Peptide-centered chimeric antigen receptors for cancer self-peptides

Abstract

Peptides derived from proteins essential for tumorigenesis, e.g., HLA-A, derived from PHOX2B, a neuroblastoma dependency gene and master transcription factor * The non-mutated peptide QYNPIRTTF (SEQ ID NO:1) discovered on 24:02 is enriched in the neuroblastoma immunopeptidome. To target QYNPIRTTF, we developed a peptide-centric chimeric antigen receptor (PC-CAR) via a counter-panning strategy using peptides with predicted potential cross-reactivity. Based on information from computational modeling, the PHOX2B peptide-centric CAR binds to HLA-A. * 23:01 and highly divergent HLA-B * They were also shown to recognize QYNPIRTTF (SEQ ID NO:1), presented by 14:02. Potent and specific killing of neuroblastoma cells expressing these HEAs in vitro was demonstrated, along with complete tumor regression in mice. TIFF2024532258000023.tif79166

Description

[Technical field]

[0001] Statement regarding federally funded research This invention was made with Government support under Grants U54 CA232568 and R35 CA220500 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] Priority claim This application claims priority to U.S. Provisional Application No. 63,236,556, filed August 24, 2021, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing Reference This application contains an electronically submitted Sequence Listing XML, which is incorporated herein by reference in its entirety. This XML Sequence Listing, created on August 23, 2022, is named CHOPP0049WO.xml and is 68,758 bytes in size.

[0004] Field The present disclosure relates generally to binding agents and methods of use thereof for the diagnosis and / or treatment of cancer. In particular, the present disclosure relates to binding agents capable of specifically binding to peptide:MHC complexes presenting peptides derived from human PHOX2B. [Background technology]

[0005] background In humans, the major histocompatibility (MHC) system, also known as human leukocyte antigen (HLA), presents peptide antigens to T cells. Recognition by T cells of specific peptide:MHC (pMHC) complexes is mediated by the T cell receptor (TCR). This recognition contributes to antigen-specific expansion of T cells and other immunological effects. From a biomedical perspective, pMHC complexes can be markers of disease states and targets for immunotherapy. It has proven difficult to generate binders that recognize pMHC complexes by traditional methods. One reason for this difficulty is the nature of the epitope. A pMHC-specific binder must recognize structural features of the peptide antigen as well as structural features of the MHC molecule. A peptide antigen presented by a typical pMHC is only 9-12 residues long, and some of these residues are buried within the pMHC complex. Thus, the number of surface-accessible residues that can freely interact with the binder is limited. A typical antibody epitope contains a larger number of amino acid residues than a single peptide antigen would provide. In order for a binder to achieve specific binding to a pMHC complex, the epitope must be smaller than typically required for specific binding, or the epitope must encompass a portion of the MHC molecule in addition to the peptide. In other words, an antibody that binds only to a peptide without extending the epitope to the MHC molecule will in most cases lack the affinity required for a useful pMHC binder. Thus, the epitope for a pMHC binder will in most cases span the MHC molecule.

[0006] However, if the interaction of the binding agent with the pMHC complex is too dependent on the interaction with the MHC molecule, the binding agent will non-specifically bind to pMHC complexes presenting non-target peptide antigens. Ultimately, a binding agent that binds to MHC independent of the peptide presented will not be able to distinguish MHC complexes presenting other peptides from the pMHC complex of interest.

[0007] From a theoretical standpoint, native TCRs should overcome this specificity problem and prove useful as binders for pMHC complexes. However, in practice this has proven not to be the case. Native TCR receptors have been cloned from epitope-reactive T cell populations. Studies of these cloned TCRs have shown that they have surprisingly low affinity, typically micromolar (μM) dissociation constants, for their cognate pMHC complexes. Also, native TCRs rarely have the binding specificity required for practical use as binders for pMHC complexes; a single TCR can recognize many different epitopes, and selectivity within the immune system is achieved by deletion of autoreactive T cells.

[0008] In contrast to TCRs, antibodies generally have a much higher affinity for their targets, but efforts to generate antibodies to pMHC complexes have similarly failed. Traditional techniques for antibody discovery, i.e., animal vaccination or library screening, are rarely successful in generating pMHC binders with useful binding characteristics.

[0009] Neuroblastoma is a childhood cancer that originates in tissue of the developing sympathetic nervous system and is often fatal despite intensive cytotoxic therapy. 16 These tumors have a high mutational burden. 17-21 and MHC expression 22,23 Neuroblastoma has low MHC expression and is therefore a difficult tumor to target by MHC-based immunotherapy. Previous studies have shown that PHOX2B is highly and specifically expressed in neuroblastoma tissues. 83 , identified as mutated in familial cases of the disease. 84,85 .

[0010] Consistent with a function in orchestrating neural crest precursor development, PHOX2B 48,49PHOX2B is expressed exclusively during fetal development and is completely silenced prenatally in normal tissues. PHOX2B expression is routinely used in diagnostic assays for neuroblastoma. 51,52 , one of two highly penetrant susceptibility genes for neuroblastoma 53 , the third most important dependency in neuroblastoma as reported in the DepMap 3,54 Taken together, we propose that PHOX2B is a highly specific tumor antigen in neuroblastoma and an ideal candidate for therapeutic targeting.

[0011] Patent WO2019 / 178081 analyzed the immunopeptidome (the repertoire of peptides presented as peptide-MHC complexes) of 16 neuroblastoma tumors and determined a large number of peptides presented by various human peptide-MHC alleles, among which were HLA-A * The PHOX2B-derived peptide QYNPIRTTF (SEQ ID NO:1) presented by the 24:02 subtype was included.

