Chimeric antigen receptor targeting interleukin- 13 receptor subunit alpha-2 and optionally b7h3 for treating glioblastoma

Abstract

Chimeric antigen receptors (CARs) specific to interleukin-13 receptor subunit alpha-2 (IL-13Rα2) and bispecific CARs specific to IL-13Rα2 and B7H3, as well as genetically engineered immune cells expressing such, optionally in combination with an armor polypeptide for enhancing CAR-T cell activity. Also provided herein are methods for treating tumors such as glioblastoma using the genetically engineered immune cells.

Description

[0001] CHIMERIC ANTIGEN RECEPTOR TARGETING INTERLEUKIN- 13 RECEPTOR SUBUNIT ALPHA-2 AND OPTIONALLY B7H3 FOR TREATING GLIOBLASTOMA

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 688,401, filed August 29, 2024, the entire contents of which is incorporated by reference herein.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 27, 2025, is named 058392-509001 WO_Seq-Listing_ST26.xml and is 193,627 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Glioblastoma (GBM), also known as grade IV astrocytoma, is the most common malignant brain tumor accounting for about 47.7% of all central nervous tumors. Predominantly made up of abnormal astrocytic cells, GBM is a fast growing and aggressive tumor invading the nearby of brain. Survival of GBM is poor with approximately 40% survival in the first year post diagnosis and 17% in the second year.

[0008] GBM presents unique treatment challenges due to various reasons, including brain localization, inherent resistant to conventional therapy, limited repair capacity of the brain, high migration capacity of malignant cells, and poor drug delivery to tumor sites. It is of great importance to develop new efficient treatment for GBM.

[0009] SUMMARY OF THE INVENTION

[0010] The present disclosure is based, at least in part, on the development of monospecific chimeric antigen receptor (CAR) constructs targeting interleukin- 13 receptor subunit alpha- 2

[0011] (IL-13Rα 2), monospecific chimeric antigen receptor (CAR) constructs targeting B7H3, and bispecific CAR constructs targeting both IL- IL-13Rα 2 and B7H3, which exhibited high binding affinity to the target antigen(s). Immune cells engineered to express such monospecific or bispecific CAR constructs showed high cytotoxicity against brain tumor cells expressing IL-

[0012] 13Rα 2 and B7H3. Further, CAR-T cells expressing the CARs presented herein, specifically the bispecific CARs, successfully reduced tumor cell growth in an animal model for GBM. Co- expressing an engineered multi-mechanism armor polypeptide in such CAR-T cells enhanced CAR-T cell persistence and anti-tumor efficiency. As such, immune cells expressing the monospecific and bispecific CAR constructs provided herein are expected to benefit GBM treatment.

[0013] Accordingly, the present disclosure features, in some aspects, a chimeric antigen receptor (CAR) polypeptide, comprising: (a) an extracellular antigen binding domain, which comprises a first antigen-binding fragment specific to interleukin- 13 receptor subunit alpha-2

[0014] (IL-13Rα2); (b) a co-stimulatory signaling domain; and (c) a cytoplasmic signaling domain. In some instances, the first antigen binding fragment is a heavy chain antibody, which comprises the same heavy chain complementarity determining regions (CDRs) as a reference antibody of 13R-03, 13R-01, 13R-02, 13R-05, or 13R-04 set forth in Table 2. Alternatively, the first antigen binding fragment is a single chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as a reference antibody of 13R-06, 13R-07, 13R- 10, 13R-08, 13R-09, 13R-10, 13R-ll, or 13R-13 set forth in Table 2.

[0015] In some examples, the first antigen binding fragment is an scFv comprising the VH and the VL, which comprises the same heavy chain and light chain CDRs respectively as the reference antibody of 13R-06. In other examples, the first antigen binding fragment is an scFv comprising the VH and the VL, which comprises the same heavy chain and light chain CDRs respectively as the reference antibody of or 13R-07.

[0016] In some examples, the first antigen binding fragment is the heavy chain antibody, which comprises the amino acid sequence of any one of SEQ ID NOs: 64, 66, 68, 72, and 76.

[0017] In other examples, the first antigen binding fragment is the scFv, which comprises:

[0018] (a) a VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL comprising the amino acid sequence of SEQ ID NO: 83;

[0019] (b) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 90;

[0020] (c) a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 99;

[0021] (d) a VH comprising the amino acid sequence of SEQ ID NO: 102 and a VL comprising the amino acid sequence of SEQ ID NO: 106;

[0022] (e) a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 112; (f) a VH comprising the amino acid sequence of SEQ ID NO: 114 and a VL comprising the amino acid sequence of SEQ ID NO: 118;

[0023] (g) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 123 or

[0024] (h) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 128.

[0025] Any of the anti-IL- 13Rα 2 scFvs provided herein may comprise the amino acid sequence of any one of SEQ ID NOs: 84, 91, 100, 107, 113, 119, 124, and 129. optionally wherein the scFv comprises the amino acid sequence of SEQ ID NO: 84.

[0026] In specific examples, the anti-IL- 13Rα 2 scFv may comprise a VH comprising SEQ ID

[0027] NO: 80 and a VL of SEQ ID NO: 83. Such an anti-IL-13Rα 2 scFv may comprise the amino acid sequence of SEQ ID NO: 84. In other specific examples, the anti-IL- 13Rα 2 may comprise a VH comprising SEQ ID NO: 86 and a VL of SEQ ID NO: 90. Such an anti-IL- 13Rα 2 scFv may comprise the amino acid sequence of SEQ ID NO: 91.

[0028] The extracellular antigen binding domain (a) of any of the CAR polypeptides provided herein may further comprise a second antigen-binding fragment specific to B7 homolog 3 protein (B7H3). In some instances, the second antigen-binding fragment is a heavy chain antibody or an scFv derived from a reference antibody set forth in Table 1 (e.g., derived from BH-07). In some examples, the second antigen-binding fragment is the scFv, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as reference antibody BH-07 set forth in Table 1. In some instances, the scFv may comprise the VH comprising the amino acid sequence of SEQ ID NO: 40 and the VL comprising the amino acid sequence of SEQ ID NO: 44. In some specific examples, the anti-B7H3 scFv may comprise the amino acid sequence of SEQ ID NO: 45.

[0029] In any of the CAR polypeptides disclosed herein, the co-stimulatory domain of (b) can be from a co-stimulatory molecule, for example, from CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD40L. In some examples, the co-stimulatory domain is from 4-1BB. Alternatively or in addition, the cytoplasmic signaling domain of (c) in the CAR polypeptide may be from CD3ζ.

[0030] In some embodiments, the CAR polypeptide may further comprise a hinge domain, a transmembrane domain, or a combination thereof; wherein the hinge domain and / or the transmembrane domain optionally is located between the extracellular antigen binding moiety of (a) and the co-stimulatory domain of (b). In some instances, the CAR polypeptide may further comprise a spacer located between the hinge domain and the transmembrane domain. Exemplary spacers may comprise the amino acid sequence of SEQ ID NO: 136 or SEQ ID NO: 137. Alternatively or in addition, the CAR polypeptide may further comprise a signal peptide located at the N-terminus of the polypeptide.

[0031] In some embodiments, the CAR polypeptide provided herein is a mono-specific CAR that binds IL-13Rα 2. In one example, the mono-specific anti-IL-13Rα 2 may comprise the amino acid sequence of SEQ ID NO: 147 or 148. In another example, the mono-specific anti-

[0032] IL-13Rα 2 may comprise the amino acid sequence of SEQ ID NO: 149 or SEQ ID NO: 150.

[0033] In other embodiments, the CAR polypeptide provided herein is a biparatopic CAR that comprises two binding moieties to B7H3. In some examples, the anti-B7H3 biparatopic CAR may comprise the amino acid sequence of SEQ ID NO: 165 or 166. In other examples, the bispecific CAR may comprise the amino acid sequence of SEQ ID NO: 167 or 168. In yet other examples, anti-B7H3 biparatopic may comprise the amino acid sequence of SEQ ID NO: 169 or 170. In still other examples, the anti-B7H3 biparatopic may comprise the amino acid sequence of SEQ ID NO: 171, or 172.

[0034] In other embodiments, the CAR polypeptides provided herein are bispecific CARs capable of binding to both B7H3 and IL-13Rα 2 (anti-B7H3 / IL-13Rα 2 bispecific CARs). In some examples, the anti-B7H3 / IL-13Rα 2 bispecific CAR may comprise the amino acid sequence of SEQ ID NO: 173 or 174. In another example, the anti-B7H3Z IL-13Rα 2 bispecific CAR may comprise the amino acid sequence of SEQ ID NO: 175 or 176.

[0035] In other aspects, the present disclosure features a nucleic acid or a set of nucleic acids, comprising a first nucleotide sequence encoding any of the CAR polypeptides disclosed herein. In some embodiments, the nucleic acid or the set of nucleic acids may further comprise a second nucleotide sequence encoding an armor polypeptide, which enhances T cell functionality, and optionally a signal peptide located at the N-terminus of the armor polypeptide. In some instances, the armor polypeptide may be IL-2, IL-5, IL-15, a costimulatory ligand, and / or an anti-PDL1 antibody.

[0036] In some instances, the armor polypeptide can be a fusion polypeptide comprising an anti-PDL1 antibody, which optionally can be a single chain variable fragment (scFv). In some examples, the armor polypeptide is a fusion polypeptide comprising an anti-PDL1 antibody and an IL- 2 polypeptide. For example, the anti-PDL1 antibody may an scFv fragment, which may comprise the amino acid sequence of SEQ ID NO: 177. In one specific example, the armor polypeptide comprises the amino acid sequence of SEQ ID NO: 179.

[0037] In some examples, the first nucleotide sequence encoding the CAR polypeptide and the second nucleotide sequence encoding the armor polypeptide can be located on two separate nucleic acids. Alternatively, the first nucleotide sequence and the second nucleotide sequence can be located on one nucleic acid. In some instances, such a nucleic acid may further comprise a third nucleotide sequence encoding a self-cleaving peptide, which is located between the first and second nucleotide sequences.

[0038] Any of the nucleic acids provided herein can be vectors (e.g., viral vectors). In some examples, the vectors are expression vectors.

[0039] In another aspect, the present disclosure features a population of immune cells, comprising genetically engineered immune cells expressing any of the CAR polypeptides provided herein. In some embodiments, the genetically engineered immune cells may further express an armor polypeptide, for example, any of the armor polypeptides disclosed herein (e.g., SEQ ID NO: 179). In some instances, the genetically engineered immune cells secrete the armor polypeptide. In some instances, the genetically engineered immune cells comprise the nucleic acid or the set of nucleic acids as disclosed herein.

[0040] In some embodiments, the genetically engineered immune cells comprise T cells, NK cells, macrophages, or a combination thereof. In some examples, the immune cells comprise T cells.

[0041] In addition, the present disclosure provides a method for eliminating IL-13Rα 2- expressing disease cells in a subject, the method comprising administering to a subject in need thereof an effective amount of the population of immune cells provided herein. In some embodiments, the subject is a human patient having both IL-13Rα 2 and B7H3 positive and optionally B7H3 or IL13Rα 2 positive disease cells. In some embodiments, the subject is a human patient having a cancer, for example, glioblastoma, neuroblastoma, lung cancer, melanoma, head and neck cancer, urothelial cancer, prostate cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, esophageal cancer, ovarian cancer, breast cancer, bladder cancer, or acute myeloid leukemia (AML), etc.. In some embodiments, the immune cells (autologous or allogenic) are administered to the subject by intracavitary delivery, intravenous infusion, or a combination thereof.

[0042] Further, provided herein is a method for treating glioblastoma in a subject, the method comprising administering to a subject in need thereof an effective amount of the population of immune cells disclosed herein. In some instances, the population of immune cells is autologous to the subject. Alternatively, the population of immune cells is allogenic to the subject.

[0043] Also within the scope of the present disclosure are any of the populations of immune cells disclosed herein for use in eliminating IL-13Rα 2-expressing disease cells in a subject or for treating a cancer associated with IL-13Rα 2 and optionally B7H3, such as glioblastoma, as well as uses of the populations of immune cells disclosed herein for manufacturing a medicament for the intended medical applications.

[0044] In yet other aspects, provided herein is a method for producing genetically engineered immune cells, the method comprising: transfecting into a population of immune cells the nucleic acid or the set of nucleic acids set as disclosed herein to produce genetically engineered immune cells expressing the CAR polypeptide and optionally the armor polypeptide encoded by the nucleic acid(s).

[0045] In addition, the present disclosure provides an antibody that binds interleukin- 13 receptor subunit alpha- 2 (IL-13Rα 2) (anti-IL-13Rα 2 antibody), as well as nucleic acid(s) encoding the antibodies. The anti-IL-13Rα 2 antibody may comprise: (a) a human heavy chain antibody comprising the same heavy chain complementary determining regions (CDRs) as a reference antibody of 13R-03, 13R-01, 13R-02, 13R-05, or 13R-04 set forth in Table 2; or (b) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as a reference antibody of 13R-06, 13R-07, 13R-10, 13R-08, 13R-09, 13R-10, 13R-l l, or 13R-13 set forth in Table 2.

[0046] In some embodiments, the anti-IL-13R«2 antibody comprises the human heavy chain antibody of (a), which optionally comprises the amino acid sequence of SEQ ID NO: 64, 66, 68, 72, or 76.

[0047] In other embodiments, the anti-IL-13Rα 2 antibody comprises the VH and VL of (b) as set forth below:

[0048] (a) a VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL comprising the amino acid sequence of SEQ ID NO: 83:

[0049] (b) a Vn comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 90;

[0050] (c) a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 99;

[0051] (d) a VH comprising the amino acid sequence of SEQ ID NO: 102 and a VL comprising the amino acid sequence of SEQ ID NO: 106;

[0052] (e) a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 112;

[0053] (f) a VH comprising the amino acid sequence of SEQ ID NO: 114 and a VL comprising the amino acid sequence of SEQ ID NO: 118;

[0054] (g) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 123; or

[0055] (h) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 128.

[0056] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0059] FIGs. 1A-1B are graphs showing the supernatant binding activity screening of anti- IL13Rα 2 clones to recombinant full-length IL13Rα 2 by ELISA. FIG. 1A: binding activity to IL13Rα 2 VH binding domain. FIG. IB: binding activity to IL13Rα 2 scFv binding domain.

[0060] FIGs. 2A-2B are graphs showing the binding activity of exemplary anti-IL13Rα 2 scFv clones to full-length recombinant IL13Rα 2 receptors by ELISA and IL13Rα 2 on cell surface by FACS analysis. FIG. 1A: binding activity of anti-IL13Rα 2 scFv clones to recombinant

[0061] IL13Rα 2. FIG. IB: binding activity to anti-IL13Rα 2 scFv clones on IL13Rα 2 / HEK293 cells.

[0062] FIG. 3 is a schematic showing exemplary anti-IL13Rα 2 chimeric antigen receptor constructs and their co-expression with either EGFRt or an armor polypeptide.

[0063] FIGs. 4A-4D are bioactivities of CAR-T cells expressing anti-IL13Rα 2 scFv CAR.

[0064] FIG. 4A: proliferation of CAR-T cells expressing EPLV167 upon target cell engagement.

[0065] FIG. 4B: proliferation of CAR-T cells expressing EPLV168 upon target cell engagement. FIG.

