 (T) with T-cell (E-M40) and BITE in 48 hours. FIG. 63B is a plot depicting apoptosis induced cell death at various E:T ratios, at various concentrations. MV4-11 (T) with T-cell (E-M40) and BITE in 48 hours.

[0091] FIG. 64A is a plot depicting apoptosis induced cell death at various E:T ratios, at various concentrations. Molm 13 (T) with T-cell (E-M40) and BITE in 48 hours. FIG. 64B is a plot depicting apoptosis induced cell death at various E:T ratios, at various concentrations. MV4-11 (T) with T-cell (E-M40) and BITE in 48 hours. [0092] FIGS. 65A-65K show cytotoxic activity of BIF002 with T cell (M45). FIG. 65A shows the effect of concentration on apoptosis induced cell death percentages in Moml3 with E:T ratio of

5: 1. FIG. 65B shows the effect of concentration on apoptosis induced cell death percentages in HL60 with E:T ratio of 5: 1. FIG. 65C shows the effect of concentration on apoptosis induced cell death percentages in Kgla with E:T ratio of 5:l. FIG. 65D shows the effect of concentration on apoptosis induced cell death percentages in K562 with E:T ratio of 5: 1. FIG. 65E shows the effect of concentration on cell viability in Mom 13 with E:T ratio of 5: 1. FIG. 65F shows the effect of concentration on cell viability in HL-60 with E:T ratio of 5: 1. FIG. 65G shows the effect of concentration on cell viability in Kgla with E:T ratio of 5: 1. FIG. 65H shows the effect of concentration on cell viability in Raji with E:T ratio of 5: 1. FIG. 651 shows IC50 values in Moml3 with E:T ratio of 5: 1 after 24 hours, and 48 hours, respectively. FIG. 65 J shows IC50 values in HL60 with E:T ratio of 5: 1 after 24 hours, and 48 hours, respectively. FIG. 65K shows IC50 values in Kgla with E:T ratio of 5: lafter 24 hours, and 48 hours, respectively.

[0093] FIGS. 66A-66D are plots depicting the effect of BIF002 on killing on HL CD34+ cell at 48h. FIG. 66A shows the effect of concentration of BIF002 (48 h)on apoptotis induced cell death percentages in NL-2 CD34+ with E:T 5: 1. FIG. 66B shows the effect of concentration of BIF002 (48 h) on apoptotis induced cell death percentage in NL-4 CD34+ with E:T 5: 1. FIG. 66C shows the effect of concentration of BIF002 (48 h) on apoptotis induced cell death percentage in NL-5 CD34+ with E:T 5: 1 FIG. 66D shows the effect of concentration of BIF002 (48 h) on apoptotis induced cell death percentage in NL-6 CD34+ with E:T 5: 1.

[0094] FIGS. 67A-67E show T cell activation(CD69,CD25); T cell (F30) as effector cells; E:T Ratio 5: 1, (cell lines with first T ). FIG. 67A is plot depicting the effect of concentration of BIF002 on T-cell activation percentages in THP-1 E:T 5: 1(M36) in 48 hours. FIG. 67B shows the effect of concentration of BIF002 on T-cell activation percentages in Molml3 E:T 5: 1(M36) in 48 hours. FIG. 67C shows the effect of concentration of BIF002 on T-cell activation percentages in MV4-11 E:T 5: 1(M36) in 48 hours. FIG. 67D shows the effect of concentration of BIF002 on T-cell activation percentages in Kgla E:T 5: 1(M36) in 48 hours. FIG. 67E shows the effect of concentration of BIF002 on T-cell activation percentages in K562 E:T 5: 1(F36) in 48 hours.

[0095] FIGS. 68A-68G show T cell activation(CD69,CD25); T cell (F30) as effector cells; E:T Ratio 5:1, (cell lines with first T ). FIG. 68A is a plot depicting the effect of concentration of BIF002 on T-cell activation percentages in THP-1 E:T 5: 1(F36) in 48 hours. FIG. 68B shows the effect of concentration of BIF002 on T-cell activation percentages in Molml3 E:T 5: l(F30) in 48 hours. FIG. 68C shows the effect of concentration of BIF002 on T-cell activation percentages in MV4-11 E:T 5:1(F3O) in 48 hours. FIG. 68D shows the effect of concentration of BIF002 on T-cell activation percentages in U937 E:T 5: l(F30) in 48 hours. FIG. 68E shows the effect of concentration of BIF002 on T-cell activation percentages in HL60 E:T 5: 1(F36) in 48 hours. FIG. 68F shows the effect of concentration of BIF002 on T-cell activation percentages in Kgla E:T 5: l(F30) in 48 hours. FIG. 68G shows the effect of concentration of BIF002 on T-cell activation percentages in K562 E:T 5:1(F3O) in 48 hours.

[0096] FIGS. 69A-69G show T cell activation(CD69,CD25); E:T Ratio 5: 1, (blasts ). FIG. 69A is plot depicting the effect of concentration of BIF002 on T-cell activation percentages in 21-1222 E:T 5:1(M58) in 48 hours. FIG. 69B shows the effect of concentration of BIF002 on T-cell activation percentages in 22-1244 E:T 5: 1(M36) in 48 hours. FIG. 69C shows the effect of concentration of BIF002 on T-cell activation percentages in 22-596 E:T 5: 1(F3O) in 48 hours. FIG. 69D shows the effect of concentration of BIF002 on T-cell activation percentages in 3448 E:T 5: l(F30) in 48 hours. FIG. 69E shows the effect of concentration of BIF002 on T-cell activation percentages in 22-0148 E:T 5:1(M58) in 48 hours. FIG. 69F shows the effect of concentration of BIF002 on T-cell activation percentages in 1160P E:T 5: l(F30) in 48 hours. FIG. 69G shows the effect of concentration of BIF002 on T-cell activation percentages in 21-710 E:T 5:l(F30) in 48 hours.

[0097] FIGS. 70A-70E show intracellular cytokines; T cell (M36) as effector cell; E:T Ratio 5: 1 (by Flow). FIG. 70A shows percentages of THP-1 intracellular cytokines expressed with various concentrations of interleukins and interferons. FIG. 70B shows percentages of molml3 intracellular cytokines expressed with various concentrations of interleukins and interferons. FIG. 70C shows percentages of MV41-11 intracellular cytokines expressed with various concentrations of interleukins and interferons. FIG. 70D shows percentages of Kgla intracellular cytokines expressed with various concentrations of interleukins and interferons. FIG. 70E shows percentages of K562 intracellular cytokines expressed with various concentrations of interleukins and interferons.

[0098] FIG. 71A depicts the effect of concentration of BIF002 on interferon gamma expression in Kgla, E:T 5: 1 (M-58) after 48 hours. FIG. 71B depicts the effect of concentration of BIF002 on interferon gamma expression in THP-1, E:T 5: 1 (M-58) after 48 hours. FIG. 71C depicts the effect of concentration of BIF002 on interferon gamma expression in 21-1222, E:T 5:1 (M-58) after 48 hours. FIG. 71D depicts the effect of concentration of BIF002 on interferon gamma expression in 22-0148, E:T 5: 1 (M-58) after 48 hours.

[0099] FIG. 72A depicts the effect of concentration of BIF002 on interferon gamma expression in THP-1, E:T 5: 1 (M-36) after 48 hours. FIG. 72B depicts the effect of concentration of BIF002 on interferon gamma expression in Molm-13, E:T 5:1 (M-36) after 48 hours. FIG. 72C depicts the effect of concentration of BIF002 on interferon gamma expression in 22-1244, E:T 5:1 (M-36) after 48 hours. FIG. 72D depicts the effect of concentration of BIF002 on interferon gamma expression in MV4-11, E:T 5: 1 (M-36) after 48 hours. FIG. 72E depicts the effect of concentration of BIF002 on interferon gamma expression in Kgla, E:T 5: 1 (M-36) after 48 hours. FIG. 72F depicts the effect of concentration of BIF002 on interferon gamma expression in K562, E:T 5: 1 (M-36) after 48 hours.

[0100] FIG. 73 depcits the effect ofBIF002 concentration with varying E:T ratios (2.5:1 and 5: l)on T-cell proliferation (CD4 and CD8) in Thp-1, Molml3, MV4-11, Kgla, K562, T cell (M36) as effector cell.

[0101] FIG. 74A shows the effect of BIF002 concentration with varying E:T ratios (2.5: 1 and 5: 1) on T-cell proliferation in Thp-1, Molml3, MV4-11, Kgla, K562. FIG. 74B is a bar graph depicting the effect of concentration of BIF002 on division index and proliferation index in Thp-1, Molml3, MV4-l l, Kgla, and K562. [0102] FIG. 75A shows the effect of target cells when Tcells and BiF002 are mixed with E:T ratio 5: 1 on day 3 on T cell proliferation in Thp-1, Molml3, Kgla, K562and T cell (M48) acting as effector cell. FIG. 75B shows IL1RAP expression and BIF002 concentration dependant on T cell proliferation in Molm 13 and kgla.

[0103] FIG. 76A shows the effect of target cells when Tcells and BiF002 are mixed with varying E:T ratios (2.5: 1 and 5: 1) on T cell proliferation and T cell (M48) acting as effector cell, (blasts). FIG. 76B shows a graph depicting the effect of concentration in various ratios on different days.

[0104] FIGS. 77A-77C show agilent real time cell analysis. FIG. 77A shows normalized GFP positive cell count plot for Molm 13 cells incubated with T(F30) at E:T ratios 5 to 1, with BiF002(0, 0.1, l,10,100nM). FIG. 77B shows normalized GFP positive cell count plot for thpl cells incubated with T(F30) at E:T ratios 5 to 1, with BiF002(0, 0.1, l,10,100nM). FIG. 77C shows normalized GFP positive cell count plot for MV411 cells incubated with T(F30) at E:T ratios 5 to 1, with BiF002(0, 0.1, l,10,100nM).

[0105] FIG. 78A shows long term cyotoxic activity of BIF002 with T cell (M45) specifically Raji killing with low E:T ratio in 5 days targeting 10,000 cells. FIG. 78B shows long time killing of BIF002 with T cell (M45) specifically Molml3 killing with low E:T ratio in 5 days targeting 10,000 cells.

[0106] FIG. 79A shows BIF002 lOnM with T cell activation markers at 72 hours. T cells were similarly activated with E:T 5 or E:T 1. FIG. 79B shows BIF002 lOnM with T cell exhausted markers at 72 hours. BiF002 by itself will not exhaust T cells. BiF002 along with the target cells exhaust the T cells. E:T 1 is increases T cell exhaustion more than 5: 1.

[0107] FIG. 80 shows IL1RAP and CD3 bispecific (BIF002) in vivo data when Molml3 luciferous model is studied exhibiting antileukemic effects in vivo.

[0108] FIG. 81 shows effects on NSG mice when injected with Molm- 13 luc + 0.6M on different days and in different concentrations. [0109] FIG. 82A shows the effects of IgG with different concentrations of BiF002 + active T cells injected in mice. FIG. 82B shows engraftment of PB by flow at day 25 of the study. FIG. 82C shows the effect on a mouse when injected with 0.6M Molm-13 luc+IV injection. The mice are NSGs. FIG. 82D shows engraftment change of the mice which exhibits total emission of of IgG plus T cells along with BiFOOl. FIG. 82E shows engraftment change of the mice which exhibits a probability of survival of of IgG plus T cells along with BiFOOl.

[0110] FIG. 83 shows IL1RAP and CD3 bispecific (BIF002) in vivo data when Molm 13 luciferous model is studied exhibiting active T cells versus resting T cells effects.

[0111] FIG. 84 shows the effects of injected Molm-13 luc + 0.6M on NSG mice with 6 doses of T-cells and 17 doses of antibodies.

[0112] FIG. 85A shows the effects of IgG with different concentrations of BiFOOl along with active T cells injected in mice. FIG. 85B shows the effects on a mouse when injected with IM Molm-13 luc+IV injection. The mice are NSGs-female. FIG. 85C shows the percentage of human T% in PB- D14. FIG. 85D shows the percentage of Molm 13% in PB-D14. FIG. 85E shows the percentage of human T% in PB-D22. FIG. 86F shows the percentage of Molm 13% in PB-D22.

[0113] FIG. 86 shows the total emission photons of different groups consisting of different concentrations of BITE and T cells.

[0114] FIG. 87 shows the probability of survival of different groups consisting of different concentrations of BITE and T cells.

[0115] FIG. 88A shows the percentage of bone marrow Molm-13% in different concentrations of BITE and T-cells. FIG. 88B shows percentage of spleen Molm 13% in different concentrations of BITE and T-cells. FIG. 88C shows percentage of peripheral blood Molm 13% in different concentrations of BITE and T-cells. FIG. 88D shows the percentage of peripheral blood T 13% in different concentrations of BITE and T-cells. FIG. 88E shows the percentage of spleen T 13% in different concentrations of BITE and T-cells. FIG. 88F shows the percentage of bone marrow T 13% in different concentrations of BITE and T-cells.

[0116] FIG. 89 shows IL1RAP and CD3 bispecific (BIF002) in vivo data when the Molml3 luciferous model is under dose exploration.

[0117] FIG. 90 shows study on NSGs mice when injected with Molm-13 luc in different concentartions on different dates.

[0118] FIG. 91A shows effects of active T cells with different concentrations of BITE injected in mice along with the Molml3 cell line. FIG. 91B shows the effect on a mouse when injected with 0.6M Molm-13 luc+IV injection. The mice are NSGs-male and the image represents after 6-8 weeks. FIG. 91C shows total emission photons of active T-cells and different concentrations of BITE in Molm 13% in PB-D24. FIG. 91D shows total emission photons of active T-cells and different concentrations of BITE in human T% in PB-D24. FIG. 91E shows emission photons of active T- cells and different concentrations of BITE in Molm 13.

[0119] FIGS. 92A-92C shows total emission photons of active T-cells and different concentrations of BITE in E-8 (FIG. 92A), E-15 (FIG. 92B) and E-22 (FIG. 92C).

[0120] FIGS. 93A-93E show IL1RAP Expression at Various Levels on the Cell Surface of AML Cell Lines and Primary AML Blasts. FIG. 93A: IL1RAP expression on AML cell lines by flow cytometry (FCM), representative histogram data (top), data from three independent experiments (bottom). FIG. 93B: ILIRAP expression compared to control on AML MNCs (n=21), AML CD34+ cells (n=14), and normal (NL) CD34+ BM cells (n=14). FIG. 93C: Comparison of ILIRAP expression in bulk cells and CD34+ population from the same AML patient (n=14). FIG. 93D: Percentage of IL1RAP+ CD33+, CD123+, and CLL-1+ cells in AML CD34+ cells by FCM (n=12). FIG. 93E: Percentage of IL1RAP+ CD33+, CD123+, and CLL-1+ in normal (NL) CD34+ bone marrow cells by FCM (n=8). [0121] FIGS. 94A-94H show Generation and Selection of an Exemplary Antibody Provided Herein. FIG. 94A: Binding of IL1RAP Fabs to immobilized IL1RAP by Surface Plasma Resonance (SPR). FIG. 94B: Profde of the derivative of fluorescence emission as a function of temperature (Upper), kD, and Tm of anti-ILlRAP Fabs (Lower). FIG. 94C: Binding of anti-ILlRAP Fab #24 to IL 1RAP -positive leukemia cell lines. FIG. 94D: SDS-PAGE of anti-ILlRAP mAb #24 antibody under reducing and non-reducing conditions. FIG. 94E: Size exclusion chromatography (SEC) of anti-ILlRAP mAb #24. FIG. 94F: Schematic design of BIF002, wild type (wt), mutant (mut). The image was created with BioRender.com. FIG. 94G: Ion exchange chromatography (IEC) of IL1RAP/CD3 T-cell engager. FIG. 94H: Binding of IL1RAP antibodies to IL 1 RAP -positive myeloid leukemia cells or T-cells, commercial anti-Human IL1RAP PE antibody was used for myeloid leukemia cells, or anti-Human CD3 PE antibody was used for T-cells.

[0122] FIGS. 95A-95J show BIF002 Demonstrates Potent Dose- and Effector-to-Target Cell Ratio-Dependent Activity. FIG. 95A: Cell lysis of THP-1 and M0LM13 with healthy donor resting T-cells at different E:T ratios and various concentrations of BIF002 at 48h (normalized to no Ab at same E:T ratio; technical triplicate; one representative experiment out of two). FIG. 95B: Real-time quantification of GFP-expressing target cell numbers (Upper) based on fluorescence plots (Lower- THP-1) during co-culture with BIF002 (0, 0.1, 1, 10, lOOnM) and T-cells (E:T ratio 5: 1), recorded by Agilent xCELLigence (one health donor T-cells with technical duplicate; one representative experiment out of two). FIG. 95C: Cell lysis (Left, technical triplicate; one representative experiment out of two) and IC50 of BIF002 on AML cell lines at E:T ratio 5: 1 in 48h (Right, from two independent experiments with different health donor T-cell). FIG. 95D) Cell lysis of BIF002 on primary AML bulk cells at E:T ratio 5: 1 in 48h (one health donor T-cells with technical duplicate or triplicate). FIG. 95E: Cell lysis of BIF002 on AML CD34+ cells at E:T ratio 5: 1 in 48h (one health donor T-cells with technical triplicate). FIG. 95F: BIF-mediated specific killing with healthy donor resting T-cells at E:T ratio 5: 1 in 48h on two AML CD34+ cells (technical triplicate). FIG. 95G: BIF-mediated specific killing with healthy donor PBMC at E:T ratio 5: 1 in 48h on two AML cell lines (technical triplicate, one representative experiment out of two). FIG. 95H: Representative primary AML bulk cell apoptotic plots when co-cultured with T-cells at E:T ratio 5 and BIF002 or control BIF026 by FCM Lefty, Primary AML bulk apoptotic cell percent with T-cells at E:T ratio 5: 1 in 48h with BIF002 or BIF026 control (n=5) Right FIG. 951: Cell lysis of BIF002 on normal CD34+ bone marrow cells at E:T ratio 5:1 in 48h (one health donor T-cells with technical triplicate). FIG. 95 J: Representative colonies and quantification of colony-forming cells (CFC) in normal CD34+ cells (one representative experiment out of two). Apoptotic cells were evaluated by 7-amino actinomycin D (7-AAD+) or Annexin V (+) labeling. Cell death was evaluated by 7-amino actinomycin D (7-AAD+) labeling. The number of depleted target-cell specific lysis was determined with the following formula: % Lysis = 100 - (viable cells of treatment group ^x 100/viable cells of untreated control group).

[0123] FIGS. 96A-96F show BIF002 Induces IL1RAP and Dose-Dependent T-Cell Activation and Cytokine Release Resulting in T-Cell Proliferation. FIG. 96A: T-cell activation assessed by CD69 and CD25 markers after 48h co-culture with AML cell lines (BIF002 InM, from three independent experiments with different health donor T-cell) or primary AML blasts containing different IL1RAP expression at E:T ratio 5: l(BIF002 lOnM, one health donor T-cells with technical duplicate). FIG. 96B: Dose-dependent T-cell activation with BIF002 in 48h co-culture with AML cell lines or blasts (technical duplicate). FIG. 96C: IL1RAP expression and dose-dependent response to BIF002 on IFN- y release after 48h co-culture of cell lines and blasts by ELISA (technical duplicate). FIG. 96D: Intracellular TFN-y expression on CD8 and CD4 T-cell subpopulations by FCM when cultured for 48h with AML cell lines at E:T ratio of 5: 1, (technical duplicate; one representative experiment out of two). FIG. 96E) Representative histogram overlays and quantification of proliferation induced by Cell Trace Violet (CTV) dilution on CD3+ T-cells after 5 days of co-culture with AML cell lines and BIF002 InM at E:T ratio 2: 1 or 5 : 1. FIG. 96F: BIF002 dose-dependent T-cell proliferation when co-cultured with AML blasts after 5 days at E:T ratio 2: 1. [0124] FIGS. 97A-97J show In vivo Efficacy of BIF002 in the Luciferase-Expressing M0LM13 Model. FIG. 97A: Schematic design of the luciferase-expressing M0LM13 mice model. FIG. 97B: Tumor burden assessed using bioluminescent imaging. FIG. 97C: Quantitation of total bioluminescent signal (Total Emission, Photons/s) in each group at indicated times post-injection. FIG. 97D: Kaplan-Meier survival curve for MOLM 13 -engrafted mice. FIG. 97E: Weight change of mice. FIG. 97F: Schematic design of the dose finding mice model. FIG. 97G: Tumor burden assessed of the dose finding mice model. FIG. 97H: Quantitation of total bioluminescent signal of the dose finding mice model. FIG. 971: Kaplan-Meier survival curve for of the dose finding mice model. FIG. 97J: Weight change of the dose finding mice model. Significance values: *, p<0.05;

**, p<0.01; ***, p<0.001; ****, p<0.0001.

[0125] FIGS. 98A-98G show In vivo Efficacy of BIF002 in the Luciferase-Expressing AML PDX Model. FIG. 98A: Schematic design of the PDX luciferous model. FIG. 98B: Tumor burden assessed using bioluminescent imaging. FIG. 98C: Quantitation of total bioluminescent signal (Total Emission, Photons/s) in each group at indicated times post-injection. FIG. 98D: Kaplan- Meier survival curve for luciferase-expressing AML PDX. FIG. 98E: Blasts and human T-cell populations at day 25 in peripheral blood by FCM. FIG. 98F: Representative Wright-Giemsa stain of peripheral blood by microscope (2 mice per group, 1000X). FIG. 98G: Weight change of mice. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0126] FIGS. 99A-99I show In vivo Efficacy of BIF002 in the AML PDX Model. FIG. 99A: Schematic design of the PDX model. FIG. 99B: Blasts and human T-cell populations at Day 28 (Upper') and at Day 70 (Lower) by flow cytometry. FIG. 99C: Representative gating of blasts and T-cells on day 28 by flow cytometry (2 mice in each group). FIG. 99D: Kaplan-Meier survival curve on first transplantation (right, n=7), and weight change of mice in first transplantation (left, week 0-1 n=10, week 5 n=7). FIG. 99E: Spleen size and weight of different groups. FIG. 99F: Blasts and human T-cell populations in bone marrow, peripheral blood, and spleen at the time for the second transplantation by flow cytometry (n=3). FIG. 99G: Wright-Giemsa stain of peripheral blood by microscope (n=3, 1000X). FIG. 99H: Blasts and human T-cell populations at Day 21 in second transplantation mice peripheral blood by flow cytometry (day 34). FIG. 991: Kaplan-Meier survival curve of mice on the second transplantation (n=7). Results shown represent mean ± SEM. Significance values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0127] FIGS. 100A-100C show that no cell killing observed when either AML cells or T-cells alone where treated with BIF002. FIG. 100A: Cell viability and apoptosis of AML cells with BIF002 or BIF026 control (lOnM) at different time points. FIG. 100B: Cell viability and apoptosis of T-cells with BIF002 or BIF026 control (lOnM) at different time points. FIG. 100C: Cell viability and apoptosis of T-cells with various dose of BIF002 or BIF026 control for 48h. Cell growth was measured with Cell Titer-Gio Luminescent Cell Viability Assay (G7570, Promega). Apoptosis of target cells were normalized to non-treatment at Oh.

[0128] FIGS. 101A-101B show IL1RAP expression and dose-dependent response to BIF002 on TNF-u release. IL1RAP expression and dose-dependent response to BIF002 on TNF-a release after 48h co-culture of cell lines (FIG. 101A) and blasts (FIG. 101B) by ELISA (technical duplicate).

[0129] FIGS. 102A-102B show proliferation of CD4 and CD8+ T-cells. FIG. 102A: Proliferation induced by Cell Trace Violet (CTV) dilution on CD4 and CD8+ T-cells after 5 days of co-culture with AML cell lines at E:T ratios 2: 1 and 5: 1, with BIF002 InM. FIG. 102B: Proliferation induced by CTV dilution on CD4 and CD8+ T-cells after 5 days of co-culture with primary AML samples at E:T ratios 2: 1 with BIF002 InM or lOnM by flow cytometry (FCM).

[0130] FIGS. 103A-103B show in vivo pharmacokinetics (PK) test of BIF002 in healthy B6 mice. FIG. 103A: Schematic design of the in vivo PK tests. FIG. 103B: PK curve of BIF002 injected intravenously at 0.5mg/kg (lOpg/ml) and 5mg/kg (lOOpg/ml).

[0131] FIGS. 104A-104C show BIF002 Shows No Toxicity in Humanized Mice Model. FIG. 104A: Schematic design of the preliminary toxicity studies (BIF002 lOpg every 3 days). FIG. 104B: HuCD45% before and after treatment. FIG. 104C: spleen weight after treatment. [0132] FIG. 105 shows the patients’ primary AML sample information.

[0133] FIG. 106 shows flow antibodies used for the experiments described herein.

[0134] FIG. 107 shows the structure of anti-lLl RAP-24 Fab. Ribbon representation (left) of heavy chain and light chain. Stick representation (right) of variable regions of Fab. Residues for mutation are indicated by arrows.

[0135] FIGS. 108A-108C show the effects of IL1RAP antibodies and bispecific antibodies provided herein on IL-1 cell signaling. FIG. 108A: Results from a Western blot showing effect of a IL1RAP commercially available antibody (IL1RAP C) and IL1RAP antibody and bispecific clones provided herein on IL-1 cell signaling. M0LM13 cells were incubated with untreated human IgG 20 ug/ml (CON Ab), IL1RAP commercial antibody 20 ug/ml (ILIRAP C), IL1RAP-24 clone 20 ug/ml, and BIF002 5 ug/ml for 24 hours. After 24 hours, rhIL-ip (20 ng/ml) was added and incubated with cells for 30 minutes at 37 °C. The recommended concentration of the commercial antibody is 20 ug/ml. Since the IC50 for BIF002 is much lower, 5 ug/ml was used. FIG. 108B: IL1-P blocking with antibodies in the presence of IL1-P (0.5 ng/mL). FIG. 108C:ILl-a blocking with antibodies in the presence of ILl-a (100 ng/mL).

DETAILED DESCRIPTION

[0136] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0137] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

DEFINITIONS

[0138] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0139] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched non-cyclic carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkyl moiety may be fully saturated.

[0140] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, -CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. [0141] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable non-cyclic straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g. O, N, P, Si or S) and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH₂-CH₂-N(CH₃)-CH₃, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH₃, -CH₂-CH₂-S(O)2-CH₃, -CH=CH-O-CH₃, -Si(CH₃)₃, -CH₂-CH=N-OCH₃, -C H=CH-N(CH₃)-CH₃, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).

[0142] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)₂R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R", -OR', -SR', and/or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.

[0143] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, non-aromatic cyclic versions of "alkyl" and "heteroalkyl," respectively, wherein the carbons making up the ring or rings do not necessarily need to be bonded to a hydrogen due to all carbon valencies participating in bonds with non-hydrogen atoms. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3 -cyclohexenyl, cycloheptyl, 3- hydroxy-cyclobut-3-enyl-l,2, dione, lH-l,2,4-triazolyl-5(4H)-one, 4H-l,2,4-triazolyl, and the like. Examples of heterocycloalkyl include, but are not limited to, l-(l,2,5,6-tetrahydropyridyl), 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl , and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. A heterocycloalkyl moiety may include one ring heteroatom (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include two optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include three optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include four optionally different ring heteroatoms (e g., O, N, S, Si, or P). A heterocycloalkyl moiety may include five optionally different ring heteroatoms (e.g., O, N, S, Si, or P). A heterocycloalkyl moiety may include up to 8 optionally different ring heteroatoms (e.g., O, N, S, Si, or P).

[0144] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci- C4)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2- tri fluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like.