[0012] This HLA-A * 24:02 Binding agents specific for the PHOX2B peptide complex fill a large unmet need in the treatment and diagnosis of neuroblastoma in pediatric patients. Summary of the Invention

[0013] overview The present disclosure relates to an HLA PHOX2B polypeptide comprising a peptide having the sequence QYNPIRTTF (SEQ ID NO:1), an HLA alpha chain polypeptide, and a beta2 microglobulin polypeptide. QYNPIRTTF A binding agent that comprises an antigen-binding site that specifically binds to the complex. The antigen-binding site has a dissociation constant (K D ) and HLA PHOX2B QYNPIRTTFThe binding agent may be non-MHC restricted. The antigen binding site may be HLA-A * 24:02, HLA-A * 23:01, HLA-B * 14:02, HLA-C * 07:01, HLA-C * 06:02, HLA-A * 29:02, and HLA-A * HLA PHOX2B presented by 2 or more, 3 or more, or 4 or more of 32:01 QYNPIRTTF It can bind to the complex.

[0014] The antigen binding site is (a) a CDR-L1 region as set forth in SEQ ID NO:6, a CDR-L2 region as set forth in SEQ ID NO:7, and a CDR-L3 region as set forth in SEQ ID NO:8; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO:9, a CDR-H2 region as set forth in SEQ ID NO:10, and a CDR-H3 region as set forth in SEQ ID NO:11; H ; or (a) a CDR-L1 region as set forth in SEQ ID NO: 15, a CDR-L2 region as set forth in SEQ ID NO: 16, and a CDR-L3 region as set forth in SEQ ID NO: 17; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 18, a CDR-H2 region as set forth in SEQ ID NO: 19, and a CDR-H3 region as set forth in SEQ ID NO: 20; H ; or (a) a CDR-L1 region as set forth in SEQ ID NO: 26, a CDR-L2 region as set forth in SEQ ID NO: 27, and a CDR-L3 region as set forth in SEQ ID NO: 28; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 29, a CDR-H2 region as set forth in SEQ ID NO: 30, and a CDR-H3 region as set forth in SEQ ID NO: 31; H ; or (a) a CDR-L1 region as set forth in SEQ ID NO: 35, a CDR-L2 region as set forth in SEQ ID NO: 36, and a CDR-L3 region as set forth in SEQ ID NO: 37; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 38, a CDR-H2 region as set forth in SEQ ID NO: 39, and a CDR-H3 region as set forth in SEQ ID NO: 40; H ; or (a) a CDR-L1 region as set forth in SEQ ID NO: 44, a CDR-L2 region as set forth in SEQ ID NO: 45, and a CDR-L3 region as set forth in SEQ ID NO: 46; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 47, a CDR-H2 region as set forth in SEQ ID NO: 48, and a CDR-H3 region as set forth in SEQ ID NO: 49; H may include.

[0015] The binder is V H and V L where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:5, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:4; or the binding agent is H and V L where V Hhas at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:14, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:13; or the binding agent is H and V L where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:24; or the binding agent is H and V L where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:34, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:33; or the binding agent is H and V L where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:43, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:42.

[0016] The binding agent may be an antibody. H and V L where V H is V LThe binding agent may be selected from the group consisting of mAb, Fab, Fab', F(ab')2, Fv, Dab single chain antibody, scFv, chimeric antigen receptor (CAR), ADC, killer Ig-like receptor (KIR), BiTE, BsMAb, and TFP, and may be, for example, a single chain variable fragment (scFv) or a modular bispecific T cell-like inducer (BiTE). The intracellular signaling domain may be CD3ε, CD3γ, CD3δ, TCRα, TCRβ.

[0017] Also provided is an isolated polynucleotide comprising a nucleic acid sequence encoding a binding agent described herein, or an expression vector comprising such a polynucleotide operably linked to a cis-acting regulatory element.Also provided is a cell comprising such a polynucleotide or expression vector.Also provided is a pharmaceutical composition comprising the binding agent, isolated polynucleotide, expression vector, or cell.

[0018] In another embodiment, the binding agent is HLA PHOX2B QYNPIRTTF A method of detecting a cancer cell is provided, comprising contacting the cell with a binding agent described herein under conditions that permit binding to a complex, wherein the binding agent and HLA PHOX2B QYNPIRTTF The binding or level of the complex is indicative of cancer cells.In yet another embodiment, a method for diagnosing and treating cancer in a subject in need thereof is provided, comprising: (a) detecting the presence of cancer cells in the subject by such method, and diagnosing the subject as having cancer when cancer cells are detected; and optionally treating the subject with anti-cancer therapy.Cancer cells can be neuroblastoma.

[0019] A further aspect provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a binding agent, isolated polynucleotide, vector, cell, or pharmaceutical composition described herein. The cancer may be neuroblastoma.

[0020] In the claims and / or this specification, when used in conjunction with the term "comprising," the use of the words "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The word "about" means the recited number plus or minus 5%.