[0066] 4C: IFNγ secretion of CAR-T cells expressing EPLV167 upon target cell engagement. FIG.

[0067] 4D: IFNγ secretion of CAR-T cells expressing EPLV168 upon target cell engagement.

[0068] FIGs. 5A-5F are graphs showing bioactivities of CAR-T cells expressing EPLV167.

[0069] FIG. 5A: percentage of cell lysis on Donor 271. FIG. 5B: IFNγ secretion of Donor 271. FIG. 5C: percentage of cell lysis on Donor 375. FIG. 5D: IFNγ secretion of Donor 375. FIG. 5E: percentage of cell lysis on Donor 717. FIG. 5F: IFNγ secretion of Donor 717.

[0070] FIGs. 6A-6F are graphs showing bioactivities of CAR-T cells expressing anti-B7H3 scFv CAR EPLV195. FIG. 6A: percentage of cell lysis on Donor 988. FIG. 6B: IFNγ secretion of Donor 988. FIG. 6C: percentage of cell lysis on Donor 717. FIG. 6D: IFNγ secretion of Donor 717. FIG. 6E: percentage of cell lysis on Donor 104. FIG. 6F: IFNy secretion of Donor 104.

[0071] FIGs. 7A-7C are graphs showing percentage of cell lysis of target cells by IL13Ra2 and B7H3 monospecific, IL13Ra2-B7H3 bispecific and IL13Ra2-B7H3 bispecific armored CAR-T cells at 116 hours. FIG. 7A: percentage of cell lysis of target cells by D925 (donor) CAR-T cells at 116 hours at E:T=5.1. FIG. 7B: percentage of cell lysis of target cells by D925 CAR-T cells at 116 hoursat E:T=2.5.1. FIG. 7C: percentage of cell lysis of target cells by D925 CAR-T cells at 116 hours at E:T=1.1.

[0072] FIG. 8 is a graph showing cytotoxicity of CAR-T cells expressing anti-B7H3 CAR (EPLV326), anti-IL13Rα 2 CAR (EPLV167), bispecific CAR (EPLV306), or bispecific armored CAR (EPLV329) in a cancer cell killing and rechallenging assay

[0073] FIG. 9 is a graph showing cytotoxicity of CAR-T cells expressing bispecific CAR (EPLV306) or bispecific armored CAR (EPLV329) on cancer cell lines expressing B7H3 and do not expressing IL13Ra2.

[0074] FIG. 10 is a graph showing the binding activity of the armor from the supernatant collected during CAR-T expansion and from CAR-T cells expressing CAR (EPLV326) or expressing the CAR with an armor polypeptide of SEQ ID NO: 179.

[0075] FIG. 11 is a graph showing bioinformatic analysis on co-expression of B7H3 and IL13Rα 2 in patients.

[0076] FIG. 12 is a graph showing cytotoxicity of CAR-T cells expressing B7H3 VH / IL13Rα 2 VH to GBM cell lines.

[0077] FIGs. 13A-13B is a graph showing anti-tumor activity and safety of anti- IL13Ra2 / B7H3 bispecific armored CAR-T cells in U87MG (Glioblastoma cells) orthotopic model in NCG mice, via intracranial and intravenous CAR-T administration. FIG. 13A: tumor volume. FIG. 13B: body weight throughout the treatment.

[0078] FIGs 14A-14C are graphs that show expansion of CAR-T cells at day 35 post treatment. FIG. 14A: total CAR-T cells in treatment group. FIG. 14B: total CD8+ CAR-T cells in treatment group. FIG. 14C: total CD4+ CAR-T cells in treatment group. FIGs 15A-15B are graphs that show immunophenotyping of CAR-T cells at day 35 post treatment. FIG. 15A: CD8+ CAR-T cells in treatment group. FIG. 15B: CD4+ CAR-T cells in treatment group.

[0079] FIGs 16A-16C are graphs that show the analysis of PBMC T cell subpopulation at day 35 post treatment. FIG. 16A: Human total CD3+ T cells in treatment group. FIG. 16B: Human total CD8+ T cells in treatment group. FIG. 16C: Human total CD4+ T cells in treatment group.

[0080] FIGs 17A-17B are graphs that show immunophenotyping of PBMC T cells at day 35 post treatment. FIG. 17A: CDS T cells in treatment group. FIG. 17B: CD4 T cells in treatment group.

[0081] DETAILED DESCRIPTION OF THE INVENTION

[0082] Interleukin- 13 receptor subunit alpha- 2 (IL-13Rα 2), a.k.a. , CD213A2, is a membranebound protein similar to IL-13Ral, a subunit of the IL- 13 receptor complex. IL-13Rα 2 binds

[0083] IL- 13 with high affinity. While it does not function as a signal mediator due to lack of cytoplasmic domains, IL-13Rα 2 regulates the effects of both IL- 13 and IL-4. IL-13Rα 2 has been found to be over-expressed in various cancers, including pancreatic cancer, ovarian cancer, melanoma, and malignant gliomas. It was reported that expression of IL-13Rα 2 and IL-

[0084] 13Rα 2-mediated signaling has been shown to promote tumor proliferation, cell survival, tumor progression, invasion, and metastasis. As such, IL-13Rα 2 becomes an attractive target for cancer immunotherapy. Knudson et al., Front Immunol. 2022 13:878365. Anti-IL-13Rα 2 CAR-T cells showed clinical efficacy; however, the patient received the treatment relapsed after 7+ months due to tumor metastasis. Target escape and poor CAR-T cell expansion were detected in the patient.

[0085] B7 homolog 3 (B7H3), a.k.a., CD276, is a member of the B7 family that plays an immunoregulatory role in T cell responses. It is a type I transmembrane glycoprotein with two isoforms: B7H3 VC and B7H3 VCVC, depending on the structure of its extracellular domain. B7H3 VC contains a single extracellular V- and C-like Ig domain pair, a transmembrane domain, and a cytoplasmic tail. B7H3 VCVC contains two identical pairs of the V- and C-like Ig domain in the extracellular domain, which is the major isoform found in humans (the full length B7H3). The B7H3 VC isoform is also found in human cells due to alternative splicing (a splicing variant). It has been reported that B7H3 plays an inhibitory role in T cell activation and proliferation, leading to tumor immune evasion and influence both the immune response and tumor behavior through different signaling pathways. B7H3 expression is aberrantly upregulated in many different cancer types, which was found to be associated with poor prognosis. Zhao et al., J. Hematology & Oncology, 15 (153): 1-31 (2022). Anti-B7H3 CAR-T cells showed transient clinical efficacy but tumor resistant occurred due to heterogeneity and target escape was detected. Nature: Signal Transduction and Targeted Therapy (2021) 6: 125.

[0086] Provided herein are monospecific CAR constructs targeting IL-13Rα 2 or B7H3, and bispecific CAR constructs targeting both IL-13Rα 2 and B7H3, as well as CAR-T cells expressing such monospecific or bispecific CAR, engineered with a secreted armor polypeptide as those disclosed herein. The CAR-T cells, for example, those expressing the bispecific CAR, engineered with the multi-mechanism armor polypeptide, exhibited high treatment efficacy against GBM in an animal model. Use of the bispecific CAR solves the target escape problem associated with targeting either B7H3 or IL-13Rtx2 in cancer immunotherapy (e.g., immunotherapy of GBM). Co-expression of the armor polypeptide enhances CAR-T cell persistence and activity, thereby improving their anti-cancer effects, e.g. , in solid tumor microenvironment. Accordingly, the monospecific and bispecific CAR constructs provided herein are promising approaches for treating cancers that involve B7H3 and / or IL-13Rα 2, such as GBM.

[0087] GBM is heterogenous with frequent metastasis and suppressive tumor microenvironment. IL13 ligand CAR and B7H3 monospecific CAR-T demonstrated promising transient anti-tumor activity. Our anti-IL13Ra2 and B7H3 bispecific CAR-T addresses tumor heterogeneity, prevent and treat target escape, with potential increases safety. Bispecific multimechanism armor, anti-PDLl fused to engineered IL2, overcomes multiple TME suppression mechanisms. Engineered IL2 suppresses Treg activation, enhances CAR-T and bystander immune cell activation and infiltration, stimulates central memory T cells to increase the persistence. Anti-PDLl blocks immune checkpoint and localizes to tumor.

[0088] I. Chimeric Antigen Receptors

[0089] As used herein, the term “chimeric antigen receptor” or “CAR” refers to an artificial immune cell receptor that is capable of binding to an antigen expressed by undesired cells, for example, an antigen of interest (here IL-13Rα 2 and optionally B7H3). Generally, a CAR may comprise a fusion polypeptide, which comprises an extracellular antigen binding domain (e.g., a single chain variable fragment or scFv derived from an antibody specific to the target antigen), a co-stimulatory domain, and an intracellular signaling domain. In some instances, the fusion polypeptide may further comprise a hinge and transmembrane domain located at the C-terminus of the extracellular antigen binding domain. In some embodiments, the CARs disclosed herein are T cell receptors. In other embodiments, the CARs disclosed herein may be NK cell receptors.

[0090] A. Extracellular Antigen-Binding Fragment

[0091] Any of the CAR constructs provided herein comprises an extracellular antigen-binding fragment that is specific to one or more antigens of interest ((here IL-13Rα 2 and optionally B7H3). The extracellular antigen-binding fragment comprises one or more (e.g., 2) antigenbinding moieties, which can be derived from an antibody specific to the target antigen.

[0092] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody”, e.g., anti-BCMA antibody or anti-B7H3 antibody, encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab')2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., nanobody), single domain antibodies (e.g., a VH only antibody), multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody, e.g., anti-Galectin-9 antibody, includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavychain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known.

[0093] A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as “framework regions” (“FR”). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework region and CDRs can be precisely identified using methodology known in the art, for example, by the Rabat definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art. See, e.g., Rabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol.

[0094] 273:927-948; and Almagro, J. Mol. Recognit. 17: 132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0095] In some embodiments, an antibody moiety disclosed herein may share the same heavy chain and / or light chain complementary determining regions (CDRs) or the same VH and / or VL chains as a reference antibody. Two antibodies having the same VH and / or VL CDRS means that their CDRs are identical when determined by the same approach (e.g., the Rabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf.org.uk / abs / ). Such anti-BCMA or anti-B7H3 antibodies may have the same VH, the same VL, or both as compared to an exemplary antibody described herein.

[0096] In some embodiments, an antibody moiety disclosed herein may share a certain level of sequence identity as compared with a reference sequence. The “percent identity” of two amino acid sequences is determined using the algorithm of Rarlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Rarlin and Altschul Proc. Natl. Acad. Sci. USA

[0097] 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used.

[0098] In some embodiments, an antibody moiety disclosed herein may have one or more amino acid variations relative to a reference antibody. The amino acid residue variations as disclosed in the present disclosure (e.g., in framework regions and / or in CDRs) can be conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0099] In some embodiments, the CAR constructs provided herein may comprise an extracellular antigen-binding fragment that comprise an antigen-binding moiety specific to IL-

[0100] 13Rα 2. Such a CAR construct may be monospecific, i.e., comprising one or more antigenbinding moieties specific to the same target antigen. In some examples, the CAR constructs provided herein may be bispecific, having at least two antigen-binding moieties in the extracellular antigen-binding fragment specific to two different target antigens, for example,

[0101] IL-13Rα 2 and B7H3. In some instances, the antigen-binding moiety can be a heavy chain only antibody fragment. Alternatively, the antigen-binding moiety can be a single chain variable fragment (scFv) antibody fragment.

[0102] (a) Anti-B7H3 Binding Moiety

[0103] In some embodiments, the anti-B7H3 antibody for use in constructing the anti-

[0104] B7H3 / IL-13Rα 2 bispecific CAR as disclosed herein (parent anti-B7H3 antibodies) may be a human heavy chain antibody, which may comprise only a heavy chain variable region. Exemplary human heavy chain antibodies include BH-01, BH-02, and BH-03 as disclosed herein. In one example, the human heavy chain antibody for use in constructing the anti-B7H3 CAR can be BH-02 or a derivative thereof (see above disclosures). The amino acid sequences of these exemplary human heavy chain antibodies, as well as their heavy chain complementarity determining regions (CDRs) as determined following the Rabat scheme, are provided in Table 1 below.

[0105] In some embodiments, the anti-B7H3 binding moiety in any of the anti-B7H3 / IL-

[0106] 13Rα 2 bispecific CARs disclosed herein may be in an scFv format, which is a fusion polypeptide comprising the heavy chain variable domain (VH) and the light chain variable domain (VL) of an anti-B7H3 antibody connected by a peptide linker. In the scFv fragment, the

[0107] VH and VL fragments may be in any orientation. In some instances, the scFv may comprise, from the N-terminus to the C-terminus, a VL fragment, a peptide linker, and a VH fragment.

[0108] Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, a VH fragment, a peptide linker, and a VL fragment. In some examples, a scFv may further comprise an N- terminal signal peptide for directing the CAR comprising the scFv to cell surface. Exemplary anti-B7H3 scFv antibody fragments include BH-04-BH-10 as disclosed herein. In one example, the scFv antibody fragment for use in constructing the anti-B7H3 CAR can be BH-07 or a derivative thereof (see above disclosures). The amino acid sequences of these exemplary scFv antibody fragments, as well as their heavy chain and light chain CDRs as determined following the Rabat scheme, are provided in Table 1 below. All of the listed anti-B7H3 antibodies, as well as their derivatives as disclosed herein, are within the scope of the present disclosure.

[0109] Table 1. Anti-B7H3 Antibodies

[0110] An anti-B7H3 binding moiety (e.g. , heavy chain antibody fragment or scFv fragment) derived from a reference antibody (e.g., those listed in Table 1) refers to binding moieties having substantially similar structural and functional features as the reference antibody. Structurally, the binding moiety may have the same heavy and / or light chain complementary determining regions or the same VH and / or VL chains as the reference antibody. Alternatively, the binding moiety may only have a limited number of amino acid variations in one or more of the framework regions and / or in one or more of the CDRs without significantly affecting its binding affinity and binding specificity relative to the reference antibody. See descriptions below.

[0111] In some examples, the anti-B7H3 binding moiety may comprise the same heavy chain CDRs as those in any of the reference antibodies provided in Table 1 above (e.g., BH-02 or BH-07). Where applicable (e.g., in the context of scFv antibody fragments), the anti-B7H3 binding moiety may have the same light chain CDRs as those in any of the reference antibodies provided in Table 1 above (e.g., BH-07). Such an anti-B7H3 binding moiety may comprise the same VH and / or VL chains as the reference antibody. Alternatively, the anti-B7H3 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in the reference antibody. For example, the anti-B7H3 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in the reference antibody.

[0112] In some embodiments, the anti-B7H3 moiety may comprise a certain level of variations in one or more of the CDRs relative to those in any of the reference antibodies provided in Table 1 above (e.g., BH-02 or BH-07). For example, the anti-B7H3 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of the reference antibody. Alternatively, or in addition, the anti-B7H3 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as the reference antibody. As used herein, “individually” means that one CDR of an antibody shares the indicated sequence identity relative to the corresponding CDR of a reference antibody (e.g. , those in Table 1 such as BH-07). “Collectively” means that three VH or VL CDRS of an antibody in combination share the indicated sequence identity relative the corresponding three VH or VL CDRS of the reference antibody in combination.