[0145] The term "acyl" means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0146] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2 -imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An "arylene" and a "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. Non-limiting examples of aryl and heteroaryl groups include pyridinyl, pyrimidinyl, thiophenyl, thienyl, furanyl, indolyl, benzoxadiazolyl, benzodi oxolyl, benzodioxanyl, thianaphthanyl, pyrrolopyridinyl, indazolyl, quinolinyl, quinoxalinyl, pyridopyrazinyl, quinazolinonyl, benzoisoxazolyl, imidazopyridinyl, benzofuranyl, benzothienyl, benzothiophenyl, phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furylthienyl, pyridyl, pyrimidyl, benzothiazolyl, purinyl, benzimidazolyl, isoquinolyl, thiadiazolyl, oxadiazolyl, pyrrolyl, diazolyl, triazolyl, tetrazolyl, benzothiadiazolyl, isothiazolyl, pyrazolopyrimidinyl, pyrrolopyrimidinyl, benzotri azolyl, benzoxazolyl, or quinolyl. The examples above may be substituted or unsubstituted and divalent radicals of each heteroaryl example above are non-limiting examples of heteroarylene. A heteroaryl moiety may include one ring heteroatom (e.g., O, N, or S). A heteroaryl moiety may include two optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include three optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include four optionally different ring heteroatoms (e.g., O, N, or S). A heteroaryl moiety may include five optionally different ring heteroatoms (e.g., O, N, or S). An aryl moiety may have a single ring. An aryl moiety may have two optionally different rings. An aryl moiety may have three optionally different rings. An aryl moiety may have four optionally different rings. A heteroaryl moiety may have one ring. A heteroaryl moiety may have two optionally different rings. A heteroaryl moiety may have three optionally different rings. A heteroaryl moiety may have four optionally different rings. A heteroaryl moiety may have five optionally different rings.

[0147] A fused ring heterocycloalkyl-aryl is an aryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fused ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fused to a cycloalkyl. A fused ring heterocycloalky I -heterocycloalky I is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkylcycloalkyl, or fused ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substituents described herein.

[0148] As used herein, the terms "heteroatom" or "ring heteroatom" are meant to include, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0149] A "substituent group," as used herein, means a group selected from the following moieties:

(A) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -C0NH₂, -NO₂, -SH, -SO₂C1, -SO₃H, -SO4H, -SO₂NH₂, -NHNH₂, -0NH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, -NHSO2H, -NHC= (O)H, -NHC(0)-0H, -NHOH, -OCF3, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(i) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -C0NH₂, -NO₂, -SH, -SO₂C1, -SO3H, -SO4H, -SO₂NH₂, -NHNH₂, -0NH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, -NHSO2H, -NHC= (O)H, -NHC(0)-0H, -NHOH, -OCF3, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(a) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -C0NH₂, -NO₂, -SH, -SO₂C1, -SO3H, -SO4H, -SO₂NH₂, -NHNH₂, -0NH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, - NHS0₂H, -NHC= (O)H, -NHC(0)-0H, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -NO₂, -SH, -SO₂C1, -SO3H, -SO4H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHO(0)NHNH₂, -NHC=(O) NH₂, -NHSO₂H, -NHC= (O)H, -NHC(O)-OH, -NHOH, -OCF3, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0150] As used herein, the term "conjugate" refers to the association between atoms or molecules. The association can be direct or indirect. For example, a conjugate between a nucleic acid and a protein can be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels- Alder addition).

[0151] Useful reactive moieties or functional groups used for conjugate chemistries (including "click chemistries" as known in the art) herein include, for example:

(a) carboxyl groups and various derivatives thereof including, but not limited to, N- hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc. (c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels- Alder reactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold;

(h) amine or sulfhydryl groups, which can be, for example, acylated, alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc.;

(j) epoxides, which can react with, for example, amines and hydroxyl compounds;

(k) phosphoramidites and other standard functional groups useful in nucleic acid synthesis;

(l) metal silicon oxide bonding;

(m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds; and

(n) sulfones, for example, vinyl sulfone. [0152] Chemical synthesis of compositions by joining small modular units using conjugate (“click”) chemistry is well known in the art and described, for example, in H. C. Kolb, M. G. Finn and K. B. Sharpless ((2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie International Edition 40 (11): 2004-2021); R. A. Evans ((2007). "The Rise of Azide-Alkyne 1,3-Dipolar 'Click' Cycloaddition and its Application to Polymer Science and Surface Modification". Australian Journal of Chemistry 60 (6): 384-395; W.C. Guida et al. Med. Res. Rev. p 3 1996; Spiteri, Christian and Moses, John E. ((2010). "Copper-Catalyzed Azide-Alkyne Cycloaddition: Regioselective Synthesis of 1,4, 5 -Tri substituted 1,2,3-Triazoles". Angewandte Chemie International Edition 49 (1): 31-33); Hoyle, Charles E. and Bowman, Christopher N. ((2010). "Thiol-Ene Click Chemistry". Angewandte Chemie International Edition 49 (9): 1540-1573); Blackman, Melissa L. and Royzen, Maksim and Fox, Joseph M. ((2008). "Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels-Alder Reactivity". Journal of the American Chemical Society 130 (41): 13518-13519); Devaraj, Neal K. and Weissleder, Ralph and Hilderbrand, Scott A. ((2008). "Tetrazine Based Cycloadditions: Application to Pretargeted Live Cell Labeling". Bioconjugate Chemistry 19 (12): 2297-2299); Stockmann, Henning; Neves, Andre; Stairs, Shaun; Brindle, Kevin; Leeper, Finian ((2011). "Exploring isonitrile-based click chemistry for ligation with biomolecules". Organic & Biomolecular Chemistry),; “Selective Functionalization of a Genetically Encoded Alkene- Containing Protein via ‘Photoclick Chemistry’ in Bacterial Cells”. Journal of the American Chemical Society 130:9654-9655; Song, Wenjiao and Wang, Yizhong and Qu, Jun and Lin, Qing (2008). “Cu-free click cycloaddition reactions in chemical biology”. Chem Soc Rev 39(4): 1272- 1279, Jewett, John C. and Bertozzi, Carolyn R. (2010). “Light-Triggered Click Chemistry”. Chemical Reviews 2021 121 (12), 6991-7031, Srikanth Kumar, Gangnam and Lin, Qing all of which are hereby incorporated by reference in their entirety and for all purposes.

[0153] The reactive functional groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the proteins or nucleic acids described herein. By way of example, the nucleic acids can include a vinyl sulfone or other reactive moiety (e.g., maleimide). Optionally, the nucleic acids can include a reactive moiety having the formula -S-S-R. R can be, for example, a protecting group. Optionally, R is hexanol. As used herein, the term hexanol includes compounds with the formula CeHnOH and includes, 1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-l- pentanol, 3 -methyl- 1 -pentanol, 4-methyl-l -pentanol, 2-methyl-2-pentanol, 3-methyl-2-pentanol, 4- methyl-2-pentanol, 2-methyl-3 -pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-l -butanol, 2,3- dimethyl-1 -butanol, 3,3-dimethyl-l-butanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-2-butanol, and 2- ethyl-l-butanol. Optionally, R is 1-hexanol.

[0154] As used herein, the term "about" means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, the term "about" means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about means the specified value.

[0155] The terms "a" or "an," as used in herein means one or more. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C1-C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls. Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R- substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different.

[0156] A “chemical linker,” as provided herein, is a covalent linker, a non-covalent linker, a peptide or peptidyl linker (a linker including a peptide moiety), a nucleic acid linker, a polymer, a cleavable peptide linker, a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene or any combination thereof.

[0157] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -S(O)2NH-, -NH-, -NHC(O)NH-, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted alkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted cycloalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heterocycloalkylene, substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted arylene or substituted (e.g., substituted with a substituent group, a size-limited substituent or a lower substituent group) or unsubstituted heteroarylene.

[0158] The chemical linker as provided herein may be a bond, -O-, -S-, -C(O)-, -C(O)O-, -C(O)NH-, -S(O)₂NH-, -NH-, -NHC(O)NH-, -C-O-O- substituted or unsubstituted (e.g., C1-C20, C1-C10, C1-C5) alkylene, substituted or unsubstituted (e.g., 2 to 20 membered, 2 to 10 membered, 2 to 5 membered) heteroalkylene, substituted or unsubstituted (e.g., Cs-Cs, C3-C6, C3-C5) cycloalkylene, substituted or unsubstituted (e.g., 3 to 8 membered, 3 to 6 membered, 3 to 5 membered) heterocycloalkylene, substituted or unsubstituted (e.g., Ce-Cio, Ce-Cs, Ce-Cs) arylene or substituted or unsubstituted (e.g., 5 to 10 membered, 5 to 8 membered, 5 to 6 membered,) heteroarylene, or polymeric make-ups of the groups listed above such as polyamides, polyethlyneglycols, or linked alkyl chains.

[0159] In embodiments, the chemical linker is a covalent linker. In embodiments, the chemical linker is a hydrocarbon linker. In embodiments, the chemical linker is a cleavable peptide linker.

[0160] Thus, a chemical linker as provided herein may include a plurality of chemical moieties, wherein each of the plurality of chemical moieties is chemically different. Alternatively, the chemical linker may be a non-covalent linker. Examples of non-covalent linkers include without limitation, ionic bonds, hydrogen bonds, halogen bonds, van der Waals interactions (e.g. dipoledipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), and hydrophobic interactions. In embodiments, a chemical linker is formed using conjugate chemistry including, but not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).

[0161] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0162] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. Nucleic acid as used herein also refers to nucleic acids that have the same basic chemical structure as a naturally occurring nucleic acid. Such analogues have modified sugars and/or modified ring substituents, but retain the same basic chemical structure as the naturally occurring nucleic acid. A nucleic acid mimetic refers to chemical compounds that have a structure that is different the general chemical structure of a nucleic acid, but that functions in a manner similar to a naturally occurring nucleic acid. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0163] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0164] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0165] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0166] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that may be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0167] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. For example, a selected residue in a selected antibody (or Fab domain) corresponds to light chain threonine at Kabat position 40, when the selected residue occupies the same essential spatial or other structural relationship as a light chain threonine at Kabat position 40. In some embodiments, where a selected protein is aligned for maximum homology with the light chain of an antibody (or Fab domain), the position in the aligned selected protein aligning with threonine 40 is said to correspond to threonine 40. Instead of a primary sequence alignment, a three dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the light chain threonine at Kabat position 40, and the overall structures compared. In this case, an amino acid that occupies the same essential position as threonine 40 in the structural model is said to correspond to the threonine 40 residue. [0168] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations.

Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0169] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

[0170] The following eight groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

[0171] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.

[0172] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (z.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0173] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0174] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. AppL Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology ( 1995 supplement)).

[0175] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) AMC. Acids Res. 25:3389-3402, and Altschul et al. (1990) Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Set. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0176] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Set. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0177] An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[0178] Antibodies are large, complex molecules (molecular weight of -150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region, involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions (also referred to herein as light chain variable (VL) domain and heavy chain variable (VH) domain, respectively) come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

[0179] An “antibody variant” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains (e.g., light chain variable domain, heavy chain variable domain) of an antibody or fragment thereof. Non-limiting examples of antibody variants include single-domain antibodies or nanobodies, monospecific Fab2, bispecific Fab?, trispecific Faba, monovalent IgGs, scFv, bispecific antibodies, bispecific diabodies, trispecific triabodies, scFv- Fc, minibodies, IgNAR, V-NAR, hdgG, VhH, or peptibodies. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain or Fc region of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO97/49805 and WO 97/49805 which are incorporated by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in W02005/118629, which is incorporated by reference herein in its entirety and for all purposes.

[0180] The terms "CDR LI ", "CDR L2" and "CDR L3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C- terminal direction a CDR LI, a CDR L2 and a CDR L3. Likewise, the terms "CDR Hl ", "CDR H2" and "CDR H3" as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a CDR Hl, a CDR H2 and a CDR H3.

[0181] The terms "FR LI", "FR L2", "FR L3" and "FR L4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable light (L) chain of an antibody. In embodiments, the variable light chain provided herein includes in N-terminal to C-terminal direction a FR LI, a FR L2, a FR L3 and a FR L4. Likewise, the terms "FR Hl", "FR H2", "FR H3" and "FR H4" as provided herein are used according to their common meaning in the art and refer to the framework regions (FR) 1, 2, 3 and 4 of the variable heavy (H) chain of an antibody. In embodiments, the variable heavy chain provided herein includes in N-terminal to C-terminal direction a FR Hl, a FR H2, a FR H3 and a FR H4.

[0182] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL), variable light chain (VL) domain or light chain variable region and variable heavy chain (VH), variable heavy chain (VH) domain or heavy chain variable region refer to these light and heavy chain regions, respectively. The terms variable light chain (VL), variable light chain (VL) domain and light chain variable region as referred to herein may be used interchangeably. The terms variable heavy chain (VH), variable heavy chain (VH) domain and heavy chain variable region as referred to herein may be used interchangeably. The Fc (i.e. fragment crystallizable region) is the "base" or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0183] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) orthose identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552- 554 (1990)). The term “antibody” as referred to herein further includes antibody variants such as single domain antibodies. Thus, in embodiments an antibody includes a single monomeric variable antibody domain. Thus, in embodiments, the antibody, includes a variable light chain (VL) domain or a variable heavy chain (VH) domain. In embodiments, the antibody is a variable light chain (VL) domain or a variable heavy chain (VH) domain.

[0184] The term "biparatopic antibody" provided herein is used according to its common meaning in the biological arts, and refers to a bispecific antibody including two antigen binding regions, each of which recognizes unique, non-overlapping epitopes on the same target antigen. Without being bound to any particular theory, compared to monoclonal antibodies, biparatopic antibodies may exhibit a superior ability to promote receptor clustering, which may in turn result in improved receptor internalization, lysosomal trafficking, and receptor down regulation and therefore improved drug potency. In embodiments, a biparatopic antibody includes a first antibody region including a heavy chain and a light chain; and a second antibody region including a heavy chain and a light chain. In embodiments, the heavy chain of the first antibody region and the heavy chain of the second antibody region are different. In embodiments, the light chain of the first antibody region and the light chain of the second antibody region are different. In embodiments, a biparatopic antibody includes a first heavy chain, a first light chain, a second heavy chain and a second light chain, wherein the first heavy chain and the second heavy chain are different and wherein the first light chain and the second light chain are different. As provided herein the antibody regions (e.g., a first antibody region, a second antibody region) of a biparatopic antibody are different, when the first antibody region binds a first epitope of a target antigen and the second antibody region binds a second epitope of the same target antigen and the first epitope does not overlap with the second epitope. Thus, the first epitope and the second epitope form different parts of the same antigen. Likewise, the respective light chains and heavy chains (e.g., first light chain and second light chain, first heavy chain and second heavy chain) of a biparatopic antibody are different, when the first light chain and first heavy chain binds a first epitope of a target antigen and the second light chain and second heavy chain binds a second epitope of the same target antigen and the first epitope does not overlap with the second epitope.

[0185] For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 m Monoclonal Antibodies and Cancer Therapy (1985)). "Monoclonal" antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).

[0186] The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a lx, 5x, lOx, 20x or lOOx excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0187] A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of 10 to about 25 amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

[0188] For preparation of suitable antibodies of the invention and for use according to the invention, e.g., recombinant, monoclonal, or polyclonal antibodies, many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986)). The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent 4,946,778, U.S. Patent No. 4,816,567) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806;

5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121 :210 (1986)). Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Patent No. 4,676,980 , WO 91/00360; WO 92/200373; and EP 03089).

[0189] Methods for humanizing or primatizing non-human antibodies are well known in the art (e.g., U.S. PatentNos. 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87/02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321 :522; and Verhoyen et al. (1988) Science 239: 1534). Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349:293. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers (see, e.g., Morrison et al., PNAS USA, 81 :6851- 6855 (1984), Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Morrison and Oi, Adv. Immunol., 44:65-92 (1988), Verhoeyen et al., Science 239: 1534- 1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992), Padlan, Molec. Immun., 28:489- 498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. For example, polynucleotides comprising a first sequence coding for humanized immunoglobulin framework regions and a second sequence set coding for the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.

[0190] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The preferred antibodies of, and for use according to the invention include humanized and/or chimeric monoclonal antibodies.

[0191] Techniques for conjugating therapeutic agents to antibodies are well known (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”in Controlled Drug Delivery (2^nd Ed.), Robinson et al. (eds ), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies ‘84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982)). As used herein, the term “antibody-drug conjugate” or “ADC” refers to a therapeutic agent conjugated or otherwise covalently bound to to an antibody.

[0192] A "therapeutic agent" as referred to herein, is a composition useful in treating or preventing a disease such as cancer (e.g., leukemia). In embodiments, the therpaeutic agent is an anti-cancer agent. “Anti-cancer agent” is used in accordance with its plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, an anti -cancer agent is a chemotherapeutic. In embodiments, an anti-cancer agent is an agent identified herein having utility in methods of treating cancer. In embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer.

[0193] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solidphase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific i mmunoreacti vity ) .

[0194] A "ligand" refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor or antibody, antibody variant, antibody region or fragment thereof.

[0195] The term “IL1RAP” as used herein refers to any recombinant or naturally-occurring forms of interleukin- 1 receptor accessory protein (IL1RAP) or variants or homologs thereof that maintain IL1RAP activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to IL1RAP). In some aspects, the variants or homologs have at least 90%, 95%, 96%,

97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 10, 20, 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring IL1RAP polypeptide. In embodiments, IL1RAP is substantially identical to the protein identified by the UniProt reference number Q9NPH3 or a variant or homolog having substantial identity thereto.

[0196] The term “effector cell ligand” as provided herein refers to a cell surface molecule expressed on an effector cell of the immune system (e.g., a cytotoxic T cell, a helper T cell, a B cell, a natural killer cell). Upon binding of the first antibody region to the effector cell ligand expressed on the effector cell, the effector cell is activated and able to exert its function (e.g., selective killing or eradication of malignant, infected or otherwise unhealthy cells). In embodiments, the effector cell ligand is a CD3 protein. In embodiments, the effector cell ligand is a CD 16 protein. In embodiments, the effector cell ligand is a CD32 protein. In embodiments, the effector cell ligand is a NKp46 protein. The first antibody region as provided herein may be an antibody, an antibody variant, a fragment of an antibody or a fragment of an antibody variant.

[0197] A "CD3 protein" as referred to herein includes any of the recombinant or naturally- occurring forms of the Cluster of Differentiation 3 (CD3) proteins or variants or homologs thereof that comprise the CD3 complex that mediates signal transduction and maintains CD3 complex activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the CD3 complex). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3 proteins in the CD3 complex.

[0198] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego.

[0199] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. antibodies and antigens) to become sufficiently proximal to react, interact, or physically touch. It should be appreciated; however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture.

[0200] The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a pharmaceutical composition as provided herein and a cell. In embodiments contacting includes, for example, allowing a pharmaceutical composition as described herein to interact with a cell.

[0201] An “inhibitor” refers to a compound (e.g. antibodies or recombinant protein compositions described herein) that reduces activity when compared to a control, such as absence of the compound or a compound with known inactivity.

[0202] As defined herein, the term “activation”, “activate”, “activating”, “activator” and the like in reference to a protein-inhibitor interaction means positively affecting (e.g. increasing) the activity or function of the protein relative to the activity or function of the protein in the absence of the activator. In embodiments activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator. The terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein

[0203] The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein (e.g., IL1RAP or IL-1 receptor). The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 10% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 20% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 30% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 40% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 50% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 60% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 70% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 80% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 90% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 95% in comparison to a control in the absence of the agonist. In embodiments, agonist can increase expression or activity 95% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 1.5-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 2-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 3-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 4-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 5-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 10-fold higher than the expression or activity in the absence of the agonist. In embodiments, expression or activity is 10- fold or higher than the expression or activity in the absence of the agonist.

[0204] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein protein (e.g., IL1RAP or IL-1 receptor) relative to the activity or function of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein protein (e.g., IL1RAP or IL-1 receptor) relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein protein (e.g., IL1RAP or IL-1 receptor) resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein protein (e.g., IL1RAP or IL-1 receptor) from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).

[0205] The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein protein (e.g., IL1RAP or IL-1 receptor). The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 10% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 20% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 30% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 40% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 50% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 60% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 70% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 80% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 90% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 95% in comparison to a control in the absence of the antagonist. In embodiments, the antagonist can decrease expression or activity 95% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10- fold or lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 1.5-fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 2-fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 3-fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 4- fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 5-fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 10-fold lower than the expression or activity in the absence of the antagonist. In embodiments, expression or activity is 10-fold or lower than the expression or activity in the absence of the antagonist.

[0206] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.). [0207] A "cell" as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include, but are not limited to, yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells.

[0208] A "stem cell" as provided herein refers to a cell characterized by the ability of self-renewal through mitotic cell division and the potential to differentiate into a tissue or an organ. Among mammalian stem cells, embryonic stem cells (ES cells) and somatic stem cells (e.g., HSC) can be distinguished. Embryonic stem cells reside in the blastocyst and give rise to embryonic tissues, whereas somatic stem cells reside in adult tissues for the purpose of tissue regeneration and repair. In embodiments, the stem cell is a leukemia stem cell (LSC). A “leukemia stem cell or “LSC” as provided herein refers to a cell capable of initiating the disease (leukemia) when transplanted into immunodeficient animals and can self-renew by giving rise to leukemia in serial transplantations and also partially differentiate into non-LSC bulk blasts that resemble the original disease but are unable to self-renew. An LSC may carry a gene mutation and be able to self-renew through mitotic cell division and differentiate into the hematopoietic lineage carrying said gene mutant or an LSC may remain as immature progenitor cells, also known as blast cells. In embodiments, the LSC expresses CD34.

[0209] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0210] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0211] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the cell or organism it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0212] As defined herein, the term "inhibition", "inhibit", "inhibiting" and the like in reference to cell proliferation (e.g., cancer cell proliferation) means negatively affecting (e.g., decreasing proliferation) or killing the cell. In some embodiments, inhibition refers to reduction of a disease or symptoms of disease (e.g., cancer, cancer cell proliferation). Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly an "inhibitor" is a compound or protein that inhibits a receptor or another protein, e.g.,, by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating activity (e.g., a receptor activity or a protein activity).

[0213] “Biological sample” or “sample” refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

[0214] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. cancer) and compared to a known normal (non-diseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier- obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, synoviocytes, synovial fluid, synovial tissue, fibroblast-like synoviocytes, macrophagelike synoviocytes, etc).

[0215] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.

[0216] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

[0217] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. In some further instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B- acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including acute myeloid leukemia (AML), ALL, and CML), or multiple myeloma.

[0218] As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g., humans), including leukemia, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer or a sarcoma.

[0219] The term “leukemia” refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy -cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.

[0220] The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma. [0221] The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.

[0222] The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.

[0223] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non -metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e g., in the breast. [0224] The term "associated" or "associated with" in the context of a substance or substance activity or function associated with a disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)) means that the disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. Alternatively, the substance (e.g., IL1RAP) may be an indicator of the disease (e.g., cancer (e.g. leukemia, acute myeloid leukemia)). Thus, an associated substance may serve as a means of targeting disease tissue (e.g., cancer cells (e.g., leukemia stem cells, acute myeloid leukemia cells)).

[0225] As used herein, “treating” or “treatment of’ a condition, disease or disorder or symptoms associated with a condition (e g., AML), disease or disorder refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of condition, disorder or disease, stabilization of the state of condition, disorder or disease, prevention of development of condition, disorder or disease, prevention of spread of condition, disorder or disease, delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset, amelioration or palliation of the condition, disorder or disease state, and remission, whether partial or total. “Treating” can also mean prolonging survival of a subject beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the condition, disorder or disease, slowing the progression of the condition, disorder or disease temporarily, although in some instances, it involves halting the progression of the condition, disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition characterized by expression of the protease or symptom of the disease or condition characterized by expression of the protease. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. Further, as used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level and such terms can include but do not necessarily include complete elimination.

[0226] The terms “dose” and “dosage” are used interchangeably herein. A dose refers to the amount of active ingredient given to an individual at each administration. The dose will vary depending on a number of factors, including the range of normal doses for a given therapy, frequency of administration; size and tolerance of the individual; severity of the condition; risk of side effects; and the route of administration. One of skill will recognize that the dose can be modified depending on the above factors or based on therapeutic progress. The term “dosage form” refers to the particular format of the pharmaceutical or pharmaceutical composition, and depends on the route of administration. For example, a dosage form can be in a liquid form for nebulization, e.g., for inhalants, in a tablet or liquid, e.g., for oral delivery, or a saline solution, e.g., for injection.

[0227] By “therapeutically effective dose or amount” as used herein is meant a dose that produces effects for which it is administered (e.g. treating or preventing a disease such as AML). The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control. A therapeutically effective dose or amount may ameliorate one or more symptoms of a disease. A therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.

[0228] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini- osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies, for example cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy. The compounds of the invention can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present invention can be delivered by transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0229] The compositions of the present invention may additionally include components to provide sustained release and/or comfort. Such components include high molecular weight, anionic mucomimetic polymers, gelling polysaccharides and finely-divided drug carrier substrates. These components are discussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions of the present invention can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres, which slowly release subcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, as microspheres for oral administration (see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In embodiments, the formulations of the compositions of the present invention can be delivered by the use of liposomes which fuse with the cellular membrane or are endocytosed, i.e., by employing receptor ligands attached to the liposome, that bind to surface membrane protein receptors of the cell resulting in endocytosis. By using liposomes, particularly where the liposome surface carries receptor ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the compositions of the present invention into the target cells in vivo. (See, e.g., Al -Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Cnrr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. EIosp. Pharm. 46: 1576-1587, 1989). The compositions of the present invention can also be delivered as nanoparticles.

[0230] As used herein, the term “pharmaceutically acceptable” is used synonymously with “physiologically acceptable” and “pharmacologically acceptable”. A pharmaceutical composition will generally comprise agents for buffering and preservation in storage, and can include buffers and carriers for appropriate delivery, depending on the route of administration.

[0231] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present invention without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, di si nt egrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the invention. One of skill in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0232] The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

[0233] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0234] The pharmaceutical preparation is optionally in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The unit dosage form can be of a frozen dispersion.

ANTIBODY COMPOSITIONS

[0235] Provided herein are, inter alia, antibodies and antibody variants (e.g., humanized antibodies, bispecific antibodies, biparatopic antibodies or multivalent recombinant proteins) capable of binding Interleukin-1 receptor accessory protein (IL1RAP). The antibodies provided herein include novel light chain and heavy chain sequences and bind IL1RAP with high efficiency and specificity, thereby effectively targeting IL1RAP expressing cells. The anti-ILlRAP antibodies and recombinant proteins including an antibody region (first or second antibody binding region) that binds an interleukin- 1 receptor accessory protein (IL 1 RAP) provided herein including embodiments thereof, bind specifically to IL 1 RAP without activating signaling of IL1 receptor alpha or IL1 receptor beta relative to a standard control. The anti-ILlRAP antibodies and recombinant proteins including an antibody region (first or second antibody binding region) that binds an interleukin- 1 receptor accessory protein (IL1RAP) provided herein including embodiments thereof do not induce antibody-dependent cell cytotoxicity (ADCC) relative to a standard control. The term “ADCC” as provided herein refers to the immune mechanism through which Fc-receptor expressing effector cells (e.g., T cells) target and kill cells, which are bound by an antibody (e.g., an antibody specific to a cancer antigen or a pathogen-specific antigen). The antibodies and recombinant proteins including an antibody region (first or second antibody binding region) that binds an interleukin- 1 receptor accessory protein (IL1RAP) provided herein including embodiments thereof include Fc portions that do not trigger an ADCC in the presence of an effector cell. In embodiments, the anti-ILlRAP antibody or recombinant protein does not induce ADCC relative to a standard control (e.g., wildtype Fc dimerzing domain or an non-activatable Fc dimerzing domain).

[0236] The light and heavy chains of the antibodies provided herein may form part of recombinant proteins (e.g., bispecific antibodies or biparatopic antibodies) thereby providing for effective means to target malignant IL1RAP expressing cells for cytotoxic cell activity. Through the recruitment of effector cells (e.g., cytotxic T cells expressing CD3), the anti-ILRAPl antibodies and antibody variants (e.g., humanized antibodies, bispecific antibodies, biparatopic antibodies or multivalent recombinant proteins) provided herein are able to induce cell killing of ILlRAP-expressing cells. IL1RAP is expressed on a variety of cell types, for example, on candidate leukemic stem cells acute myeloid leukemia (AML) patients, but not on normal hematopoietic stem cells. Thus, the anti- IL1RAP antibodies and antibody variants (e.g., humanized antibodies, bispecific antibodies, biparatopic antibodies or multivalent recombinant proteins) provided herein are, inter alia, useful for the treatment of ILlRAP-expressing cancers such as AML.