[0021] It is contemplated that any method or composition described herein may be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] [Figure 1]Kinetic analysis of clones 10 and 302. The figure shows the association and dissociation of scFv of clone 10 or clone 302 with soluble PHOX2B(43-51)A*24:02 complex as measured by biolayer interferometry (BLI). Binding to PHOX2B(43-51) complex (200 nM) is overlaid with a non-specific complex (3 x 200 nM) control, showing specific binding to PHOX2B(43-51) complex only. The arrows marked "Target" show binding of scFv to PHOX2B(43-51)MHC complex. The arrows marked "Control" show binding of scFv to three pMHC complexes containing irrelevant peptides. Figure 1A is clone 10 and Figure 1B is clone 302. [Diagram 2] Specificity array analysis of clones 10 and 302. This figure shows a bead-based binding assay demonstrating the pMHC specificity of each scFv for the target PHOX2B(43-51) complex compared to 95 unrelated complexes. Binding is normalized to the target complex (indicated by arrow). Figure 2A is clone 10 and Figure 2B is clone 302. [Diagram 3] Cross-reactivity analysis of clones 10 and 302 against homologous peptides. This figure shows bead-based binding assays demonstrating the pMHC specificity of each scFv for the target PHOX2B(43-51) complex compared to peptides derived from the proteome and immunopeptidome that are highly homologous to the target peptide. Binding is normalized to the target complex (indicated by arrows). Figure 3A is clone 10 and Figure 3B is clone 302. [Figure 4] Binding of PHOX2B(43-51) X-scan peptide to clone 10 scFv. This figure shows a bead-based binding assay demonstrating binding to complexes loaded with single residue variants of the PHOX2B(43-51) target peptide. To demonstrate the tolerance of clone 10 to substitutions at each position, each non-anchor position of the target peptide was substituted with every other natural amino acid (except cysteine). Binding is normalized to the target complex and the control complex. [Diagram 5] Binding of PHOX2B(43-51) X-scan peptide to clone 302 scFv. This figure shows a bead-based binding assay demonstrating binding to complexes loaded with single residue variants of the PHOX2B(43-51) target peptide. To demonstrate the tolerance of clone 302 to substitutions at each position, each non-anchor position of the target peptide was substituted with every other natural amino acid (except cysteine). Binding is normalized to the target complex and the control complex. [Figure 6] sCRAP specificity testing of clone 10 and clone 302 CARs. This figure shows the prediction of peptides potentially cross-reactive with PHOX2B epitopes by the sCRAP algorithm, and testing of clone 10 and 302 CAR constructs as scFvs in VL-VH and VH-VL orientations. Peptides were loaded onto fluorophore-labeled MHC tetramers and used to stain cells transduced with the CAR constructs. Clones that retained selectivity were further prioritized (selective receptors marked by arrows). Peptide scores represent predicted cross-reactivity based on amino acid sequences of normal tissue peptides; overall scores were calculated based on peptide scores, binding affinity, and normal tissue expression; T means peptide reported in normal tissue immunopeptidome, F means absence. [Figure 7] Functional proof of target selectivity by Clone 10 LH CAR. This figure shows the binding of Clone 10 LH CAR to predicted cross-reactive peptides. Functional screening in peptide-pulsed SW620 cells for two peptides that showed tetramer binding, ABCA8 and MYO7B, shows CAR killing only through supraphysiological concentrations of ABCA8 at 50 μM compared to killing by PHOX2B at 0.1 μM. ABCA8 and MYO7B are not detected in normal tissue immunopeptidomes, and none of the sCRAP predicted peptides (FDFTI, SLC23A2, and TNS4) detected in the normal immunopeptidome show binding to 10LH. [Figure 8] Structural basis of CAR binding to PHOX2B peptides presented on multiple HLAs. Figure 8A shows the crystal structure of PHOX2B / HLA-A24 and a model of PHOX2B in complex with HLA-A*23:01, HLA-B*14:02, and HLA-C*07:02. Figure 8B shows that the charged polar residues of HLA-C07, R151, Q155, and R69, align with the key 10LH interacting residues, I5, R6, and I7 (MHC residues in blue, PHOX2B / 10LH interacting residues in red). R151, Q155, and R69 create steric and charge hindrance at the key peptide binding residues. Figure 8C shows staining of PHOX2B PC-CAR 10LH (bottom) revealing strong binding to HLA-A*24:02, HLA-A*23:01, and HLA-B*14:02, but not to HLA-C07.10LH; PHOX2B PC-CAR; CD19; CAR against CD19; UT; untransduced T cells. [Figure 9] Cross-HLA recognition of PHOX2B peptide. Figure 9A shows the binding of 10LH and 302LH CARs to labeled PHOX2B 43-51 HLA-A*23:01 tetramer. Figure 9B shows that 10LH CAR kills HLA-A*23:01 / PHOX2B-WM873 cells when pulsed with PHOX2B peptide, but not with CHRNA3 peptide. [Figure 10]PHOX2B-specific PC-CAR T cells induce potent tumor killing in neuroblastoma lines in vitro. This figure shows that the clone 10LH CAR induces specific killing and IFNγ release in HLA-A*24:02 and HLA-A*23:01 and PHOX2B-expressing neuroblastoma cells (SKNAS, NBSD, and SKNFI), but not in HLA-A*24:02 / PHOX2B-non-neuroblastoma tumor cells (SW620, HEPG2, and KATO III) unless PHOX2B peptide is added. No T cell activity was observed in SW620 when pulsed with 10 μM of the predicted cross-reactive peptides ABCA8 or MYO7B. Cytotoxicity was visualized by loss of green fluorescence in GFP-transduced cancer cells and IFNγ release measured by ELISA. The order of cells listed on the right is the same as the right-most data point for each. [Figure 11] Antibodies specific for PHOX2B 43-51 MHC stain PHOX2B-positive neuroblastoma cells in vitro. This figure shows that staining of cancer cells with tetramerized 10LH scFv allows detection of PHOX2B pMHC on neuroblastoma cells but not in HLA-matched controls. [Figure 12] PHOX2B-specific PC-CAR T cells induce potent tumor killing in vivo and subvert traditional HLA restriction. PHOX2B-specific PC-CAR T cells induce potent tumor killing in mice engrafted with neuroblastoma PDX tumors including the extremely fast-growing line COG-564x and the HLA-A*23:01 line NBSD. Six mice were enrolled in each arm; for each PDX line, data from one of two in vivo studies are shown. [Figure 13]PHOX2B-specific PC-CAR T cells induce MHC upregulation in an in vivo MHC-low neuroblastoma model. Treatment with 10LH and 302LH PC-CARs strongly upregulates HLA expression in PDX tumors harvested from mice that reached tumor burden compared to mice treated with untransduced T cells (COG-564x harvested 11 days after treatment; NBSD harvested 14 days after treatment for UT and 17 days after treatment for 10LH and 302LH). [Figure 14A] Various chimeric receptor constructs. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] See legend to Figure 14A. [Figure 14H] See legend to Figure 14A. [Figure 15A-1] Sequence of chimeric receptor. [Figure 15A-2] See description of Figure 15A-1. [Figure 15B-1] Sequence of chimeric receptor. [Figure 15B-2] See legend to Figure 15B-1. [Figure 15B-3] See legend to Figure 15B-1. [Figure 16]Kinetic analysis of clones 9 and 1114. The figure shows the association and dissociation of soluble PHOX2B(43-51)A*24:02 complex with scFv of clone 9 or clone 1114 as measured by biolayer interferometry (BLI). Binding to the PHOX2B(43-51) complex (200 nM) is overlaid with a non-specific complex (200 nM) control that differs from the target PHOX2B complex by a single residue. The arrow marked "target" indicates binding of the scFv to the PHOX2B(43-51) MHC complex. The arrows marked "Control" indicate binding of scFv to control peptides (NYTPIRTTF (SEQ ID NO: 52), LYNPIRTTF (SEQ ID NO: 53), QYQPLRTTF (SEQ ID NO: 54), QYNPIKTTF (SEQ ID NO: 55), QYNPLQTTF (SEQ ID NO: 56), QYNPLKTTF (SEQ ID NO: 57), QYNAIRTTF (SEQ ID NO: 58)). Figure 16A is clone 9 and Figure 16B is clone 1114. [Figure 17] Kinetic analysis of clone 1113. This figure shows the association and dissociation of the scFv of clone 1113 with the soluble PHOX2B(43-51)A*24:02 complex as measured by biolayer interferometry (BLI). Binding to the PHOX2B(43-51) complex (200 nM) is overlaid with a non-specific complex (3 x 200 nM) control, showing specific binding to the PHOX2B(43-51) complex only. The arrows marked "Target" indicate binding of the scFv to the PHOX2B(43-51)MHC complex. The arrows marked "Control" indicate binding of the scFv to three pMHC complexes containing irrelevant peptides. [Figure 18] Specificity array analysis of clones 9 and 1113. This figure shows a bead-based binding assay showing the pMHC specificity of each scFv for the target PHOX2B(43-51) complex compared to 95 unrelated complexes. Binding is normalized to the target complex (indicated by arrow). Figure 18A is clone 9 and Figure 18B is clone 1113. [Figure 19] Specificity array analysis of clone 1114. This figure shows a bead-based binding assay demonstrating the pMHC specificity of clone 1114 scFv for the target PHOX2B(43-51) complex compared to 95 unrelated complexes. Binding is normalized to the target complex (indicated by the arrow). [Figure 20] Cross-reactivity analysis of clones 9 and 1113 against homologous peptides. This figure shows bead-based binding assays demonstrating the pMHC specificity of each scFv for the target PHOX2B(43-51) complex compared to peptides derived from the proteome and immunopeptidome that are highly homologous to the target peptide. Binding is normalized to the target complex (indicated by arrows). Figure 20A is clone 9 and Figure 20B is clone 1113. [Figure 21] Cross-reactivity analysis of clone 1114 against homologous peptides. This figure shows a bead-based binding assay demonstrating the pMHC specificity of clone 1114 scFv for the target PHOX2B(43-51) complex compared to peptides derived from the proteome and immunopeptidome that are highly homologous to the target peptide. Binding is normalized to the target complex (indicated by the arrow). [Figure 22] Binding of PHOX2B(43-51) X-scan peptide to clone 9 scFv. This figure shows a bead-based binding assay demonstrating binding to complexes loaded with single residue variants of the PHOX2B(43-51) target peptide. To demonstrate the tolerance of clone 9 to substitutions at each position, each non-anchor position of the target peptide was substituted with every other natural amino acid (except cysteine). Binding is normalized to the target complex and the control complex. [Diagram 23]Binding of PHOX2B(43-51) X-scan peptide to clone 1113 scFv. This figure shows a bead-based binding assay demonstrating binding to complexes loaded with single residue variants of the PHOX2B(43-51) target peptide. To demonstrate the tolerance of clone 1113 to substitutions at each position, each non-anchor position of the target peptide was substituted with every other natural amino acid (except cysteine). Binding is normalized to the target complex and the control complex. [Figure 24] Binding of PHOX2B(43-51) X-scan peptide to clone 1114 scFv. This figure shows a bead-based binding assay demonstrating binding to complexes loaded with single residue variants of the PHOX2B(43-51) target peptide. To demonstrate the tolerance of clone 1114 to substitutions at each position, each non-anchor position of the target peptide was substituted with every other natural amino acid (except cysteine). Binding is normalized to the target complex and the control complex. [Diagram 25] sCRAP cross-reactivity algorithm. Figure 25A shows a cross-reactivity algorithm developed to identify peptides presented in normal tissues with similar biophysical properties as tumor antigens, for example, to preemptively predict cross-reactivity and screen for specificity. Figure 25B is an illustration of the peptide scoring system described in the method. Figure 25C is a schematic diagram of the algorithm workflow that describes how tumor peptides are scored against each peptide predicted to be presented from the normal proteome (a total of 92.4 x 106 possible MHC peptides). Binding affinity is predicted for each normal peptide, and the maximum gene expression of the parent genes is taken into account in the overall score of each peptide. Peptides are queried against a normal tissue immunopeptidomics database. [Figure 26]The pMHC cross-reactivity algorithm sCRAP predicts MAGE-A3 toxicity through TITIN. Figure 26A is a top-list of peptides predicted to be cross-reactive with the MAGE-A3 peptide EVDPIGHLY (SEQ ID NO:59), revealing cross-reactivity with Titin peptide ESDPIVAQY (SEQ ID NO:60), ranked 4th out of 1,143,861 possible peptides presented on HLA-A*01:01. Figure 26B shows that TITIN is highly expressed in cardiac and muscle tissues. [Figure 27] Schematic representation of the data and construct design of 10LH BiTE. The IgG leader is followed by the 10LH scFv, a GDDDDKS linker, followed by the OKT3 (anti-CD3) scFv and a 6xHis tag. [Figure 28] Cytotoxicity mediated by a PHOX2B-specific bispecific antibody in HLA-A*24:02-expressing cell lines in vitro. Clone 10 is shown to induce specific killing of K562 cells stably transfected with HLA-A*24:02 treated with exogenous PHOX2B-targeting peptide (white bars), but not in conditions treated with a non-relevant peptide (black bars) or DMSO negative control (grey bars). Cytotoxicity was quantified by the loss of GFP-expressing target cells. [Figure 29]Cytotoxicity mediated by PHOX2B-specific bispecific antibodies of cell lines expressing HLA-A*24:02 or HLA-A*23:01 in vitro. Figure 29A shows that clone 10 and clone 302 induce specific killing of K562 cells stably transfected with HLA-A*24:02 treated with exogenous PHOX2B targeting peptide (white bars), but not in conditions treated with unrelated peptide (black bars) or DMSO negative control (gray bars). Figure 29B shows that clone 302 does not induce specific killing of K562 cells stably transfected with HLA-A*23:01 treated with exogenous PHOX2B targeting peptide (white bars), and clone 10 does, but not in conditions treated with unrelated peptide (black bars) or DMSO negative control (gray bars). Cytotoxicity was quantified by the loss of GFP-expressing target cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description The curative potential of chimeric antigen receptor (CAR) T cell-based cancer immunotherapy has been established in leukemia, but its application to solid tumors is limited by the paucity of known tumor-specific membrane proteins. 1,2 Membrane proteins account for as much as one-quarter of the proteome, but only a small proportion of these proteins are specifically expressed in tumor cells and not in normal tissues, and even less are essential for tumor homeostasis. 3 Rather, the majority of cancer driver proteins reside in the cytoplasm or cell nucleus and become accessible to the immune system only through peptide presentation on the major histocompatibility complex (MHC).