[0113] In some instances, the anti-B7H3 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of a reference antibody provided in Table 1 (e.g., BH-02 or BH-07). In some instances, the anti-B7H3 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of the reference antibody and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.

[0114] In some examples, the anti-B7H3 moiety disclosed herein may be any of the anti-B7H3 heavy chain antibody provided in Table 1 above. For example, the anti-B7H3 binding moiety may comprise the amino acid sequence of SEQ ID NO: 4, 8, 9, or 13. In specific examples, the anti-B7H3 binding moiety may comprise the amino acid sequence of SEQ ID NO: 8 or 9 (e.g., SEQ ID NO: 8). Alternatively, the anti-B7H3 binding moiety may comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 98%, or above) identical to any of the heavy chain antibody fragments provided in Table 1, e.g., SEQ ID NO: 4, 8, 9, or 13. Such an anti-B7H3 binding moiety may comprise variations in framework regions only as compared with the parent counterpart.

[0115] In some examples, the anti-B7H3 binding moiety disclosed herein may be any of the scFv fragments provided in Table 1 above. For example, the anti-B7H3 scFv fragments may comprise the amino acid sequence of SEQ ID NO: 20, 29, 36, 45, 50, 56, or 61, or comprise the same VH and VL fragments thereof. In specific examples, the anti-B7H3 scFv fragment may comprise the VH of SEQ ID NO: 40 and / or the VL of SEQ ID NO: 44. In one example, the anti-B7H3 scFv fragment may comprise the amino acid sequence of SEQ ID NO: 45. Alternatively, the anti-B7H3 moiety may comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 98%, or above) identical to any of the scFv fragments provided in Table 1, e.g., SEQ ID NO: 20, 29, 36, 45, 50, 56, or 61 (e.g., SEQ ID NO: 45). Such an anti-B7H3 binding moiety may comprise variations in framework regions only as compared with the parent counterpart. In other examples, the anti-B7H3 moiety disclosed herein may comprise the same VH and VL sequences as in those listed in Table 1, e.g., SEQ ID NO: 20, 29, 36, 45, 50, 56, or 61, but has a reversed orientation of the VH and VL fragments.

[0116] Any of the anti-B7H3 binding moieties disclosed herein (e.g., the heavy chain antibody fragment provided in Table 1 such as SEQ ID NO: 8 or 9, the scFv fragment provided in Table 1 such as SEQ ID NO: 45, or their counterparts having reversed VH and VL orientation, or derivatives thereof as also disclosed herein) may be used for constructing the bispecific anti-

[0117] B7H3 / IL-13Rα 2 CAR constructs as disclosed herein.

[0118] (b ) Anti-IL-13Rα 2 Moiety

[0119] In some embodiments, the anti-IL-13Rα 2 antibody for use in constructing the anti-IL-

[0120] 13Rα 2 CAR or the bispecific anti-IL-13Rα 2 / B7H3 CAR as disclosed herein (parent anti-IL-

[0121] 13Rα 2 antibodies) may be a human heavy chain antibody, which may comprise only a heavy chain variable region. Exemplary human heavy chain antibodies include 13R-01, 13R-02, 13R-03, 13R-04, and 13R-05 as disclosed herein. The amino acid sequences of these exemplary human heavy chain antibodies, as well as their heavy chain complementarity determining regions (CDRs) as determined following the Rabat scheme, are provided in Table 2 below.

[0122] In some embodiments, the anti-IL-13R«2 binding moiety in any of the anti-IL- 13Rα 2

[0123] CARs or the bispecific anti-IL-13Rα 2 / B7H3 CARs disclosed herein may be in an scFv format, which is a fusion polypeptide comprising the heavy chain variable domain (VH) and the light chain variable domain (VL) of an anti-IL-13Rα 2 antibody connected by a peptide linker. In the scFv fragment, the VH and VL fragments may be in any orientation. In some instances, the scFv may comprise, from the N-terminus to the C-terminus, a VL fragment, a peptide linker, and a VH fragment. Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, a VH fragment, a peptide linker, and a VL fragment. In some examples, a scFv may further comprise an N-terminal signal peptide for directing the CAR comprising the scFv to cell surface. Exemplary anti-IL-13Rα 2 scFv antibody fragments include 13R-06, 13R-07, 13R-10, 13R-08, 13R-09, 13R-10, 13R-11, or 13R-13 as disclosed herein. In one example, the scFv antibody fragment for use in constructing the anti-IL- ! 3Rcx2 CARs or the bispecific anti-IL-

[0124] 13Rα 2 / B7H3 CARs can be 13R-06 or 13R-07 or a derivative thereof (see above disclosures).

[0125] The amino acid sequences of these exemplary scFv antibody fragments, as well as their heavy chain and light chain CDRs as determined following the Rabat scheme, are provided in Table

[0126] 2 below. All of the listed anti-IL-13Rα 2 antibodies, as well as their derivatives as disclosed herein, are within the scope of the present disclosure.

[0127] Table 2. Anti-IL-13Rα 2 Antibodies

[0128] An anti-IL-13Rα 2 binding moiety (e.g., heavy chain antibody fragment or scFv fragment) derived from a reference antibody (e.g., those listed in Table 2) refers to binding moieties having substantially similar structural and functional features as the reference antibody. Structurally, the binding moiety may have the same heavy and / or light chain complementary determining regions or the same VH and / or VL chains as the reference antibody. Alternatively, the binding moiety may only have a limited number of amino acid variations in one or more of the framework regions and / or in one or more of the CDRs without significantly affecting its binding affinity and binding specificity relative to the reference antibody. See descriptions below.

[0129] In some examples, the anti-IL-13R«2 binding moiety may comprise the same heavy chain CDRs as those in any of the reference antibodies provided in Table 2 above (e.g., 13R

[0130] 06). Where applicable (e.g., in the context of scFv antibody fragments), the anti-IL-13Rcx2 binding moiety may have the same light chain CDRs as those in any of the reference antibodies provided in Table 2 above (e.g., 13R-06). Such an anti-IL- 13Rα 2 binding moiety may comprise the same VH and / or VL chains as the reference antibody. Alternatively, the anti¬

[0131] IL-13Rα 2 binding moiety may comprise amino acid variations in one or more of the framework regions relative to the corresponding framework regions in the reference antibody.

[0132] For example, the anti-IL-13Rα 2 binding moiety may comprise, collectively, up to 15 amino acid variations (e.g. , up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more framework regions relative to the corresponding framework regions in the reference antibody.

[0133] In some embodiments, the anti-IL-13Rα 2 moiety may comprise a certain level of variations in one or more of the CDRs relative to those in any of the reference antibodies provided in Table 2 above (e.g., 13R-06). For example, the anti-IL- 13Rα 2 moiety may comprise heavy chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VH CDRS of the reference antibody.

[0134] Alternatively, or in addition, the anti-IL- 13Rα 2 antibody may comprise light chain CDRs that are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity, individually or collectively, as compared with the VL CDRS as the reference antibody. As used herein, “individually” means that one CDR of an antibody shares the indicated sequence identity relative to the corresponding CDR of a reference antibody (e.g. , those in Table 2 such as 13R-06). “Collectively” means that three VH or VL CDRs of an antibody in combination share the indicated sequence identity relative the corresponding three VH or VL CDRS of the reference antibody in combination.

[0135] In some instances, the anti-IL- 13Rα 2 moiety may comprise up to 10 amino acid variations (e.g., up to 9, 8, 7. 6, 5, 4, 3, 2, or 1 amino acid variations) in one or more of the heavy chain and light chain CDRs collectively relative to those in the CDRs of a reference antibody provided in Table 2 (e.g., 13R-06). In some instances, the anti-IL- 13Rct2 moiety may comprise the same heavy chain CDR3 as the heavy chain CDR3 of the reference antibody and comprise one or more amino acid variations in one or more of the other heavy chain and light chain CDRs.

[0136] In some examples, the anti-IL-13R«2 moiety disclosed herein may be any of the anti-

[0137] IL-13Rα 2 heavy chain antibody provided in Table 2 above. For example, the anti-IL- 13Rα 2 binding moiety may comprise the amino acid sequence of SEQ ID NO: 64, 66,68, 72, 76, or 80. Alternatively, the anti-B7H3 binding moiety may comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 98%, or above) identical to any of the heavy chain antibody fragments provided in Table 2, e.g., SEQ ID NO: 64, 66,68, 72, 76, or 80. Such an 64, 66,68, 72, 76, or 80 binding moiety may comprise variations in framework regions only as compared with the parent counterpart.

[0138] In some examples, the anti-IL-13Rα 2 binding moiety disclosed herein may be any of the scFv fragments provided in Table 2 above. For example, the anti-IL- 13Rα 2 scFv fragments may comprise the amino acid sequence of SEQ ID NO: 84, 91, 100, 107, 113, 1 19, 124, or 129, or comprise the same VH and VL fragments thereof. In specific examples, the anti-IL-13Rα 2 scFv fragment may comprise the VH of SEQ ID NO: 80 and the VL of SEQ ID

[0139] NO: 83. In one example, the anti-IL- 13Rα 2 scFv fragment may comprise the amino acid sequence of SEQ ID NO: 84. Alternatively, the anti-IL- 13Rα 2 moiety may comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 98%, or above) identical to any of the scFv fragments provided in Table 2, e.g., SEQ ID NO: 84, 91, 100, 107, 113, 119,

[0140] 124, or 129 (e.g., SEQ ID NO: 84). Such an anti-IL- 13Rα 2 binding moiety may comprise variations in framework regions only as compared with the parent counterpart. In other examples, the anti-IL- 13Rα 2 moiety disclosed herein may comprise the same VH and VL sequences as in those listed in Table 2, e.g., SEQ ID NO: 84, 91, 100, 107, 113, 119, 124, or 129, but has a reversed orientation of the VH and VL fragments.

[0141] Any of the anti-IL- 13Rα 2 binding moieties disclosed herein (e.g., the heavy chain antibody fragment provided in Table 2, the scFv fragment provided in Table 2 such as SEQ ID NO: 84, or their counterparts having reversed VH and VL orientation, or derivatives thereof as also disclosed herein) may be used for constructing the anti-IL- 13Rα 2 CAR constructs

[0142] (including biparatopic CARs) or the bispecific anti-IL 13Rα 2 / B7H3 CARs as disclosed herein.

[0143] Any of the parent anti-IL- 13Rα 2 antibodies provided herein is also within the scope of the present disclosure. In this context, the term “antibody” (interchangeably used in plural form) refers to an immunoglobulin molecule capable of specific binding to a target (human IL-

[0144] 13Rα 2 here), through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The anti-IL- 13Rα 2 antibodies provided herein encompasses not only intact (e.g., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single-chain antibody (scFv), fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, single domain antibody (e.g., nanobody), single domain antibodies (e.g., a VH only antibody), multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies (e.g., antibody-drug conjugates or ADCs). An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0145] In some embodiments, the anti-IL- 13Rα 2 antibodies may be formulated into a pharmaceutical composition for therapeutic applications, for example, treating a disease (e.g., cancer) involving IL-13Rα 2+disease cells or IL-13Rα 2-mediated signaling pathways. To perform such a treatment method, an effective amount of the anti-IL- 13Rα 2 antibody or a pharmaceutical composition comprising such can be administered to a subject (e.g., a human patient) who needs the treatment via suitable route, e.g., intravenous infusion or subcutaneous injection.

[0146] Alternatively, such anti-IL- 13Rα 2 antibodies may be used for detecting and optionally quantifying IL- 13Rα 2 levels or IL-13Rα 2+cell levels in a biological sample using a conventional method, for example, any immunohistological method known to those of skill in the art (see, e.g., Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol.

[0147] 105:3087-3096 (1987)). Other antibody-based methods useful for detecting IL-13Rα 2 expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable assays are described in more detail elsewhere herein. The term “biological sample" means any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing IL-

[0148] 13Rα 2. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0149] In some examples, the anti-IL- 13Rα 2 antibodies as described herein can be conjugated to a detectable label, which can be any agent capable of releasing a detectable signal directly or indirectly. The presence of such a detectable signal or intensity of the signal is indicative of presence or quantity of the target antigen in the sample. Alternatively, a secondary antibody specific to the anti-IL- 13Rα 2 antibody or specific to the target antigen may be used in the methods disclosed herein.

[0150] B. Other Components of Chimeric Antigen Receptor Constructs

[0151] In addition to the extracellular antigen binding domains disclosed herein, any of the anti-IL- 13Rα 2 CARs or the bispecific anti-IL- 13Rα 2 / B7H3 CARs may further comprise one or more intracellular signaling domains (e.g., co- stimulatory and cytoplasmic signaling domains), and optionally a hinge domain, a spacer, a transmembrane domain, an N-terminal signal peptide, or a combination thereof. Exemplary components for CAR construction are provided in Table 3 below. Table 3. Exemplary Components of Chimeric Antigen Receptor Constructs

[0152] ( i ) Signaling Domains

[0153] Any of the anti-IL- 13Rα 2 CAR or the bispecific anti-IL- 13Rα 2 / B7H3 CAR constructs disclosed herein may comprise one or more intracellular signaling domains, which typically contain a co- stimulatory domain and a cytoplasmic signaling domain. A “co-stimulatory signaling domain” refers to at least a fragment of a co-stimulatory signaling protein that mediates signal transduction within a cell to induce an immune response such as an effector function (a secondary signal). A cytoplasmic signaling domain may be any signaling domain involved in triggering cell signaling (primary signaling) that leads to immune cell proliferation and / or activation. The cytoplasmic signaling domain as described herein is not a co-stimulatory signaling domain, which, as known in the art, relays a co-stimulatory or secondary signal for fully activating immune cells.

[0154] In some embodiments, the co-stimulatory signaling domain and the cytoplasmic signaling domain are for use in CAR constructs disclosed herein that are to be introduced into

[0155] T cells. In some instances, a co-stimulatory signaling domain may be derived from a costimulatory protein involved in T cell responses, for example, a member of the B7 / CD28 family, a member of the TNF superfamily, a member of the SLAM family, or any other co- stimulatory molecules. Examples include, but are not limited to, 4-1BB, CD28, OX40, ICOS,

[0156] CD40, CD40L, CD27, GITR, HVEM, TIM1, LFAl(CDl la) or CD2. In specific examples, the co-stimulatory signaling domain is a 4-1BB signaling domain (e.g., SEQ ID NO: 139 in Table 4 above).

[0157] The cytoplasmic signaling domain may comprise an immunoreceptor tyrosine-based activation motif (IT AM) domain or may be IT AM free. An “ITAM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. Exemplary cytoplasmic signaling domains include the signaling domain of CD3ζ, e.g., SEQ ID NO: 140.

[0158] ( ii ) Hinge and Transmembrane Domains

[0159] In some instances, the anti-IL- 13Rα 2 CAR or the bispecific anti-IL- 13Rα 2 / B7H3 CAR constructs disclosed herein may contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. A “transmembrane domain” can be a peptide fragment that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability of the CAR containing such. Exemplary transmembrane domains may be a CDS transmembrane domain, or a CD28 transmembrane domain. In one example, the transmembrane domain can comprise SEQ ID NO: 138 shown in Table 4 above.