[0237] In an aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

[0238] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:73. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:74.

[0239] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:73 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24.

[0240] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 73 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Fab.

[0241] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:386 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:386 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Y54S.

[0242] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:386 and the light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:386 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Y54S Fab.

[0243] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:387 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:387 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Q99E.

[0244] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:387 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:387 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Q99E Fab.

[0245] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:388 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:388 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 T101E.

[0246] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:388 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:388 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 T101E Fab.

[0247] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:389 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:389 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Q99ET101E.

[0248] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:389 and the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:389 and the light chain variable domain is the sequence of SEQ ID NO:74. In one further embodiment, the antibody is IL1RAP-24 Q99ET101E Fab.

[0249] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:73 and the light chain variable domain includes the sequence of SEQ ID NO:390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is IL1RAP-24 LIOS.

[0250] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 73 and the light chain variable domain includes the sequence of SEQ ID NO:390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is IL1RAP-24 KOS Fab.

[0251] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 386 and the light chain variable domain includes the sequence of SEQ ID NO: 390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:386 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is ILlRAP-24 I30SY54S.

[0252] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:386 and the light chain variable domain includes the sequence of SEQ ID NO:390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:386 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is IL1RAP-24 I30SY54S Fab.

[0253] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 387 and the light chain variable domain includes the sequence of SEQ ID NO: 390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:387 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is ILlRAP-24 I30SQ99E.

[0254] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:387 and the light chain variable domain includes the sequence of SEQ ID NO:390. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:387 and the light chain variable domain is the sequence of SEQ ID NO:390. In one further embodiment, the antibody is IL1RAP-24 I30SQ99E Fab.

[0255] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5 and a CDR H3 as set forth in SEQ ID NO:6; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:40, a CDR L2 as set forth in SEQ ID NO:41, and a CDR L3 as set forth in SEQ ID NO:42.

[0256] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:77. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:77. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:78. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:78.

[0257] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:77 and the light chain variable domain includes the sequence of SEQ ID NO:78. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:77 and the light chain variable domain is the sequence of SEQ ID NO:78. In one further embodiment, the antibody is IL1RAP-2.

[0258] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 77 and the light chain variable domain includes the sequence of SEQ ID NO:78. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:77 and the light chain variable domain is the sequence of SEQ ID NO:78. In one further embodiment, the antibody is IL1RAP-2 Fab.

[0259] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 7, a CDR H2 as set forth in SEQ ID NO:8 and a CDR H3 as set forth in SEQ ID NO:9; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:43, a CDR L2 as set forth in SEQ ID NO:44, and a CDR L3 as set forth in SEQ ID NO:45.

[0260] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:79. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:79. In embodiments, light chain variable domain includes the sequence of SEQ ID NO:80. In embodiments, light chain variable domain is the sequence of SEQ ID NO: 80.

[0261] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 79 and the light chain variable domain includes the sequence of SEQ ID NO:80. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:79 and the light chain variable domain is the sequence of SEQ ID NO:80. In one further embodiment, the antibody is IL1RAP-10. [0262] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:79 and the light chain variable domain includes the sequence of SEQ ID NO: 80. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:79 and the light chain variable domain is the sequence of SEQ ID NO:80. In one further embodiment, the antibody is IL1RAP-10 Fab.

[0263] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 10, a CDR H2 as set forth in SEQ ID NO: 11 and a CDR H3 as set forth in SEQ ID NO: 12; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:46, a CDR L2 as set forth in SEQ ID NO:47, and a CDR L3 as set forth in SEQ ID NO:48.

[0264] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:81. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:81. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:82. In embodiments, the light chain variable domain is the sequence of SEQ ID NO: 82.

[0265] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 81 and the light chain variable domain includes the sequence of SEQ ID NO:82. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:81 and the light chain variable domain is the sequence of SEQ ID NO:82. In one further embodiment, the antibody is IL1RAP-17.

[0266] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:81 and the light chain variable domain includes the sequence of SEQ ID NO: 82. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:81 and the light chain variable domain is the sequence of SEQ ID NO:82. In one further embodiment, the antibody is IL1RAP-17 Fab. [0267] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14 and a CDR H3 as set forth in SEQ ID NO: 15; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:49, a CDR L2 as set forth in SEQ ID NO:50, and a CDR L3 as set forth in SEQ ID NO:51.

[0268] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:83. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:83. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:84. In embodiments, the light chain variable domain is the sequence of SEQ ID NO: 84.

[0269] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 83 and the light chain variable domain includes the sequence of SEQ ID NO:84. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:83 and the light chain variable domain is the sequence of SEQ ID NO:84. In one further embodiment, the antibody is IL1RAP-22.

[0270] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 83 and the light chain variable domain includes the sequence of SEQ ID NO: 84. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:83 and the light chain variable domain is the sequence of SEQ ID NO:84. In one further embodiment, the antibody is IL1RAP-22 Fab.

[0271] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 16, a CDR H2 as set forth in SEQ ID NO: 17 and a CDR H3 as set forth in SEQ ID NO: 18; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:52, a CDR L2 as set forth in SEQ ID NO:53, and a CDR L3 as set forth in SEQ ID NO:54. [0272] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:85. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:85. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:86. In embodiments, the light chain variable domain is the sequence of SEQ ID NO: 86.

[0273] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 85 and the light chain variable domain includes the sequence of SEQ ID NO:86. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:85 and the light chain variable domain is the sequence of SEQ ID NO:86. In one further embodiment, the antibody is IL1RAP-25.

[0274] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:85 and the light chain variable domain includes the sequence of SEQ ID NO: 86. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:85 and the light chain variable domain is the sequence of SEQ ID NO:86. In one further embodiment, the antibody is IL1RAP-25 Fab.

[0275] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO: 19, a CDR H2 as set forth in SEQ ID NO:20 and a CDR H3 as set forth in SEQ ID NO:21; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:55, a CDR L2 as set forth in SEQ ID NO:56, and a CDR L3 as set forth in SEQ ID NO:57.

[0276] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:87. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:87. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:88. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:88.

[0277] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 87 and the light chain variable domain includes the sequence of SEQ ID NO:88. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:87 and the light chain variable domain is the sequence of SEQ ID NO:88. In one further embodiment, the antibody is IL1RAP-33.

[0278] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 87 and the light chain variable domain includes the sequence of SEQ ID NO:88. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:87 and the light chain variable domain is the sequence of SEQ ID NO:88. In one further embodiment, the antibody is IL1RAP-33 Fab.

[0279] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:22, a CDR H2 as set forth in SEQ ID NO:23 and a CDR H3 as set forth in SEQ ID NO:24; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:58, a CDR L2 as set forth in SEQ ID NO:59, and a CDR L3 as set forth in SEQ ID NO:60.

In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:89. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:89. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:90. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:90.

[0280] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO: 89 and the light chain variable domain includes the sequence of SEQ ID NO:90. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:89 and the light chain variable domain is the sequence of SEQ ID NO:90. In one further embodiment, the antibody is IL1RAP-46.

[0281] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:89 and the light chain variable domain includes the sequence of SEQ ID NO:90. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:89 and the light chain variable domain is the sequence of SEQ ID NO:90. In one further embodiment, the antibody is IL1RAP-46 Fab.

[0282] In another aspect is provided an anti-interleukin-1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:25, a CDR H2 as set forth in SEQ ID NO:26 and a CDR H3 as set forth in SEQ ID NO:27; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:61, a CDR L2 as set forth in SEQ ID NO: 62, and a CDR L3 as set forth in SEQ ID NO: 63.

In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:91. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:91. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:92. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:92.

[0283] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:91 and the light chain variable domain includes the sequence of SEQ ID NO:92. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:91 and the light chain variable domain is the sequence of SEQ ID NO:92. In one further embodiment, the antibody is IL1RAP-49.

[0284] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:91 and the light chain variable domain includes the sequence of SEQ ID NO:92. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:91 and the light chain variable domain is the sequence of SEQ ID NO:92. In one further embodiment, the antibody is IL1RAP-49 Fab.

[0285] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:28, a CDR H2 as set forth in SEQ ID NO:29 and a CDR H3 as set forth in SEQ ID NO:30; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:64, a CDR L2 as set forth in SEQ ID NO:65, and a CDR L3 as set forth in SEQ ID NO:66.

In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:93. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:93. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:94. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:94.

[0286] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:93 and the light chain variable domain includes the sequence of SEQ ID NO:94. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:93 and the light chain variable domain is the sequence of SEQ ID NO:94. In one further embodiment, the antibody is IL1RAP-53.

[0287] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:93 and the light chain variable domain includes the sequence of SEQ ID NO:94. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:93 and the light chain variable domain is the sequence of SEQ ID NO:94. In one further embodiment, the antibody is IL1RAP-53 Fab.

[0288] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:31, a CDR H2 as set forth in SEQ ID NO:32 and a CDR H3 as set forth in SEQ ID NO:33; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO: 67, a CDR L2 as set forth in SEQ ID NO:68, and a CDR L3 as set forth in SEQ ID NO:69.

[0289] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:95. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:95. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:96. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:96. [0290] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:95 and the light chain variable domain includes the sequence of SEQ ID NO:96. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:95 and the light chain variable domain is the sequence of SEQ ID NO:96. In one further embodiment, the antibody is IL1RAP-58.

[0291] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:95 and the light chain variable domain includes the sequence of SEQ ID NO:96. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:95 and the light chain variable domain is the sequence of SEQ ID NO:96. In one further embodiment, the antibody is IL1RAP-58 Fab.

[0292] In another aspect is provided an anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody including a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:34, a CDR H2 as set forth in SEQ ID NO:35 and a CDR H3 as set forth in SEQ ID NO:36; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:70, a CDR L2 as set forth in SEQ ID NO:71, and a CDR L3 as set forth in SEQ ID NO:72.

[0293] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:97. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:97. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO: 98. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:98.

[0294] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:97 and the light chain variable domain includes the sequence of SEQ ID NO: 98. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:97 and the light chain variable domain is the sequence of SEQ ID NO:98. In one further embodiment, the antibody is IL1RAP-61.

[0295] In embodiments, the anti-ILlRAP antibody is a Fab fragment. In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:97 and the light chain variable domain includes the sequence of SEQ ID NO:98. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:97 and the light chain variable domain is the sequence of SEQ ID NO:98. In one further embodiment, the antibody is IL1RAP-61 Fab.

[0296] In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor signaling relative to a standard control.

[0297] In embodiments, the anti-interleukin-1 receptor accessory protein (TL1RAP) antibody does not inhibit ILl-receptor alpha signaling relative to a standard control.

[0298] In emebodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor beta signaling relative to a standard control.

[0299] In embodiments, anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control.

[0300] In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

[0301] In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody does not block signaling of one or more co-receptors of IL1RAP.

[0302] In embodiments, the one or more co-receptors of IL1RAP include IL1R1, ST2, C-KIT or IL1RL2. In embodiments, the one or more co-receptors of IL1RAP include IL1RL In embodiments, the one or more co-receptors of IL 1 RAP include ST2. In embodiments, the one or more co-receptors of IL1RAP include C-KIT. In embodiments, the one or more co-receptors of IL1RAP include IL I RL2

[0303] In embodiments, a standard control as provided herein includes, without limitation, an TL-1 receptor binding ligand (e.g., ILl-alpha or ILl-beta protein, peptide or fragment thereof, or an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab) at a detectable level. In embodiments, the standard control includes a ILl-alpha protein, peptide or fragment thereof. In embodiments, the standard control includes a IL 1 -beta protein, peptide or fragment thereof. In embodiments the standard control includes an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab). In embodiments, the standard control includes nidanilimab.

[0304] In embodiments, the standard control inhibits IL-1 receptor signaling or IL-1 receptor activity relative to the anti-interleukin-1 receptor acecessory protein (IL1RAP) antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 20% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 30% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 40% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 50% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 60% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 70% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 80% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 90% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% or more in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof.

[0305] In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold lower in comparison to the anti- IL1RAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 2-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 3 -fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 4- fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 5-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold or lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof.

[0306] As described above, a "light chain variable (VL) domain" as provided herein refers to the variable region of the light chain of an antibody, an antibody variant or fragment thereof. Likewise, the "heavy chain variable (VH) domain" as provided herein refers to the variable region of the heavy chain of an antibody, an antibody variant or fragment thereof. The light chain variable domain and the heavy chain variable domain together form the paratope, which binds an antigen (epitope). The paratope or antigen-binding site is formed at the N-terminus of an antibody, an antibody variant or fragment thereof. In embodiments, the light chain variable (VL) domain includes CDR LI, CDR L2, CDR L3 and FR LI, FR L2, FR L3 and FR L4 (framework regions) of an antibody light chain. In embodiments, the heavy chain variable (VH) domain includes CDR Hl, CDR H2, CDR H3 and FR Hl, FR H2, FR H3 and FR H4 (framework regions) of an antibody heavy chain. In embodiments, the light chain variable (VL) domain and a light chain constant (CL) domain form part of an antibody light chain. In embodiments, the heavy chain variable (VH) domain and a heavy chain constant (CHI) domain form part of an antibody heavy chain. In embodiments, the heavy chain variable (VH) domain and one or more heavy chain constant (CHI, CH2, or CH3) domains form part of an antibody heavy chain. Thus, in embodiments, the light chain variable (VL) domain forms part of an antibody. In embodiments, the heavy chain variable (VH) domain forms part of an antibody. In embodiments, the light chain variable (VL) domain forms part of a therapeutic antibody. In embodiments, the heavy chain variable (VH) domain forms part of a therapeutic antibody. In embodiments, the light chain variable (VL) domain forms part of a human antibody. In embodiments, the heavy chain variable (VH) domain forms part of a human antibody. In embodiments, the light chain variable (VL) domain forms part of a humanized antibody. In embodiments, the heavy chain variable (VH) domain forms part of a humanized antibody. In embodiments, the light chain variable (VL) domain forms part of a chimeric antibody. In embodiments, the heavy chain variable (VH) domain forms part of a chimeric antibody. In embodiments, the light chain variable (VL) domain forms part of an antibody fragment. In embodiments, the heavy chain variable (VH) domain forms part of an antibody fragment. In embodiments, the light chain variable (VL) domain forms part of an antibody variant. In embodiments, the heavy chain variable (VH) domain forms part of an antibody variant. In embodiments, the light chain variable (VL) domain forms part of a Fab. In embodiments, the heavy chain variable (VH) domain forms part of a Fab. In embodiments, the light chain variable (VL) domain forms part of a scFv. In embodiments, the heavy chain variable (VH) domain forms part of a scFv. In embodiments, the light chain variable (VL) domain forms part of a single domain antibody. In embodiments, the heavy chain variable (VH) domain forms part of a single domain antibody.

[0307] The ability of an antibody to bind a specific epitope (e.g., IL1RAP) can be described by the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD) as defined herein is the ratio of the dissociation rate (K-off) and the association rate (K-on) of an antibody to IL1RAP. It is described by the following formula: KD = K-off/K-on.

[0308] In embodiments, the antibody binds IL1RAP with a binding affinity (KD) of about 1 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 2 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 3 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 4 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 5 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 6 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 7 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 8 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 9 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 10 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 20 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 30 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 40 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 50 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 60 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 70 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 80 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 90 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 100 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 150 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 200 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 250 nM to about 440 nM. In embodiments, the antibody binds IL1RAP with a KD of about 300 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 350 nM to about 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 400 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 410 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 420 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 430 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 435 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 436 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 437 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 438 nM to about 440 nM. In embodiments, the antibody binds ILIRAP with a KD of about 439 nM to about 440 nM.

[0309] In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 439 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 438 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 437 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 436 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 435 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 430 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 420 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 410 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 400 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 350 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 300 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 250 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 200 nM. In embodiments, the antibody binds IL1RAP with a KD of about 1 nM to about 150 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 1 nM to about 100 nM. In embodiments, the antibody binds IL 1 RAP with a KD of about 1 nM to about 90 nM. In embodiments, the antibody binds IL1RAP with a KD of about 1 nM to about 80 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 70 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 60 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 50 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 40 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 30 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 20 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 10 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 9 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 8 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 7 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 6 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 5 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 4 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 3 nM. In embodiments, the antibody binds ILIRAP with a KD of about 1 nM to about 2 nM.

[0310] In embodiments, the antibody binds ILIRAP with a binding affinity (KD) of 1 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 2 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 3 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 4 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 5 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 6 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 7 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 8 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 9 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 10 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 20 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 30 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 40 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 50 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 60 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 70 nM to 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of 80 nM to 440 nM. In embodiments, the antibody binds IL 1 RAP with a KD of 90 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 100 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 150 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 200 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 250 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 300 nM to 440 nM. In embodiments, the antibody binds IL1RAP with a KD of 350 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 400 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 410 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 420 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 430 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 435 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 436 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 437 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 438 nM to 440 nM. In embodiments, the antibody binds ILIRAP with a KD of 439 nM to 440 nM.

[0311] In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 439 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 438 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 437 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 436 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 435 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 430 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 420 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 410 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 400 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 350 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 300 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 250 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 200 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 150 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 100 nM. In embodiments, the antibody binds IL 1 RAP with a KD of 1 nM to 90 nM. In embodiments, the antibody binds IL 1 RAP with a KD of 1 nM to 80 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 70 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 60 nM. In embodiments, the antibody binds IL1RAP with a KD of 1 nM to 50 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 40 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 30 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 20 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 10 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 9 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 8 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 7 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 6 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 5 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 4 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 3 nM. In embodiments, the antibody binds ILIRAP with a KD of 1 nM to 2 nM.

[0312] In embodiments, the antibody has a binding affinity (KD) of about 2.2 nM, about 2.5 nM, about 3.4 nM, about 74 nM or about 437 nM. In embodiments, the antibody has a binding affinity (KD) of about 2.2 nM. In embodiments, the antibody has a binding affinity (KD) of about 2.5 nM. In embodiments, the antibody has a binding affinity (KD) of about 3.4 nM. In embodiments, the antibody has a binding affinity (KD) of about 74 nM. In embodiments, the antibody has a binding affinity (KD) of about 437 nM.

[0313] In embodiments, the antibody has a binding affinity (KD) of 2.2 nM, 2.5 nM, 3.4 nM, about 74 nM or 437 nM. In embodiments, the antibody has a binding affinity (KD) of 2.2 nM. In embodiments, the antibody has a binding affinity (KD) of 2.5 nM. In embodiments, the antibody has a binding affinity (KD) of 3.4 nM. In embodiments, the antibody has a binding affinity (KD) of 74 nM. In embodiments, the antibody has a binding affinity (KD) of 437 nM.

[0314] In embodiments, the antibody is a chimeric antibody, a ‘Fab fragment or a single chain antibody (scFv). In embodiments, the antibody is a chimeric antibody. In embodiments, the antibody is a Fab' fragment. In embodiments, the antibody forms part of a Fab' fragment. In embodiments, the antibody is a single chain antibody (scFv). In embodiments, the light chain variable domain and the heavy chain variable domain form part of an scFv. In embodiments, the antibody is a single domain antibody. In embodiments, the single domain antibody includes a light chain variable domain. In embodiments, the single domain antibody includes a heavy chain variable domain.

[0315] In embodiments, the antibody is a humanized antibody. In embodiments, the humanized antibody includes a heavy chain variable domain including the sequence of SEQ ID NO:75, SEQ ID NO: 100 or SEQ ID NO: 102; and a light chain variable domain including the sequence of SEQ ID NO:76, SEQ ID NO: 101 or SEQ ID NO: 103.

[0316] In embodiments, the humanized antibody includes a heavy chain variable domain including the sequence of SEQ ID NO:75; and a light chain variable domain including the sequence of SEQ ID NO:76. In one further embodiment, the humanized antibody is 5k59.

[0317] In embodiments, the humanized antibody includes a heavy chain variable domain including the sequence of SEQ ID NO: 100; and a light chain variable domain including the sequence of SEQ ID NO: 101. In one further embodiment, the humanized antibody is 5k59 K41T.

[0318] In embodiments, the humanized antibody includes a heavy chain variable domain including the sequence of SEQ ID NO: 102; and a light chain variable domain including the sequence of SEQ ID NO: 103. In one further embodiment, the humanized antibody is 4nzr.

[0319] In embodiments, the humanized antibody binds IL 1 RAP with a binding affinity (KD) of about 1.0 nM to about 6 nM. In embodiments, the humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.06 nM, about 1.49 nM or 5.36 nM. [0320] The anti-ILlRAP antibodies provided herein including embodiments thereof exhibit improved binding affinity and specificity compared to known anti IL 1 RAP antibodies and most importantly have the benefit of being cross-reactive towards human and non-human primate IL1RAP (e.g, cynomolgus I 1RAP), thereby facilitating production and pre-clinical testing. Thus, in embodiments, the antibody binds to human IL1RAP or cynomolgus IL1RAP.

[0321] In embodiments, the antibody includes a heavy chain variable domain including a serine at a position corresponding to position 30 of SEQ ID NO: 73; a glutamic acid at a position corresponding to position 99 of SEQ ID NO: 73; or a glutamic acid at a positon corresponding to position 101 of SEQ ID NO:73. In embodiments, the antibody includes a heavy chain variable domain including a serine at a position corresponding to position 30 of SEQ ID NO: 73. In embodiments, the antibody includes a heavy chain variable domain including a glutamic acid at a position corresponding to position 99 of SEQ ID NO: 73. In embodiments, the antibody includes a heavy chain variable domain including a glutamic acid at a positon corresponding to position 101 of SEQ ID NO: 73. In embodiments, the antibody includes a light chain variable domain including a serine at a position corresponding to position 30 of SEQ ID NO:74. In embodiments, the antibody does not bind IL1RAP at a detectable level.

[0322] In embodiments, the antibody further includes a central cavity formed by a heavy chain variable (VH) region, a light chain variable (VL) region, a heavy chain constant region (CH) and a light chain constant region (CL), wherein the central cavity forms a peptide binding site including framework region amino acid residues. In embodiments, a compound including a peptidyl moiety is bound to the peptide binding site.

[0323] The "central cavity" with respect to the three-dimensional structure of a Fab, refers to the internal cavity of the Fab lined by portions of the heavy and light chain variable and constant regions and including amino acids lining a hole within the cavity. In embodiments, where the antibody region includes a Fab, the central cavity thus is lined by residues of the VH, VL, CHI, and CL regions. The central cavity does not include the antigen binding site. Thus, in embodiments the compound that binds to the central cavity does not impact (e.g. measurably impact) the binding of the antibody region to the epitope. In other words, in embodiments, occupancy of this site does not affect antigen binding. In embodiments, the central cavity is lined by amino acid residues capable of interacting with a compound including a peptidyl moiety (e.g. a meditope) provided herein including embodiments thereof (e.g., a peptide of formula (I) or (II)). The amino acids residues capable of interacting with the compound including a peptidyl moiety (e.g. a meditope) may from part of the peptide binding site (also referred to herein as a meditope binding site). The peptide binding site may be engineered into any appropriate antibody thereby forming an antibody or antibody region with the peptide binding site (also referred to herein as a meditope enabled antibody or meditope enabled antibody region).

[0324] The term “meditope-enabled” antibody and “meMAb” refer to an antibody or functional fragment thereof that is able to bind to a meditope, via a meditope binding site. Examples of meditope-enabled antibodies include, but are not limited to, cetuximab and others described herein. A “meditope binding site” is a region of the meditope-enabled antibody containing the amino acid residues that interact with a bound meditope, which residues include framework region (FR) residues of the heavy and light chains. With reference to a Fab fragment or a Fab portion of an antibody, the meditope binding site is located within the central cavity of the Fab fragment or portion.

[0325] In some embodiments, the meditope binding site includes residues 40, 41, 83, and 85 of the light chain of a meditope-enabled antibody, according to Kabat numbering, and/or residues 39, 89, 105, and 108 of the heavy chain of the meditope-enabled antibody, according to Kabat numbering.

[0326] In some embodiments, the meditope binding site is located within a cavity formed by residues 8, 9, 10, 38, 39, 40, 41 42, 43, 44, 45, 82, 83, 84, 85, 86, 87, 99, 100, 101, 102, 103, 104, 105, 142, 162, 163, 164, 165, 166, 167, 168, and 173 of the light chain and 6, 9, 38, 39, 40, 41, 42, 43, 44, 45, 84, 86, 87, 88, 89, 90, 91, 103, 104, 105, 106, 107, 108, 111, 110, 147, 150, 151, 152, 173, 174, 175, 176, 177, 185, 186, and 187 of the heavy chain of the antibody, according to Kabat numbering.

[0327] With respect to a Fab portion of a meditope-enabled antibody, the meditope binding site includes residues within the central cavity. The meditope-binding site typically further includes constant region residues.

[0328] The terms “meditope”, as used herein, refers to a peptide or peptides that binds to a meditope-binding site of a meditope-enabled antibody, which antibody has a threonine at position 40, an asparagine at position 41, and an aspartage at position 85 of its light chain, according to Kabat numbering, or contains a meditope binding site containing residues that correspond to those within the meditope-binding site of cetuximab, meditope-enabled trastuzumab, or meditope-enabled M5A, disclosed herein. Exemplary meditopes include, but are not limited to, the cQFD and cQYN peptides and variants thereof (“meditope variants” or “variant meditopes”), as well as multivalent and labeled meditopes. Other molecules may also bind to meditope binding sites of meditope- enabled antibodies, with functional characteristics similar to those of a meditope. Such molecules, meditope analogs, may include, but are not limited to, small molecules, aptamers, nucleic acid molecules, peptibodies and any other substance able to bind to the same meditope binding site as a meditope.

[0329] In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 83, a residue at a position corresponding to Kabat position 30 or a residue at a position corresponding to Kabat position 52. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 40, a residue at a position corresponding to Kabat position 41, a residue at a position corresponding to Kabat position 30, a residue at a position corresponding to Kabat position 52, a residue at a position corresponding to Kabat position 83, or a residue at a position corresponding to Kabat position 85. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 40. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 41. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 30. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 52. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat 83. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 85.

[0330] In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 30. In embodiments, the residue at a position corresponding to Kabat position 30 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 30 is aspartic acid. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 52. In embodiments, the residue at a position corresponding to Kabat position 52 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 52 is aspartic acid. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 83. In embodiments, the residue at a position corresponding to Kabat position 83 is a negatively charged amino acid residue. In embodiments, the residue at a position corresponding to Kabat position 83 is glutamic acid. In embodiments, the residue at a position corresponding to Kabat position 83 is isoleucine. In embodiments, the amino acid residues lining the central cavity include a residue at a position corresponding to Kabat position 85.

[0331] In embodiments, the central cavity is lined by (formed by) a light chain residue at a position corresponding to Kabat position Gln38, Thr40, Gln41, Gly42, Ser43, Asp 52, Asp85, Ile83, Tyr87, Lysl03, Vall63, Thrl64, or Glul65. A "light chain residue" as provided herein refers to a residue forming part of a light chain of an antibody or antibody fragment. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Gln38. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Thr40 In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Gln41. In embodiments, the central cavity is lined (e g., formed) by a light chain residue at a position corresponding to Kabat position Gly42. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position to Ser43. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Asp85. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Tyr87. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Lysl03. In embodiments, the central cavity is lined (e g., formed) by a light chain residue at a position corresponding to Kabat position Vall63. In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Thrl64 In embodiments, the central cavity is lined (e.g., formed) by a light chain residue at a position corresponding to Kabat position Glut 65.

[0332] In embodiments, the central cavity is lined by (formed by) a heavy chain residue at a position corresponding to Kabat position Asp 30, Gln39, Pro40, Thr91, Ala92, Ile93, Tyr95, Glnl l2, Leul l5, Glul55, Prol56, Prol74, Alal75, or Tyrl83. A "heavy chain residue" as provided herein refers to a residue forming part of a heavy chain of an antibody or antibody fragment. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Gln39. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Pro40. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Thr91. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Ala92. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Ile93. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Tyr95. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Glnl 12. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Leul 15. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Glul55. In embodiments, the central cavity is lined (e g., formed) by a heavy chain residue at a position corresponding to Kabat position Prol56. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Prol74. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Alal75. In embodiments, the central cavity is lined (e.g., formed) by a heavy chain residue at a position corresponding to Kabat position Tyrl83.