[0026] MHC class I proteins, encoded by the highly polymorphic human leukocyte antigen (HLA) A, B, and C genes, present a snapshot of the intracellular proteome on the cell surface (the immunopeptideome), where T cells monitor peptide-MHC complexes (pMHC) for antigens derived from foreign pathogens. 4 T cell recognition of mutant pMHC (neoantigens) as non-self mediates a healing response achieved through immune checkpoint blockade 5 and complete remission using adoptive transfer of tumor-infiltrating lymphocytes (TILs). 6 Despite this, only about 5% of these neoantigens are predicted to bind to a given HLA allotype. 7 However, only 1.6% of neoantigens have been reported to be immunogenic. 8 Subclonal mutations and downregulation of mutated non-essential genes further restrict the pool of therapeutically relevant neoantigens, and therefore, in most cancers, the mutational threshold for effective neoantigen-based therapy is never crossed. 9,10 Tumor cells also present numerous non-mutated self-peptides on MHC. 11 , many of which are immunogenically silent due to negative thymic selection of T cells. We hypothesized that a subset of the immunopeptideome consists of tumor-specific peptides derived from essential tumor proteins, and that these could be targeted using synthetic peptide-centered chimeric antigen receptors (PC-CARs).

[0027] Peptides presented in the MHC groove constitute only a small portion of the surface of the extracellular pMHC molecule. Typical 8-14 residue long peptides presented on MHC class I constitute only about 2-3% of the amino acids in the pMHC complex and are spatially confined to the adjacent α-helices of the MHC groove, making it extremely difficult to generate peptide-specific single-chain antibody variable fragment (scFv) binders. 12Furthermore, cross-reactivity of the modified receptor with peptides bearing biophysically similar molecular surfaces displayed in normal tissues led to significant toxicity and death. 13-15 .

[0028] Here, the inventors identify the PHOX2B peptide QYNPIRTTF (SEQ ID NO:1) in the context of HLA-A * Not only the complex with 24:02, but also HLA-A * 23:01 and HLA-B * We present immunotherapeutic targeting of the neuroblastoma CRC master regulator PHOX2B using PC-CAR, which is also specific for its complex with 14:02. These and other aspects of the disclosure are presented in detail below.

[0029] I. Definition Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include singulars.In general, the nomenclature utilized in connection with the antibodies and related molecules, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein, and the techniques thereof, are well known and commonly used in the art.

[0030] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art, as described in various general and more specific references cited and described throughout this specification.For example, see B. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory (1988); and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0031] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies, and fragments thereof herein may be further clarified by the descriptions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991); Bork et al., J Mol. Biol. 242, 309-320 (1994); Chothia and Lesk J. Mol Biol. 196:901-917 (1987), Chothia et al. Nature 342, 877-883 (1989), and / or Al-Lazikani et al. J Mol Biol 273, 927-948 (1997).