[0160] Alternatively or in addition, the CAR construct disclosed herein may also comprise a hinge domain, which may be located between the extracellular antigen binding domain and the transmembrane domain, or between the transmembrane domain and the intracellular signaling domain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof. A hinge domain may contain 5-20 amino acid residues. In some embodiments, the hinge domain may be a CDS hinge domain or an IgG hinge. Other hinge domains may be used. In one example, the hinge domain can comprise SEQ ID NO: 135 shown in Table 4 above.

[0161] In some examples, the anti-IL- 13 Rα 2 CAR or the bispecific anti-IL- 13Rα 2 / B7H3 CAR may further comprise a spacer between the hinge domain and the transmembrane domain. Examples of such spacers are provided in Table 4 above (e.g., SEQ ID NO: 136 or SEQ ID

[0162] NO: 137). Alternatively, the anti-IL- 13Rα 2 CAR or the bispecific anti-IL- 13Rα 2 / B7H3 CAR CAR may lack such a spacer fragment between the hinge domain and the transmembrane domain.

[0163] C. Anti-IL- 13Rα 2 Chimeric Antigen Receptors

[0164] In some aspects, provided herein are anti-IL- 13Rα 2 CAR constructs, including biparatopic CARs, comprising the anti-IL- 13Rα 2 binding moieties and the additional components as disclosed herein, nucleic acids encoding such, and host cells expressing such.

[0165] The anti-IL- 13Rα 2 CAR may comprise (a) an extracellular binding domain comprising one or more of any of the anti-IL- 13Rα 2 binding moieties, e.g., an anti-IL- 13Rα 2 human heavy chain antibody fragment or an anti-IL- 13Rα 2 scFv derived from any of the reference antibodies provided in Table 2 above (e.g., 13R-06), or a combination thereof; (b) a co-stimulatory signaling domain such as those disclosed herein; and (c) a cytoplasmic signaling domain such as those disclosed herein. The anti-IL- 13Rα 2 CAR may further comprise a hinge domain and a transmembrane domain located at the C-terminal of the extracellular antigen binding domain.

[0166] Optionally, the anti-IL- 13Rα 2 CAR may further comprise a spacer between the hinge and transmembrane domains. In some examples, the anti-IL- 13 Rα 2 CAR may comprise the amino acid sequence of SEQ ID NO: 148 or 150.

[0167] In some instances, the anti-IL- 13Rα 2 CAR construct disclosed herein may further comprise an N-terminus signal peptide, e.g., SEQ ID NO: 130 or 131. Exemplary anti-B7H3

[0168] CAR constructs in full length (containing the signal peptide) may comprise the amino acid sequence of SEQ ID NO: 147 or 149.

[0169] Exemplary anti-IL- 13Rα 2 CAR constructs (as well as anti-B7H3 CAR constructs) are provided in Table 4 below, all of which are within the scope of the present disclosure.

[0170] Table 4. Exemplary Anti-IL13Rα 2 or Anti-B7H3 Chimeric Antigen Receptors (CARs)

[0171] D. Bispecific CARs Targeting IL-13Rα 2 and / or B7H3

[0172] In some aspects, provided herein are anti-IL-13Rα 2 and / or B7H3 bispecific CARs at least two binding moieties, one or more intracellular signaling domains such as co-stimulatory signaling domains and cytoplasmic signaling domains, and optionally a hinge and a transmembrane domain as disclosed herein.

[0173] In some embodiments, provided herein are anti-IL- 13Rα 2 / B7H3 bispecific CARs each comprising an anti-IL- 13Rα 2 binding moiety (e.g., an IL- 13Rα 2 scFv such as those disclosed herein; see Table 2 above, such as SEQ ID NO: 84), an anti-B7H3 moiety (e.g., an anti-B7H3 scFv such as those disclosed; see Table 1 above, such as SEQ ID NO: 45), one or more intracellular signaling domains such as co-stimulatory signaling domains and cytoplasmic signaling domains, and optionally a hinge domain and a transmembrane domain as disclosed herein. In some instances, the anti-IL- 13Rα 2 / B7H3 bispecific CAR may be a single polypeptide comprising both the anti-IL- 13Rα 2 moiety and the anti-B7H3 moiety. In other instances, the anti-IL- 13Rα 2 / anti-B7H3 bispecific CAR may be a multiple-chain (e.g., 2- chain) molecule. The anti-IL- 13Rα 2 moiety and the anti-B7H3 moiety may be located on separate polypeptides.

[0174] In some embodiments, the anti-IL- 13Rα 2 / anti-B7H3 bispecific CAR disclosed herein may comprise an anti-IL-13Rα 2 binding moiety (e.g. , scFv) derived from 13R-06 and an anti

[0175] B7H3 binding moiety (e.g., scFv) derived from BH-07. In other embodiments, the anti-IL-

[0176] 13Rα 2 / anti-B7H3 bispecific CAR disclosed herein may comprise an anti-IL- 13Rα 2 binding moiety (e.g., scFv) derived from H7 and optionally an anti-B7H3 binding moiety (e.g., scFv) derived from BH-07.

[0177] The anti-IL- 13Rα 2 binding moiety derived from 13R-06 (e.g., scFv) may be any of the anti-IL- 13Rα 2 moieties relating to 13R-06 disclosed above. In some instances, it may comprise the same heavy chain and / or light chain CDRs as 13R-06. In specific examples, the scFv may comprise the same VH and / or same VL as 13R-06. In some instances, the scFv may comprise, from the N-terminus to the C-terminus, a VL fragment (e.g., SEQ ID NO: 83), a peptide linker (e.g., SEQ ID NO: 133), and a VH fragment (e.g., SEQ ID NO: 80).

[0178] Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, a VH fragment (e.g., SEQ ID NO: 80), a peptide linker (e.g., SEQ ID NO: 133), and a VL fragment (e.g., SEQ

[0179] ID NO: 83). In one specific example, the anti-IL-13Rα 2 binding moiety may comprise SEQ ID NO: 84.

[0180] The anti-B7H3 binding moiety derived from BH-07 (e.g., scFv) may be any of the anti- B7H3 binding moieties relating to BH-07 disclosed above. In some instances, it comprises the same heavy chain and / or light chain CDRs as BH-07. In specific examples, the scFv may comprise the same VH and / or same VL as BH-07. In some instances, the scFv may comprise, from the N-terminus to the C-terminus, a VL fragment (e.g., SEQ ID NO: 44), a peptide linker (e.g., SEQ ID NO: 133), and a VH fragment (e.g., SEQ ID NO: 40). Alternatively, the scFv may comprise, from the N-terminus to the C-terminus, a VH fragment (e.g., SEQ ID NO: 40), a peptide linker (e.g., SEQ ID NO: 133), and a VL fragment (e.g. , SEQ ID NO: 44). In one specific example, the anti-B7H3 moiety may comprise SEQ ID NO: 45.

[0181] In some instances, the anti-IL-13Rα 2 / anti-B7H3 bispecific CAR disclosed herein may comprise an anti-B7H3 binding moiety derived from BH-02 VH (e.g., SEQ ID NO: 8 or SEQ ID NO: 9) and an anti-IL-13Rα 2 binding moiety derived from R13-02 VH (e.g. , SEQ ID NO: 66). In some examples, the bispecific CAR may comprise the anti-B7H3 binding moiety N- terminal to the anti- IL- 13Rα 2 binding moiety. Alternatively, the bispecific CAR may comprise the anti-B7H3 binding moiety C-terminal to the anli-IL- 13Rα 2 binding moiety. In one specific example, such a bispecific CAR may comprise the amino acid sequence of SEQ ID NO: 184 or 185.

[0182] In some embodiments, the bi-specific anti-IL-13Rα 2 / anti-B7H3 binding moieties may be located on a single polypeptide. In some examples, the single polypeptide contains a spacer

[0183] (a peptide linker) between the anti-IL- 13Rα 2 binding moiety and the anti-B7H3 binding moiety. Examples are provided in Table 3. See also Examples below.

[0184] Any of the fusion polypeptide comprising the anti-IL-13Rα 2 and anti-B7H3 moieties may further comprise a co-stimulatory signaling domain and a cytoplasmic signaling domain such as those disclosed herein. Optionally, the fusion polypeptide may further comprise a hinge domain and a transmembrane domain as also disclosed herein. In some examples, the bispecific CAR can be included in a multi-cistronic expression cassette with an armor gene (e.g., those listed in Table 6 below) via a self-cleavage peptide linker.

[0185] Exemplary anti-IL-13Rα 2 / anti-B7H3 bispecific CAR constructs are provided in Table

[0186] 5 below, each of which is within the scope of the present disclosure. In one example, the bispecific CAR comprises the amino acid sequence of SEQ ID NO: 175 or 176.

[0187] In other embodiments, provided herein are biparatopic CAR polypeptides comprising two binding moieties to two different epitopes in B7H3 or IL- 13Rα 2. Biparatopic CARs are a class of bispecific CAR constructs that target two non-overlapping epitopes on the same target.

[0188] In some examples, provided herein are anti-B7H3 biparatopic CARs comprising any of the two anti-B7H3 fragments disclosed in Table 1 above. In other examples, provided herein are anti-

[0189] IL-13Rα 2 biparatopic CARs comprising any of the two anti-IL-13Rα 2 fragments disclosed in

[0190] Table 2 above. Examples of such biparatopic CARs are also provided in Table 5 below, each of which is within the scope of the present disclosure.

[0191] Table 5. Exemplary Biparatopic and Bi-Specific CAR Targeting B7H3 and IL13Rα 2

[0192] IL Engineered Immune Cells Expressing Anti-IL- 13R«2 CAR or Anti-IL- 13Rα 2 / B7H3 Bispecific CAR

[0193] In some aspects, provided herein are genetically engineered immune cells such as T cells NK cells, or macrophages having surface expression of any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL- 13Rα 2 / B7H3 bispecific CAR constructions disclosed herein.

[0194] In some embodiments, such engineered immune cells may further express an armor polypeptide as disclosed herein.

[0195] (a) CAR-T Cells and Armored CAR-T Cells

[0196] The genetically engineered immune cells can express any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL- 13Rα 2 / B7H3 bispecific CAR constructions disclosed herein.

[0197] In some embodiments, the engineered immune cells may express an anti-IL- 13Rα 2 CAR may comprise an anti-IL- 13 Rα 2 binding moiety derived from any of the anti-IL- 13Ra2 antibody provided in Table 2 above, for example, derived from clone 13R-06. In some embodiments, the engineered immune cells may express an anti-IL- 13Rα 2 / B7H3 bispecific CAR may comprise an anti-IL- 13Rα 2 binding moiety derived from any of the anti-IL- 13Ra2 antibody provided in Table 2 above, for example, derived from clone 13R-06, and an anti-B7H3 binding moiety derived from any of the anti-B7H3 antibodies provided in Table 1 above, for example, derived from clone BH-H7. In some examples, the engineered immune cells may express a bispecific CAR listed in Table 5 above, e.g., SEQ ID NO: 175 or 176.

[0198] Any of the CAR-expression immune cells disclosed herein may be engineered to include additional mechanisms to reprogram the CAR-expressing cells so as to enhance their bioactivity and / or persistence, thereby enhancing overall therapeutic effects. For example, the CAR-expressing immune cells may be further engineered to express an armor polypeptide to enhance physical and / or biological features of the CAR-T cells. Such CAR-T cells are known as armored CAR-T cells, which co-express one or more CAR constructs and an armor polypeptide that is capable of enhancing CAR-T cell features, e.g., improving growth and / or persistence, enhancing efficacy, reducing toxicities, etc., or a combination thereof.

[0199] Exemplary armor polypeptides include, but are not limited to, a suitable cytokine such as IL-2, IL-5, IL-15, IL-12, IL-17, IL-21, a co-stimulatory ligand (e.g., CD80, or CD86), a checkpoint inhibitor (e.g., an anti-PDl or anti-PDL1 antibody fragment), a soluble receptor such as a soluble PD1, TGFR2 trap, or VEGFR2 trap, and / or an immune cell activation ligand (e.g., 4-1BBL). Such an armor polypeptide may be a naturally-occurring polypeptide. Alternatively, it can be an engineered polypeptide, for example, an engineered IL-2 polypeptide. In some embodiments, the armor polypeptide may be a fusion polypeptide comprising, e.g., a cytokine or a fragment thereof (e.g., IL2 or IL 15 or a fragment thereof), and a checkpoint inhibitor (e.g., an anti-PDLl fragment).

[0200] In some instances, the CAR can be co-expressed with an armor polypeptide in a host immune cell, which enhances physical and / or biological features of the host immune cells. See, e.g., disclosures herein and relevant disclosures in WO2021 / 030633, WO2022 / 159771, and WO2023 / 220597, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

[0201] In some instances, the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL 13Rα 2 / B7H3 bispecific CAR may be co-expressed with a truncated EGFR, e.g., as disclosed in Table 6 below.

[0202] Specific examples of armor polypeptides are provided in Table 6 below.

[0203] Table 6: Exemplary Armor Polypeptides and Others for Co-Expression with Anti-B7H3 CAR

[0204] In some instances, the coding sequences of the CAR constructs and the armor polypeptides may be located in a same expression cassette. The two coding sequences may be separated by a ribosome entry site (IRES) or a coding sequence for a self-cleavage peptide (e.g., P2A or T2A) so as to produce two separate polypeptides (CAR and the armor polypeptide). In other instances, two separate expression cassettes may be used to express the CAR construct and the armor polypeptide in armored CAR-T cells. In some examples, the armor polypeptide contains a N-terminus signal peptide so that the polypeptide can be secreted from the CAR-T cells. Alternatively, the armor polypeptide may be expressed as an intracellular protein or a membrane-bound protein.

[0205] In specific examples, the CAR-T cells disclosed herein are CAR-T cells co-expressing anti-IL13Ra2 and anti-B7H3 scFvs in tandem format (e.g., comprising the amino acid sequence of 175 or 176) and an anti-PDLl -engineered IL2 armor polypeptide, which may comprise the amino acid sequence of SEQ ID NO: 179.

[0206] (b) Preparation of CAR-Expressing Immune Cells

[0207] The genetically engineered immune cells disclosed herein may be prepared by introducing one or more expression cassettes encoding any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL-13Rα 2 / B7H3 bispecific CAR constructs disclosed herein, optionally one or more armor polypeptides and / or a truncated EGFR such as those disclosed herein into suitable immune cells and collecting the resultant engineered immune cells that express the CAR on cell surface.

[0208] A population of immune cells, as the starting parent cells, can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, or tissues such as spleen, lymph node, thymus, stem cells, or tumor tissue. A source suitable for obtaining the type of host cells desired would be evident to one of skill in the art. In some embodiments, the population of immune cells is derived from PBMCs. The type of host cells desired (e.g., T cells, NK cells, macrophages, or a combination thereof) may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules. As a nonlimiting example, anti-CD3 and anti-CD28 antibodies may be used for expansion of T cells. In some embodiments, a specific type of cells (e.g., T cells, NK cells, or macrophages) may be enriched from the immune cell population. Such enriched cell subpopulation may be expanded and / or activated in vitro prior to the genetic engineered for introduction of the CAR-encoding expression cassette and / or the armor polypeptide-encoding expression cassette (may be the same expression cassette).