[0333] The central cavity provided herein includes a peptide binding site (also referred to herein as a meditope binding site) including framework region amino acid (FR) residues. In embodiments, the peptide binding site does not include CDR residues of the heavy chain or the light chain. In embodiments, the peptide binding site includes FR residues of the heavy chain or the light chain. In embodiments, the peptide binding site includes FR residues of the heavy chain and the light chain. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 83, a residue at a position corresponding to Kabat position 30 or a residue at a position corresponding to Kabat position 52. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 40, a residue at a position corresponding to Kabat position 41, a residue at a position corresponding to Kabat position 30, a residue at a position corresponding to Kabat position 52, a residue at a position corresponding to Kabat position 83, or a residue at a position corresponding to Kabat position 85. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 40. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 41. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 30. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 52. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 83. In embodiments, the peptide binding site includes a residue at a position corresponding to Kabat position 85. In embodiments, residues forming a peptide binding site are described in Donaldson et al., 2013 (“Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies,” PNAS, 2013, vol. 110, no. 43, pp. 17456-17461) and in published US application US20120301400 Al, both of which are hereby incorporated by reference in their entireties and for all purposes.

[0334] In embodiments, the central cavity is lined by amino acid residues capable of binding a compound including a peptidyl moiety. Thus, in embodiments, the peptide binding site provided herein is capable of binding a compound including a peptidyl moiety. In embodiments, the peptide binding site is capable of binding the peptidyl moiety. In embodiments, the peptide binding site provided herein is bound to a compound including a peptidyl moiety. In embodiments, the peptide binding site is bound to the peptidyl moiety. In embodiments, the peptidyl moiety is a moiety as described in published US application US20120301400 Al and Avery et al. 2015 (Scientific Reports 5:7817) which are hereby incorporated by reference in their entirety and for all purposes.

[0335] In embodiments, the compound that binds to the peptide binding site is a peptide or includes a peptidyl moiety. In embodiments, the compound is a substituted peptide. In embodiments, the peptide is between 5 and 16 amino acids in length. In embodiments, the compound includes a substituted peptidyl moiety. In embodiments, the peptidyl moiety is between 5 and 16 amino acids in length. The peptide or peptidyl moiety provided herein may also be referred to as a "meditope." In embodiments, the peptide or peptidyl moiety has the formula:

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 (I).

Where the sequence of Formula (I) is a peptidyl moiety, a person having ordinary skill in the art will immediately understand that the peptidyl moiety is attached to the remainder of the compound at one or more attachments points. In formula (I), XI is Cys, Gly, P-alanine, 2,3-diaminopropionic acid, -azidoalanine, or null; X2 is Gin or null; X3 is Phe, Tyr, P-P’-diphenyLAla, His, Asp, 2- bromo-L-phenylalanine, 3-bromo-L-phenylalanine, 4-bromo-L-phenylalanine, Asn, Gin, a modified Phe, a hydratable carbonyl-containing residue or a boronic acid-containing residue; X4 is Asp or Asn; X5 is Leu; P-P’-diphenyl-Ala, Phe, a non-natural analog of phenylalanine, tryptophan, tyrosine, a hydratable carbonyl-containing residue or a boronic acid-containing residue; X6 is Ser or Cys; X7 is Thr, Ser or Cys; X8 is Arg, a modified (substituted) Arg, a hydratable carbonyl or a boronic acidcontaining residue; X9 is Arg or Ala; X10 is Leu, Gin, Glu, P-P’-diphenyl-Ala, Phe, a non-natural analog of phenylalanine, tryptophan, tyrosine, a hydratable carbonyl-containing residue or a boronic acid-containing residue; Xl lis Lys; and X12 is Cys, Gly, 7-aminoheptanoic acid, P-alanine, diaminopropionic acid, propargylglycine, isoaspartic acid or null; wherein the modified Phe is a Phe with one or more halogen incorporated into the phenyl ring and wherein the modified Arg has a structure of the formula: formula (IA), R, R’ and R” are independently substituted or unsubstituted alkyl, substituted or un substituted aryl, substituted or un substituted heteroaryl, or NHR’” and R’” is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl.

[0336] In embodiments, the peptide is a cyclic peptide. In embodiments, the peptidyl moiety is a cyclic peptidyl moiety. In embodiments, the peptide or peptidyl moiety includes a disulfide bridge, a thioether bridge, a lactam linkage, cycloaddition. In embodiments, the cyclic portion of the cyclic peptide or cyclic peptidyl moiety is formed through binding between XI and X12, XI and XI 1, X3 and XI 1, X4 and XI 1, or X2 and X12. In embodiments, the non-natural amino acid is P-P’- diphenyl-Ala, branched alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, each of the one or more halogen is an ortho-, meta-, or para-bromo phenyl substituent.

[0337] In embodiments, the peptide or peptidyl moiety has the formula: [0338] In formula (II), R³ is hydrogen, R^3A-substituted or unsubstituted aryl, wherein R^3A is hydrogen, halogen or Ci-4 unsubstituted alkyl. R³ is hydrogen, R^3A -substituted or unsubstituted aryl, wherein R^3A is hydrogen, halogen or Ci-4 unsubstituted alkyl. R⁵ is R^5A-substituted or unsubstituted Ci-s (e.g., C1-4) alkyl. R^5A is oxo, acetal, ketal, -B(OH)2, boronic ester, phosphonate ester, ortho ester, -CO2C1-4 alkyl, -CH=CH-CHO, -CH=CH-C(O)R^5A , -CH=CH-CO2R^5A -CO2H, -CONH2, or R^5A -substituted or unsubstituted aryl, R^5A -substituted or unsubstituted heteroaryl (e.g., naphthyl, imidazole, indole), wherein R^5A is substituted or unsubstituted C1-4 alkyl and R^5A is -OH, fluoro, chloro, bromo or iodo. R⁶ is -L⁶ OH or -L⁶ SH, wherein L⁶ is substituted or unsubstituted C1-4 alkylene. R⁷ is -L⁷ OH or -L⁷ SH, wherein L⁷ is substituted or unsubstituted C1-4 alkyl. The symbol m is 0, 1, 2, 3, 4, or 5. [0339] In formula (II), R⁸ is -OH, -NR^aR^b, -N(R^c)C(O)R^e, or -N(R^c)C(=NR^d)R^e. R^a is H. R^b is H or C1-8 alkyl optionally substituted with one or more substituents selected from the group consisting of oxo, acetal, and ketal, -B(0H)2, -SH, boronic ester, phosphonate ester, ortho ester, -CH CH- CHO, -CH=CH-C(0)CI-₄ alkyl, -CH=CH-CO₂CI-₄ alkyl, -C0₂H, or -CO₂Ci-₄ alkyl group. R^c is H, Ci-8 alkyl, C3-8 cycloalkyl, branched alkyl, or aryl. R^d is H or a C1-8 alkyl, C₂-8 alkenyl, C₂-8 alkynyl, C3-8 cycloalkyl, branched alkyl, or aryl group, each optionally substituted with one or more substituents selected from the group consisting of -N3, -NH₂, -OH,

-SH, halogen, oxo, acetal, ketal, -B(0H)₂, boronic ester, phosphonate ester, ortho ester, -CH=CH-CH0, -CH=CH-C(O)Ci-₄alkyl, -CH=CH-CO₂Ci-₄alkyl, -C0₂H, and

-CO₂C1.₄ alkyl group. R^e is H, -NHR^d; or a C i-i2 alkyl, C3-8 cycloalkyl, C2-12 alkenyl, C₂-8 alkynyl, or aryl group, each optionally substituted with one or more substituents selected from the group consisting of -N3, -NH₂, -OH, -SH, oxo, C_2.4 acetal, C_2.4 ketal, -B(0H)₂, boronic ester, phosphonate ester, ortho ester, -CH=CH-CH0, -CH=CH-C(O)CI-₄ alkyl, -CH=CH-CO2CI-₄ alkyl, and -CO₂Ci-₄ alkyl group.

[0340] In formula (II), R⁹ is substituted or unsubstituted Ci-₄ alkyl. R¹⁰ is R^10A- substituted or unsubstituted C1-8 alkyl, wherein R^10A is oxo, acetal, ketal, -B(0H)₂, boronic ester, phosphonate ester, ortho ester, -CH=CH-CH0, -CH=CH-C(0)CI-₄ alkyl, -CH=CH-CO₂CI-₄ alkyl, -CO₂Ci-₄ alkyl, -C0₂H, -C0NH₂, R^10B-substituted or unsubstituted phenyl, R^10B-substituted or unsubstituted naphthyl, R^10B-substituted or unsubstituted imidazolyl, or R^10B-substituted or unsubstituted indolyl, wherein R^10B is -OH or halogen. The symbol n is 0 or 1. The symbol p is 0 or 1.

[0341] In formula (II), X is R^x-substituted or unsubstituted Ci-8 alkylene, R^x-substituted or unsubstituted C2-8 alkenylene, R^x is oxo, -C(O), -NH2, -NHC(O) or -NHC(0)R^y, wherein one carbon of the alkenylene is optionally replaced with -C(0)NH, a 5-membered heteroarylene, or -S-S, and R^y is -Ci-₄ alkyl, -CH(R^Z)C(O) or -CH(R^z)C02H, wherein R^z is -H or R^z -substituted or unsubstituted -Ci-₄ alkyl, wherein R^z is -OH, -SH, or -NH₂. Formula (I) or (II) includes all appropriate pharmaceutically acceptable salts. More information regarding the concepts of peptide binding sites (meditope binding sites) and peptides (meditopes) can be found in international application serial no. PCT/US2011/055656, PCT/US2015/053880, PCT/US2012/032938 and US application serial no. US 14/453,586, which are hereby incorporated in their entirety and for all purposes.

[0342] In embodiments, the antibody is bound to an IL1RAP. In embodiments, the IL1RAP is a human IL1RAP. In embodiments, the IL1RAP forms part of a cell. In embodiments, the IL1RAP is expressed on the surface of the cell. In embodiments, the cell is a cancer cell. In embodiments, the cancer cell is a leukemia stem cell (LSC). In embodiments, the cancer cell is an acute myeloid leukemia (AML) cell. In embodiments, the cancer cell is a chronic myeloid leukemia (CML) cell. In embodiments, the cancer cell is a lung cancer cell. In embodiments, the cancer cell is a non-small cell lung cancer (NSCLC) cell. In embodiments, the cancer cell is a pancreatic cancer cell. In embodiments, the cancer cell is a melanoma cell. In embodiments, the cancer cell is a breast cancer cell. In embodiments, the cancer cell is a colon cancer cell.

RECOMBINANT PROTEIN COMPOSITIONS

[0343] As described above, the light chain variable (VL) domain and the heavy chain variable (VH) domain provided herein including embodiments thereof, may each independently form part of an antibody, an antibody variant, a fragment of an antibody, a fragment of an antibody variant, or a recombinant protein (e.g., abispecific antibody, biparatopic antibody or multivalent recombinant protein provided herein). Provided herein are, inter alia, recombinant proteins (e.g., a bispecific antibody, biparatopic antibody or multivalent recombinant protein provided herein), which include the light chain variable (VL) domain and/or the heavy chain variable (VH) domain as provided herein and are therefore capable of binding IL1RAP and recruiting effector cells to IL1RAP- expressing cells (e.g., LSCs) thereby eliminating the ILlRAP-expressing cells. In embodiments, the recombinant protein is a chimeric antigen receptor (CAR). In embodiments, the recombinant protein is a bispecific antibody. In embodiments, the recombinant protein is a biparatopic anti- IL1RAP antibody. The recombinant proteins including an antibody region (first or second antibody binding region) that binds an interleukin-1 receptor accessory protein (IL1RAP) provided herein including embodiments thereof do not induce antibody-dependent cell cytotoxicity (ADCC) relative to a standard control. The anti-ILlRAP antibodies and recombinant proteins including an antibody region (first or second antibody binding region) that binds an interleukin- 1 receptor accessory protein (IL1RAP) provided herein including embodiments thereof, bind specifically to IL1RAP without activating signaling of IL1 receptor alpha or IL1 receptor beta relative to a standard control.

Bispecific Antibody Compositions

[0344] In an aspect is provided a recombinant protein including: (i) a first antibody region; and (ii) a second antibody region, including: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO: 39.

[0345] In embodiments, the second heavy chain variable domain includes the sequence of SEQ ID NO:73. In embodiments, the second light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:73. In embodiments, the second light chain variable domain is the sequence of SEQ ID NO: 74.

[0346] In embodiments, the second heavy chain variable domain includes the sequence of SEQ ID NO:75. In embodiments, the second light chain variable domain includes the sequence of SEQ ID NO:76. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:75. In embodiments, the second light chain variable domain is the sequence of SEQ ID NO:76.

[0347] In embodiments, the first antibody region binds a cell effector ligand. In embodiments, the cell effector ligand is CD3, CD70, or CD123. In embodiments, the cell effector ligand is CD3. In embodiments, the cell effector ligand is CD70. In embodiments, the cell effector ligand is CD123.

[0348] In embodiments, the first antibody region includes: (a) a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO: 242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247.

[0349] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248. In embodiments, the first light chain variable domain includes the sequence of SEQ ID NO:249. In embodiments, the first light chain variable domain is the sequence of SEQ ID NO:249.

[0350] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248 and the first light chain variable domain includes the sequence of SEQ ID NO:249. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248 and the first light chain variable domain is the sequence of SEQ ID NO:249.

[0351] In embodiments, the first antibody region includes the sequence of SEQ ID NO:251. In embodiments, the first antibody region is the sequence of SEQ ID NO:251.

[0352] In embodiments, the second heavy chain variable domain inlcudes the sequence of SEQ ID NO:73 and the second light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:73 and the second light chain variable domain is the sequence of SEQ ID NO: 74. In embodiments, the second antibody region binds IL1RAP with a binding affinity (KD) of about 6.03 nM.

[0353] In embodiments, the second heavy chain variable domain includes the sequence of SEQ ID NO:75 and the second light chain variable domain includes the sequence of SEQ ID NO:76. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:75 and the second light chain variable domain is the sequence of SEQ ID NO:76. In embodiments, the second antibody region binds IL1RAP with a binding affinity (KD) of about 7.16 nM.

[0354] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248, the first light chain variable domain includes the sequence of SEQ ID NO:249, the second heavy chain variable domain includes the sequence of SEQ ID NO:73, and the second light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248, the first light chain variable domain is the sequence of SEQ ID NO:249, the second heavy chain variable domain is the sequence of SEQ ID NO:73, and the second light chain variable domain is the sequence of SEQ ID NO:74.

[0355] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248, the first light chain variable domain includes the sequence of SEQ ID NO:249, the second heavy chain variable domain includes the sequence of SEQ ID NO: 75, and the second light chain variable domain includes the sequence of SEQ ID NO:76. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248, the first light chain variable domain is the sequence of SEQ ID NO:249, the second heavy chain variable domain is the sequence of SEQ ID NO:75, and the second light chain variable domain is the sequence of SEQ ID NO:76.

[0356] In embodiments, the first antigen binding region further includes a first Fc dimerizing domain; and wherein the second antigen binding region further includes a second Fc dimerizing domain. In embodiments, the first Fc dimerizing domain and the second Fc dimerizing domain form a Fc region thereby binding the first antigen binding region to the second antigen binding region.

[0357] An "antibody region" as provided herein refers to a monovalent or multivalent protein moiety that forms part of an antibody or recombinant protein (e.g., a bispecific antibody, biparatopic antibody or multivalent recombinant protein provided herein) provided herein including embodiments thereof. A person of ordinary skill in the art will therefore immediately recognize that the antibody region is a protein moiety capable of binding an antigen (epitope). Thus, the antibody region provided herein may include a domain of an antibody (e.g., a light chain variable (VL) domain, a heavy chain variable (VH) domain) or a fragment of an antibody (e.g., Fab). In embodiments, the antibody region is a protein conjugate. A "protein conjugate" as provided herein refers to a construct consisting of more than one polypeptide, wherein the polypeptides are bound together covalently or non-covalently. In embodiments, the protein conjugate includes a Fab moiety (a monovalent Fab) covalently attached to an scFv moiety (a monovalent scFv). In embodiments, the protein conjugate includes a plurality (at least two) Fab moieties. In embodiments, the polypeptides of a protein conjugate are encoded by one nucleic acid molecule. In embodiments, the polypeptides of a protein conjugate are encoded by different nucleic acid molecules. In embodiments, the polypeptides are connected through a linker. In embodiments, the polypeptides are connected through a chemical linker. In embodiments, the antibody region is an scFv. The antibody region may include a light chain variable (VL) domain and/or a heavy chain variable (VH) domain. Thus, in embodiments, the antibody region includes a single domain antibody. In embodiments, the antibody region includes a light chain variable (VL) domain. In embodiments, the antibody region includes a heavy chain variable (VH) domain. In embodiments, the antibody region is a single domain antibody. In embodiments, the single domain antibody includes a heavy chain variable (VH) domain. In embodiments, the single domain antibody includes a light chain variable (VL) domain. In embodiments, the single domain antibody is a heavy chain variable (VH) domain. In embodiments, the single domain antibody is a light chain variable (VL) domain.

[0358] In embodiments, the first Fc dimerizing domain includes a lysine at a position corresponding to position 405; and wherein the second Fc dimerizing domain includes an arginine at a position corresponding to position 409.

[0359] In embodiments, the anti-ILlRAP antibody or recombinant protein does not induce ADCC relative to a standard control (e.g., wildtype Fc dimerzing domain or active Fc dimerzing domain that induces ADCC). In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, or a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297. In embodiments, the first Fc dimerizing domain includes a cysteine at a position corresponding to position 292. In embodiments, the first Fc dimerizing domain includes a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain is a non-active Fc dimerizing domain. In embodiments, the first Fc dimerizing domain does not bind an Fc receptor. In embodiments, the first Fc dimerizing domain does not induce ADCC.

[0360] In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, or a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297. In embodiments, the second Fc dimerizing domain includes a cysteine at a position corresponding to position 292. In embodiments, the second Fc dimerizing domain includes a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain is a non-active Fc dimerizing domain. In embodiments, the second Fc dimerizing domain does not bind an Fc receptor. In embodiments, the second Fc dimerizing domain does not induce ADCC.

[0361] In embodiments, the first Fc dimerizing domain includes: a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302; and the second Fc dimerizing domain includes: a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain and the second Fc dimerizing domain are non-active Fc dimerizing domains. In embodiments, the first Fc dimerizing domain and the second Fc dimerizing domain do not bind an Fc receptor. In embodiments, the first Fc antibody binding domain and the second Fc binding domain are non-active Fc antibody binding domains.

[0362] In embodiments, the first antigen binding region and the second antigen binding region independently are Fab fragments. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248; the first light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO:73; the second light chain variable domain includes the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes an arginine at a position corresponding to position 409.

[0363] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248; the light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:73; the second light chain variable domain is the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes an arginine at a position corresponding to position 409. In one further embodiment, the recombinant protein is ILR24X3.1. In one embodiment, the recombinant protein is BiFOOl .

[0364] In embodiments, the first antigen binding region and the second antigen binding region independently are Fab fragments. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248; the first light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297 a cysteine at a position corresponding to position 292, a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO:73; the second light chain variable domain includes the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0365] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248; the light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:73; the second light chain variable domain is the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409. In one further embodiment, the recombinant protein is ILR24X3.1 FcEN. In one embodiment, the recombinant protein is BiF002.

[0366] In embodiments, the first antigen binding region and the second antigen binding region independently are Fab fragments. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248 with a serine at a position corresponding to position 33; the first light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO: 73; the second light chain variable domain includes the sequence of SEQ ID NO: 74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0367] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248 with a serine at a position corresponding to position 33; the light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:73; the second light chain variable domain is the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0368] In embodiments, the first antigen binding region is a CD3 -antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:433; the first light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO: 73; the second light chain variable domain includes the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0369] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:433; the light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:73; the second light chain variable domain is the sequence of SEQ ID NO:74; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409. In one further embodiment, the recombinant protein is ILR24X3.2 FcEN. In one embodiment, the recombinant protein is BiF018.

[0370] In embodiments, the first antigen binding region and the second antigen binding region independently are Fab fragments. In embodiments, the first antigen binding region is a CD3-antigen binding region, and the second antigen binding region is an ILlRAP-antigen binding region. In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248 with a serine at a position corresponding to position 33; the first light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO: 73 with a glutamic acid at a position corresponding to position 99; the second light chain variable domain includes the sequence of SEQ ID NO: 74 with a serine at a position corresponding to position 30; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0371] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248 with a serine at a position corresponding to position 33; the light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:73 with a glutamic acid at a position corresponding to position 99; the second light chain variable domain is the sequence of SEQ ID NO:74 with a serine at a position corresponding to position 30; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0372] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:433, the light chain variable domain includes the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain includes the sequence of SEQ ID NO:387; the second light chain variable domain includes the sequence of SEQ ID NO:390; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409.

[0373] In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:433; the first light chain variable domain is the sequence of SEQ ID NO:249; the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and a lysine at a position corresponding to position 405; the second heavy chain variable domain is the sequence of SEQ ID NO:387; the second light chain variable domain is the sequence of SEQ ID NO:390; and the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302, and an arginine at a position corresponding to position 409. In one further embodiment, the recombinant protein is ILR24.3X3.2 FcEN. In one embodiment, the recombinant protein is BiF026.

[0374] In embodiments, the recombinant protein is bound to an IL1RAP. In embodiments, the IL1RAP is a human IL1RAP. In embodiments, the IL1RAP forms part of a cell. In embodiments, the IL1RAP is expressed on the surface of the cell. In embodiments, the cell is a cancer cell. In embodiments, the cancer cell is an acute myeloid leukemia (AML) cell. second antibody region does not inhibit IL 1 -receptor signaling relative to a standard control.

[0376] In embodiments, the second antibody region does not inhibit IL 1 -receptor alpha signaling relative to a standard control.

[0377] In embodiments, the second antibody region does not inhibit IL 1 -receptor beta signaling relative to a standard control.

[0378] In embodiments, the second antibody region does not inhibit binding of an ILl-beta protein to its cognate receptor relative to a standard control.

[0379] In embodiments, the second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

[0380] In embodiments, the second antibody region does not block signaling of one or more coreceptors of IL 1 RAP .

[0381] In embodiments, the second antibody region wherein said one or more co-receptors of IL1RAP include IL1R1, ST2, C-KIT or IL1RL2. In embodiments, the second antibody region wherein said one or more co-receptors of IL1RAP include IL1R1. In embodiments, the second antibody region wherein said one or more co-receptors of IL1RAP include ST2. In embodiments, the second antibody region wherein said one or more co-receptors of IL1RAP include C-KIT. In embodiments, the second antibody region wherein said one or more co-receptors of IL1RAP include IL1 RL2 [0382] In embodiments, a standard control as provided herein includes, without limitation, an IL-1 receptor binding ligand (e.g., ILl-alpha or ILl-beta protein, peptide or fragment thereof, or an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab) at a detectable level. In embodiments, the standard control includes a ILl-alpha protein, peptide or fragment thereof. In embodiments, the standard control includes a ILl-beta protein, peptide or fragment thereof. In embodiments the standard control includes an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab). In embodiments, the standard control includes nidanilimab.

[0383] In embodiments, the standard control inhibits IL-1 receptor signaling or IL-1 receptor activity relative to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 20% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 30% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 40% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 50% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 60% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 70% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 80% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 90% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% or more in comparison to the second antibody region provided herein including embodiments thereof.

[0384] In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 2-fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 3- fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 4-fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 5-fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold lower in comparison to the second antibody region provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold or lower in comparison to the second antibody region provided herein including embodiments thereof. Biparatopic Antibody Compositions

[0385] In aspect is provided a recombinant protein including a first antigen binding domain and a second antigen binding domain independently including a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain includes a CDR Hl sequence, a CDR H2 sequence and a CDR H3 sequence of any one of the antibody clones set forth by Table 1; and wherein said light chain variable domain includes a CDR LI sequence, a CDR L2 sequence and a CDR L3 sequence of any one of the antibody clones set forth by Table 2.

[0386] In embodiments, the second antigen binding region is an ILlRAP-antigen binding region includes: (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO: 37, a second CDR L2 as set forth in SEQ ID NO: 38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0387] In embodiments, the second heavy chain variable domain inlcudes the sequence of SEQ ID NO:73 and the second light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:73 and the second light chain variable domain is the sequence of SEQ ID NO: 74. In embodiments, the second antibody region binds IL1RAP with a binding affinity (KD) of about 6.03 nM.

[0388] In embodiments, the first antigen binding region is an ILlRAP-antigen binding region including a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO:4, a first CDR H2 as set forth in SEQ ID NO:5 and a first CDR H3 as set forth in SEQ ID NO:6; and a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:40, a first CDR L2 as set forth in SEQ ID NO:41, and a first CDR L3 as set forth in SEQ ID NO:42.

[0389] In embodiments, the first antigen binding region includes a first heavy chain variable domain including the sequence of SEQ ID NO:77 and a light chain variable domain including the sequence of SEQ ID NO:78. In embodiments, the first antigen binding region includes a first heavy chain variable domain with the sequence of SEQ ID NO:77 and a light chain variable domain with the sequence of SEQ ID NO:78. In one further embodiment, the recombinant protein is ILR2xILR24

[0390] In embodiments, the first antigen binding region is an ILlRAP-antigen binding region including a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO: 13, a first CDR H2 as set forth in SEQ ID NO: 14 and a first CDR H3 as set forth in SEQ ID NO: 15; and a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:49, a first CDR L2 as set forth in SEQ ID NO:50, and a first CDR L3 as set forth in SEQ ID NO:51.

[0391] In embodiments, the first antigen binding region includes a first heavy chain variable domain including the sequence of SEQ ID NO: 83 and a light chain variable domain including the sequence of SEQ ID NO:84. In embodiments, the first antigen binding region includes a first heavy chain variable domain with the sequence of SEQ ID NO: 83 and a light chain variable domain with the sequence of SEQ ID NO:84. In one further embodiment, the recombinant protein is ILR22xILR24.

[0392] In embodiments, the first antigen binding region is an ILlRAP-antigen binding region including a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO:28, a first CDR H2 as set forth in SEQ ID NO:29 and a first CDR H3 as set forth in SEQ ID NO: 30; and a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:64, a first CDR L2 as set forth in SEQ ID NO:65, and a first CDR L3 as set forth in SEQ ID NO:66.

[0393] In embodiments, the first antigen binding region includes a first heavy chain variable domain including the sequence of SEQ ID NO:94 and a light chain variable domain including the sequence of SEQ ID NO: 95. In embodiments, the first antigen binding region includes a first heavy chain variable domain with the sequence of SEQ ID NO: 94 and a light chain variable domain with the sequence of SEQ ID NO:95. In one further embodiment, the recombinant protein is ILR53xILR24.

[0394] In embodiments, the first antigen binding region further includes a first Fc dimerizing domain; and wherein the second antigen binding region further includes a second Fc dimerizing domain. In embodiments, the first Fc dimerizing domain and the second Fc dimerizing domain form a Fc region thereby binding the first antigen binding region to the second antigen binding region.

[0395] An "antibody region" as provided herein refers to a monovalent or multivalent protein moiety that forms part of an antibody or recombinant protein (e.g., a bispecific antibody, biparatopic antibody or multivalent recombinant protein provided herein) provided herein including embodiments thereof. A person of ordinary skill in the art will therefore immediately recognize that the antibody region is a protein moiety capable of binding an antigen (epitope). Thus, the antibody region provided herein may include a domain of an antibody (e.g., a light chain variable (VL) domain, a heavy chain variable (VH) domain) or a fragment of an antibody (e.g., Fab). In embodiments, the antibody region is a protein conjugate. A "protein conjugate" as provided herein refers to a construct consisting of more than one polypeptide, wherein the polypeptides are bound together covalently or non-covalently. In embodiments, the protein conjugate includes a Fab moiety (a monovalent Fab) covalently attached to an scFv moiety (a monovalent scFv). In embodiments, the protein conjugate includes a plurality (at least two) Fab moieties. In embodiments, the polypeptides of a protein conjugate are encoded by one nucleic acid molecule. In embodiments, the polypeptides of a protein conjugate are encoded by different nucleic acid molecules. In embodiments, the polypeptides are connected through a linker. In embodiments, the polypeptides are connected through a chemical linker. In embodiments, the antibody region is an scFv. The antibody region may include a light chain variable (VL) domain and/or a heavy chain variable (VH) domain. Thus, in embodiments, the antibody region includes a single domain antibody. In embodiments, the antibody region includes a light chain variable (VL) domain. In embodiments, the antibody region includes a heavy chain variable (VH) domain. In embodiments, the antibody region is a single domain antibody. In embodiments, the single domain antibody includes a heavy chain variable (VH) domain. In embodiments, the single domain antibody includes a light chain variable (VL) domain. In embodiments, the single domain antibody is a heavy chain variable (VH) domain. In embodiments, the single domain antibody is a light chain variable (VL) domain.