[0032] As used herein, "PHOX2B" is defined as the sequence disclosed in Uniprot ID Q99453, shown below: TIFF2024532258000002.tif36149. The PHOX2B epitope targeted by the claimed sequences of the present invention comprises residues 43-51 of Uniprot ID Q99453, and may hereafter be referred to as PHOX2B 43-51.

[0033] As used herein, "antigen-binding site" shall be taken to mean a structure formed by a protein, such as an antibody, that can bind or specifically bind to an antigen. An antigen-binding site does not necessarily have to be a series of consecutive amino acids, or even amino acids within a single polypeptide chain. For example, in an Fv, which comprises two different polypeptide chains from an antibody, the antigen-binding site is generally a V domain that interacts with an antigen, but is not necessarily present in one or more of the CDRs of each variable region. L and V HIn some embodiments, the antigen-binding site is an antibody antigen-binding site. In such embodiments, the antigen-binding site may comprise one or more complementarity determining regions or "CDRs". In some embodiments, the antibody antigen-binding site is comprised of a series of amino acids of V H or V L or at least a portion of Fv.

[0034] As used herein, the terms "complementarity determining region" or "CDR" are used interchangeably to refer to the antigen-binding regions found within the variable regions of the heavy and light chain polypeptides. Generally, antibodies are H 3 each (CDR H1 or H1; CDR H2 or H2; and CDR H3 or H3), V L Each of these contains three CDRs (CDR L1 or L1; CDR L2 or L2; and CDR L3 or L3).

[0035] As used herein, "variable region" and "CDR" can refer to variable regions and CDRs defined by any approach, e.g., a combination of approaches, known in the art. According to a specific embodiment, the CDRs are determined according to Kabat et al. (supra).

[0036] As used herein, "binding" or "binding" or "specifically binding" refers to an antibody:antigen binding mode, which preferably has a K of less than 1 μM or less than 500 nM in the case of clinically relevant binders. D The binding agents of the present disclosure can bind to the PHOX2B:pMHC complex with high affinity. For example, in some embodiments, the binding agent has a binding affinity of about 10 -6 M or less, e.g., 1×10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , or 10-14 Dissociation constant (K D ) to bind to PHOX2B:pMHC. The specificity of the binding is determined with reference to a non-target protein, e.g., bovine serum albumin (BSA). In some embodiments, the binding agent is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 1 ... 4 Double, 10 5 Double, or 10 6 fold lower dissociation constant (K D ) to the PHOX2B:pMHC complex. In some cases, specificity is determined by measuring binding of the binder to MHC loaded with a non-target peptide or empty MHC. In some cases, specificity is determined by measuring binding of the binder to the target peptide alone or to the target peptide loaded onto MHC of a different allotype. In specific embodiments of the disclosure, the binder is MHC-restricted, i.e., the binder is MHC-restricted, i.e., the dissociation constant of the binder is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 1 ... 4 Double, 10 5 Double, or 10 6 fold lower dissociation constant (K D ) and the MHC (e.g., HLA-A * 24:02) specifically binds to a target peptide (e.g., PHOX2B peptide) loaded onto the antibody.

[0037] As used herein, the phrase "chimeric antigen receptor (CAR)" refers to a recombinant or synthetic molecule that combines antibody-based specificity for a desired antigen with an intracellular domain that activates the T cell receptor to generate a chimeric protein that exhibits cellular immune activity against a particular antigen.

[0038] As used herein, the term "T cell receptor" or "TCR" refers to soluble and non-soluble recombinant T cell receptors.

[0039] As used herein, "T cell receptor (TCR) fusion proteins" or "TFPs" generally include recombinant polypeptides derived from various polypeptides that include a TCR, which are (i) capable of binding to a surface antigen on a target cell, and (ii) typically capable of interacting with other polypeptide components of an intact TCR complex when co-located on a T cell or T cell surface.

[0040] As used herein, "T cell receptor-like antibody" or "TCRL" refers to an antibody that binds to MHC presenting HLA-restricted peptide antigens. TCRL binding to a target typically has MHC-restricted specificity: TCRL does not bind to MHC in the absence of complexed peptide, and TCRL does not bind to peptide in the absence of MHC. TCRL is characterized by sufficient affinity to allow specific binding to tumor antigens even when TCRL is provided in a soluble form rather than a membrane-bound form. TCRL is being developed as a new therapeutic class to target and mediate specific killing of tumor cells. In addition, TCRL is a valuable research reagent that allows the study of human class I peptide-MHC ligand presentation and TCR-peptide-MHC interactions. In one embodiment, the binding agent of the present disclosure is a TCRL.

[0041] As used herein, the phrase "MHC (or HLA) restricted peptide" refers to a peptide that may be presented on an MHC molecule. Such peptides may be identified by experimental techniques such as mass spectrometry, reverse-immunology, or in silico analysis. MHC (or HLA) presented peptide refers to a peptide that has been confirmed in vitro or in vivo to be presented by an MHC molecule.

[0042] The term "cancer," as used herein, is defined as a disease characterized by the rapid, unregulated growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0043] "Compound" refers to any molecule, including small molecules, polypeptides, and other macromolecules. In some embodiments, the compound is a low molecular weight compound having a molecular weight of less than about 2000 daltons.

[0044] The term "naturally occurring" (or "native") as used herein as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a natural source and that has not been intentionally modified by man in the laboratory or otherwise is naturally occurring.

[0045] The term "operably linked," as used herein, refers to the positioning of the components so described in a relationship permitting them to function in their intended manner. For example, a regulatory sequence "operably linked" to a coding sequence is connected such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

[0046] The term "protein" shall be taken to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., a polypeptide complex) that are covalently or non-covalently linked to one another. For example, a series of polypeptide chains may be covalently linked, for example, using a suitable chemical linker or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0047] It will be understood from the above paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0048] The term "polynucleotide," as referred to herein, means a polymer of at least 10 bases in length of nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide, or an RNA-DNA heteroduplex. The term includes single- and double-stranded forms of DNA.