[0209] To construct the immune cells that express any of the anti-IL- 13Rα 2 CAR polypeptides or any of the anti-IL- 13Rα 2 / B7H3 bispecific CAR polypeptides described herein (e.g. , those provided in Tables 4 and 5), optionally one or more armor polypeptides such as those disclosed herein (e.g., those provided in Table 6), expression vectors for stable or transient expression of the CAR polypeptide and optionally the armor polypeptide may be created via conventional methods and introduced into immune host cells. For example, nucleic acids encoding the CAR polypeptides and optionally the armor polypeptide may be cloned into one or more suitable expression vector(s), such as a viral vector(s) in operable linkage to a suitable promoter. Non-limiting examples of useful vectors of the disclosure include viral vectors such as, e.g., retroviral vectors including gamma retroviral vectors, adeno-associated virus vectors (AAV vectors), and lenti viral vectors. The nucleic acids and the vector may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding the CAR polypeptides, and optionally the armor polypeptide. The synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors / plasmids / viral vectors would depend on the type of host cells for expression of the CAR polypeptides and optionally the armor polypeptide but should be suitable for integration and replication in eukaryotic cells. Any of such nucleic acids encoding the CAR and optionally the armor polypeptide and expression vectors comprising such are also within the scope of the present disclosure.

[0210] A variety of promoters can be used for expression of the CAR polypeptides and optionally the armor polypeptide described herein, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, or herpes simplex tk virus promoter. Additional promoters for expression of the CAR polypeptides and optionally the armor polypeptide include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within an immune cell. In some embodiments, the promoter can be the pEF la promoter.

[0211] Additionally, the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene or the kanamycin gene for selection of stable or transient transfectants in host cells; enhancer / promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; S V40 polyomavirus origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g. , HSV thymidine kinase or an inducible caspase such as iCasp9), and reporter gene for assessing expression of the CAR polypeptide.

[0212] In one specific embodiment, such vectors may also include a suicide gene. As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and caspases such as caspase 8.

[0213] The nucleic acid disclosed herein may comprise two coding sequences, one for any of the anti-IL-13Rα 2 CAR constructs or any of the anti-IL-13Rα 2 / B7H3 bispecific CAR constructs disclosed herein (e.g., (e.g., those provided in Table 4 and Table 5) and the other for the armor polypeptide or a truncated EGER (e.g., those provided in Table 6). The two coding sequences may be configured such that the polypeptides encoded by the two coding sequences can be expressed as independent (and physically separate) polypeptides. To achieve this goal, the nucleic acid described herein may contain a third nucleotide sequence located between the first and second coding sequences. This third nucleotide sequence may, for example, encode a ribosomal skipping site. A ribosomal skipping site is a sequence that impairs normal peptide bond formation. This mechanism results in the translation of additional open reading frames from one messenger RNA. This third nucleotide sequence may, for example, encode a self-cleavage peptide such as P2A, T2A, or F2A peptide (see, for example, Kim et al. , PLoS One. 2011 ;6(4):el8556). See also FIG. 3.

[0214] Any of the vectors comprising a nucleic acid sequence that encodes an anti-IL-13R«2

[0215] CAR polypeptide or an anti-IL-13Rα 2 / B7H3 bispecific CAR polypeptide and optionally an armor polypeptide and / or a truncated EGFR described herein is also within the scope of the present disclosure.

[0216] Such a vector, or the sequence encoding a CAR polypeptide and optionally the armor polypeptide or the truncated EGFR contained therein, may be delivered into host cells such as host immune cells (e.g., T cells, NK cells, or macrophages) by any suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection using reagents such as liposomes, or viral transduction (e.g., retroviral transduction such as lentiviral transduction).

[0217] Following introduction into the host cells a vector encoding any of the anti-IL-13Rα 2

[0218] CAR polypeptide or the anti-IL-13Rα 2 / B7H3 bispecific CAR polypeptides provided herein (e.g., those provided in Tables 4 and 5), optionally the armor polypeptide and / or the truncated EGFR as also provided herein (e.g., those provided in Table 6), the cells may be cultured under conditions that allow for expression of the CAR polypeptide and optionally the armor polypeptide and / or the truncated EGFR. When expression of the CAR polypeptide and / or the armor polypeptide / truncated EGFR is regulated by a regulatable promoter, the host cells may be cultured in conditions wherein the regulatable promoter is activated. In some embodiments, the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or in conditions in which the inducing molecule is produced. Determining whether the CAR polypeptide and / or the armor polypeptide is expressed will be evident to one of skill in the art and may be assessed by any known method, for example, detection of the CAR polypeptide-encoding and / or armor polypeptide-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of the CAR or armor polypeptide protein by methods including Western blotting, fluorescence microscopy, and flow cytometry. Alternatively, expression of functional CAR may be determined by binding activity and / or CTL activity against cells expressing the target antigen, e.g., full length B7H3 and / or splicing variant B7H3, and / or IL-13Rα 2.

[0219] Methods for preparing host cells expressing any of the CAR polypeptides, and optionally an armor polypeptide / truncated EGFR, as described herein, may also comprise activating the host cells ex vivo. Activating a host cell means stimulating a host cell into an activated state in which the cell may be able to perform effector functions. Methods of activating a host cell will depend on the type of host cell used for expression of the CAR polypeptides and optionally the armor polypeptide and / or the truncated EGFR. For example, T cells may be activated ex vivo in the presence of one or more molecules including, but not limited to: an anti-CD3 antibody, an anti-CD28 antibody, IL-2, and / or phytohemoagglutinin. In other examples, NK cells may be activated ex vivo in the presence of one or molecules such as a 4-1BB ligand, an anti-4-lBB antibody, IL-15, an anti-IL-15 receptor antibody, IL-2, IL12, IL-21, and / or K562 cells. In some embodiments, the host cells expressing any of the CAR polypeptides (CAR-expressing cells), and optionally the armor polypeptide (armored CAR cells) described herein are activated ex vivo prior to administration to a subject. Determining whether a host cell is activated will be evident to one of skill in the art and may include assessing expression of one or more cell surface markers associated with cell activation, expression or secretion of cytokines, and cell morphology.

[0220] Methods for preparing host cells expressing any of the CAR polypeptides, and optionally the armor polypeptide, described herein may comprise expanding the host cells ex vivo. Expanding host cells may involve any method that results in an increase in the number of cells expressing CAR polypeptides and optionally the armor polypeptide, for example, allowing the host cells to proliferate or stimulating the host cells to proliferate. Methods for stimulating expansion of host cells will depend on the type of host cell used for expression of the CAR polypeptides, and optionally the armor polypeptide, and will be evident to one of skill in the art. In some embodiments, the host cells expressing any of the CAR polypeptides, optionally the armor polypeptide, described herein are expanded ex vivo prior to administration to a subject.

[0221] In some embodiments, the host cells expressing the CAR polypeptides and optionally the armor polypeptide are expanded and activated ex vivo prior to administration of the cells to the subject. Host cell activation and expansion may be used to allow integration of a viral vector into the genome and expression of the gene encoding a CAR polypeptide and optionally the armor polypeptide as described herein. If mRNA electroporation is used, no activation and / or expansion may be required, although electroporation may be more effective when performed on activated cells.

[0222] In some instances, a CAR polypeptide and / or an armor polypeptide is transiently expressed in a suitable host cell (e.g., for 3-5 days). Transient expression may be advantageous if there is a potential toxicity and should be helpful in initial phases of clinical testing for possible side effects.

[0223] (c) Pharmaceutical Compositions

[0224] Any of the genetically engineered immune cells expressing an anti-IL-13Rα 2 CAR construct or an anti-IL-13Rα 2 / B7H3 bispecific CAR construction, and optionally an armor polypeptide and / or a truncated EGER as disclosed herein may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0225] The phrase “pharmaceutically acceptable”, as used in connection with compositions of the present disclosure, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans. “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered. Any of the pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.

[0226] Pharmaceutically acceptable carriers, including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g. Remington: The Science and Practice of Pharmacy 20thEd. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0227] For examples of additional useful agents, see also Physician's Desk Reference, 59thedition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0228] III. Therapeutic Applications

[0229] Any of the genetically engineered immune cells (e.g., T cells, NK cells, and / or macrophages) expressing an anti-IL- 13Rα 2 CAR constructs or an anti-IL- 13Rα 2 / B7H3 bispecific CAR as disclosed herein (e.g., those provided in Tables 4 and 5), and optionally an armor polypeptide and / or truncated EGFR as also disclosed herein (e.g., those provided in Table 6) may be used for therapeutic purposes, for example, to eliminate undesired cells expressing IL-13Rα 2 and optionally B7H3 as well (full length and / or splicing variant). In some examples, the genetically engineered immune cells are armored CAR-T cells expressing any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL- 13Rα 2 / B7H3 bispecific CAR such as those provided in Tables 4 and 5 above, together with an armor polypeptide such as those provided in Table 6 above.

[0230] To practice the method described herein, an effective amount of the immune cells (e.g.,

[0231] T lymphocytes, NK cells, or macrophages) expressing any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL- 13Rα 2 / B7H3 bispecific CAR constructs described herein (e.g., those provided in Tables 4 and 5 above), and optionally an armor polypeptide and / or truncated EGER (e.g., those provided in Table 6 above) or pharmaceutical compositions thereof may be administered to a subject in need of the treatment via a suitable route, such as intravenous administration. As used herein, an effective amount refers to the amount of the respective agent

[0232] (e.g., the CAR-T cells expressing the anti-IL-13Rα 2 CAR or the anti-IL- 13Rα 2 / B7H3 bispecific CAR and optionally the armor polypeptide) that upon administration confers a therapeutic effect on the subject. Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender, sex, and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject in need of treatment is a human cancer patient.

[0233] As used herein, the term “therapeutically effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a compound or pharmaceutical composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0234] In some embodiments, the methods of the disclosure may be used for eliminating or inhibiting disease cells expressing IL- 13Rα 2 and optionally B7H3 as well. Accordingly, any of the immune cells disclosed herein may be used for treating a disease associated with IL- 13Rα 2+and optionally B7H3+disease cells, such as IL-13Rα 2+and optionally B7H3+cancer cells. The method disclosed herein may be used for treating a cancer involving IL-13Rα 2+and optionally B7H3+cancer cells, for example, lung cancer, breast cancer, colon cancer, pancreatic cancer, ovary cancer, glioblastoma, HCC, RCC, gastric cancer, esophageal cancer, prostate cancer, bladder cancer, head & neck cancer, squamous carcinoma, and leukemia.

[0235] In some embodiments, an effective amount of any of the genetically engineered immune cells express a bispecific anti-IL- 13Rα 2 / B7H3 CAR as disclosed herein (e.g., those provided in Table 5 such as EPLV306), and optionally an armor polypeptide (e.g., those provided in Table 6 such as the fusion polypeptide of SEQ ID NO: 179) may be given to a subject in need of the treatment via a suitable route, for example, intravenous infusion, and / or intracavitary injection. The subject may be a human patient having a disease associated with IL-13Rα 2+and / or B7H3+disease cells, such as IL-13Rα 2+and / or B7H3+cancer cells. In some instances, the human patient has a cancer involving IL-13Rα 2+and / or B7H3+cancer cells. In some instances, the human patient may have a brain tumor, for example, glioblastoma (GBM) or neuroblastoma. In other instances, the human patient may have a solid tumor, for example, glioblastoma, neuroblastoma, lung cancer, melanoma, head and neck cancer, urothelial cancer, prostate cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, esophageal cancer, ovarian cancer, breast cancer, bladder cancer, or acute myeloid leukemia (AML). In some aspects, provided herein is a method for treating glioblastoma in a subject (e.g., a human GBM patient) in need thereof using any of the genetically engineered immune cells provided herein. In some specific examples, the genetically engineered immune cells may express an anti-IL- 13Rα 2 CAR as disclosed herein. In other specific examples, the genetically engineered immune cells may express an anti-IL- 13Rα 2 / B7H3 bispecific CAR as disclosed herein (e.g., SEQ ID NO: 175 or 176). Such genetically engineered immune cells (e.g., comprising T cells and / or NK cells) may be armored - further expressing an armor polypeptide as also provided herein, e.g., the armor polypeptide comprising SEQ ID NO: 179. The genetically engineered immune cells may be administered to the GBM patient via intravenous infusion, intracavitary injection, or a combination thereof.

[0236] In some embodiments, the immune cells (e.g. , NK and / or T cells) for use in the treatment disclosed herein may be autologous to the subject, i.e., the immune cells may be obtained from the subject in need of the treatment, genetically engineered for expression of the CAR polypeptides, and then administered to the same subject. In one specific embodiment, prior to re-introduction into the subject, the autologous immune cells (e.g. , T lymphocytes, NK cells, or macrophages) are activated and / or expanded ex vivo. Administration of autologous cells to a subject may result in reduced rejection of the host cells as compared to administration of non- autologous cells.

[0237] Alternatively, the genetically engineered immune cells (e.g. , T cells, NK cells, or macrophages) can be allogeneic cells, i.e., the cells are obtained from a first subject, genetically engineered for expression of the anti-IL- 13Rα 2 CAR polypeptide or the anti-IL-

[0238] 13Rα 2 / B7H3 bispecific CAR polypeptide, and administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor. In a specific embodiment, the T lymphocytes are allogeneic T lymphocytes, in which the expression of the endogenous T cell receptor has been inhibited or eliminated. In one specific embodiment, prior to introduction into the subject, the allogeneic T lymphocytes are activated and / or expanded ex vivo. T lymphocytes can be activated by any method known in the art, e.g., in the presence of anti-CD3 / CD28, IL-2, and / or phytohemoagglutinin.

[0239] NK cells can be activated by any method known in the art, e.g. , in the presence of one or more agents selected from the group consisting of CD 137 ligand protein, CD 137 antibody, IL-L5 protein, IL-15 receptor antibody, IL-2 protein, IL-12 protein, IL-21 protein, and K562 cell line. See, e.g. , U.S. Patents Nos. 7,435,596 and 8,026,097 for the description of useful methods for expanding NK cells. For example, NK cells used in the methods of the disclosure may be preferentially expanded by exposure to cells that lack or poorly express major histocompatibility complex I and / or II molecules and which have been genetically modified to express membrane bound IL- 15 and 4- IBB ligand (CDI37L). Such cell lines include, but are not necessarily limited to, K562 [ATCC, CCL 243; Lozzio et al., Blood 45(3): 321-334 (1975); Klein et al., Int. J. Cancer 18: 421-431 (1976)], and the Wilms tumor cell line HFWT (Fehniger et al., Int Rev Immunol 20(3-4):503-534 (2001); Harada H, et al., Exp Hematol 32(7):614-621 (2004)), the uterine endometrium tumor cell line HHUA, the melanoma cell line

[0240] HMV-II, the hepatoblastoma cell line HuH-6, the lung small cell carcinoma cell lines Lu-130 and Lu- 134- A, the neuroblastoma cell lines NB 19 and N1369, the embryonal carcinoma cell line from testis NEC 14, the cervix carcinoma cell line TCO-2, and the bone marrow- metastasized neuroblastoma cell line TNB 1 [Harada, et al., Jpn. J. Cancer Res 93: 313-319 (2002)]. Preferably the cell line used lacks or poorly expresses both MHC I and II molecules, such as the K562 and HFWT cell lines. A solid support may be used instead of a cell line. Such support should preferably have attached on its surface at least one molecule capable of binding to NK cells and inducing a primary activation event and / or a proliferative response or capable of binding a molecule having such an affect thereby acting as a scaffold. The support may have attached to its surface the CD137 ligand protein, a CD137 antibody, the IL-15 protein or an IL- 15 receptor antibody. Preferably, the support will have IL- 15 receptor antibody and CD 137 antibody bound on its surface.