[0396] In embodiments, the first Fc dimerizing domain includes a lysine at a position corresponding to position 405; and wherein the second Fc dimerizing domain includes an arginine at a position corresponding to position 409. In embodiments, the first Fc antibody binding domain and the second Fc binding domain are non-active Fc antibody binding domains.

[0397] In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, or a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain includes a glycine at a position corresponding to position 297. In embodiments, the first Fc dimerizing domain includes a cysteine at a position corresponding to position 292. In embodiments, the first Fc dimerizing domain includes a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain is a non-active Fc dimerizing domain.

[0398] In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, or a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain includes a glycine at a position corresponding to position 297. In embodiments, the second Fc dimerizing domain includes a cysteine at a position corresponding to position 292. In embodiments, the second Fc dimerizing domain includes a cysteine at a position corresponding to position 302. In embodiments, the second Fc dimerizing domain is a non-active Fc dimerizing domain.

[0399] In embodiments, the first Fc dimerizing domain includes: a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302; and the second Fc dimerizing domain includes: a glycine at a position corresponding to position 297, a cysteine at a position corresponding to position 292, and a cysteine at a position corresponding to position 302. In embodiments, the first Fc dimerizing domain and the second Fc dimerizing domain are non-active Fc dimerizing domains. In embodiments, the first Fc antibody binding domain and the second Fc binding domain are non- active Fc antibody binding domains.

[0400] In embodiments, the recombinant protein is bound to an IL1RAP. In embodiments, the IL1RAP is a human IL1RAP. In embodiments, the IL1RAP forms part of a cell. In embodiments, the IL1RAP is expressed on the surface of the cell. In embodiments, the cell is a cancer cell. In embodiments, the cancer cell is an acute myeloid leukemia (AML) cell.

MULTIVALENT RECOMBINANT PROTEIN COMPOSITIONS

[0401] Provided herein are, inter alia recombinant proteins, wherein the recombinant protein is a multivalent recombinant protein. An antibody region of the multivalent recombinant protein may include any of the light chain and heavy chain variable domains provided herein including embodiments thereof. The light chain variable (VL) domain and/or the heavy chain variable (VH) domain as provided herein may form part of a multivalent recombinant protein.

[0402] Thus, in an aspect is provided a multivalent recombinant protein including: (i) a first Fc dimerizing domain bound to a first antibody region through a first chemical linker; (ii) a second Fc dimerizing domain bound to a second antibody region through a second chemical linker; wherein the first Fc dimerizing domain is covalently attached to the second Fc dimerizing domain thereby forming a Fc region binding the first antibody region to the second antibody region; and (iii) a third antibody region bound through a third chemical linker to the first antibody region, the second antibody region or the Fc region. In embodiments, the third antibody region is bound through a third chemical linker to the first antibody region, the second antibody region or the Fc region. In embodiments, the third antibody region is bound through a third chemical linker to the second antibody region. In embodiments, the third antibody region is bound through a third chemical linker to the Fc region.

[0403] An “Fc dimerizing domain” or “Fc domain” as referred to herein is a polypeptide including an antibody CH2 domain or fragment thereof bound (covalently and/or non-covalently) to an antibody CH3 domain or fragment thereof. Upon binding of two Fc domains or Fc dimerizing domains an antibody Fc region is formed. Thus, an Fc region may include a first Fc dimerzing domain non-covalently or covalently bound to a second dimerizing domain. In embodiments, the Fc region may bind an effector cell (e.g., Nk cell or cytotoxic T cell) thereby activating said effector cell. In embodiments, the CH3 domain of the first Fc dimerizing domain is non-covalently bound to the CH3 domain of the second Fc dimerizing domain. In embodiments, the CH2 domain of the first Fc dimerizing domain is covalently bound to the CH2 domain of the second Fc dimerizing domain. In embodiments, the CH2 domain of the first Fc dimerizing domain is bound to the CH2 domain of the second Fc dimerizing domain through a disulfide linkage. In embodiments, the Fc dimerizing domain includes a CH2 domain and a CH3 domain. In embodiments, the Fc dimerizing domain includes from the N-terminus to the C-terminus a CH2 domain and a CH3 domain.

[0404] In embodiments, the multivalent recombinant protein further includes a fourth antibody region bound through a fourth chemical linker to the first antibody region, the second antibody region or the Fc region. In embodiments, the fourth antibody region is bound through a fourth chemical linker to the first antibody region. In embodiments, the fourth antibody region is bound through a fourth chemical linker to the second antibody region. In embodiments, the fourth antibody region is bound through a fourth chemical linker to the Fc region.

[0405] In embodiments, the first, the second, the third and the fourth antibody region are independently a ‘Fab fragment or scFv. In embodiments, the first, the second, the third and the fourth antibody region are independently a ‘Fab fragment. In embodiments, the first antibody region is a ‘Fab fragment. In embodiments, the second antibody region is a ‘Fab fragment. In embodiments, the third antibody region is a ‘Fab fragment. In embodiments, the fourth antibody region is a ‘Fab fragment. In embodiments, the first, the second, the third and the fourth antibody region are independently a scFv. In embodiments, the first antibody region is a scFv. In embodiments, the second antibody region is a scFv. In embodiments, the third antibody region is a scFv. In embodiments, the fourth antibody region is a scFv.

[0406] In embodiments, the third antibody region is bound to the N-terminus of the second antibody region. In embodiments, the third antibody region is a scFv. In embodiments, the third antibody region binds CD3. In embodiments, the third antibody region includes: (a) a third heavy chain variable domain including a third CDR Hl as set forth in SEQ ID NO:242, a third CDR H2 as set forth in SEQ ID NO:243 and a third CDR H3 as set forth in SEQ ID NO:244; and (b) a third light chain variable domain including a third CDR LI as set forth in SEQ ID NO:245, a third CDR L2 as set forth in SEQ ID NO:246, and a third CDR L3 as set forth in SEQ ID NO:247.

[0407] In embodiments, the first antibody region is a first anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody region, the second antibody region is a second anti-ILlRAP antibody region and the third antibody region is an anti-CD3 antibody region.

[0408] In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is an anti-CD123 antibody region and the third antibody region is an anti- CD3 antibody region.

[0409] In embodiments, the third antibody region is a ‘Fab fragment. In embodiments, the third antibody region binds to IL1RAP. In embodiments, the first antibody region is an anti-CD3 antibody region, the second antibody region is an anti-CD123 antibody region and the third antibody region is an anti-ILlRAP antibody region. In embodiments, the first antibody region is an anti-CD3 antibody region, the second antibody region is a first anti-ILlRAP antibody region and the third antibody region is a second anti-ILlRAP antibody region. [0410] In embodiments, the third antibody region is bound to the C-terminus of the second antibody region. In embodiments, the third antibody region is a scFv. In embodiments, the third antibody region binds to CD3. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region and the third antibody region is an anti-CD3 antibody region. In embodiments, the first antibody region is an anti- CD123 antibody region, the second antibody region is an anti-ILlRAP antibody region and the third antibody region is an anti-CD3 antibody region.

[0411] In embodiments, the third antibody region is bound to the C-terminus of the Fc region. In embodiments, the third antibody region is a scFv. In embodiments, the third antibody region binds to CD3. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region and the third antibody region is an anti- CD3 antibody region. In embodiments, the first antibody region is an anti-CD123 antibody region, the second antibody region is an anti-ILlRAP antibody region and the third antibody region is an anti-CD3 antibody region.

[0412] In embodiments, the third antibody region is bound to the C-terminus of the first Fc dimerizing domain and the fourth antibody region is bound to the C-terminus of the second Fc dimerizing domain. In embodiments, the third antibody region and the fourth antibody region are indepedently a scFv. In embodiments, the third antibody region and the fourth antibody region independently bind to CD3. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region, the third antibody region is a first anti-CD3 antibody region, and the fourth antibody region is a second anti- CD3 antibody region.

[0413] In embodiments, the third antibody region is bound to the N-terminus of the first antibody region and the fourth antibody region is bound to the N-terminus of the second antibody region. In embodiments, the first antibody region is a first ’Fab fragment and the second antibody region is a second ’Fab fragment. In embodiments, the third antibody region is bound to the N-terminus of the variable light chain of the first ’Fab fragment and the fourth antibody region is bound to the N- terminus of the variable light chain of the second ’Fab fragment.In embodiments, the third antibody region and fourth antibody region are independently a scFv. In embodiments, the third antibody region and fourth antibody region independently bind CD3. In embodiments, the first is a first anti- IL1RAP ‘Fab fragment and the second ‘Fab fragment is a second anti-ILlRAP ‘Fab fragment. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region, the third antibody region is a first anti-CD3 antibody region, and the fourth antibody region is a second anti-CD3 antibody region..

[0414] In embodiments, the third antibody region is bound to the C-terminus of the first antibody region and the fourth antibody region is bound to the C-terminus of the second antibody region. In embodiments, the first antibody region is a first ‘Fab fragment and the second antibody region is a second ‘Fab fragment. In embodiments, the third antibody region is bound to the C-terminus of the variable light chain of the first ‘Fab fragment and the fourth antibody region is bound to the C- terminus of the variable light chain of the second ‘Fab fragment. In embodiments, the third antibody region and the fourth antibody region are independently a scFv. In embodiments, the third antibody region and the fourth antibody region independently bind to CD3. In embodiments, the first ‘Fab fragment is a first anti-ILlRAP ‘Fab fragment and the second ‘Fab fragment is a second anti- IL1RAP ‘Fab fragment. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region, the third antibody region is a first anti-CD3 antibody region, and the fourth antibody region is a second anti-CD3 antibody region..

[0415] In embodiments, the third antibody region is bound to the N-terminus of the first antibody region and the fourth antibody region is bound to the N-terminus of the second antibody region. In embodiments, the first antibody region is a first ‘Fab fragment and the second antibody region is a second ‘Fab fragment. In embodiments, the third antibody region is bound to the N-terminus of the variable heavy chain of the first ‘Fab fragment and the fourth antibody region is bound to the N- terminus of the variable heavy chain of the second ‘Fab fragment. In embodiments, the third antibody region and the fourth antibody region are independently a scFv. In embodiments, the third antibody region and the fourth antibody region independently bind to CD3. In embodiments, the first ‘Fab fragment is a first anti-ILlRAP ‘Fab fragment and the second ‘Fab fragment is a second anti-ILlRAP ‘Fab fragment. In embodiments, the first antibody region is a first anti-ILlRAP antibody region, the second antibody region is a second anti-ILlRAP antibody region, the third antibody region is a first anti-CD3 antibody region, and the fourth antibody region is a second anti- CD3 antibody region.

NUCLEIC ACID COMPOSITIONS

[0416] The compositions provided herein include nuceic acid molecules encoding the anti- IL1RAP antibodies and recombinant proteins provided herein including embodiments thereof. Thus, in an aspect, an isolated nucleic acid encoding an antibody as provided herein including embodiments thereof is provided.

[0417] In another aspect, an isolated nucleic acid encoding a recombinant protein as provided herein, including embodiments thereof, is provided.

PHARMACEUTICAL COMPOSITIONS

[0418] The compositions provided herein include pharmaceutical compositions including the anti IL1RAP antibodies and recombinant proteins provided herein including embodiments thereof. Thus, in an aspect is provided a pharmaceutical composition including a therapeutically effective amount of an antibody as provided herein including embodiments thereof and a pharmaceutically acceptable excipient.

[0419] In another aspect is provided a pharmaceutical composition including a therapeutically effective amount of a recombinant protein as provided herein, including embodiments thereof, and a pharmaceutically acceptable excipient. METHODS OF TREATMENT

[0420] The compositions (e.g., the anti IL1RAP antibodies and recombinant proteins) provided herein, including embodiments thereof, are contemplated as providing effective treatments for diseases such as cancer (e.g., leukemia [e.g., AML]). Thus, in an aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of an antibody as provided herein including embodiments thereof, thereby treating cancer in the subject.

[0421] In another aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of a recombinant protein as described herein, including embodiments thereof, thereby treating cancer in the subject. In embodiments, the cancer is leukemia. In embodiments, the cancer is acute myeloid leukemia. In embodiments, the cancer is chronic myeloid leukemia (CML). In embodiments, the cancer is lung cancer. In embodiments, the cancer is non-small cell lung cancer (NSCLC). In embodiments, the cancer is pancreatic cancer. In embodiments, the cancer is melanoma. In embodiments, the cancer is breast cancer. In embodiments, the cancer is colon cancer. In embodiments, the method further includes administering to the subject a second therapeutic agent.

[0422] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0423] In another aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of an anti- interleukin- 1 receptor accessory protein (IL1RAP) antibody, wherein the antibody does not inhibit IL 1 -receptor signaling relative to a standard control, thereby treating cancer in the subject. [0424] In embodiments, a standard control as provided herein includes, without limitation, an IL-1 receptor binding ligand (e.g., ILl-alpha or ILl-beta protein, peptide or fragment thereof, or an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab) at a detectable level. In embodiments, the standard control includes a ILl-alpha protein, peptide or fragment thereof. In embodiments, the standard control includes a ILl-beta protein, peptide or fragment thereof. In embodiments the standard control includes an antibody known to induce signaling of IL-1 receptor (e.g., nidanilimab). In embodiments, the standard control includes nidanilimab.

[0425] In embodiments, the standard control inhibits IL-1 receptor signaling or IL-1 receptor activity relative to the anti-interleukin-1 receptor acecessory protein (IL1RAP) antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 20% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 30% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 40% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 50% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 60% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 70% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 80% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 90% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 95% or more in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof

[0426] In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 1.5-fold lower in comparison to the anti- IL1RAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 2-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 3 -fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 4- fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 5-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. In embodiments, the standard control can decrease IL-1 receptor signaling or IL-1 receptor activity 10-fold or lower in comparison to the anti-ILlRAP antibody provided herein including embodiments thereof. [0427] In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody includes a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain includes: a CDR Hl as set forth in SEQ ID NO:1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein the light chain variable domain includes: a CDR LI as set forth in SEQ ID NO:37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO: 39.

[0428] In embodiments, the heavy chain variable domain includes the sequence of SEQ ID NO:73. In embodiments, the heavy chain variable domain is the sequence of SEQ ID NO:73. In embodiments, the light chain variable domain includes the sequence of SEQ ID NO:74. In embodiments, the light chain variable domain is the sequence of SEQ ID NO:74.

[0429] In another aspect is provided a method of treating cancer in a subject in need thereof, the method including administering to a subject a therapeutically effective amount of a recombinant protein including: (i) a first antibody region, wherein the first antibody binding region binds an effector cell ligand; and (ii) a second antibody region, wherein the second antibody region binds an interleukin-1 receptor accessory protein (IL1RAP) and does not inhibit ILl-receptor signaling relative to a standard control, thereby treating cancer in the subject.

[0430] In embodiments, the effector cell ligand is CD3, CD70 or CD123. In embodiments, the effector cell ligand is CD3. In embodiments, the effector cell ligand is CD70. In embodiments, the effector cell ligand is CD 123.

[0431] In embodiments, the first antibody region includes a first heavy chain variable domain including a first CDR Hl as set forth in SEQ ID NO: 242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain including a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247. [0432] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248. In embodiments, the first light chain variable domain includes the sequence of SEQ ID NO:249. In embodiments, the first light chain variable domain is the sequence of SEQ ID NO:249.

[0433] In embodiments, the first heavy chain variable domain includes the sequence of SEQ ID NO:248 and the first light chain variable domain includes the sequence of SEQ ID NO:249. In embodiments, the first heavy chain variable domain is the sequence of SEQ ID NO:248 and the first light chain variable domain is the sequence of SEQ ID NO:249.

[0434] In embodiments, the first antibody region includes the sequence of SEQ ID NO:251. In embodiments, the first antibody region is the sequence of SEQ ID NO:251.

[0435] In embodiments, the second antibody region includes (a) a second heavy chain variable domain including a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain including a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0436] In embodiments, the second heavy chain variable domain includes the sequence of SEQ ID NO:73 and the second light chain variable domain includes the sequence of SEQ ID NO: 74. In embodiments, the second heavy chain variable domain is the sequence of SEQ ID NO:73 and the second light chain variable domain is the sequence of SEQ ID NO: 74.

[0437] In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not inhibit ILl-receptor signaling relative to a standard control. In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) does not inhibit ILl- receptor signaling relative to a standard control. In embodiments, the second antibody region does not inhibit ILl-receptor signaling relative to a standard control. [0438] In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not inhibit IL 1 -receptor alpha signaling relative to a standard control. In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit IL 1 -receptor alpha signaling relative to a standard control. In embodiments, the second antibody region does not inhibit IL 1 -receptor alpha signaling relative to a standard control.

[0439] In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not inhibit ILl-receptor beta signaling relative to a standard control. In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor beta signaling relative to a standard control. In embodiments, the second antibody region does not inhibit ILl-receptor beta signaling relative to a standard control.

[0440] In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control. In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control. In embodiments, the second antibody region does not inhibit binding of an IL 1- beta protein to its cognate receptor relative to a standard control.

[0441] In embodiments, the anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control. In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control. In embodiments, the second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

[0442] In embodiments, the cancer is acute myeloid leukemia (AML).

[0443] In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody or the second antibody region does not block signaling of one or more co-receptors of IL1RAP. In embodiments, the anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody does not block signaling of one or more co-receptors of IL1RAP. In embodiments, the second antibody region does not block signaling of one or more co-receptors of IL1RAP.

[0444] In embodiments, the one or more co-receptors of IL1RAP include IL1R1, ST2, C-KIT or IL1RL2. In embodiments, the one or more co-receptors of IL1RAP include IL1R1. In embodiments, the one or more co-receptors of IL1RAP include ST2. In embodiments, the one or more co-receptors of IL1RAP include C-KIT. In embodiments, the one or more co-receptors of IL1RAP include IL I RL2

EXAMPLES Example 1 : CDR SEQUENCES OF THE HEAVY CHAIN VARIABLE DOMAINS AND LIGHT CHAIN VARIABLE DOMAINS FOR ANTI-IL1RAP ANTIBODY CLONES

[0445] Table 1. CDR sequences of the heavy chain variable domains of anti-ILlRAP antibodies provided herein.

[0446] Table 1. CDR sequences of the light chain variable domains of anti-ILlRAP antibodies provided herein.

Example 2: SEQUENCES OF THE HEAVY CHAIN VARIABLE DOMAINS AND LIGHT

CHAIN VARIABLE DOMAINS FOR ANTI-IL1RAP ANTIBODY CLONES

[0447] Table 2. Sequences of heavy variable chain domains and light chain variable domains of IL 1RAP antibodies provided herein.

Example 3: SEQUENCES OF THE HEAVY CHAINS AND LIGHT CHAINS FOR ANTI-IL1RAP

ANTIBODY CLONES

[0448] Table 3. Sequences of heavy chains and light chains of IL1RAP antibodies provided herein.

Example 4: FRAMEWORK SEQUENCES OF HEAVY CHAIN VARIABLE DOMAINS AND

LIGHT CHAIN VARIABLE DOMAINS FOR ANTLIL1RAP ANTIBODY CLONES

[0449] Table 4. The framework sequences of heavy chain variable domains and light chain variable domains of IL1RAP antibodies provided herein

Example 5: SEQUENCES OF SINGLE CHAIN anti-cd3 ANTIBODIES AND FRAGMENTS

THEREOF

[0450] Table 5. The sequences of single chain anti-CD3 antibodies and fragments thereof provided herein Example 6: xcelllGENCE REAL-TIME CELL ANALYSIS

[0451] The Agilent xCELLigence instrument allows for longitudinal acquisition of fluorescent and brightfield images from a single well. In these studies, the target (T) AML cells carried the gene for green fluorescent protein (GFP) so they could be visualized by fluorescent imaging. MOLM-13 and THP1 cells were tested. The effector (E) T cells, derived from healthy platelet donors, were unlabeled or labeled with a nuclear dye, DAPI (data not shown). They are identified by sex (M or F) and age (e.g., M58 is from a 58 yr old male). T cells are enriched from the mononuclear cell mixture by negative immunophenotype selection using a kit from StemCells Inc. The BiF002 construct has one arm from IL1RAP-24 that recognizes IL1RAP and the other arm which recognizes CD3. BiF002 carries mutation(s) that abolish Fc receptor binding. Wells are seeded at different E:T ratios. Preliminary studies were conducted to determine which donor and ratio yield the best signal to noise ratio, i.e., the best Ab-dependent killing and lowest Ab independent (basal) cell killing. Images are collected every 15 minutes and concatenated to make a “movie” (data not shown).

[0452] Antibody-dependent cell cytotoxicity (ADCC) experiments of THP1 cells by healthy donor T cells (M58) at E:T ratio 5:1 were performed. BiF002 concentrations tested were 0 nM (control), 0.1 nM, 1 nM, 10 nM, 100 nM. In the absence (control) or at very low concentrations of BiF002 ( 0.1 nM), the green AML cells clump together and appear to increase in fluorescence over time (they are proliferating). In wells with sufficient BiF002, green fluorescence diminishes to nearly invisible levels over time indicative of cytotoxicity. Images were captured at 48h (Agilent xCELLigence). The same experiment was repeated using MOLM-13 cells as the target cell.

[0453] Bright field images of ADCC incubations demonstrate increased clumping of T cells when the target AML has more IL1RAP expression (U937, HL60 > KGla) and with increasing concentrations of BiF002 (images not shown).

[0454] Merged bright field and fluorescent images of ADCC incubations (images not shown) at 12 hrs of incubation demonstrate loss of AML cells marked by GFP/green fluorescence only in cells that have adequate IL1RAP expression (MOLM-13) and added BIF002. When only fluorescence is imaged, AML cells are shown to clump and disappear with increasing BiF002 concentrations. K562 cells lack IL1RAP surface expression and are not targeted by T cells as shown by the presence of GFP when BiF002 is present at 10 nM.

Example 7: BiF002 in vivo efficacy study

[0455] A pilot efficacy study was performed to establish in vivo efficacy of BiF002 by assessment of tumor burden and survival (FIGS. 80-81). On Study Day 0, 0.6 x 10^A6 MOLM-13 (Luc+) cells were injected intravenously into NSGs mice. On Study Day 9, and weekly starting on 26, resting T cells (3 x 10^A6) were administered by IV injection for a total of 6 doses. T cells all originated from one donor and were previously cryopreserved. BiF002 was administered every 3 days starting at Study Day 10 at 100 microG/mouse for a total of 18 doses. Control groups consisted of human IgG, BiF002, and huIgG + T cells. Bioluminescence imaging was performed weekly starting on Study Day 8. Images were collected and quantification of luminescence also performed for statistical analyses (FIGS. 82A-82D). Other correlative studies included weekly weight measurements and quantitation of human CD45+ in peripheral blood mononuclear cells to determine human cell engraftment. This study demonstrated the superiority of BiF002 when administered with T cells compared to BiF002 alone as well as all other groups.

Example 8: Exploration of Donor T Cell Dose and Activation State

[0456] The dose and activation state of T cells were explored in this study (FIGS. 83-84). On Study Day 0, 1 x 10^A6 MOLM-13 (Luc+) cells were injected intravenously into NSGs mice. Starting on Study Day 3 and weekly thereafter, T cells (6 doses of 3 x 10^A6 resting or 5 doses of 1.5 x 10^A6 activated) were administered by IV injection. T cells originated from one donor and were previously cryopreserved. Five days prior to administration, resting T cells were non-specifically activated using CD3/CD8 beads in culture and allowed to proliferate; these were used in the activated T cell groups. BiF002 was administered every 3 days starting at Study Day 4 at 100 microG/mouse except in one group where 33 microG was administered. Control groups consisted of human IgG, huIgG + resting T cells and BiF002 alone with no T cells. Bioluminescence imaging was performed weekly starting on Study Day 8. Images were collected and quantification of luminescence also performed for statistical analyses (FIGS. 85A-85F).

Example 9: biF002 DOSE EXPLORATION

[0457] A dose exploration study was performed to determine the optimum dose of BiF002 and demonstrate a dose-response relationship to tumor burden and survival (FIGS. 89-90). On Study Day 0, 1 x 10^A6 MOLM-13 (Luc+) cells were injected intravenously into NSGs mice. Starting on Study Day 1 and weekly thereafter, 5 doses of in vitro activated T cells (3 x 10^A6) were administered by IV injection. T cells originated from one donor and were previously cryopreserved. Five days prior to administration, resting T cells were non-specifically activated using CD3/CD8 beads in culture and allowed to proliferate. BiF002 was administered every 3 days starting at Study Day 1 at 0.1, 1 and 10 microG/mouse. Control groups consisted of no treatment and activated T cells only. Bioluminescence imaging was performed weekly starting on Study Day 8. Images were collected and quantification of luminescence also performed for statistical analyses (FIGS. 91A- 92C). In this ongoing study, BiF002 at 1 microG appears most active. Example 10: ADDITIONAL CDR SEQUENCES OF THE HEAVY CHAIN VARIABLE DOMAINS AND LIGHT CHAIN VARIABLE DOMAINS FOR ANTLIL1RAP ANTIBODY CLONES

[0458] Table 6. CDR sequences of the heavy chain variable domains of anti-ILlRAP antibodies provided herein.

[0459] Table 7. CDR sequences of the light chain variable domains of anti-ILlRAP antibodies provided herein.

Example 11 : additional SEQUENCES OF THE HEAVY CHAIN VARIABLE DOMAINS AND

LIGHT CHAIN VARIABLE DOMAINS FOR ANTLIL1RAP ANTIBODY CLONES

[0460] Table 7. Sequences of heavy variable chain domains and light chain variable domains of IL1RAP antibodies provided herein.