[0049] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical in a comparison window (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis). The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences in a comparison window, determining the number of positions at which identical nucleic acid bases (e.g., A, T, C, G, U, or I) or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The term "substantial identity," as used herein, refers to a feature of a polynucleotide or amino acid sequence that includes a sequence having at least 85 percent sequence identity, preferably at least 90-95 percent sequence identity, and more preferably at least 99 percent sequence identity, compared to a reference sequence in a comparison window of at least 18 nucleotide (6 amino acid) positions, and often in a window of at least 24-48 nucleotide (8-16 amino acid) positions, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that may contain deletions or additions that account for 20 percent or less of the reference sequence in the comparison window. The reference sequence may be a subset of a larger sequence.

[0050] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991).

[0051] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants generally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but do not necessarily, have specific three-dimensional structural characteristics and specific charge characteristics.

[0052] The term "agent" is used herein to mean a chemical compound, a mixture of compounds, a biological macromolecule, or an extract made from biological material.

[0053] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.In case of conflict, the present application, including the definitions herein, shall prevail.However, the mention of references, papers, publications, patents, patent publications, and patent applications cited in this specification is not, and should not be interpreted as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0054] Any concentration range, percentage range, ratio range, or integer range herein should be understood to include any integer value contained in the stated range, and fractions thereof (e.g., tenths and hundredths of an integer), as appropriate, unless otherwise indicated. The term "about," when immediately preceding a number or numeral, means that the number or numeral is within a range of ±10%. The terms "a" and "an," as used herein, should be understood to refer to "one or more" of the listed components, unless otherwise indicated. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0055] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0056] III. Binders The term "binding agent" as used herein refers to any molecule capable of binding to a PHOX2B:HLA complex. Specifically, a binding agent can bind to a PHOX2B:HLA complex comprising the sequence QYNPIRTTF (SEQ ID NO:1). In some embodiments, a binding agent is or comprises a polypeptide. In some embodiments, binding agents of the present disclosure comprise the sequences provided and variants thereof. The present disclosure specifically contemplates binding agents having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity, or even at least 96%, 97%, 98%, or 99% sequence identity, to the full-length variable region of the constructs disclosed herein, so long as binding affinity to the PHOX2B:HLA complex is maintained. In some embodiments, the binding agents of the present disclosure further include dimeric binding agents obtained by splitting a single chain variable fragment (scFv) sequence into light and heavy chains, respectively, with a polyG / S linker, and also include homologs or variants of such dimeric binding agents. Optionally, the specificity or affinity of binding to the PHOX2B:HLA complex is maintained or even improved. In a specific embodiment, the binding agent comprises a heavy chain and a light chain that respectively comprise the three heavy chain CDR sequences and the three light chain CDR sequences of the present disclosure, which maintain or improve binding. In an embodiment of the present disclosure, the binding agent is an antibody or an antigen-binding fragment thereof, an artificial protein that is soluble (e.g., a bispecific antibody), or an artificial protein that is membrane-bound (e.g., a chimeric antibody receptor or a TCR fusion protein).

[0057] For binders derived from immunoglobulin (Ig) variable domains, where the target contact surface (complementarity determining regions or CDRs) is created through the loops connecting the β-strands, the binding activity to the target may be transferable through grafting of the CDR loops to related Ig domains (e.g., other human Ig family members) or even to non-Ig β-sheet scaffolds. This is particularly true when the structure of the binder in complex with the target is similar to that of the V L Domains and V H This is the case when one or a few of the six CDRs of the domain combination are shown to contribute primarily to binding. The present disclosure specifically contemplates binding agents with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a single CDR region of the construct disclosed herein, so long as the binding affinity to the PHOX2B:HLA complex is functionally maintained. CDR grafting has been widely used for the "humanization" of antibodies, where CDR loops from an antibody derived from a non-human host are grafted onto a human Ig scaffold to reduce immunogenicity. Many antibodies approved for therapeutic use have been humanized through CDR grafting from mouse antibodies onto a human scaffold. Examples of grafting CDR loops from antibody scaffolds to non-Ig alternative scaffolds have also been reported (Nicaise M., et al. Protein Sci 13:1882-1891 (2004); Petrovskaya LE, et al. Biochemistry (Mosc) 77:62-70 (2012); Pacheco et al. Protein Eng Des Sel 27:431-438 (2014)).

[0058] K of binding agents to targets, including enzyme-linked immunosorbent (ELISA) assays and surface plasmon resonance (SPR) assays DVarious means of determining are known. In some cases, the affinity and specificity of binding is determined by optical interference, for example, by the Pall ForteBio BLItz® system, as described in Sultana A. Lee J. Curr Protoc Protein Sci, 79: 19.25.1-19.25.26 (2015). The affinity of the binder can be measured using a soluble form of the binder or using a membrane-bound form, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR) fusion protein (TFP). Conversely, pMHC complexes can be tested in soluble form or bound to native cell membranes.

[0059] A.Antibodies In some embodiments, the binding agent is an antibody or antibody fragment. Suitable antibody fragments for carrying out some embodiments of the present disclosure include one to three complementarity determining regions (CDRs) of an immunoglobulin light chain (herein referred to as a "light chain") and one to three CDRs of an immunoglobulin heavy chain (herein referred to as a "heavy chain"). Optionally, the binding agent includes a light chain variable region, a heavy chain variable region, a light chain, or a heavy chain.