[0241] In accordance with the present disclosure, patients can be treated by infusing therapeutically effective doses of immune cells such as T lymphocytes and / or NK cells expressing an anti-IL- 13Rα 2 CAR polypeptide, e.g., those listed in Table 4 above, or an anti-

[0242] IL-13Rα 2 / anti-B7H3 bispecific CAR polypeptide, e.g., as listed in Table 5 above (e.g., EPLV306), and optionally an armor polypeptide as listed in Table 6 (e.g., the fusion polypeptide comprising SEQ ID NO: 179) in the range of about 105to 109CAR+ cells to a patient. The infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. The appropriate infusion dose and schedule will vary from patient to patient but can be determined by the treating physician for a particular patient. In some examples, initial doses of approximately 106cells / Kg can be infused, escalating to 108or more cells / Kg.

[0243] The particular dosage regimen, i.e., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history. The appropriate dosage of the CAR-expressing immune cells (e.g., armored CAR-T cells) used will depend on the type of cancer to be treated, the severity and course of the disease, previous therapy, the patient's clinical history and response to the immune cell therapy, and the discretion of the attending physician.

[0244] In some embodiments, the genetically engineered immune cells (e.g., armored CAR-T cells) expressing any of the anti-IL- 13Rα 2 CAR constructs or any of the anti-IL- 13Rα 2 / anti- B7H3 bispecific CAR constructs disclosed herein may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth. Such therapies can be administered simultaneously or sequentially (in any order) with the immunotherapy according to the present disclosure. When co- administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0245] IV. Kits for Therapeutic Applications

[0246] The present disclosure also provides kits for use of the genetically engineered immune cells (e.g., T lymphocytes, NK cells, or macrophages) expressing an anti-IL- 13Rα 2 CAR as disclosed herein (see, e.g., Table 4), or an anti-IL- 13Rα 2 / B7H3 bispecific CAR (see, e.g., Table 5) and optionally an armor polypeptide and / or truncated EGFR as described herein. See, e.g., Table 6. Such kits may include one or more containers comprising the genetically engineered immune cells, which may be formulated in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0247] In some embodiments, the kit described herein comprises genetically engineered immune cells, which may be expanded in vitro. The immune cells may express any of the CAR disclosed herein, for example, any of the anti-IL- 13Rα 2 CARs such as those provided in Table

[0248] 4 above or any of the anti-IL- 13Rα 2 / B7H3 bispecific CARs such as those provided in Table 5 above. The immune cells may be armored CAR-T cells, which further express an armor polypeptide (e.g., those provided in Table 6 above).

[0249] In some embodiments, the kit can additionally comprise instructions for use in any of the methods described herein. The included instructions may comprise a description of administration of the genetically engineered immune cells disclosed herein to achieve the intended activity, e.g., eliminating the target disease cells such as cancer cells expressing IL-

[0250] 13Rα 2 and optionally B7H3 (full length and / or splicing variant) in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.

[0251] The instructions relating to the use of the genetically engineered immune cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the genetically engineered immune cells are used for treating, delaying the onset, and / or alleviating a disease or disorder associated with IL- 13Rα 2-positive and optionally B7H3-positive disease cells in a subject.

[0252] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port.

[0253] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0254] General techniques

[0255] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987);

[0256] PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. ( 1986»; Immobilized Cells and Enzymes (IRL Press, ( 1986» ; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0257] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0258] EXAMPLES

[0259] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

[0260] Example 1: Discovery of Human Anti-IL13R«2 Antibody

[0261] This example illustrates development of exemplary human anti-IL13RA2 antibodies, including heavy chain antibodies (VH antibodies) and single chain variable fragment (scFv) antibodies. A. Human Anti-IL13Rα 2 VH and scFv Selection by mRNA Display mRNA display technology was used for the identification of IL13Rα 2 VH and scFv binders from natural human VH and scFv libraries. Briefly, the 108VH and 1012-13scFv DNA libraries were first transcribed into mRNA library and then translated into mRNA-VH or mRNA-scFv fusion libraries by covalent coupling through a puromycin linker, similar to the reported procedure (see, e.g., US6258558B1, the relevant disclosures of which are incorporated by reference for the subject matter and purpose referenced herein). The fusion libraries were first counter selected with human IgGs (negative proteins) multiple times to remove non-specific binders, followed by selection against recombinant IL13Rα 2-Fc fusion protein and captured on Protein G magnetic beads. IL13Rα 2 full length (4 Ig domains) and isoform (2 Ig domains) were selected against at alternative rounds. Binders were eluted off and then enriched by PCR amplification with library specific oligos. At round 3-5, the VH and scFv libraries were selected on recombinant IL13Rα 2 / HEK293 cell lines.

[0262] B. Identifications and Characterization of Anti-IL13Rα 2 VH and scFv Antibodies

[0263] After 4-6 rounds of selections, the IL13Rα 2 enriched VH and scFv library was cloned into bacterial periplasmic expression vector pET22b and transformed into TOP 10 competent cells. Each of the VH and scFv molecule was engineered to have a C-terminal flag and 6xHis tag for purification and assay detection. Clones from TOP 10 cells were pooled and the miniprep DNA were prepared and subsequently transformed into bacterial Rosetta II strain for expression. Single clone was picked, grown and induced with 0.1 -0.5 mM IPTG in 96 well plate for expression. The supernatant was collected after 16-24 hours induction at 30°C for assays to identify anti-IL13Rα 2 antibodies.

[0264] IL13Rα 2 binding screening ELISA was developed for the identification of individual anti-IL13Rα 2 antibodies. Briefly, a 384 well plate was immobilized with human Fc, human IL13Rα 2-Fc (full length) respectively, at a final concentration of 2 ug / mL in lx PBS in a total volume of 25 uL per well. The plate was incubated overnight at 4°C followed by blocking with 80 uL of superblock per well for 1 hour. 25 uL of supernatant was added to Fc and human IL13Rα 2 immobilized wells and incubated for 1 hour with shaking. The IL13Rα 2 binding was detected by adding 25 uL of anti-Flag HRP diluted at 1:5000 in 1 x PBST. In between each step, the plate was washed 3 times with 1 x PBST in a plate washer. The plate was then developed with 20 uL of TMB substrate for 5 mins and stopped by adding 20 uL of 2 N sulfuric acid. The plate was read at OD450 nm Biotek plate reader and the binding and selectivity was analyzed with Excel bar graph. Clones with IL13Rα 2 target binding over human Fc >2-fold were subjected for DNA sequencing. Their binding activities to IL 13Rα 2 are shown in FIGs. 1A and IB. The clones with unique sequences were produced and purified for further characterization.

[0265] C. Anti-IL13Rα 2 VH and scFv Antibody Production in E.coli

[0266] The selective anti-IL 13Rα 2 VH and scFv clones were picked from a glycerol stock plate and grown overnight into a 5 mL culture in a Thomson 24- well plate with a breathable membrane. This culture, and all subsequent cultures described below were grown at 37°C and shaking at 225 RPM in Terrific Broth Complete plus 100 ug / mL carbenicillin and 34 ug / mL chloramphenicol, with 1 :5,000 dilution of antifoam-204 also added, unless specified otherwise. This overnight starter culture was then used to inoculate the larger culture, 1: 100 dilution of starter culture into the designated production culture and grown until OD600 was between 0.5- 0.8. At this point, the culture was induced with a final concentration of IPTG at 0.1mM and incubated over night at 30°C. The following day, the cultures were spun for 30 min at 5,000 x g, to pellet the cells and then the supernatant was filter sterilized through a 0.2 um sterilizing Polyethersulfone (PES) membrane.

[0267] For the purification, 3 uL GE Ni Sepharose Excel resin per 1 mL of filtered supernatant was used. Disposable 10 mL or 20 mL BioRad Econo-Pac columns were used. The resin was equilibrated with at least 20 column volume (CV) buffer A (1xPBS, pH7.4 with extra NaCl added to 500 mM). The filter sterilized supernatant was purified by gravity flow by either controlling the flow to 1 mL / min or was poured over two times, over same packed resin bed. The column was then washed with the following buffers: 10 CV buffer A, 20 CV buffer B (IxPBS, pH7.4 with extra NaCl to 500mM, and 30mM imidazole). The two Detox buffers were used to remove endotoxin as optional step if needed. For 250 mL expression culture purifications, antibody bound column was washed sequentially with 20 CV buffer C (IxPBS pH7.4 with extra NaCl to 500mM, 1% Txl 14), 20 CV buffer D (1x PBS pH7.4 with extra NaCl to 500mM, 1% Tx100 + 0.2% TNBP) and 40 CV buffer E (1xPBS pH7.4 with extra NaCl to 500mM). The protein was eluted with Eluting buffer F (1xPBS pH7.4 with extra NaCl to 500mM, and 500mM imidazole) in a total of six fractions (0.5 CV pre elute, 5 x 1 CV elute). Fractions were run on a Bradford assay (100ul diluted Bradford solution + 10ul sample) and the fractions with bright blue color were pooled. Protein concentration was measured by A280 extension coefficient. SDS-PAGE gel to analyze the purity of the purified antibodies. D. Anti-ILI3Rα 2 VH and scFv Antibody Binding Affinity Determination by ELISA An ELISA assay was developed to determine the EC50 of anti-ILI3Rα 2 antibodies. Briefly, 384 well plate was immobilized with human IL13Rα 2-Fc recombinant protein at final concentration of 2 ug / mL in lx PBS in total volume of 25 uL per well. The plate was incubated overnight at 4°C followed by blocking with 80 uL of superblock per well for 1 hour. Purified anti-IL13Rα 2 VH or scFv antibodies were 2-fold serial titrated from 200 nM. 25 uL of diluted

[0268] VH or scFv antibodies were added to human IL13Rα 2 immobilized wells and incubated for I hour with shaking. The IL 13Rα 2 binding was detected by adding 25 uL of anti-Flag HRP diluted at 1:5000 in 1 x PBST. In between each step, the plate was washed 3 times with 1 x PBST in a plate washer. The plate was then developed with 20 uL of TMB substrate for 5 mins and stopped by adding 20 uL of 2 N sulfuric acid. The plate was read at OD450 nm Biotek plate reader and then plotted in Prism 8. 1 software. EC50 was calculated and showed in the Table 7 below.

[0269] Table 7. EC50 of Purified Human Anti-IL13Rα 2 VH and scFv Clones

[0270] E. VH and ScFv Antibody Binding to IL13Rα 2 Cell Lines by Fluorescence-Activated Cell Sorting (FACS)

[0271] (i) Recombinant and Endogenous Cell line Characterization

[0272] HEK293 cells were transfected with a construct encoding the full-length and splice variant isoform of human IL13Rα 2 sequence with C-terminal flag and Myc tags in pCMV6-Entry vector. G418 drug selection process yielded a polyclonal, drug resistant pool of IL 13Rα 2 target-expressing cells. The IL13Rα 2 target-expressing cells were sorted by FACS to yield a IL13Rα 2 target expressing polyclonal pool. The high expression IL13Rα 2 / HEK293 and splice variant IL13Rα 2 / HEK293 cell lines were then used for selection and screening assays. The IL13Rα 2 expression level of IL13Rα 2 / HEK293 and a panel of IL13Rα 2 expressing cancer cell lines were quantified by FACS using MESF microsphere beads (Bangs Laboratories, Inc., 647) for standard calibration following manufacturer’s protocol. The IL13Rα 2 receptor counts were summarized in Table 8.

[0273] Table 8. Receptor Characterization of Various Cell Lines

[0274] ( ii ) VH and scFv Cell Binding Activities on Two Isoform of Recombinant Cell Lines by FACS

[0275] To determine the binding selectivity and affinity of anti-IL13Rα 2 VHs and scFvs bind to IL13Rα 2 expressing cells, 200 nM of purified VH and scFv antibodies were diluted in full medium and incubated with recombinant IL13Rα 2 / HEK293 cell lines (full length, splice variant) and HEK293 cells in 96 wells plate on ice for 1 hour. Cells were spun down at 1200 rpm for 6 minutes at 4°C to remove primary antibodies. Cells were then washed once with 200 uL of full medium per well. Samples were detected with premixed anti-His Biotin Streptavidin Alexa fluor 647 by adding 100 uL of diluted secondary antibody and incubated at 4°C for 30 minutes in the dark. Samples were spun down at 1200 rpm for 5 minutes at 4°C and washed twice with 200 uL of lx PBS per well. Reconstituted samples in 200 uL of lx PBS are read on Attune NxT cytometer. Analysis was done by Attune NxT software plotting the overlaying the histogram of anti-IL13Rα 2 VHs and scFvs binding onto both negative and target cell lines. The anti-IL13Rα 2 VHs and scFvs showed selective binding to IL13Rα 2 / HEK293 cells and not to HEK293 parental cells (not shown). The full length and isoform IL13Rq2 cell binding affinity for VH and ScFv antibodies was also generated with serial diluted VHs and scFvs as described above. All the anti-IL13Rα 2 scFvs showed binding to isoforms of IL13Rα 2 cell lines with similar affinity (FIGs. 2A and 2B). The EC50 was calculated and showed in the Table 9.

[0276] Table 9. ECso of IL13R«2 VH and scFvs binders to IL13R«2 / HEK293 cell lines

[0277] F. Anti-IL13Rq2 VH and scFv Binding to Endogenous IL 13Rα 2 Expressing Cancer Cell lines

[0278] Cell binding activity with IL13Rq2 expressing cancer cell lines were also assayed by FACS following the method described above. 200 nM of anti-IL13Rα 2VHs or scFvs were tested with negative cell line and cancer lines. The VHs and scFvs demonstrated specific binding with different cell lines and the binding intensity correlated with IL13Rα 2 receptor number on each cell line.

[0279] G. Anti-IL 13Rα 2 VH Antibodies Epitope Binning with IL13 Ligand by Fluorescence- Activated Cell Sorting (FACS)

[0280] 200 nM of purified anti-IL13Rq2 VH antibodies were evaluated binding to IL13 / HEK293 and parental HEK cells in the presence or absence of 1 uM of IL13 ligand for 1 hour at 4°C. Cells were spun down at 1200 rpm for 5 minutes at 4°C. Samples were detected with premixed anti-His Biotin Streptavidin Alexa fluor 647 by adding 100 uL of diluted secondary antibody and incubated at 4°C for 30 minutes in the dark. The samples were spun down at 1200 rpm for 5 minutes at 4°C and washed twice with 200 uL of lx PBS per well. The resultant samples were then reconstituted in 200uL of 1x PBS and read on Attune NxT cytometer. Analysis was done by Attune NxT software and then plotted in Prism 8. 1 software.

[0281] The results of this study show that the IL 13 ligand compete against 2 of the 3 the anti- IL 13Rα 2 VH antibody clones from binding to IL13Rα 2 / HEK293 cells, 1 VH binds to distinct epitope from IL13 ligand The result shown in Table 10.