Example 12: ADDITIONAL SEQUENCES FOR ANTIBODY AND RECOMBINANT PROTEINS PROVIDED HEREIN

[0461] SEQ ID NO:433 (H26H8 Y33S) CD3 VH QVQLQESGPGLVKPSETLSLTCTASGYTFTNYSIHWVRQSPGKGLEWIGWIYCGDGSTKFN EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSS

[0462] SEQ ID NO:434 (H26H8 Y33S) CD3 HC

QVQLQESGPGLVKPSETLSLTCTASGYTFTNYSIHWVRQSPGKGLEWIGWIYCGDGSTKFN

EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQTYICNVNHKP SNTK VDKKVEPKSC

[0463] SEQ ID NO:435 (H26H8) CD3 Fab LC

DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRES GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCKQSYILRTFGGGTKVEIKRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0464] SEQ ID NO:436 (H26H8) CD3 Fab HC

QVQLQESGPGLVKPSETLSLTCTASGYTFTNYYIHWVRQSPGKGLEWIGWIYCGDGSTKFN EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQTYICNVNHKP SNTK VDKKVEPKSC

[0465] SEQ ID NO:437 ILR24X3.1FcEN - CD3 HC

QVQLQESGPGLVKPSETLSLTCTASGYTFTNYYIHWVRQSPGKGLEWIGWIYCGDGSTKFN EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQTYICNVNHKP SNTK VDKKVEPKSCDKTHTCPPCP APELLGGPS VFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL

TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

[0466] SEQ ID NO:438 ILR24X3.1FcEN - CD3 LC

DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRES GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCKQSYILRTFGGGTKVEIKRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0467] SEQ ID NO:439 ILR24X3.1FcEN - IL1RAP-24 HC

QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHGKGLEWIGIISTYSGNTKFH QKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARQGTWYFDVWGAGTTVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

[0468] SEQ ID NO:440 ILR24X3.1FcEN - IL1RAP-24 LC

QIVLTQSPAIMSASPGEKVTMTCSASSTIIYIHWYQQKPGTSPKGWIYDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQRTSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLS SPVTKSFNRGEC

[0469] SEQ ID NO:441 ILR24X3.2FcEN - CD3 HC

QVQLQESGPGLVKPSETLSLTCTASGYTFTNYSIHWVRQSPGKGLEWIGWIYCGDGSTKFN EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQTYICNVNHKP SNTK VDKK VEPKSCDKTHTCPPCP APELLGGPS VFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL

TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK [0470] SEQ ID NO:442 ILR24X3.2FcEN - CD3 LC

DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRES GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCKQSYILRTFGGGTKVEIKRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0471] SEQ ID NO:443 ILR24X3.2FcEN - IL1RAP-24 HC

QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHGKGLEWIGIISTYSGNTKFH

QKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARQGTWYFDVWGAGTTVTVSSAST

KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK

[0472] SEQ ID NO:445 ILR24X3.2FcEN - IL1RAP-24 LC

QIVLTQSPAIMSASPGEKVTMTCSASSTIIYIHWYQQKPGTSPKGWIYDTSKLASGVPARFSG SGSGTSYSLTISSMEAEDAATYYCHQRTSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLS SPVTKSFNRGEC

[0473] SEQ ID NO:446 ILR24.3X3.2FcEN - CD3 HC

QVQLQESGPGLVKPSETLSLTCTASGYTFTNYSIHWVRQSPGKGLEWIGWIYCGDGSTKFN EQFKDRTTLSADHSKTQASLKLSSVTAADTAIYFCARDWAGSYFFDFWGQGTMVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS L S SWT VP S S SLGTQTYICNVNHKP SNTK VDKK VEPKSCDKTHTCPPCP APELLGGPS VFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK

[0474] SEQ ID NO:447 ILR24.3X3.2FcEN - CD3 LC

DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNYLAWYQQKPGKAPKLLIYWASTRES

GVPSRFSGSGSGTDFTFTISSLQPEDIATYYCKQSYILRTFGGGTKVEIKRRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0475] SEQ ID NO:448 ILR24.3X3.2FcEN - IL1RAP-24 HC

QVQLQQSGPELVRPGVSVKISCKGSGYTFTDYAMHWVKQSHGKGLEWIGIISTYSGNTKFH

QKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAREGTWYFDVWGAGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVL

TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

[0476] SEQ ID NO:449 ILR24.3X3.2FcEN - IL1RAP-24 LC

QIVLTQSPAIMSASPGEKVTMTCSASSTISYIHWYQQKPGTSPKGWIYDTSKLASGVPARFS

GSGSGTSYSLTISSMEAEDAATYYCHQRTSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC Example 13: ILlRAP-specific T-cell Engager Depletes Acute Myeloid Leukemia Leukemic Stem Cells

[0477] Abstract

[0478] The interleukin-1 receptor accessory protein (IL1RAP) is expressed at high levels on Acute Myeloid Leukemia (AML) blasts including leukemic stem cells (LSCs), but not on hematopoietic stem cells (HSCs). To target IL1RAP in AML, we generated BIF002, an anti-ILlRAP/CD3 bispecific T-cell engager (TCE) with a mutated human IgGl Fc to diminish FcyR binding. We submitted BIF002 through a comprehensive characterization of its antileukemic activity. When incubated with healthy donor T-cells and AML cell lines or primary AML blasts at subnanomolar concentrations, BIF002 triggered IL1RAP dependent T-cell activation and Ab-dependent T-cell lysis of leukemic cells in a time-, dose- and effector-to-target (E: T) ratio-dependent manner. In murine xenograft studies, BIF002 administered intravenously along with human T cells led to depletion of leukemia cells, and significantly survival advantage for mice transplanted with ILlRAP-expressing M0LM13 cells and patient-derived AML blasts without inducing off-target side effects, as compared with isotype control antibody administered together with T cells.

Furthermore, BiF002 selectively and effectively redirects T-cells to eliminate LSCs, as evidenced by the absence of disease initiation in secondary recipients of bone marrow (BM) from donors treated with BIF002 and T-cells (median survival not reached, all survive over 200 days) compared with recipients from donors treated with vehicle (median survival 26 days; p=0.0004) or those treated with T cells + control (26 days; p=0.0002). Taken altogether, these results support BIF002 as a novel anti-ILlRAP/CD3 TCE that efficiently targets LSCs underscoring its potential as a promising therapeutic approach for AML patients.

[0479] Introduction

[0480] Acute myeloid leukemia (AML) is a cytogenetically and molecularly heterogeneous clonal disease characterized by the accumulation of undifferentiated leukemic cells (blasts), which culminates in bone marrow (BM) failure¹. Despite progress, the 5-year overall survival (OS) of AML patients remains only ~30%^2-4. The persistence of highly treatment-resistant leukemic stem cells (LSCs), primitive cells capable of unlimited self-renewal and initiation and maintenance of the disease is often recognized as a root cause of the treatment failures in AML patients^5-7. The lack of specific membrane surface antigens that distinguish LSCs from normal hematopoietic stem cells (HSCs) have made challenging to target and eliminate these cells. While allogenic hematopoietic stem cell transplantation is the only curative approach for most AML patients as it provides a graft- vs-leukemia effect that potentially eliminates LSCs, its applicability is limited only to fit and younger patients. Thus, novel, more effective and safer therapies are highly needed for AML patients.

[0481] Recently, immune checkpoint inhibitors, monoclonal antibodies, bispecific T-cell engager antibodies (TCEs), and chimeric antigen receptor T-cells (CAR-T cells) have emerged as novel immune-based approaches for cancer treatment^8-11. While these approaches show promise for lymphoid leukemias, but they are still investigational for AML. TCEs (anti-CD33/CD3, anti- CD123/CD3, anti-CLL-l/CD3, anti-FLT3/CD3), and CD123 or CD33 CAR-T cells are undergoing clinical trials to support the feasibility of this approach in AML^12-19. However, these targets are also frequently expressed on heathy donor-derived hematopoietic stem/progenitor cells (HSC/HSPC), potentially leading to significant on-target, off-leukemia toxicity^20-22.

[0482] The interleukin 1 receptor accessory protein (IL1RAP) is a member of the IL-1 superfamily and that is mostly expressed in the liver, the placenta, and white blood cells (NCBI Gene ID 3556; UniProtKB ID Q9NPH3; Bgee Gene ID ENSG00000196083), and is emerging as a novel therapeutic target of interest in AML and other solid tumors²³’²⁴. This protein is a co-receptor of type 1 interleukin 1 receptor (IL1R1) with a central role in amplification and transmission of the IL1 downstream signal activating of the MAPK pathway via MyD88²⁵. High levels of IL1RAP have been found on the surface of AML, chronic myeloid leukemia (CML) and high risk-myelodysplastic syndrome (MDS) cells and have been associated with rapid disease growth and poor outcome^26,27. Of note, IL1RAP was also reportedly expressed at significantly higher levels in LSCs compared to normal HSCs, thereby offering an AML-specific immunotherapeutic target^{28 29}. To this end, blocking IL1 signaling with IL1 receptor antagonists³⁰ or monoclonal antibody has resulted in inhibition of leukemia growth, without off-target effects on normal hematopoiesis^24,28’³¹. Currently, monoclonal IL1RAP antibodies are being tested in clinical trials of both solid tumor and leukemic patients³², and IL1RAP CAR-T cells have demonstrated significant activity in preclinical models and are also being tested in clinical trials^{33 34}., but to our knowledge an IL1RAP TCE has not been reported yet.

[0483] Herein, we describe the development and functional characterization of BIF002, a novel anti-ILlRAP/CD3 TCE. Fab arm exchange was used to create an IgG-like format with high affinity to human(h) IL1RAP and relatively weak affinity to CD3. Mutations in the Fc were incorporated to reduce FcyR binding. We observe significant preclinical activity of BIF002, with no evidence of early hematologic or non-hematologic toxicity. Importantly, using AML patient-derived xenografts (PDXs), in secondary transplant experiments, we showed that BIF002 can eliminate LSCs.

[0484] Materials and methods

[0485] Generation of BIF002 and controls

[0486] To develop an anti-hILlRAP antibody for leukemia immunotherapy, Balb/c mice were immunized with the recombinant extracellular domain (ECD) of human(h) IL1RAP (ECD, S21- E359). Plasma B cells from spleens of immunized mice were screened for IL1RAP antibody production using the Beacon® optofluidic system. Cells secreting anti-hILlRAP antibodies were identified, and their variable heavy (VH) and light (VL) chain sequences were determined from reverse transcription and cDNA synthesis. Twelve sets of VH/VL sequences were cloned into a vector containing human constant domains to create murine-human chimeric anti-ILlRAP antibodies. These antibodies were produced in ExpiCHO cells and purified, and their purity (>90%) confirmed by size exclusion chromatography (SEC) and SDS-PAGE (Figures 1A-1L and 3A-3L). [0487] The selected variant IL1RAP mAb 24 was used to generate the TCE by Fab-arm exchange (FAE)35. K409R and F405L mutations in the Fc CH3 domain were introduced into the anti-ILlRAP and anti-hCD3(H26H8) mAbs, respectively. H26H8 is a humanized, human / non-human primate, cross-reactive anti-hCD3 mAb developed at City of Hope (manuscript in preparation). Moreover, Fc mutations abrogating CD16a binding were also introduced to parental mAbs to eliminate the Fc receptor binding36. The Fab exchange product was verified on ion exchange chromatography (IEC). The resulting TCE, BIF002 (anti-ILlRAP/CD3), is a full-length IgGl with the inclusion of Fc mutations and is the lead TCE candidate for in vitro and in vivo evaluation.

[0488] As a control, point mutations were introduced into the CDRs of each arm based on crystal structure of IL1RAP Fab24 (Figures 26A-28B and 107) and H26H8. BiF018 consists of anti- hlLlRAP partnered with an anti-hCD3 arm incorporating a mutation to abrogate CD3 binding, and a wildtype IgGl Fc. BiF026 includes a mutation (mut) in the anti-hILlRAP arm to abrogate IL1RAP binding paired with the previous mut. anti-hCD3 and Fc (mut). BiF018 and BiF026 served as control constructs.

[0489] Human samples collection

[0490] All primary AML (Figure 105) and healthy donor samples were obtained from the City of Hope National Medical Center (COHNMC). Sample acquisition was approved by the Institutional Review Boards at COHNMC and met Declaration of Helsinki requirements. Mononuclear cells (MNCs) were isolated using Ficoll separation. T-cells were isolated from PBMCs using EasySep™ human T-cell enrichment kit (Stem cell Technologies) and will expand with anti-CD3/CD28 Dynabeads (Gibco) for in vivo experiments. CD34+ cells were isolated using magnetic bead selection (Miltenyi Biotec).

[0491] In vitro cytotoxicity analysis, T-cell activation, cytokine release and proliferation analysis

[0492] Redirected T-cell cytotoxicity was assayed at 48h using purified T-cells as effector cells with various cell lines or patient AML blasts as target cells, at different effector-to-target (E: T) ratios, and serial dilutions of BIF002. Target cells were stained with PKH67 (Sigma-Aldrich) one day before co-incubation and distinguished from effector cells using GFP channel. Target cells were seeded at a density of 2xl0⁴/200 pl. Cell death and cell apoptosis was evaluated by Flow cytometry (FCM) with 7-amino actinomycin D (7-AAD) and Annexin V labeling, respectively. Cell lysis ability was determined with the following formula: % Lysis = 100 - (viable cells of treatment group x 100/viable cells of untreated control group).

[0493] Real time T-cell mediated cell cytotoxicity of M0LM13^Luci/GFP+ or THP-1^LUCI/GFP+ cells were conducted using the xCELLigence RTCA eSight platform (Agilent). E:T ratio 5 : 1 was used for in vitro IC50 determination, T-cell activation and IFN-y cytokine release analysis. In T-cell proliferation analysis, T-cells were labeled with cell trace TM violet (CTV, Thermo Fisher) before co-culture, after 5 days of co-culture with target cells at an E:T ratio of 5: 1 or 2: 1, proliferating T cells were identified as CTV^LOW T cells by FCM.

[0494] Mouse studies

[0495] C57BL/6J mice (Jackson Laboratory) were used to evaluate the pharmacokinetics (PK) of BIF002. The mice received a single bolus administration of either 0.5 mg/kg (10 pg) or 5 mg/kg (100 pg). After administration, serum of 3 mice/timepoint/dose group were collected at 0.5, 2, 8, 24,48, 72, 120 and 168-hours post-dose. IL1RAP levels were measured using an indirect ELISA assay, and PK parameters were analyzed by the Analytical Pharmacology Core at COHNMC.

[0496] The antileukemic efficacy of BIF002 was evaluated in 6-8-week-old NSG-SGM3 (NSGS) mice (The Jackson Laboratory) engrafted with either M0LM13^LUCI/GFP+ cells(lxl0⁶) or patients’ AML Luci+ blasts (0.5xl0⁶) and treated with BIF002 10 pg every 3 days and in vitro expanded healthy donor T-cells given weekly with E:T ratio 3: 1. Mouse treated with normal h-IgG (R&D, M0LM13^{LUCI GFP+} model) and isotype BIF026 (PDX models), BIF002 along, and control antibody with T-cells were used as control s(n=5/group). Mouse care and experimental procedures were performed in accordance with Institutional Animal Care and Use Committee (IACUC) approved protocol (#15005) at City of Hope. The mice were injected intraperitoneally with d-Luciferin (150mg/kg) (Promega), and the tumor burden (measured as photons) was detected using a bioluminescence imaging (BLI) system. The survival rate of each group was recorded and plotted in a Kaplan-Meier survival curve. Additionally, the mice's body weight change rate was calculated.

[0497] To further characterize the impact of BIF002 on LSCs, a separate PDX model was created by engrafting blasts(lxl0⁶) from a relapsed AML patient into NOD/Scid/Il-2rg (NSG). The mice were divided into 3 groups (Vehicle, T-cells + control (BIF026), and T-cells + BIF002, n=10). Health donor human T-cells (3xl0⁶) were administered weekly for 3 doses starting on Day 10, and BIF002 or control antibody treatment began on Day 11 for 3 weeks. On Day 34, BMMNC were harvested from 3 mice/group and injected into NSGS recipient mice (n=7/group) for a second transplantation to detect LSCs, without further treatment. PB of the first set (Day 28, hCD45/CD2) and recipient mice (Day 21, hCD45/CD3) were sampled for engraftment by FCM.

[0498] To assess the impact of BIF002 on the normal hematopoietic system, human CD34+ BM cells were injected into NSGS mice. After 4 weeks, mice were treated with BIF002 10 pg or vehicle for 3 weeks, and sacrificed one week later to compared the engraftments in PB and BM. Analysis of human leukocyte markers (hCD45+, hCD34, CD3, CD19, CD14, CD33) was performed on collected mouse samples.

[0499] Statistical analysis

[0500] Statistical analyses were conducted using GraphPad Prism V.9.0.5. Group comparisons utilized two-tailed, unpaired Student t-tests. Survival comparisons employed the Log-rank test. In vitro experiments were performed at least twice with duplicates or triplicates. In vivo experiments involved 5-7 mice per group. Significance was set at P < 0.05. Results are presented as mean ± s.e.m. with significance levels indicated as *P < 0.05, **P < 0.01, ***p < 0.001, and ****p < 0.0001.

[0501] Results

[0502] IL1RAP expression in AML cell lines and primary patients’ blasts. [0503] We assessed IL1RAP expression on representative AML cell lines, primary AML blasts and normal CD34+ BM cells by FCM. High levels of IL1RAP expression were detected on all the AML cell lines (ILlRAP^pos), except for KG-la cells, which were IL1RAP^1OW, and K562, which were ILlRAP^neg (Figure 93 A). AML MNCs (65 ± 5%, n=21) and CD34⁺ blasts (76 ± 6%, n=14) also expressed significantly higher levels of IL1-RAP than normal CD34¹ BM cells (10 ± 2%, n=14, both p<0.0001; Figure 93B). Notably, the IL1RAP expression was higher in LSC-enriched CD34+ population than the bulk blasts from the same donor (n=14, p=0.011; Figure 93C).

[0504] Compared with other AML immunotherapeutic targets i.e., CD33 (93 ± 4%), CD 123 (85 ± 4%) or CLL-1(78 ± 6%), IL1RAP (80 ± 6%) was expressed at a similarly high levels on AML CD34⁺ cells (n=12; Figure 93D). However, the IL1RAP expression (8 ± 2%) was significantly lower than that of CD33 (64 ± 6%), CD123 (58 ± 9%), CLL-1 (35 ± 6%) on normal HSC-enriched CD34+ cells (n=8; Figure 93E).

[0505] Characterization of the novel anti-ILlRAP antibodies and BIF002.

[0506] From an array of 56 anti-ILlRAP fragment antigen-bindings (Fabs), we selected six (#2, #22, #24, #46, #53 and #58) candidates based on kinetic parameters that included antigen binding by SPR (dissociation constants in the range 2 nM to 400 nM; Figure 94A) and protein melting temperatures (Tm) by protein thermal shift assay (Figure 94B). Compared to Trastuzumab, a clinically available anti-Her2 antibody with a Tm of 75 °C, the Fab #24 had a Tm of 77 °C and was the most stable among the six candidate fragments. Compared to isotype control, Fab #24 showed a significant binding to IL 1 RAP-positive leukemia cell line, MV4-11 and M0LM13 (Figure 94C). The high purity of anti-ILlRAP Fab #24 was demonstrated as a major single band on SDS-PAGE (Figure 94D), and as a major single symmetric peak on the SEC (Figure 94E).

[0507] Based on these results, the anti-ILlRAP Fab #24 was selected to produce the following anti-ILlRAPxCD3 TCEs: BIF002, BIF018, BIF026, which differed for mutations in the Fab or Fc moieties (Figure 94F. and methods for details). To confirm the heterodimer configuration of the anti-ILlRAPxCD3 TCEs, the final reaction was verified on ion exchange chromatography (IEC), we observed a significant shift of the elution of the anti-ILlRAPxCD3 TCEs from both parental antibodies (Figure 94G). We also tested the TCEs binding to each of the two targets, CD3 and IL1RAP. We used ILlRAP^pos AML blasts and M0LM13 cell line to test the IL1RAP binding of anti-ILlRAP #24, BIF002, BIF018, BIF026, and normal human T-cell and T-ALL cell lines Jurkat to test the CD3 binding. FACS analysis demonstrated that BIF002 bound both to ILlRAP^pos AML cells, and CD3+ T-cells. Of note, BIF026 binding to both targets was abolished by the mutations introduced in the constructs (Figure 94H).

[0508] BiF002 activity on AML cells.

[0509] Next, to test the antileukemic activity of BIF002, we firstly co-cultured healthy donor T- cells with AML cell lines M0LM13 or THP-1 cells at E:T ratios ranging from 1 :10 to 10: 1 and BIF002 serial dilutions (range 0.01-10 nM) for 48h. We observed a dose- and E:T ratio- dependent cell lysis activity (Figure 95A). At 48h, a near complete target cell lysis was observed at an E:T ratio of 5:1 or higher with BIF002 1 nM or higher.

[0510] To observe the BIF002-dependent cell lysis in real time, M0LM13^LUCI/GFP and THP- JLUCI GFP ceils ^_rea^_ec| _wj^ BIF002 (0.1-100 nM) and T cells was imaged using xCELLigence RTCA eSight platform and the dynamic changes of the GFP signals was recorded over time (Figure 95B). At E:T ratio 5: 1, in the absence of the BIF002, T-cells displayed no cytolytic activity, while in O.lnM or higher BIF002, the cytolytic activity of the T-cells increased in a dose- and timedependent manner, as shown by the decreased GFP signals from the target AML cells. Interestingly, when we co-culture BIF002 10 nM with the T-cells and target cells (M0LM13, THP-1) with different E:T ratio (1: 1 and 5: 1), the real time monitoring imaging also clearly show larger differences in cytolysis efficiency, i.e., the more effector cells the better T-cell cytolytic ability and proliferation (Video 1. M0LM13 E:T ratio 1 : 1, Video 2. M0LM13 E:T ratio 5: 1, Video 3. THP-1 E:T ratio 1 : 1, Video 4. THP-1 E:T ratio 5: 1). Within the video, we can observe that BIF002 guides T-cells to 1L1RAP expressing AML cells ultimately leading to AML cells lysis. [0511] Using ILlRAP^pos AML cell lines we then calculated BIF002 IC50s. After 48-hour exposure, starting with resting T cells and an E:T ratio 5: 1, the BIF002 IC50 was calculated to be within a subnanomolar range (Figure 95C), and correlated inversely with IL1RAP expression levels. While ILlRAP^pos AML cells were killed in the presence of BIF002, ILlRAP^neg K562 cells remained viable. The median IC50s of ILlRAP^pos AML bulk and CD34+ blasts were also in a subnanomolar range, i.e., 0.56 nM (range 0.09-2.69 nM, n=5) and 0.53 nM (range: 0.31-3.78 nM, n=5), Figure 95D, E

[0512] The specificity of BIF002 was tested by introducing mutations in the IL1RAP and CD3 arms. After 48h coculture, at E:T ratio 5:1, while BIF002 increased selective lysis of AML cells, no killing was detected for the BIF018 and BIF026 mutants (Figure 95F). Similar results were observed using PBMC (E: T at 5: 1) as source of T effector cells (Figure 95G). We also observed an increasing apoptosis rate in ILlRAP^pos primary AML blasts (n=5) after treatment with BIF002, but not with BIF026 control (Figure 95H). Of note, no cell killing was observed when either AML or T-cells alone were treated with BIF002 or the mutants (Figure S2). Importantly, no significant changes in the viability of normal CD34+ BM cells (n=4) were observed when cocultured with T cells and increasing doses of BIF002 (Figure 951). Furthermore, no decrease in colony formation was observed in normal CD34+ BM cells after co-cultured with BIF002 and T cells compared to coculture with T cells along or with BIF026 and T cells (Figure 95J), supporting that BIF002 spared normal HSCs given their virtual lack of ILIRAP expression.

[0513] BIF002 induces dose-dependent T-cell activation, cytokine release and proliferation.

[0514] To determine if BIF002-induced T cell activation, AML cell lines or primary blasts were incubated with T-cells in the presence of BIF002 (E:T at 5: 1, 48h). Compared to untreated control group, BIF002-treated T-cells presented with increased CD25 and CD69 expression on both CD4 and CD8 T subpopulations, both of which showed a dose-dependent response to BIF002 (range 0.01-10nM; Figures 96A and 96B). A dose-dependent T cell secretion of IFN-y was also demonstrated in culture supernatant by ELISA (Figure 96C) and intracellular T-cell by FCM (Figure 96D). A BIF002-dose dependent TNF-a level increased was also found in ILlRAP^pos AML cell lines or primary blasts and T cells co-cultures supernatant (Figures 101A-101B).

[0515] To assess BIF002-induced T-cell proliferation, we labeled T-cells with cell trace TM violet (CTV, Invitrogen) and incubated them with AML cell lines or primary blasts at E:T ratio of 2: 1 or 5: 1 in the presence of BIF002. On day 5, significant CD3+ T-cell proliferation was observed, indicated by a multipeaked CTV signal (Figures 96E and 96F). Both CD4 and CD8+ T cell subpopulation proliferated under the same conditions (Figures 102A-102B). Treatment of IL1RAP- negative cells or with BIF026 control did not induce the described activities, confirming that binding to both CD3 and IL1RAP is essential for T-cell activation.

[0516] Pharmacokinetics (PK) indirect ELISA

[0517] We conducted preliminary pharmacokinetics (PK) of BIF002. Healthy C57BL/6J mice were treated with 0.5 mg/kg (10 pg) and 5 mg/kg (100 pg) BIF002 doses, that were empirically selected. The Cmax and CAUC were dose proportional. Cmax was 2.7 pg/ml (18nM) at 2h vs. 27.9 pg/ml (186nM) at 8h, and AUC0-168h was 340.4 pg/mLxhr vs. 4031.1 pg/mLxhr, for 10 pg and 100 pg doses, respectively. And the terminal half-lives for the BIF002 10 pg and 100 pg were of 18 days and 23 days, respectively (Figures 103A-103B). The PK data confirms that in the absence of antigen, a slower decay of BIF002 from circulation.

[0518] In vivo BIF002 activity

[0519] Considering the PK results and that the Cmax of BIF002 10 pg was well above the IC50s, we selected this dosage to the in vivo B F002 antileukemic activity. M0LM13^LUCI/GFP+ cells were engrafted into immunodeficient NSGS mice (Figure 97A). The engrafted mice were divided in 4 groups receiving lOpg control antibody (human IgG), BIF002, T cells + IgG, T cells + BIF002 (n=4-5). Mice treated with T cells + BIF002 exhibited a significant tumor reduction on Day 33 than other groups, as measured by BLI (Figures 97B-97C), and had a significantly longer overall survival (OS; median 54.5 days) compared to IgG (27 days, p=0.0045), BIF002 alone (36 days, p=0.0049), or T cells + IgG (33 days, p=0.0062) treated controls (Figure 97D).

[0520] In a different experiment, BIF002 was also tested at lower doses. M0LM13^LUCI/GFP+ engrafted mice were divided in 4 groups receiving BIF002 (0.1, I pg) with in vitro CD3-bead expanded and activated healthy donor T-cells, and T-cell alone with vehicle (n=5) and untreated mice (n=2) were the control groups (Figure 97F). On Day 29 of treatment, mice treated with T cells + 1 pg of BIF002 had 20 to 100-fold tumor reduction (Figures 97G-97H) and survived longer OS (median 57 days) compared with mice that received T cells + 0.1 pg of BIF002 (median 31 days, p=0.0018) or with T cells alone (median 34 days, p=0.0019) or vehicle (median 27.5 days, p =0.0082) (Figure 971).

[0521] In vivo activity of BIF002 was then evaluated in PDX models. Luciferase-expressing primary blasts from a relap sed/refractory complex karyotype AML patient were injected into NSGS mice and were treated for 3 weeks with lOpg BIF002 or control antibody (BIF026) (Figure 98A). For this experiment we selected the dose of 10 pg considering the 10 times higher IC50 compared to cell lines (see Figures 94C-94E). Mice treated with T cells + BIF002 had 20 to 1000-fold reduction in disease burden and survived longer OS than control antibody or BIF002 alone or T-cells + BIF026 treated mice (median 46 days vs 26, 25 or 30 days, respectively, all p=0.0003) (Figures 98B-98D). On day 25, PB was collected from mice to assess circulating blasts and T-cell percentages. The T-cells + BIF002 group had a significantly lower % circulating blast (0-0.2%) compared with the BIF026 group (23.5 ± 6.32%, p=0.0069), the BIF002 alone group (24.6 ± 7.68%, p=0.0047), and the T-cells + BIF026 group (4.5 ± 1.46%, p=0.8931) (Figures 98E-98F). We also observed T cells in the PB of mice treated with normal human T cells, 12.9 ± 3.58% in T + BIF026 group vs. 3.7+2.1% in T+ BIF002 group on Day 25 (p=0.0395), as shown in Figure 98E.

[0522] To determine the impact of BIF002 on LSCs, we engrafted primary blasts from a relapsed complex karyotype AML patient into NSG mice, (see treatment schema Figure 99 A). Mice treatment was initiated on Day 10 after transplant, T cells were given weekly along with 10 pg of BIF002 or control antibody (BIF026) for 3 weeks. On day 28, we observed a significant reduction of circulating blasts in mice receiving T-cells + BIF002 (0%) compared with those receiving vehicle (34.7 ± 6.9%, p=0.0007) or T-cells + BIF026 (35.1 ± 7.4%, p=0.0006) (Figures 99B-99C). After 70 days, all mice treated with T cells + BIF002 were alive, with no detectable blasts in PB or BM, while all vehicle (median survival 38 days, p=0.0002) and T cells + BIF026 (38 days, p=0.0001) treated controls had died of leukemia (Figures 99B, 99D).

[0523] To determine if BIF002 had eradicated LSCs, we randomly selected 3 female mice from the first set of each group on Day 34, harvest the BM mononuclear cells (MNC), and transplanted same number of cells into second set NSGS recipients (n=7). At the time of harvest, compared with donors treated with T + BIF002 (spleen weight 0.0723g), donors treated with vehicle (0.4473g, p=0.0007) or T + control (0.5653g, p=0.0024) had significant splenomegaly (Figure 99E). Close to 100% blasts in PB, BM, and 70% in SP were detected in mice treated with Vehicle and T-cells + control, but near complete (PB 1.3%, BM 0.2%, SP 0.4%) leukemia clearance was found in T-cells + BIF002 group by FCM (Figure 99F), and blood smear (Figure 99G). More importantly, we show that LSCs elimination since recipients from T+ BIF002-treated donors had a significant decrease in tumor burden, On Day 21, mice transplanted with T-cells + BIF002 showed no detected blasts, while the vehicle group (34.3±5.6%, p < 0.0001) and T+ BIF026 group (32.5±5.2%, p=0.0002) exhibited high blast percentages (Figure 99H), and survive significantly longer (median survival not reached, all survive over 200 days ) compared with recipients from donors treated with vehicle (median survival 26 days; p=0.0004) or those treated with T cells + control (26 days; p=0.0002) (Figure 991).

[0524] Taken together, these data show that human AML-engrafted mice treated with the combination of human T-cells and BIF002 can significantly reduce tumor burden and prolong overall survival with minimal side effects, and no toxify to normal hematopoietic system.

[0525] Preliminary toxicity studies [0526] No obvious weight changes or other toxicities were observed after BIF002 administration alone or with T cells in all experiments described above (Figures 97E, 97J, Figure 98G, Figure 99D). To investigate further the z/7 vivo toxicity of BIF002 on the normal hematopoietic system, we administered human CD34+ HSC into sub-lethally irradiated NSGS mice. After 4 weeks of injection, we confirmed the expression of hCD45 in mouse PB. Subsequently, mice were subjected to a 3-week treatment with either BIF002 10 pg or a vehicle control. Following the treatment, mice exhibited normal chimerism between groups in bone marrow and similar spleen size (Figures 104A- 104C), which indicated that the hematopoietic potential of these cells remained relatively unaffected.

[0527] Discussion

[0528] Recently, IL1RAP has emerged as an ideal target for cancer immunotherapy as it is significantly upregulated on the surface of malignant cells in leukemia and solid tumors compared to the corresponding normal tissues³⁸’⁴⁰. This protein presumably mediates malignant growth through the amplification of inflammatory cancer-driving mechanisms and is associated with worse clinical outcomes⁴¹. Importantly, IL1RAP is reportedly highly expressed on LSCs, suggesting that it is a potential target for AML and other myeloid neoplasms. Of note, when compared to other AML surface markers (i.e., CD33, CD123 and CLL-1), IL1RAP shows low expression on normal HSCs, making it less likely an on-target, off-leukemia hematologic toxicity.

[0529] Herein, we report on the design, selection, and characterization of BIF002, a novel CD3- IL1RAP TCE. We show BIF002 efficiently binds to IL1RAP positive AML cells and CD3 positive T cells, activates T cells in the presence of IL1RAP only, and redirects the engaged T cells to lysis IL1RAP positive AML cells. Importantly, we not only showed the antileukemic activity on “bulk” blasts when BIF002 was administrated along with T cells to PDX models, but also its ability to eradicate LSCs, as shown in secondary transplantation experiments. Mice that received BMMNCs from primary donors treated with BIF002 and T-cells, remained alive more than 200 days in the absence of further treatment, while those that received BM from primary donors treated with T+ BIF026 (mutant BIF002) or vehicle all succumbed to disease with a median OS of only 26 days. Furthermore, we also observe no evidence of in vivo toxicity. Mice treated with BIF002 alone or in combination with T-cells had no weight loss or behavioral changes or signs of GVHD in all three models. Importantly, we also did not observe any preliminary negative impact on normal HSC activity in vitro and in vivo.

[0530] To our knowledge, IL1RAP TCEs other than BIF002 have not been reported yet. Other groups have reported on IL1RAP antibodies that activate the antibody-dependent-cell-mediated cytotoxicity (ADCC) and induce direct blockade of IL 1 RAP signaling and chimeric antigen receptor T-cells (CAR-T) therapy have been reported and are currently in clinical trials. Nadunolimab (alternative names Nidanilimab and CAN04)⁴², is a monoclonal antibody that induce both ADCC and the blockade of IL1RAP signaling and is being currently tested as a single agent (NCT03267316) or in combination with other immunotherapeutic (NCT04452214) or chemotherapy (NCT04990037; NCT05116891; NCT05181462) in solid tumors. IL1RAP CAR-T-cells, which have been also being previously reported^33,34, are being explored in clinical trials for patients with CML (NCT02842320) or AML (NCT04169022). TCEs, CAR-T, and monoclonal antibodies are emerging as significantly effective immunotherapeutics for cancer treatment.

[0531] While BIF002 demonstrates no toxicity to T cells and normal hematopoiesis in vitro its effects on HSC at the mouse model level remain to be fully elucidated. Nevertheless, our preliminary data using NSGS mouse engrafted with normal human CD34 cells, showed that BIF002 was well tolerated, with a limited hematopoietic toxicity likely due to the relatively modest expression of IL1-RAP on normal HSCs.

[0532] In summary, the preclinical data herein presented support the antileukemic activity of a novel CD3-IL1RAP TCE, BIF002 that selectively redirects T-cells to AML bulk blasts and LSCs, with a stable serum half-life, thereby offering an opportunity for a novel treatment for AML patients. IND-enabling studies to rapidly translate BIF002 from the bench to the bed side are ongoing. [0533] Reference

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Haematologica. 2022; 107(8): 1786-1795. [0553] 20. Haubner S, Perna F, Kohnke T, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019;33(l):64-74.

[0554] 21. Taussig DC, Pearce DJ, Simpson C, et al. Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood. 2005;106(13):4086-4092.

[0555] 22. Bakker AB, van den Oudenrijn S, Bakker AQ, et al. C-type lectin-like molecule-1 : a novel myeloid cell surface marker associated with acute myeloid leukemia. Cancer Res. 2004;64(22):8443-8450.

[0556] 23. Jaras M, Johnels P, Hansen N, et al. Isolation and killing of candidate chronic myeloid leukemia stem cells by antibody targeting of IL-1 receptor accessory protein. Proc Natl Acad Sci USA. 2010;107(37):16280-16285.

[0557] 24. Agerstam H, Karlsson C, Hansena N, et al. Antibodies targeting human IL1RAP (IL1R3) show therapeutic effects in xenograft models of acute myeloid leukemia. Proceedings of the National Academy of Sciences of the United States of America. 2015; 112(34): 10786-10791.

[0558] 25. Boraschi D, Italiani P, Weil S, Martin MU. The family of the interleukin-1 receptors. Immunol Rev. 2018;281(l): 197-232.

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[0560] 27. De Boer B, Sheveleva S, Apelt K, et al. The IL1-IL1RAP axis plays an important role in the inflammatory leukemic niche that favors acute myeloid leukemia proliferation over normal hematopoiesis. Haematologica. 2021;106(12):3067-3078.

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[0564] 31 Agerstam H, Hansen N, von Palffy S, et al. IL1RAP antibodies block IL-1 -induced expansion of candidate CML stem cells and mediate cell killing in xenograft models. Blood. 2016;128(23):2683-2693.

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[0570] 37. Han L, Dong L, Leung K, et al. METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism. Cell Stem Cell. 2023;30(l):52-68.el3. [0571] 38. Frenay J, Bellaye PS, Oudot A, et al. IL-1RAP, a Key Therapeutic Target in Cancer. IntJMolSci. 2022;23(23).

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P EMBODIMENTS

[0576] P Embodiment 1. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

[0577] P Embodiment 2. The antibody of P embodiment 1, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:73.

[0578] P Embodiment 3. The antibody of P embodiment 1 or 2, wherein said light chain variable domain comprises the sequence of SEQ ID NO:74. [0579] P Embodiment 4. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5 and a CDR H3 as set forth in SEQ ID NO:6; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:40, a CDR L2 as set forth in SEQ ID NO:41, and a CDR L3 as set forth in SEQ ID NO:42.

[0580] P Embodiment 5. The antibody of P embodiment 4, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:77.

[0581] P Embodiment 6. The antibody of P embodiment 4 or 5, wherein said light chain variable domain comprises the sequence of SEQ ID NO:78.

[0582] P Embodiment 7. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8 and a CDR H3 as set forth in SEQ ID NO:9; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:43, a CDR L2 as set forth in SEQ ID NO:44, and a CDR L3 as set forth in SEQ ID NO:45.

[0583] P Embodiment 8. The antibody of P embodiment 7, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:79.

[0584] P Embodiment 9. The antibody of P embodiment 7 or 8, wherein said light chain variable domain comprises the sequence of SEQ ID NO:80.

[0585] P Embodiment 10. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 10, a CDR H2 as set forth in SEQ ID NO: 11 and a CDR H3 as set forth in SEQ ID NO: 12; and wherein said light chain variable domain comprises: a CDR I as set forth in SEQ ID NO: 46, a CDR L2 as set forth in SEQ ID NO:47, and a CDR L3 as set forth in SEQ ID NO:48.

[0586] P Embodiment 11. The antibody of P embodiment 10, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:81.

[0587] P Embodiment 12. The antibody of P embodiment 10 or 11, wherein said light chain variable domain comprises the sequence of SEQ ID NO:82.

[0588] P Embodiment 13. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14 and a CDR H3 as set forth in SEQ ID NO: 15; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:49, a CDR L2 as set forth in SEQ ID NO: 50, and a CDR L3 as set forth in SEQ ID NO:51.

[0589] P Embodiment 14. The antibody of P embodiment 13, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:83.

[0590] P Embodiment 15. The antibody of P embodiment 13 or 14, wherein said light chain variable domain comprises the sequence of SEQ ID NO:84.

[0591] P Embodiment 16. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 16, a CDR H2 as set forth in SEQ ID NO: 17 and a CDR H3 as set forth in SEQ ID NO: 18; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:52, a CDR L2 as set forth in SEQ ID NO:53, and a CDR L3 as set forth in SEQ ID NO:54.

[0592] P Embodiment 17. The antibody of P embodiment 16, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO: 85. [0593] P Embodiment 18. The antibody of P embodiment 16 or 17, wherein said light chain variable domain comprises the sequence of SEQ ID NO:86.

[0594] P Embodiment 19. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 19, a CDR H2 as set forth in SEQ ID NO:20 and a CDR H3 as set forth in SEQ ID NO:21; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:55, a CDR L2 as set forth in SEQ ID NO:56, and a CDR L3 as set forth in SEQ ID NO:57.

[0595] P Embodiment 20. The antibody of P embodiment 19, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:87.

[0596] P Embodiment 21. The antibody of P embodiment 19 or 20, wherein said light chain variable domain comprises the sequence of SEQ ID NO:88.

[0597] P Embodiment 22. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:22, a CDR H2 as set forth in SEQ ID NO:23 and a CDR H3 as set forth in SEQ ID NO:24; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:58, a CDR L2 as set forth in SEQ ID NO:59, and a CDR L3 as set forth in SEQ ID NO:60.

[0598] P Embodiment 23. The antibody of P embodiment 22, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:89.

[0599] P Embodiment 24. The antibody of P embodiment 22 or 23, wherein said light chain variable domain comprises the sequence of SEQ ID NO:90.

[0600] P Embodiment 25. An anti -interleukin- 1 receptor accessory protein (IL 1 RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:25, a CDR H2 as set forth in SEQ ID NO:26 and a CDR H3 as set forth in SEQ ID NO:27; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:61, a CDR L2 as set forth in SEQ ID NO: 62, and a CDR L3 as set forth in SEQ ID NO: 63.

[0601] P Embodiment 26. The antibody of P embodiment 25, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:91

[0602] P Embodiment 27. The antibody of P embodiment 25 or 26, wherein said light chain variable domain comprises the sequence of SEQ ID NO:92.

[0603] P Embodiment 28. An anti -interleukin- 1 receptor accessory protein (IL1 RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:28, a CDR H2 as set forth in SEQ ID NO:29 and a CDR H3 as set forth in SEQ ID NO:30; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:64, a CDR L2 as set forth in SEQ ID NO:65, and a CDR L3 as set forth in SEQ ID NO:66.

[0604] P Embodiment 29. The antibody of P embodiment 28, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:93.

[0605] P Embodiment 30. The antibody of P embodiment 28 or 29, wherein said light chain variable domain comprises the sequence of SEQ ID NO:94.

[0606] P Embodiment 31. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:31, a CDR H2 as set forth in SEQ ID NO:32 and a CDR H3 as set forth in SEQ ID NO:33; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:67, a CDR L2 as set forth in SEQ ID NO:68, and a CDR L3 as set forth in SEQ ID NO:69. [0607] P Embodiment 32. The antibody of P embodiment 31, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:95.

[0608] P Embodiment 33. The antibody of P embodiment 31 or 32, wherein said light chain variable domain comprises the sequence of SEQ ID NO:96.

[0609] P Embodiment 34. An anti -interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:34, a CDR H2 as set forth in SEQ ID NO:35 and a CDR H3 as set forth in SEQ ID NO:36; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:70, a CDR L2 as set forth in SEQ ID NO:71, and a CDR L3 as set forth in SEQ ID NO:72.

[0610] P Embodiment 35. The antibody of P embodiment 34, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:97.

[0611] P Embodiment 36. The antibody of P embodiment 34 or 35, wherein said light chain variable domain comprises the sequence of SEQ ID NO:98.

[0612] P Embodiment 37. The antibody of any one of P embodiments 1-36, wherein said antibody binds IL1RAP with a binding affinity (KD) of about 1 nM to about 440 nM.

[0613] P Embodiment 38. The antibody of any one of P embodiments 1-37, wherein said antibody has a binding affinity (KD) of about 2.2 nM, about 2.5 nM, about 3.4 nM, about 74 nM or about 437 nM.

[0614] P Embodiment 39. The antibody of any one of P embodiments 1-38, wherein said antibody is a chimeric antibody, a ‘Fab fragment or a single chain antibody (scFv).

[0615] P Embodiment 40. The antibody of P embodiment 1, wherein said antibody is a humanized antibody. [0616] P Embodiment 41. The antibody of P embodiment 40, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75, SEQ ID NO: 100 or SEQ ID NO: 102; and a light chain variable domain comprising the sequence of SEQ ID NO:76, SEQ ID NO: 101 or SEQ ID NO: 103.

[0617] P Embodiment 42. The antibody of P embodiment 40 or 41, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75; and a light chain variable domain comprising the sequence of SEQ ID NO:76.

[0618] P Embodiment 43. The antibody of P embodiment 40 or 41, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 100; and a light chain variable domain comprising the sequence of SEQ ID NO: 101.

[0619] P Embodiment 44. The antibody of P embodiment 40 or 41, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 102; and a light chain variable domain comprising the sequence of SEQ ID NO: 103.

[0620] P Embodiment 45. The antibody of any one of P embodiments 40-44, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.0 nM to about 6 nM.

[0621] P Embodiment 46. The antibody of any one of P embodiments 40-45, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.06 nM, about 1.49 nM or 5.36 nM.

[0622] P Embodiment 47. The antibody of any one of P embodiments 1-46, wherein said antibody binds to human IL1RAP or cynomolgus IL1RAP.

[0623] P Embodiment 48. The antibody of any one of P embodiments 1-3, wherein said antibody comprises a heavy chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO:73; a glutamic acid at a position corresponding to position 99 of SEQ ID NO:73; or a glutamic acid at a positon corresponding to position 101 of SEQ ID NO:73. [0624] P Embodiment 49. The antibody of P embodiment 48, wherein said antibody comprises a light chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO:74.

[0625] P Embodiment 50. The antibody of any one of P embodiments 48-49, wherein said antibody does not bind IL1RAP at a detectable level.

[0626] P Embodiment 51 . The antibody of any one of P embodiments 1 -49, further comprising a central cavity formed by a heavy chain variable (VH) region, a light chain variable (VL) region, a heavy chain constant region (CH) and a light chain constant region (CL), wherein said central cavity forms a peptide binding site comprising framework region amino acid residues.

[0627] P Embodiment 52. The antibody of P embodiment 51, wherein a compound comprising a peptidyl moiety is bound to said peptide binding site.

[0628] P Embodiment 53. The antibody of any one of P embodiments 1-52, wherein said antibody is bound to an IL1RAP protein.

[0629] P Embodiment 54. The antibody of P embodiment 53, wherein said IL1RAP protein is expressed on a cell.

[0630] P Embodiment 55. The antibody of P embodiment 54, wherein said cell is a cancer cell.

[0631] P Embodiment 56. The antibody of P embodiment 55, wherein said cancer cell is an acute myeloid leukemia (AML) cell.

[0632] P Embodiment 57. A recombinant protein comprising: (i) a first antibody region; and (ii) a second antibody region, comprising: (a) a second heavy chain variable domain comprising a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain comprising a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39. [0633] P Embodiment 58. The recombinant protein of P embodiment 57, wherein said first antibody region binds a first effector cell ligand.

[0634] P Embodiment 59. The recombinant protein of P embodiment 57 or 58, wherein said effector cell ligand is CD3, CD70 or CD 123.

[0635] P Embodiment 60. The recombinant protein of P embodiment 57-59, wherein said first antibody region comprises a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247.

[0636] P Embodiment 61. The recombinant protein of P embodiment 60, wherein said first antibody region comprises the sequence of SEQ ID NO:251.

[0637] P Embodiment 62. The recombinant protein of any one of P embodiments 57-61, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO: 73 and said second light chain variable domain comprises the sequence of SEQ ID NO: 74.

[0638] P Embodiment 63. The recombinant protein of any one of P embodiments 57-62, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 6.03 nM.

[0639] P Embodiment 64. The recombinant protein of any one of P embodiments 57-61, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:75 and said second light chain variable domain comprises the sequence of SEQ ID NO:76.

[0640] P Embodiment 65. The recombinant protein of any one of P embodiments 57-61 or 64, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 7.16 nM.

[0641] P Embodiment 66. The recombinant protein of any one of P embodiments 57-65, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:4, a first CDR H2 as set forth in SEQ ID NO: 5 and a first CDR H3 as set forth in SEQ ID NO: 6; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:40, a first CDR L2 as set forth in SEQ ID NO:42, and a first CDR L3 as set forth in SEQ ID NO:43.

[0642] P Embodiment 67. The recombinant protein of any one of P embodiments 57-66, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:77 and a light chain variable domain comprising the sequence of SEQ ID NO:78.

[0643] P Embodiment 68. The recombinant protein of any one of P embodiments 57-65, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO: 13, a first CDR H2 as set forth in SEQ ID NO: 14 and a first CDR H3 as set forth in SEQ ID NO: 15; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:49, a first CDR L2 as set forth in SEQ ID NO:50, and a first CDR L3 as set forth in SEQ ID NO:51

[0644] P Embodiment 69. The recombinant protein of any one of P embodiments 57-65 or 68, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:83 and a light chain variable domain comprising the sequence of SEQ ID NO:84.

[0645] P Embodiment 70. The recombinant protein of any one of P embodiments 57-65, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:28, a first CDR H2 as set forth in SEQ ID NO:29 and a first CDR H3 as set forth in SEQ ID NO:30; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:64, a first CDR L2 as set forth in SEQ ID NO:65, and a first CDR L3 as set forth in SEQ ID NO:66. [0646] P Embodiment 71. The recombinant protein of any one of P embodiments 57-65 or 70, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:94 and a light chain variable domain comprising the sequence of SEQ ID NO:95

[0647] P Embodiment 72. The recombinant protein of any one of P embodiments 57-71, wherein said first antigen binding region further comprises a first Fc dimerizing domain; and wherein said second antigen binding region further comprises a second Fc dimerizing domain.

[0648] P Embodiment 73. The recombinant protein of P embodiment 72, wherein said first Fc dimerizing domain and said second Fc dimerizing domain form a Fc region thereby binding said first antigen binding region to said second antigen binding region.

[0649] P Embodiment 74. The recombinant protein of P embodiment 72 or 73, wherein said first Fc dimerizing domain comprises a lysine at a position corresponding to position 405; and wherein said second Fc dimerizing domain comprises an arginine at a position corresponding to position 409.

[0650] P Embodiment 75. The recombinant protein of any one of P embodiments 72-74, wherein said first Fc antibody binding domain and said second Fc binding domain are non-active Fc antibody binding domains.

[0651] P Embodiment 76. The recombinant protein of any one of P embodiments 57-75, wherein said antibody is bound to an IL1RAP protein.

[0652] P Embodiment 77. The recombinant protein of P embodiment 76, wherein said IL1RAP protein is expressed on a cell.

[0653] P Embodiment 78. The recombinant protein of P embodiment 77, wherein said cell is a cancer cell.

[0654] P Embodiment 79. The recombinant protein of P embodiment 78, wherein said cancer cell is an acute myeloid leukemia (AML) cell. [0655] P Embodiment 80. An isolated nucleic acid encoding an antibody of any one of P embodiments 1-55 or a recombinant protein of any one of P embodiments 57-78.

[0656] P Embodiment 81. A pharmaceutical composition comprising (i) a therapeutically effective amount of an antibody of any one of P embodiments 1-55 and a pharmaceutically acceptable excipient or (ii) a therapeutically effective amount of a recombinant protein of any one of P embodiments 57-78, and a pharmaceutically acceptable excipient.

[0657] P Embodiment 82. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of (i) an antibody of any one of P embodiments 1-55 or (ii) a recombinant protein of any one of P embodiments 57-78, thereby treating cancer in said subject.

[0658] P Embodiment 83. The method of P embodiment 82, wherein said cancer is acute myeloid leukemia (AML).

EMBODIMENTS

[0659] Embodiment 1. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of an anti-interleukin- 1 receptor accessory protein (IL 1 RAP) antibody, wherein said antibody does not inhibit IL 1 -receptor signaling relative to a standard control, thereby treating cancer in said subject.

[0660] Embodiment 2. The method of embodiment 1, wherein said anti -interleukin- 1 receptor accessory protein (IL1RAP) antibody comprises a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO: 2 and a CDR H3 as set forth in SEQ ID NO: 3; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

[0661] Embodiment 3. The method of any one of embodiments 1-2, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:73. [0662] Embodiment 4. The method of any one of embodiments 1-3, wherein said light chain variable domain comprises the sequence of SEQ ID NO:74.

[0663] Embodiment 5. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of a recombinant protein comprising: (i) a first antibody region, wherein said first antibody binding region binds an effector cell ligand; and (ii) a second antibody region, wherein said second antibody region binds an interleukin-1 receptor accessory protein (IL1RAP) and does not inhibit ILl-receptor signaling relative to a standard control, thereby treating cancer in said subject.

[0664] Embodiment 6. The method of embodiment 5, wherein said effector cell ligand is CD3, CD70 or CD 123.

[0665] Embodiment 7. The method of any one of embodiments 5-6, wherein said first antibody region comprises a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247.

[0666] Embodiment 8. The method of any one of embodiments 5-7, wherein said first antibody region comprises the sequence of SEQ ID NO:251.

[0667] Embodiment 9. The method of any one of embodiments 5-8, wherein said second antibody region comprises (a) a second heavy chain variable domain comprising a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain comprising a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO: 39. [0668] Embodiment 10. The method of embodiment 9, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:73 and said second light chain variable domain comprises the sequence of SEQ ID NO: 74.

[0669] Embodiment 11. The method of any one of embodiments 1-10, wherein said antiinterleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit IL 1 -receptor signaling relative to a standard control.

[0670] Embodiment 12. The method of any one of embodiments 1-10, wherein said antiinterleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit ILl-receptor alpha signaling relative to a standard control.

[0671] Embodiment 13. The method of any one of embodiments 1-10, wherein said antiinterleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit ILl-receptor beta signaling relative to a standard control.

[0672] Embodiment 14. The method of any one of embodiments 1-10, wherein said antiinterleukin-1 receptor accessory protein (IL 1 RAP) antibody or said second antibody region does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control.

[0673] Embodiment 15. The method of any one of embodiments 1-10, wherein said antiinterleukin-1 receptor accessory protein (IL 1 RAP) antibody or said second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

[0674] Embodiment 16. The method any one of embodiments 1-1 , wherein said cancer is acute myeloid leukemia (AML).

[0675] Embodiment 17. The method any one of embodiments 1-16, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not block signaling of one or more co-receptors of IL1RAP. [0676] Embodiment 18. The method of embodiment 17, wherein said one or more co-receptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1RL2.

[0677] Embodiment 19. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

[0678] Embodiment 20. The antibody of embodiment 19, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:73.

[0679] Embodiment 21. The antibody of embodiment 19 or 20, wherein said light chain variable domain comprises the sequence of SEQ ID NO:74.

[0680] Embodiment 22. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5 and a CDR H3 as set forth in SEQ ID NO:6; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:40, a CDR L2 as set forth in SEQ ID NO:41, and a CDR L3 as set forth in SEQ ID NO:42.

[0681] Embodiment 23. The antibody of embodiment 22, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:77.

[0682] Embodiment 24. The antibody of embodiment 22 or 23, wherein said light chain variable domain comprises the sequence of SEQ ID NO:78.

[0683] Embodiment 25. An anti-interleukin-1 receptor accessory protein (IL 1 RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8 and a CDR H3 as set forth in SEQ ID NO:9; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:43, a CDR L2 as set forth in SEQ ID NO:44, and a CDR L3 as set forth in SEQ ID NO 45.

[0684] Embodiment 26. The antibody of embodiment 25, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:79.

[0685] Embodiment 27. The antibody of embodiment 25 or 26, wherein said light chain variable domain comprises the sequence of SEQ ID NO:80.

[0686] Embodiment 28. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 10, a CDR H2 as set forth in SEQ ID NO:11 and a CDR H3 as set forth in SEQ ID NO: 12; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:46, a CDR L2 as set forth in SEQ ID NO:47, and a CDR L3 as set forth in SEQ ID NO:48.

[0687] Embodiment 29. The antibody of embodiment 28, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:81.

[0688] Embodiment 30. The antibody of embodiment 28 or 29, wherein said light chain variable domain comprises the sequence of SEQ ID NO:82.

[0689] Embodiment 31. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID NO: 14 and a CDR H3 as set forth in SEQ ID NO: 15; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:49, a CDR L2 as set forth in SEQ ID NO:50, and a CDR L3 as set forth in SEQ ID NO:51. [0690] Embodiment 32. The antibody of embodiment 31, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:83.

[0691] Embodiment 33. The antibody of embodiment 31 or 32, wherein said light chain variable domain comprises the sequence of SEQ ID NO:84.

[0692] Embodiment 34. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 16, a CDR H2 as set forth in SEQ ID NO: 17 and a CDR H3 as set forth in SEQ ID NO: 18; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:52, a CDR L2 as set forth in SEQ ID NO:53, and a CDR L3 as set forth in SEQ ID NO:54.

[0693] Embodiment 35. The antibody of embodiment 34, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO: 85.

[0694] Embodiment 36. The antibody of embodiment 34 or 35, wherein said light chain variable domain comprises the sequence of SEQ ID NO:86.

[0695] Embodiment 37. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 19, a CDR H2 as set forth in SEQ ID NO:20 and a CDR H3 as set forth in SEQ ID NO:21; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:55, a CDR L2 as set forth in SEQ ID NO:56, and a CDR L3 as set forth in SEQ ID NO:57.

[0696] Embodiment 38. The antibody of embodiment 37, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:87.

[0697] Embodiment 39. The antibody of embodiment 37 or 38, wherein said light chain variable domain comprises the sequence of SEQ ID NO:88. [0698] Embodiment 40. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:22, a CDR H2 as set forth in SEQ ID NO:23 and a CDR H3 as set forth in SEQ ID NO:24; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:58, a CDR L2 as set forth in SEQ ID NO:59, and a CDR L3 as set forth in SEQ ID NO:60.

[0699] Embodiment 41. The antibody of embodiment 40, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:89.

[0700] Embodiment 42. The antibody of embodiment 40 or 41, wherein said light chain variable domain comprises the sequence of SEQ ID NO:90.

[0701] Embodiment 43. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:25, a CDR H2 as set forth in SEQ ID NO:26 and a CDR H3 as set forth in SEQ ID NO:27; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:61, a CDR L2 as set forth in SEQ ID NO: 62, and a CDR L3 as set forth in SEQ ID NO: 63.