[0282] Table 10. Competition of IL13Ra VH Binders with IL13 Ligand

[0283] H. Anti-IL 13Rα 2 VH and scFv Epitope Binning by SPR

[0284] To develop biparatopic IL13RaCARs, binding epitopes of anti-IL13Ra VHs and scFvs were evaluated. An epitope binning assay was developed with Biacore T200. Briefly, human IL13Rα -Fc tag protein was immobilized on the Protein A sensor chip FC2 at 1 ug / mL at flow rate of 10 ul / min for 60 seconds. VH1 or scFvl at 300 nM was injected to FC1 and FC2 for 90 sec at flow rate of 30 ul / min to reach binding saturation, followed by injection of 300 nM of VH2 or scFv2 at the same flow rate and time. The data was analyzed with Biacore T200 evaluation software version 3.0. The dual baseline was set for the analysis. The binding response unit was calculated from subtraction of FC1 from FC2. The epitope binning data is summarized in Tables 11.

[0285] Table 11. Competition of IL13Ra VH Binders with IL13Ra scFv Binders

[0286] I. Anti-IL 13Rα 2 VH and scFv Thermostability Studies scFv melting temperature was measured with Protein Thermal ShiftTM Dye (Thermo Fisher, 4461146). The testing VH and scFv sample reactions were prepared according to manufacturer’s instructions. The plate or strips was then put into a Quant Studio 3 instrument with the proceeding method being run. Step 1: 100% ramp rate to 25.0° with time 2 min and finally Step 2: 1% ramp rate to 99.0°C with time 2 min. The samples and subsequent Tm were then analyzed (and Tm calculated) using the QuantStudio Design and Analysis Software and the Protein Thermal Shift Software 1.3. The Tm was summarized in Table 12.

[0287] Table 12. Tm of IL13Ra VH Binders

[0288] Example 2: Chimeric Antigen Receptor (CAR) Conversion and Characterization

[0289] This example reports construction of chimeric antigen receptors using the anti-

[0290] IL13Rα 2 antibodies provided herein and characterization of immune cells expressing the CAR constructs (monospecific or bi-specific), either alone or in combination with exemplary armor polypeptides.

[0291] A. Construction of CAR Constructs and Expression of Such in Host Cells

[0292] (i) CAR Construct

[0293] Anti-IL13Rα 2 VHs (SEQ ID NOs: 64, 66, 68, 72, and 76) and anti-IL13Rα 2 scFv candidates (SEQ ID NOs: 84, 91, 100, 107, 113, 119, 124, and 129) were converted into IL13Rα 2 monospecific CARs. From N terminal orientation of anti-IL13Rα 2 VH or scFv, flag tag, IgG4 hinge, spacer, CD28 transmembrane, 41BB and CD3z intracellular signaling domains. The spacers have variable length as short, medium, long for candidate screening and optimization. For VH CAR, membrane bound EGFRt was added at C terminus through a T2A cleavage linker and a secretion signal. A multi-mechanism bispecific secreted armor comprising of anti-PDLl scFv and engineered IL2 fusion was engineered to the C-terminus of the scFv or VH CARs. In addition, several biparatopic CARs have been engineered based on the epitope binning data. Exemplary CAR constructs and expression cassettes for such, either alone or in combination with armor polypeptides, are shown in FIG. 3. The sequences were cloned into lend virus vectors following standard molecular biology methods.

[0294] (ii) Lentivirus Production and Characterization

[0295] Lentiviral vectors were co-transfected with LV-MAX packaging mix using polyethylenimine (PEI) transfection reagents to Expi HEK293 following manufacture’ s protocol. Transfected cells were grown for 72 hrs at 37 °C shaking with 8% CO2level. Supernatant were harvested by centrifugation at 3200 rpm at RT for 10 mins and vacuum filtration using 0.45um PES membrane. Virus was concentrated by ultracentrifugation (Beckman Coultier) at 12,000 rpm for Ihr at 4C. The pellet was then resuspended in Lentivirus stabilizer, aliquoted immediately and stored at -80°C.

[0296] (iii) IL13Ra2 Mono-specific CAR Transduction and Expansion

[0297] PBMCs were isolated from fresh healthy donor’s LRS chamber using density gradient centrifugation lymphoprep and SepMate 50 kit from Stemcell Technology. CD3+ Pan T cells were then isolated from PBMCs using EasySep human T cell isolation kit following Stemcell technology protocols. Pan T cells were activated with human T-activator CD3 / CD28 dynabeads at 1 : 1 bead to cell ratio 24 hours and then transduced with lentivirus in the presence of dynabeads and Img / mL protamine sulfate. Spinoculation was done at 300g for 2 hours at 25 °C. Cells and viruses were incubated for 24 hours at 37 °C. Next day, cells were removed from beads and viruses. Cells were grown for 4-9 days in 5% human serum containing recombinant human IL15 and IL7 (Peprotech) in X-vivo 15 (Lonza) media. Media were changed every 2-3 days with added fresh cytokines.

[0298] B. Anti-ILl 3Rα 2 Mono-Specific CAR Characterization

[0299] (i) Expression of Anti-ILl 3Ra2 CAR Constructs in T Cells

[0300] CAR surface expression was assessed by surface staining of anti-flag tag antibody directly conjugated with Mix-n-stain AF647. Briefly, 100,000 lentivirus transduced T cells were incubated with 0.1 ul of anti-flag- AF647 for 1 hour in dark at 4°C shaking. Cells were spun down at 1 ,300rpm for 5 minutes, supernatant removed and washed with 200uL 1x PBS. The resultant samples were reconstituted in 200uL of lx PBS. The percentage surface expression was quantified by reading the fluorescence-stained cells on Attune NxT Flow Cytometer. CAR-expression of different CAR constructs ranged from 50-85%.

[0301] (ii) Anti-ILl 3Ra2 VH CAR Activity Analysis

[0302] Human PBMCs and Pan T cell isolation, virus transduction and T cell expansion were described above. VH CARs with different spacers have been created. To screen different CAR activity, real time image-based CTL activity assay was performed with a glioblastoma cell line U87 MG engineered with GFP. Briefly, CAR transduced T cells and same donor un-transduced T cells were incubated with 10,000 U87MG-GFP respectively at effector (CAR-T) to target cell (cancer cell line) ratio of 10:1 in RPMI1640 media with 10% FBS. No cytokines were added. The assay was run for 42 hours and GFP of target cells was imaged and quantified by Cytation 5 scanner. The data was further analyzed by Prism 8 software.

[0303] The CTL activity with different sequence and spacer of VH CAR-Ts and the end point of percentage of target cell killing by different CAR-Ts were calculated.

[0304] (iii) IL13Ra2 scFv CAR Activity Screening

[0305] IL13Rα 2 scFv CAR-Ts were produced, CTL activity, cytokine screening was performed as described above with multiple donors. EPLV167 and EPLV168 (see Table 5 above) were used in this study as examples.

[0306] IL13Rα 2 scFv CAR-Ts showed robust surface expression, all constructs showed >80% expression. CAR-T cells were co-incubated with GFP engineered HEK293, recombinant IL13Rα 2 / HEK293(full length and splice variant), U87MG and U251MG that expressing different IL13Rα 2 receptor number at the effector to target cell ratio of 10: 1, 5:1, and 2.5: 1 for 64 hours. Un-transduced T cells from the same donor was used as control. The potent and specific CAR-T mediated CTL activities were observed at all tested E:T ratios. The activity is correlated with IL13Rα 2 receptor number.

[0307] (iv) scFv CAR-T Cell Proliferation Upon Target Cell Engagement

[0308] To further test the CAR-T cell expansion upon target cell engagement, 3 day proliferation assay was performed using EPLV167 and EPLV168 as examples. The transduced T cells were labelled with Cell Trace Far Red at final concentration of luM. 20,000 labeled T cells were co-cultured with 20,000 of HEK293, IL13Rα 2 / HEK293(full length and splice variant), U87MG, and U251MG target cells at E:T ratio 1 :1 respectively. The assay was set up with RPMI media with 10% FBS and fresh media was added to cells every two days. No cytokine added to the media during the assay. The CAR-T proliferation was analyzed on Attune NxT Flow Cytometer. The CAR-T cells demonstrated target cell specific expansion upon engagement over 3 days and the fold of proliferation correlated with target expression level on cells (FIGs. 4A and 4B). CAR-T activity were also confirmed by evaluation of cytokine release. IFNy was detected with Human IFNγ Duoset ELISA kit (R&D System). Recombinant IFNy was serial diluted and included in the assay to create standard curve. Supernatant IFNγ and recombinant IFNγ were assayed following the manufacture’s protocol provided. The data was analyzed using Prism 8.0 software. The supernatant IFNγ secreted by CAR-Ts upon target cell engagement confirmed the activities observed in CTL assays (FIGs. 4C and 4D).

[0309] (v) EPLV167 VH / scFv CAR characterization

[0310] Anti-IL13Rα 2 VHs / scFvs demonstrated broad binding epitopes to IL13Rα 2.

[0311] Expressions of IL13Rα 2 CAR are provided in Table 13 below.

[0312] Table 13. Anti-IL13R«2 Mono CAR Expression

[0313] The CAR-T cells were co-incubated with GFP engineered HEK293, IL13Rα 2 / HEK293, U251 and U87 MG target cells. Then, CTL activity (FIGs. 5A, 5C, and 5E) and IFNγ (FIGs. 5B, 5D, and 5F) were evaluated.

[0314] C. Anti-B7H3 Mono CAR Characterization

[0315] (i) EPLV195 scEv CAR Characterization

[0316] To further characterize the B7H3 CAR candidate, EPLV195 expression, CTL activity and cytokine release was tested in multiple donors and multiple target and control cell lines with different effector to target cell ratio as described. Expressions of B7H3 CAR are provided in Table 14 below.

[0317] Table 14. B7H3 mono CAR expression

[0318] EPLV195 has been confirmed to have robust CAR-T surface expression, potent and specific target cell killing activities, and cytokine release, FIGs. 6A-6E as an example from one donor. ( ii ) EPLV195 scFv CAR Spacer Optimization and in vitro Persistence Assessment with Multiple Rounds of Cancer Cell Rechallenging

[0319] It is known that the scFv binding epitope and spacer between scFv binding domain and transmembrane domain is important to provide the optimal immune synapse between immune cells and tumor cells and to mediate the potent anti-tumor activity. Therefore short, medium and long spacers were engineered with the candidate scFv CAR. CAR-T cells were produced and incubated with A375-GFP cell line at effector to target cell ratio of 2.5: 1 and 1 :1 for 72 hours without additional cytokine. The CAR-T cells were rechallenged twice with more A375 cancer cells and incubated for additional 72 hours at each rechallenging without adding in more CAR-T cells and cytokine.

[0320] CAR-T cells with short, medium long spacers showed similar cell killing activity at initial 72 hours of the assay. At first rechallenging assay, CAR-T with short and medium spacer showed better CTL activity than the CAR-T with long spacer. The CAR-T with short and medium spacer showed similar potent and persistent CTL activity.

[0321] D. Analyzing Mono-Specific CAR-T Cells and Bispecific CAR-T Cells

[0322] Expression of monospecific anti-IL13Rα 2 CAR-T cells (expressing construct

[0323] EPLV167), monospecific anti-B7H3 CAR-T cells (expressing construct EPLV195) and bispecific CAR-T cells (expressing construct EPLV305 or construct EPLV306) are provided in Table 15 below.

[0324] Table 15. Expression Levels of CAR Constructs in Donor Cells

[0325] Cytotoxicity assay was performed in CHOK1 and U87MG cells with different E / T ratios (FIGs. 7A-7C). (EPLV306: IL13Ra-B7H3 Bispecific CAR; EPLV167: IL13Rα mono CAR; EPLV326: B7H3 mono CAR; EPLV329: TL13Rα 2-B7H3 Bispecific armored CAR; UT: non-transduced). The in vitro activities of the candidate CAR constructs were examined in the in the U87 cancer cell rechallenging CTL assay. In a five-rounds of killing and rechallenging assay, no significant difference was observed at the at the effector to target cell ratio of 2.5:1 with respect to cytotoxicity, indicating persistence of the CAR-T cells. (FIG. 8).

[0326] Example 3: CAR-T Cells Expressing Both CAR and Armor Polypeptide

[0327] This example explores bioactivities of immune cells expressing both a CAR polypeptide and a multi-mechanism armor polypeptide of SEQ ID NO: 179, which comprises an anti-PDLl scFv and an engineered IL2 polypeptide. The anti-PDL1 scFv can block PDL1 immune checkpoint and localizes the armor polypeptide to tumor cells, while the engineered IL2 portion can significantly reduce IL2Ra binding activity and suppress Treg cell activation. In addition, IL2 binding to IL2Rb / g was also fine-tuned by the armor polypeptide to selectively activate central memory T cells and with slower kinetics. Upon activation, CAR-T cells transduced with a coding sequence for the armor polypeptide secretes the armor polypeptide to activate CAR-T cells and other immune cells, thereby improving the potency and persistence via, e.g., overcoming suppressive tumor microenvironment.

[0328] A. CAR-T Cells Expressing Anti-B7H3 Monospecific scFv CAR and Armor Polypeptide secrets functional armor

[0329] An expression cassette comprising, from 5’ to 3’, a coding sequence for the anti-B7H3 monoclonal scFv CAR (anti-B7H3 CAR EPLV326, comprising the amino acid sequence of SEQ ID NO: 155), a coding sequence of the T2A cleavable linker, and a nucleotide sequence encoding a signal peptide and the armor polypeptide of SEQ ID NO: 179 was constructed for expressing both the CAR and the armor polypeptide in host immune cells. With the interim signal peptide, the armor polypeptide is secreted by the CAR-T cells to the surrounding environment.

[0330] To test the function of secreted armor, the supernatant of CAR-T with and without armor was also tested on binding to PDL1 / K562 cells. The supernatant was collected from Day 4-7 and anti-PLl activity was evaluated with PDL1 / K562 cell line. The supernatant from armored CAR-T showed binding to target cells, time course dependent with supernatant collected, Figure 10.

[0331] B. Bispecific CAR Activity Assessment Against Mono-target Expressing Cancer Cells To evaluate the bispecific targeting CAR in overcoming target escape mechanism,

[0332] Bispecific CAR-T cells expressing EPLV306 without armor and armored CAR-T cells expressing construct EPLV329, which produces bi-specific CAR of EPLV306 and the armor polypeptide of SEQ ID NO: 179 were produced and CTL assay was set up with cancer cell lines BxPC-3 cells or T47D cells that only express B7H3 (IL13Ra negative). Both bispecific CARs with and without armor demonstrated similar and potent CTL activity with Cell lines only expressing B7H3 (Figure 9).

[0333] The two binding moieties in the anti-B7H3 / IL13Rα 2 bispecific CAR provided herein can act in synergism to enhance cancer engagement and CAR-T cell activation. Further, CAR- T cells expressing the bispecific CAR can be activated through binding of one binding moiety to the cognate target antigen, even when the target cancer cells lose the other target antigen. As such, the bispecific CAR is expected to be more effective relative to the corresponding monospecific CARs in eliminating target disease cells expressing one or both target antigens. Even when the target disease cells lose both target antigens, the armor polypeptide secreted by the CAR-T cells can stimulate bystander immune cells and central memory cells, which can mediate killing of the target disease cells such as cancer cells. Accordingly, the CAR-T cells expressing a bispecific CAR as provided herein are expected to exhibit enhanced therapeutic effects and persistence in vivo.

[0334] Example 4: In Vivo Efficacy Study with Orthortopic U87MG glioblastoma model

[0335] This example explores in vivo efficacy of CAR-T cells transfected with construct

[0336] EPLV329, which provides the anti-IL13Rα 2 / B7H3bispecific CAR EPLV306 and the armor polypeptide of SEQ ID NO: 179 (as an example) in an animal model for glioblastoma (GBM).

[0337] A. IL-13Rα 2 and B7H3 as Treatment Targets for Glioblastoma

[0338] Bioinformatic analysis of 155 patients’ RNA sequences from TCGA data base showed patients are co-expressing both B7H3 and IL13Rα 2 (>72% GBM) (FIG. 11), suggesting that targeting B7H3 and lL13Rα 2, for example, by CAR-T cells expressing the bispecific CARs provided herein, could benefit treatment of GBM. As shown in FIG. 12, CAR-T cells expressing anti-B7H3 / IL13Rα 2 bispecific VH-VH CAR exhibited superior cytotoxicity against several GBM cell lines.

[0339] B. In Vivo Efficacy of CAR-T Cells Expressing Anti-IL13Rα 2 / B7H3 Bispecific CAR To evaluate the anti-tumor activity of CAR-T cells (armored or non-armored), expressing the bi-specific CAR of SEQ ID NO: 175 and optionally the armor polypeptide of SEQ ID NO: 179, 6-8 week old female NCG mice (Charles River Laboratories, Wilmington, MA) were inoculated subcutaneously on the right hind flank with 5 x 106A375 human melanoma tumor cells suspended in 1 : 1 serum free media and Matrigel (Corning, Glendale, Arizona). When tumors reached an average volume of -120 mm3, mice were randomized into 3 treatment groups (day 1). Treatment groups (n = 5 mice / group) were as follows: (1) vehicle, (2) CAR-T cells (2E6 CAR-T cell dose; i.c.), and (3) armored CAR-T cells (9E6 CAR-T cell dose; i.v). CAR-T cells were administered by intravenous injection on day 1 and then on day 10. Tumor volumes and mouse body weights were recorded twice per week and plotted against time for each group. All animals treated with armored CAR-T cells demonstrated complete tumor regressions 10 days after their first treatment with the CAR-T cells (FIG. 13A). No body weight loss observed and no clinical sign of toxicity observed (FIG. 13B).

[0340] These animals were subsequently rechallenged with 5 x 106A375 tumor cells inoculated subcutaneously in the left hind flank 30 days after their first CAR-T treatment, but without additional CAR-T treatment. Five naive age-matched female NCG mice were inoculated with A375 tumor cells at the same time to serve as a control cohort. Once tumors were palpable, tumor volumes and body weights were recorded twice per week. While control treated mice showed tumor growth, the armored CAR-T demonstrated persistent anti-tumor activity.

[0341] CAR-T cell phenotype is associated with the persistence of the anti-tumor activity. Blood was collected from mice at day 35 of CAR-T treatment. T cell and CAR-T cell phenotype was analyzed using FACS assay with a panel of antibodies detecting T cell differentiation markers. Briefly, anti-CD3, anti-CD4, anti-CD8 were used to stain the transduced T cells as described above. Analysis was done by Attune NxT software. The CD3, CD4, CDS positive CAR-T cells and Tn, Tscm, Tem and Tern cells were gated.

[0342] At day 35, in armored CAR-T treatment groups, dose dependent expansion of total T cells, CDS T cells and armored CAR-T cells have been observed (FIGs. 14A-14C).

[0343] Immunophenotyping of CAR-T cells were assessed and majority are TEM cells in PBMCs (FIGs. 15A and 15B). Also, the CAR-T cells demonstrated robust CD3+, CD8+, CD4+ T cells

[0344] (FIGs. 16A-16C). Furthermore, immunophenotyping of T cells analysis concluded that most of the T cells are TEFF and TEM cells (FIGs. 17A and 17B).

[0345] OTHER EMBODIMENTS

[0346] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0347] EQUIVALENTS

[0348] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0349] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0350] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0351] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0352] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or’- should be construed in the same fashion, i.e., “one or more’- of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0353] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0354] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0355] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

1. WHAT IS CLAIMED IS:

1. A chimeric antigen receptor (CAR) polypeptide, comprising:(a) an extracellular antigen binding domain, which comprises a first antigen-binding fragment specific to interleukin- 13 receptor subunit alpha- 2 (IL-13Rα 2);(b) a co-stimulatory signaling domain; and(c) a cytoplasmic signaling domain; wherein the first antigen binding fragment is a heavy chain antibody, which comprises the same heavy chain complementarity determining regions (CDRs) as a reference antibody of 13R-03, 13R-01, 13R-02, 13R-05, or 13R-04 set forth in Table 2, or wherein the first antigen binding fragment is a single chain variable fragment (scFv) comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as a reference antibody of 13R-06, 13R-07, 13R-10, 13R-08, 13R-09, I3R-10, 13R-ll, or 13R-13 set forth in Table 2.

2. The CAR polypeptide of claim 1, wherein the first antigen binding fragment is an scFv comprising the VH and the VL, which comprises the same heavy chain and light chain CDRs respectively as the reference antibody of 13R-06 or 13R-07.

3. The CAR polypeptide of claim 1, wherein the first antigen binding fragment is the heavy chain antibody, which comprises the amino acid sequence of any one of SEQ ID NOs: 64, 66, 68, 72, and 76.

4. The CAR polypeptide of claim 1 , wherein the first antigen binding fragment is the scFv, which comprises:(i) a VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL comprising the amino acid sequence of SEQ ID NO: 83:(ii) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 90;(iii) a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 99;(iv) a VH comprising the amino acid sequence of SEQ ID NO: 102 and a VL comprisingthe amino acid sequence of SEQ ID NO: 106;(v) a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 112;(vi) a VH comprising the amino acid sequence of SEQ ID NO: 114 and a VL comprising the amino acid sequence of SEQ ID NO: 118;(vii) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 123; or(viii) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 128.

5. The CAR polypeptide of claim 4, wherein the scFv comprises the amino acid sequence of any one of SEQ ID NOs: 84, 91, 100, 107, 113, 119, 124, and 129; optionally wherein the scFv comprises the amino acid sequence of SEQ ID NO: 84.

6. The CAR polypeptide of any one of claims 1-5, wherein the extracellular antigen binding domain of (a) further comprises a second antigen-binding fragment specific to B7 homolog 3 protein (B7H3).

7. The CAR polypeptide of claim 6, wherein the second antigen-binding fragment is a heavy chain antibody or an scFv derived from a reference antibody set forth in Table 1.

8. The CAR polypeptide of claim 7, wherein the second antigen-binding fragment is a single chain variable fragment (scFv), which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as reference antibody BH-07 set forth in Table 1.

9. The CAR polypeptide of claim 8, wherein the scFv comprises the VH comprising the amino acid sequence of SEQ ID NO: 40 and the VL comprising the amino acid sequence of SEQ ID NO: 44.

10. The CAR polypeptide of claim 9, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 45.

11. The CAR polypeptide of any one of claims 1-10, wherein the co- stimulatorydomain of (b) is from a co- stimulatory molecule selected from the group consisting of CD28, 4- 1BB, OX40, ICOS, CD27, CD40, and CD40L; optionally wherein the co-stimulatory domain is from 4-1BB.

12. The CAR polypeptide of any one of claims 1-11, wherein the cytoplasmic signaling domain of (c) is from CD3ζ.

13. The CAR polypeptide of any one of claims 1-12, which further comprises a hinge domain, a transmembrane domain, or a combination thereof; wherein the hinge domain and / or the transmembrane domain optionally is located between the extracellular antigen binding moiety of (a) and the co-stimulatory domain of (b).

14. The CAR polypeptide of claim 13, which further comprises a spacer located between the hinge domain and the transmembrane domain; optionally wherein the spacer comprises the amino acid sequence of SEQ ID NO: 136 or 137.

15. The CAR polypeptide of any one of claims 1-14, which further comprises a signal peptide located at the N-terminus of the polypeptide.

16. The CAR polypeptide of any one of claims 1-5 and 11-15, which is a monospecific CAR that binds IL-13Rα 2.

17. The CAR polypeptide of claim 16, which comprises the amino acid sequence of any one of SEQ ID NOs: 147, 148, 149, and 150.

18. The CAR polypeptide of any one of claims 6-15, which is a bi-specific CAR that binds both IL-13Rα 2 and B7H3.

19. The CAR polypeptide of claim 18, which comprises the amino acid sequence of any one of SEQ ID NOs: 173, 174, 175, and 176.

20. A nucleic acid or a set of nucleic acids, comprising a first nucleotide sequence encoding a CAR polypeptide set forth in any one of claims 1-19.

21. The nucleic acid or the set of nucleic acids of claim 20, further comprising a second nucleotide sequence encoding an armor polypeptide, which enhances T cell functionality.

22. The nucleic acid or the set of nucleic acids of claim 21, wherein the second nucleotide sequence further encodes a signal peptide located at the N-terminus of the armor polypeptide.

23. The nucleic acid or the set of nucleic acids of claim 21 or claim 22, wherein the armor polypeptide is selected from the group consisting of IL-2, IL-15, IL7, IL12, IL21, a costimulatory ligand, an anti-PDL1 antibody, and a fusion polypeptide comprising the anti-PDLl antibody; optionally wherein the anti-PDL1 antibody is a single chain variable fragment (scFv).

24. The nucleic acid or the set of nucleic acids of claim 21 or claim 22, wherein the armor polypeptide is a fusion polypeptide comprising an anti-PDL1 antibody and an IL-2 polypeptide, the anti-PDL1 antibody being an scFv fragment, which optionally comprises the amino acid sequence of SEQ ID NO: 177.

25. The nucleic acid or the set of nucleic acids of claim 24, wherein the armor polypeptide comprises the amino acid sequence of SEQ ID NO: 179.

26. The nucleic acid or the set of nucleic acids of any one of claims 20-25, wherein the first nucleotide sequence and the second nucleotide sequence are located on two separate nucleic acids, which optionally are vectors, preferably expression vectors.

27. The nucleic acid or the set of nucleic acids of any one of claims 20-25, wherein the first nucleotide sequence and the second nucleotide sequence are located on one nucleic acid, and wherein the nucleic acid further comprises a third nucleotide sequence encoding a self-cleaving peptide, which is located between the first and second nucleotide sequences.

28. The nucleic acid or the set of nucleic acids of claim 27, wherein the nucleic acid is a vector, optionally an expression vector.

29. A population of immune cells, comprising genetically engineered immune cells expressing a CAR polypeptide set forth in any one of claims 1-19.

30. The population of immune cells of claim 29, wherein the genetically engineered immune cells further express an armor polypeptide, which optionally is set forth in any one of claims 21-25.

31. The population of immune cells of claim 30, wherein the genetically engineered immune cells secrete the armor polypeptide.

32. The population of immune cells of any one of claims 29-31 , wherein the genetically engineered immune cells comprise the nucleic acid or the set of nucleic acids of any one of claims 20-28.

33. The population of immune cells of any one of claims 29-32, wherein the genetically engineered immune cells comprise T cells, NK cells, macrophages, or a combination thereof; optionally wherein the immune cells comprise T cells.

34. A method for eliminating IL-13Rα 2-expressing disease cells in a subject, the method comprising administering to a subject in need thereof an effective amount of the population of immune cells set forth in any one of claims 29-34.

35. The method of claim 34, wherein the subject is a human patient having both IL- 13Ra2 positive and optionally B7H3 positive disease cells.

36. The method of claim 34 or claim 35, wherein the subject is a human patient having the disease cells, which are cancer cells.

37. The method of claim 36, wherein the human patient has glioblastoma, neuroblastoma, lung cancer, melanoma, head and neck cancer, urothelial cancer, prostate cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, esophageal cancer, ovarian cancer, breast cancer, bladder cancer, or acute myeloid leukemia (AML).

38. The method of claim 37, wherein the immune cells are administered to the subject by intracavitary delivery, intravenous infusion, or a combination thereof.

39. A method for treating a solid tumor in a subject, the method comprising administering to a subject in need thereof an effective amount of the population of immune cells set forth in any one of claims 29-34; wherein the solid tumor comprises tumor cells expressing IL13Rα 2 and / or B7H3.

40. The method of claim 39, wherein the subject is a human patient having glioblastoma, neuroblastoma, lung cancer, melanoma, head and neck cancer, urothelial cancer, prostate cancer, colorectal cancer, pancreatic cancer, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, esophageal cancer, ovarian cancer, breast cancer, bladder cancer, or acute myeloid leukemia (AML).

41. The method of any one of claims 34-40, wherein the population of immune cells is autologous to the subject.

42. The method of any one of claims 34-40, wherein the population of immune cells is allogenic to the subject.

43. A method for producing genetically engineered immune cells, comprising: transfecting into a population of immune cells the nucleic acid or the set of nucleic acids set forth in any one of claims 20-28 to produce genetically engineered immune cells expressing the CAR polypeptide and optionally the armor polypeptide encoded by the nucleic acid(s).

44. An antibody that binds interleukin- 13 receptor subunit alpha-2 (IL-13Rα 2)(anti-IL- 13Rα 2 antibody), the anti-IL- 13Rα 2 antibody comprising:(a) a human heavy chain antibody comprising the same heavy chain complementary determining regions (CDRs) as a reference antibody of 13R-03, 13R- 01, 13R-02, 13R-05, or 13R-04 set forth in Table 2; or(b) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL comprises the same heavy chain and light chain CDRs as a reference antibody of 13R-06, 13R-07, 13R-10, 13R-08, 13R-09, 13R-10, 13R-ll, or13R-13 set forth in Table 2.

45. The anti-IL-13Rα 2 antibody of claim 44, wherein the antibody comprises the human heavy chain antibody of (a), which optionally comprises the amino acid sequence of SEQ ID NO: 64, 66, 68, 72, or 76.

46. The anti-IL- 13Rα 2 antibody of claim 44, wherein the antibody comprises the VH and VL of (b), and wherein:(i) a VH comprising the amino acid sequence of SEQ ID NO: 80 and a VL comprising the amino acid sequence of SEQ ID NO: 83;(j) a VH comprising the amino acid sequence of SEQ ID NO: 86 and a VL comprising the amino acid sequence of SEQ ID NO: 90;(k) a VH comprising the amino acid sequence of SEQ ID NO: 95 and a VL comprising the amino acid sequence of SEQ ID NO: 99;(l) a VH comprising the amino acid sequence of SEQ ID NO: 102 and a VL comprising the amino acid sequence of SEQ ID NO: 106;(m)a VH comprising the amino acid sequence of SEQ ID NO: 108 and a VL comprising the amino acid sequence of SEQ ID NO: 112;(n) a VH comprising the amino acid sequence of SEQ ID NO: 114 and a VL comprising the amino acid sequence of SEQ ID NO: 118;(o) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 123; or(p) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 128.

47. A nucleic acid or a set of nucleic acid, which collectively encodes the anti-IL-13Rα 2 antibody of any one of claims 43-46.

Images