[0702] Embodiment 44. The antibody of embodiment 43, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:91

[0703] Embodiment 45. The antibody of embodiment 43 or 44, wherein said light chain variable domain comprises the sequence of SEQ ID NO:92.

[0704] Embodiment 46. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:28, a CDR H2 as set forth in SEQ ID NO:29 and a CDR H3 as set forth in SEQ ID NO:30; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:64, a CDR L2 as set forth in SEQ ID NO:65, and a CDR L3 as set forth in SEQ ID NO:66.

[0705] Embodiment 47. The antibody of embodiment 46, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:93.

[0706] Embodiment 48. The antibody of embodiment 46 or 47, wherein said light chain variable domain comprises the sequence of SEQ ID NO:94.

[0707] Embodiment 49. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:31 , a CDR H2 as set forth in SEQ ID NO:32 and a CDR H3 as set forth in SEQ ID NO:33; and wherein said light chain variable domain comprises:a CDR LI as set forth in SEQ ID NO:67, a CDR L2 as set forth in SEQ ID NO:68, and a CDR L3 as set forth in SEQ ID NO:69.

[0708] Embodiment 50. The antibody of embodiment 49, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:95.

[0709] Embodiment 51. The antibody of embodiment 49 or 50, wherein said light chain variable domain comprises the sequence of SEQ ID NO:96.

[0710] Embodiment 52. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:34, a CDR H2 as set forth in SEQ ID NO:35 and a CDR H3 as set forth in SEQ ID NO:36; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:70, a CDR L2 as set forth in SEQ ID NO:71, and a CDR L3 as set forth in SEQ ID NO:72.

[0711] Embodiment 53. The antibody of embodiment 52, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:97. [0712] Embodiment 54. The antibody of embodiment 52 or 53, wherein said light chain variable domain comprises the sequence of SEQ ID NO:98.

[0713] Embodiment 55. The antibody of any one of embodiments 19-54, wherein said antiinterleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor signaling relative to a standard control.

[0714] Embodiment 56. The antibody of any one of embodiments 19-54, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor alpha signaling relative to a standard control.

[0715] Embodiment 57. The antibody of any one of embodiments 19-54, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor beta signaling relative to a standard control.

[0716] Embodiment 58. The antibody of any one of embodiments 19-54, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an ILl-beta protein to its cognate receptor relative to a standard control.

[0717] Embodiment 59. The antibody of any one of embodiments 19-54, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an ILl-alpha protein to its cognate receptor relative to a standard control.

[0718] Embodiment 60. The antibody of any one of embodiments 19-54, wherein said anti- interleukin-1 receptor accessory protein (IL1RAP) antibody does not block signaling of one or more co-receptors of IL 1 RAP.

[0719] Embodiment 61. The antibody of embodiment 60, wherein said one or more coreceptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1RL2.

[0720] Embodiment 62. The antibody of any one of embodiments 19-61, wherein said antibody binds IL1RAP with a binding affinity (KD) of about 1 nM to about 440 nM. [0721] Embodiment 63. The antibody of any one of embodiments 19-62, wherein said antibody has a binding affinity (KD) of about 2.2 nM, about 2.5 nM, about 3.4 nM, about 74 nM or about 437 nM.

[0722] Embodiment 64. The antibody of any one of embodiments 19-63, wherein said antibody is a chimeric antibody, a ‘Fab fragment or a single chain antibody (scFv).

[0723] Embodiment 65. The antibody of embodiment 19, wherein said antibody is a humanized antibody.

[0724] Embodiment 66. The antibody of embodiment 65, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75, SEQ ID NO:100 or SEQ ID NO:102; and a light chain variable domain comprising the sequence of SEQ ID NO:76, SEQ ID NO: 101 or SEQ ID NO: 103.

[0725] Embodiment 67. The antibody of embodiment 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75; and a light chain variable domain comprising the sequence of SEQ ID NO:76.

[0726] Embodiment 68. The antibody of embodiment 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 100; and a light chain variable domain comprising the sequence of SEQ ID NO: 101.

[0727] Embodiment 69. The antibody of embodiment 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 102; and a light chain variable domain comprising the sequence of SEQ ID NO: 103.

[0728] Embodiment 70. The antibody of any one of embodiments 65-69, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.0 nM to about 6 nM. [0729] Embodiment 71. The antibody of any one of embodiments 65-70, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.06 nM, about 1.49 nM or 5.36 nM.

[0730] Embodiment 72. The antibody of any one of embodiments 19-71, wherein said antibody binds to human IL1RAP or cynomolgus IL1RAP.

[0731] Embodiment 73. The antibody of any one of embodiments 19-21, wherein said antibody comprises a heavy chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO: 73; a glutamic acid at a position corresponding to position 99 of SEQ ID NO:73; or a glutamic acid at a positon corresponding to position 101 of SEQ ID NO:73.

[0732] Embodiment 74. The antibody of embodiment 73, wherein said antibody comprises a light chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO:74.

[0733] Embodiment 75. The antibody of any one of embodiments 73-74, wherein said antibody does not bind IL1RAP at a detectable level.

[0734] Embodiment 76. The antibody of any one of embodiments 19-74, further comprising a central cavity formed by a heavy chain variable (VH) region, a light chain variable (VL) region, a heavy chain constant region (CH) and a light chain constant region (CL), wherein said central cavity forms a peptide binding site comprising framework region amino acid residues.

[0735] Embodiment 77. The antibody of embodiment 76, wherein a compound comprising a peptidyl moiety is bound to said peptide binding site.

[0736] Embodiment 78. The antibody of any one of embodiments 19-77, wherein said antibody is bound to an IL1RAP protein.

[0737] Embodiment 79. The antibody of embodiment 78, wherein said IL1RAP protein is expressed on a cell. [0738] Embodiment 80. The antibody of embodiment 79, wherein said cell is a cancer cell.

[0739] Embodiment 81. The antibody of embodiment 80, wherein said cancer cell is an acute myeloid leukemia (AML) cell.

[0740] Embodiment 82. A recombinant protein comprising: (i) a first antibody region; and (ii) a second antibody region, comprising: (a) a second heavy chain variable domain comprising a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and (b) a second light chain variable domain comprising a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

[0741] Embodiment 83. The recombinant protein of embodiment 82, wherein said first antibody region binds a first effector cell ligand.

[0742] Embodiment 84. The recombinant protein of embodiment 82 or 83, wherein said effector cell ligand is CD3, CD70 or CD 123.

[0743] Embodiment 85. The recombinant protein of embodiment 82-84, wherein said first antibody region comprises a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and (b) a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247.

[0744] Embodiment 86. The recombinant protein of embodiment 85, wherein said first antibody region comprises the sequence of SEQ ID NO:251.

[0745] Embodiment 87. The recombinant protein of any one of embodiments 82-86, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:73 and said second light chain variable domain comprises the sequence of SEQ ID NO: 74. [0746] Embodiment 88. The recombinant protein of any one of embodiments 82-87, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 6.03 nM.

[0747] Embodiment 89. The recombinant protein of any one of embodiments 82-86, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:75 and said second light chain variable domain comprises the sequence of SEQ ID NO:76.

[0748] Embodiment 90. The recombinant protein of any one of embodiments 82-86 or 89, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 7.16 nM.

[0749] Embodiment 91. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not inhibit IL1 -receptor signaling relative to a standard control.

[0750] Embodiment 92. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not inhibit IL 1 -receptor alpha signaling relative to a standard control.

[0751] Embodiment 93. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not inhibit IL 1 -receptor beta signaling relative to a standard control.

[0752] Embodiment 94. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control.

[0753] Embodiment 95. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

[0754] Embodiment 96. The recombinant protein of any one of embodiments 82-90, wherein said second antibody region does not block signaling of one or more co-receptors of IL1RAP. [0755] Embodiment 97. The recombinant protein of embodiment 96, wherein said second antibody region wherein said one or more co-receptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1 RL2

[0756] Embodiment 98. The recombinant protein of any one of embodiments 82-90, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:4, a first CDR H2 as set forth in SEQ ID NO: 5 and a first CDR H3 as set forth in SEQ ID NO: 6; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:40, a first CDR L2 as set forth in SEQ ID NO:42, and a first CDR L3 as set forth in SEQ ID NO:43.

[0757] Embodiment 99. The recombinant protein of any one of embodiments 82-98, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:77 and a light chain variable domain comprising the sequence of SEQ ID NO:78.

[0758] Embodiment 100. The recombinant protein of any one of embodiments 82-90, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO: 13, a first CDR H2 as set forth in SEQ ID NO: 14 and a first CDR H3 as set forth in SEQ ID NO: 15; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:49, a first CDR L2 as set forth in SEQ ID NO:50, and a first CDR L3 as set forth in SEQ ID NO:51

[0759] Embodiment 101. The recombinant protein of any one of embodiments 82-90 or 100, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:83 and a light chain variable domain comprising the sequence of SEQ ID NO:84.

[0760] Embodiment 102. The recombinant protein of any one of embodiments 82-90, wherein said first antigen binding region is an IL 1 RAP-anti gen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:28, a first CDR H2 as set forth in SEQ ID NO:29 and a first CDR H3 as set forth in SEQ ID NO:30; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:64, a first CDR L2 as set forth in SEQ ID NO:65, and a first CDR L3 as set forth in SEQ ID NO:66.

[0761] Embodiment 103. The recombinant protein of any one of embodiments 82-90 or 102, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:94 and a light chain variable domain comprising the sequence of SEQ ID NO:95

[0762] Embodiment 104. The recombinant protein of any one of embodiments 82-103, wherein said first antigen binding region further comprises a first Fc dimerizing domain; and wherein said second antigen binding region further comprises a second Fc dimerizing domain.

[0763] Embodiment 105. The recombinant protein of embodiment 104, wherein said first Fc dimerizing domain and said second Fc dimerizing domain form a Fc region thereby binding said first antigen binding region to said second antigen binding region.

[0764] Embodiment 106. The recombinant protein of embodiment 104 or 105, wherein said first Fc dimerizing domain comprises a lysine at a position corresponding to position 405; and wherein said second Fc dimerizing domain comprises an arginine at a position corresponding to position 409.

[0765] Embodiment 107. The recombinant protein of any one of embodiments 104-106, wherein said first Fc dimerizing domain and said second Fc dimerizing domain are non-active Fc dimerizing domains.

[0766] Embodiment 108. The recombinant protein of any one of embodiments 82-107, wherein said antibody is bound to an IL1RAP protein.

[0767] Embodiment 109. The recombinant protein of embodiment 108, wherein said IL1RAP protein is expressed on a cell. [0768] Embodiment 110. The recombinant protein of embodiment 109, wherein said cell is a cancer cell.

[0769] Embodiment 111. The recombinant protein of embodiment 110, wherein said cancer cell is an acute myeloid leukemia (AML) cell. [0770] Embodiment 112. An isolated nucleic acid encoding an antibody of any one of embodiments 19-80 or a recombinant protein of any one of embodiments 82-110.

[0771] Embodiment 113. A pharmaceutical composition comprising (i) a therapeutically effective amount of an antibody of any one of embodiments 19-80 and a pharmaceutically acceptable excipient or (ii) a therapeutically effective amount of a recombinant protein of any one of embodiments 82-110, and a pharmaceutically acceptable excipient.

[0772] Embodiment 114. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of (i) an antibody of any one of embodiments 19-80 or (ii) a recombinant protein of any one of embodiments 82-110, thereby treating cancer in said subject. [0773] Embodiment 115. The method of embodiment 114, wherein said cancer is acute myeloid leukemia (AML).

Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of an anti-interleukin- 1 receptor accessory protein (IL 1 RAP) antibody, wherein said antibody does not inhibit IL1- receptor signaling relative to a standard control, thereby treating cancer in said subject.

2. The method of claim 1, wherein said anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprises a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

3. The method of any one of claims 1-2, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:73.

4. The method of any one of claims 1-3, wherein said light chain variable domain comprises the sequence of SEQ ID NO:74.

5. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of a recombinant protein comprising:

(i) a first antibody region, wherein said first antibody binding region binds an effector cell ligand; and

(ii) a second antibody region, wherein said second antibody region binds an interleukin-1 receptor accessory protein (IL1RAP) and does not inhibit ILl-receptor signaling relative to a standard control, thereby treating cancer in said subject.

6. The method of claim 5, wherein said effector cell ligand is CD3, CD70 or CD123.

7. The method of any one of claims 5-6, wherein said first antibody region comprises a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ

ID NO: 244; and

(b) a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:245, a first CDR L2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO 247.

8. The method of any one of claims 5-7, wherein said first antibody region comprises the sequence of SEQ ID NO:251.

9. The method of any one of claims 5-8, wherein said second antibody region comprises (a) a second heavy chain variable domain comprising a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and

(b) a second light chain variable domain comprising a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

10. The method of claim 9, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:73 and said second light chain variable domain comprises the sequence of SEQ ID NO: 74.

11. The method of any one of claims 1-10, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit IL 1 -receptor signaling relative to a standard control.

12. The method of any one of claims 1-10, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit IL 1 -receptor alpha signaling relative to a standard control.

13. The method of any one of claims 1-10, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit IL 1 -receptor beta signaling relative to a standard control.

14. The method of any one of claims 1-10, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control.

15. The method of any one of claims 1-10, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

16. The method any one of claims 1-15, wherein said cancer is acute myeloid leukemia (AML).

17. The method any one of claims 1-16, wherein said anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody or said second antibody region does not block signaling of one or more co-receptors of IL1RAP.

18. The method of claim 17, wherein said one or more co-receptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1RL2.

19. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 1, a CDR H2 as set forth in SEQ ID NO:2 and a CDR H3 as set forth in SEQ ID NO:3; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 37, a CDR L2 as set forth in SEQ ID NO:38, and a CDR L3 as set forth in SEQ ID NO:39.

20. The antibody of claim 19, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:73.

21. The antibody of claim 19 or 20, wherein said light chain variable domain comprises the sequence of SEQ ID NO:74.

22. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:4, a CDR H2 as set forth in SEQ ID NO:5 and a CDR H3 as set forth in SEQ ID NO:6; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:40, a CDR L2 as set forth in SEQ ID NO:41, and a CDR L3 as set forth in SEQ ID NO:42.

23. The antibody of claim 22, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:77.

24. The antibody of claim 22 or 23, wherein said light chain variable domain comprises the sequence of SEQ ID NO:78.

25. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:7, a CDR H2 as set forth in SEQ ID NO:8 and a CDR H3 as set forth in SEQ ID NO:9; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:43, a CDR L2 as set forth in SEQ ID NO:44, and a CDR L3 as set forth in SEQ ID NO:45.

26. The antibody of claim 25, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:79.

27. The antibody of claim 25 or 26, wherein said light chain variable domain comprises the sequence of SEQ ID NO:80.

28. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 10, a CDR H2 as set forth in SEQ ID

NO: 11 and a CDR H3 as set forth in SEQ ID NO: 12; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:46, a CDR L2 as set forth in SEQ ID NO:47, and a CDR L3 as set forth in SEQ ID NO:48.

29. The antibody of claim 28, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:81.

30. The antibody of claim 28 or 29, wherein said light chain variable domain comprises the sequence of SEQ ID NO:82.

31. An anti-interleukin-1 receptor accessory protein (IL 1 RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 13, a CDR H2 as set forth in SEQ ID

NO: 14 and a CDR H3 as set forth in SEQ ID NO: 15; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:49, a CDR L2 as set forth in SEQ ID NO:50, and a CDR L3 as set forth in SEQ ID NO:51.

32. The antibody of claim 31, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:83.

33. The antibody of claim 31 or 32, wherein said light chain variable domain comprises the sequence of SEQ ID NO:84.

34. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 16, a CDR H2 as set forth in SEQ ID NO:17 and a CDR H3 as set forth in SEQ ID NO:18; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 52, a CDR L2 as set forth in SEQ ID NO:53, and a CDR L3 as set forth in SEQ ID NO:54.

35. The antibody of claim 34, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:85.

36. The antibody of claim 34 or 35, wherein said light chain variable domain comprises the sequence of SEQ ID NO:86.

37. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO: 19, a CDR H2 as set forth in SEQ ID NO:20 and a CDR H3 as set forth in SEQ ID NO:21; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:55, a CDR L2 as set forth in SEQ ID NO:56, and a CDR L3 as set forth in SEQ ID NO:57.

38. The antibody of claim 37, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:87.

39. The antibody of claim 37 or 38, wherein said light chain variable domain comprises the sequence of SEQ ID NO:88.

40. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:22, a CDR H2 as set forth in SEQ ID NO:23 and a CDR H3 as set forth in SEQ ID NO:24; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:58, a CDR L2 as set forth in SEQ ID NO:59, and a CDR L3 as set forth in SEQ ID NO:60.

41. The antibody of claim 40, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:89.

42. The antibody of claim 40 or 41, wherein said light chain variable domain comprises the sequence of SEQ ID NO:90.

43. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:25, a CDR H2 as set forth in SEQ ID NO:26 and a CDR H3 as set forth in SEQ ID NO:27; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:61, a CDR L2 as set forth in SEQ ID NO:62, and a CDR L3 as set forth in SEQ ID NO:63.

44. The antibody of claim 43, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:91 .

45. The antibody of claim 43 or 44, wherein said light chain variable domain comprises the sequence of SEQ ID NO:92.

46. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:28, a CDR H2 as set forth in SEQ ID NO:29 and a CDR H3 as set forth in SEQ ID NO:30; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 64, a CDR L2 as set forth in SEQ ID NO:65, and a CDR L3 as set forth in SEQ ID NO:66.

47. The antibody of claim 46, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:93.

48. The antibody of claim 46 or 47, wherein said light chain variable domain comprises the sequence of SEQ ID NO:94.

49. An anti-interleukin- 1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:31, a CDR H2 as set forth in SEQ ID

NO:32 and a CDR H3 as set forth in SEQ ID NO:33; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO: 67, a CDR L2 as set forth in SEQ ID NO:68, and a CDR L3 as set forth in SEQ ID NO:69.

50. The antibody of claim 49, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:95.

51 . The antibody of claim 49 or 50, wherein said light chain variable domain comprises the sequence of SEQ ID NO:96.

52. An anti-interleukin-1 receptor accessory protein (IL1RAP) antibody comprising a heavy chain variable domain and a light chain variable domain, wherein said heavy chain variable domain comprises: a CDR Hl as set forth in SEQ ID NO:34, a CDR H2 as set forth in SEQ ID

NO:35 and a CDR H3 as set forth in SEQ ID NO:36; and wherein said light chain variable domain comprises: a CDR LI as set forth in SEQ ID NO:70, a CDR L2 as set forth in SEQ ID NO:71, and a CDR L3 as set forth in SEQ ID NO:72.

53. The antibody of claim 52, wherein said heavy chain variable domain comprises the sequence of SEQ ID NO:97.

54. The antibody of claim 52 or 53, wherein said light chain variable domain comprises the sequence of SEQ ID NO:98.

55. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL 1 RAP) antibody does not inhibit IL 1 -receptor signaling relative to a standard control.

56. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor alpha signaling relative to a standard control.

57. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit ILl-receptor beta signaling relative to a standard control.

58. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an ILl-beta protein to its cognate receptor relative to a standard control.

59. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not inhibit binding of an ILl-alpha protein to its cognate receptor relative to a standard control.

60. The antibody of any one of claims 19-54, wherein said anti-interleukin-1 receptor accessory protein (IL1RAP) antibody does not block signaling of one or more coreceptors of IL 1 RAP.

61. The antibody of claim 60, wherein said one or more co-receptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1RL2.

62. The antibody of any one of claims 19-61, wherein said antibody binds IL1RAP with a binding affinity (KD) of about 1 nM to about 440 nM.

63. The antibody of any one of claims 19-62, wherein said antibody has a binding affinity (KD) of about 2.2 nM, about 2.5 nM, about 3.4 nM, about 74 nM or about 437 nM.

64. The antibody of any one of claims 19-63, wherein said antibody is a chimeric antibody, a ‘Fab fragment or a single chain antibody (scFv).

65. The antibody of claim 19, wherein said antibody is a humanized antibody.

66. The antibody of claim 65, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75, SEQ ID NO: 100 or SEQ ID NO: 102; and a light chain variable domain comprising the sequence of SEQ ID NO:76, SEQ ID NO: 101 or SEQ ID NO: 103.

67. The antibody of claim 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75; and a light chain variable domain comprising the sequence of SEQ ID NO:76.

68. The antibody of claim 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 100; and a light chain variable domain comprising the sequence of SEQ ID NO: 101.

69. The antibody of claim 65 or 66, wherein said humanized antibody comprises a heavy chain variable domain comprising the sequence of SEQ ID NO: 102; and a light chain variable domain comprising the sequence of SEQ ID NO: 103.

70. The antibody of any one of claims 65-69, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.0 nM to about 6 nM.

71. The antibody of any one of claims 65-70, wherein said humanized antibody binds IL1RAP with a binding affinity (KD) of about 1.06 nM, about 1.49 nM or 5.36 nM.

72. The antibody of any one of claims 19-71, wherein said antibody binds to human IL1RAP or cynomolgus IL1 RAP

73. The antibody of any one of claims 19-21, wherein said antibody comprises a heavy chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO:73; a glutamic acid at a position corresponding to position 99 of SEQ ID NO:73; or a glutamic acid at a positon corresponding to position 101 of SEQ ID NO: 73.

74. The antibody of claim 73, wherein said antibody comprises a light chain variable domain comprising a serine at a position corresponding to position 30 of SEQ ID NO:74.

75. The antibody of any one of claims 73-74, wherein said antibody does not bind IL1RAP at a detectable level.

76. The antibody of any one of claims 19-74, further comprising a central cavity formed by a heavy chain variable (VH) region, a light chain variable (VL) region, a heavy chain constant region (CH) and a light chain constant region (CL), wherein said central cavity forms a peptide binding site comprising framework region amino acid residues.

77. The antibody of claim 76, wherein a compound comprising a peptidyl moiety is bound to said peptide binding site.

78. The antibody of any one of claims 19-77, wherein said antibody is bound to an IL 1 RAP protein.

79. The antibody of claim 78, wherein said IL1RAP protein is expressed on a cell.

80. The antibody of claim 79, wherein said cell is a cancer cell.

81. The antibody of claim 80, wherein said cancer cell is an acute myeloid leukemia (AML) cell.

82. A recombinant protein comprising: (i) a first antibody region; and

(ii) a second antibody region, comprising:

(a) a second heavy chain variable domain comprising a second CDR Hl as set forth in SEQ ID NO: 1, a second CDR H2 as set forth in SEQ ID NO:2 and a second CDR H3 as set forth in SEQ ID NO:3; and

(b) a second light chain variable domain comprising a second CDR LI as set forth in SEQ ID NO:37, a second CDR L2 as set forth in SEQ ID NO:38, and a second CDR L3 as set forth in SEQ ID NO:39.

83. The recombinant protein of claim 82, wherein said first antibody region binds a first effector cell ligand.

84. The recombinant protein of claim 82 or 83, wherein said effector cell ligand is CD3, CD70 or CD123.

85. The recombinant protein of claim 82-84, wherein said first antibody region comprises a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:242, a first CDR H2 as set forth in SEQ ID NO:243 and a first CDR H3 as set forth in SEQ ID NO:244; and

(b) a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:245, a first CDRL2 as set forth in SEQ ID NO:246, and a first CDR L3 as set forth in SEQ ID NO:247.

86. The recombinant protein of claim 85, wherein said first antibody region comprises the sequence of SEQ ID NO:251.

87. The recombinant protein of any one of claims 82-86, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:73 and said second light chain variable domain comprises the sequence of SEQ ID NO: 74.

88. The recombinant protein of any one of claims 82-87, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 6.03 nM.

89. The recombinant protein of any one of claims 82-86, wherein said second heavy chain variable domain comprises the sequence of SEQ ID NO:75 and said second light chain variable domain comprises the sequence of SEQ ID NO:76.

90. The recombinant protein of any one of claims 82-86 or 89, wherein said second antibody region binds IL1RAP with a binding affinity (KD) of about 7.16 nM.

91. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not inhibit ILl-receptor signaling relative to a standard control.

92. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not inhibit ILl-receptor alpha signaling relative to a standard control.

93. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not inhibit ILl-receptor beta signaling relative to a standard control.

94. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not inhibit binding of an IL 1 -beta protein to its cognate receptor relative to a standard control.

95. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not inhibit binding of an IL 1 -alpha protein to its cognate receptor relative to a standard control.

96. The recombinant protein of any one of claims 82-90, wherein said second antibody region does not block signaling of one or more co-receptors of IL1RAP.

97. The recombinant protein of claim 96, wherein said second antibody region wherein said one or more co-receptors of IL1RAP comprise IL1R1, ST2, C-KIT or IL1RL2.

98. The recombinant protein of any one of claims 82-90, wherein said first antigen binding region is an ILlRAP-antigen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:4, a first CDR H2 as set forth in SEQ ID NO:5 and a first CDR H3 as set forth in SEQ ID NO:6; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:40, a first CDR L2 as set forth in SEQ ID NO:42, and a first CDR L3 as set forth in SEQ ID NO:43.

99. The recombinant protein of any one of claims 82-98, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:77 and a light chain variable domain comprising the sequence of SEQ ID NO:78.

100. The recombinant protein of any one of claims 82-90, wherein said first antigen binding region is an ILlRAP-antigen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO: 13, a first CDR H2 as set forth in SEQ ID NO: 14 and a first CDR H3 as set forth in SEQ ID NO: 15; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:49, a first CDR L2 as set forth in SEQ ID NO:50, and a first CDR L3 as set forth in SEQ ID NO:51.

101. The recombinant protein of any one of claims 82-90 or 100, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:83 and a light chain variable domain comprising the sequence of SEQ ID NO:84.

102. The recombinant protein of any one of claims 82-90, wherein said first antigen binding region is an ILlRAP-antigen binding region comprising a first heavy chain variable domain comprising a first CDR Hl as set forth in SEQ ID NO:28, a first CDR H2 as set forth in SEQ ID NO: 29 and a first CDR H3 as set forth in SEQ ID NO: 30; and a first light chain variable domain comprising a first CDR LI as set forth in SEQ ID NO:64, a first CDR L2 as set forth in SEQ ID NO:65, and a first CDR L3 as set forth in SEQ ID NO:66.

103. The recombinant protein of any one of claims 82-90 or 102, wherein said first antigen binding region comprises a first heavy chain variable domain comprising the sequence of SEQ ID NO:94 and a light chain variable domain comprising the sequence of SEQ ID NO:95.

104. The recombinant protein of any one of claims 82-103, wherein said first antigen binding region further comprises a first Fc dimerizing domain; and wherein said second antigen binding region further comprises a second Fc dimerizing domain.

105. The recombinant protein of claim 104, wherein said first Fc dimerizing domain and said second Fc dimerizing domain form a Fc region thereby binding said first antigen binding region to said second antigen binding region.

106. The recombinant protein of claim 104 or 105, wherein said first Fc dimerizing domain comprises a lysine at a position corresponding to position 405; and wherein said second Fc dimerizing domain comprises an arginine at a position corresponding to position 409.

107. The recombinant protein of any one of claims 104-106, wherein said first Fc dimerizing domain and said second Fc dimerizing domain are non-active Fc dimerizing domains.

108. The recombinant protein of any one of claims 82-107, wherein said antibody is bound to an IL1RAP protein.

109. The recombinant protein of claim 108, wherein said IL1RAP protein is expressed on a cell.

110. The recombinant protein of claim 109, wherein said cell is a cancer cell.

111. The recombinant protein of claim 110, wherein said cancer cell is an acute myeloid leukemia (AML) cell.

112. An isolated nucleic acid encoding an antibody of any one of claims 19-80 or a recombinant protein of any one of claims 82-110.

113. A pharmaceutical composition comprising (i) a therapeutically effective amount of an antibody of any one of claims 19-80 and a pharmaceutically acceptable excipient or (ii) a therapeutically effective amount of a recombinant protein of any one of claims 82-110, and a pharmaceutically acceptable excipient.

114. A method of treating cancer in a subject in need thereof, said method comprising administering to a subject a therapeutically effective amount of (i) an antibody of any one of claims 19-80 or (ii) a recombinant protein of any one of claims 82-1 10, thereby treating cancer in said subject.

115. The method of claim 114, wherein said cancer is acute myeloid leukemia (AML).