[0060] The identity of the amino acid residues of a particular antibody that make up a variable region or CDR can be determined using methods well known in the art, including, for example, the sequence variability defined by Kabat et al. (see, e.g., Kabat et al., 1992 Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC), the locations of structural loop regions defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883 (1989)), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, Martin et al. Proc Natl Acad Sci USA. 86:9268 (1989); and see the worldwide website www.bioinf-org.uk / abs), and available complex crystal structures defined by contact definition (MacCallum et al. al. J. Mol. Biol. 262:732-745 (1996)), "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166 (2008)), and the IMGT method (Lefranc MP, et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev Comp Immunol 27:55-77 (2003)).

[0061] In certain embodiments, the binding agent is a functional antibody fragment comprising the entire or essentially the entire variable region of both the light and heavy chains, including, but not limited to, those defined as follows: (i) an Fv, defined as a fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains; (ii) a single chain variable fragment or single chain Fv ("scFv"), which is a genetically engineered single chain molecule comprising the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule; (iii) a disulfide stabilized Fv ("dsFv"), which is a genetically engineered antibody comprising the variable region of the light chain and the variable region of the heavy chain linked by a genetically engineered disulfide bond; (iv) an intact light chain and (v) Fab', a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, which can be obtained by treating an intact antibody with the enzyme papain to give a variable domain and an Fd fragment of the heavy chain consisting of the CH1 domain; (vi) F(ab')2 (i.e., a dimer of Fab' fragments joined by two disulfide bonds), a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule, which can be obtained by treating an intact antibody with the enzyme pepsin, followed by reduction (to give two Fab' fragments per antibody molecule); and (vii) a single V fragment that exhibits sufficient affinity for the antigen. H Domain or V L Single domain antibodies or nanobodies, which are composed of domains.

[0062] Methods for producing polyclonal and monoclonal antibodies and fragments thereof are well known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0063] Antibody fragments according to some embodiments of the disclosure can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in E. coli cells or mammalian cells (e.g., Chinese hamster ovary cell culture system or other protein expression system). Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent, optionally with a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce 3.5S Fab' monovalent fragments. Alternatively, enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein. See also Porter, RR, Biochem J. 73:119-126 (1959). Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, can also be used so long as the fragments bind to the antigen recognized by the intact antibody.

[0064] In embodiments where the binding agent is an antibody, the heavy and light chains of the antibody of the present disclosure can be full length (e.g., the antibody can include at least one, and preferably two, complete heavy chains and at least one or two complete light chains). In some embodiments, the antibody heavy chain constant region is selected from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the immunoglobulin isotype is selected from IgG1, IgG2, IgG3, and IgG4, more specifically, IgG1 (e.g., human IgG1) or IgG4 (e.g., human IgG4). The choice of the type of antibody depends on the immune effector function that the antibody is designed to elicit. In one embodiment, the binding agent elicits antibody-dependent cellular cytotoxicity. In one embodiment, the binding agent elicits complement-dependent cytotoxicity.

[0065] Bispecific antibody configurations are also contemplated herein. Bispecific monoclonal antibodies (BsMAb, BsAb) are artificial proteins or protein complexes that are composed of fragments of two different monoclonal antibodies and therefore bind to two different types of antigens. According to a specific embodiment, BsMAb is engineered to simultaneously bind to effector cells (e.g., using receptors such as CD3) and to targets such as tumor cells to be destroyed. Anti-CD3 antibodies known in the art that can be used to direct bispecific antibody engagement with CD3-positive effector cells include SP-34 (Pessano et al., EMBO J (1985) 4:337-344), OKT3 (Kung et al., Science (1979) 206:347-349), UCHT1 (Beverley PCL, Callard RE Eur J Immunol (1981) 11:329), 12F6 (Wong JT and Colvin RB, J Immunol (1987) 139:1369-1374), and all humanized and / or affinity engineered variants (e.g., Shalaby et al., J Exp Med (1992) 175:217-225). Other affinity scaffolds, such as VHH domains, can also be used to modify CD3 binding (e.g., WO / 2015 / 095412). Other configurations, such as triabodies or tetrabodies, are also contemplated.

[0066] B. Single-chain variable fragments (scFv) The Fv fragment is V H Chain and V L The association may be non-covalent, as described in Inbar et al. Proc Natl Acad. Sci. USA 69:2659-62 (1972). Alternatively, the variable chains may be linked by intermolecular disulfide bonds or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragment comprises a V-chain linked by a peptide linker. H Chain and V LThese single-chain antigen-binding proteins (scFv) consist of V chains connected by oligonucleotides. H Domains and V L scFvs are prepared by constructing a structural gene containing a DNA sequence encoding the domain. The structural gene is inserted into an expression vector, which is then introduced into a host cell, such as E. coli. The recombinant host cell synthesizes a single polypeptide chain containing a linker peptide bridging the two V domains. Methods for making scFvs are described, for example, by Whitlow and Filpula, Methods 2:97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio / Technology 11:1271-77 (1993); U.S. Patent No. 4,946,778. The resulting polypeptide may fold back on itself to form an antigen-binding monomer or may form multimers (e.g., dimers, trimers, or tetramers) depending on the length of a flexible linker between the two variable domains (Kortt et al., Prot Eng 10:423 (1997); Kortt et al, Biomol Eng 18:95-108 (2001)). L and V HBy combining polypeptides containing the above, multimeric scFvs that bind different epitopes can be formed (Kriangkum et al., (2001) Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, Science 242:423 (1988); Huston et al. Proc Natl Acad Sci USA 85:5879 (1988); Ward et al. Nature 334:544 (1989), de Graaf et al. Methods Mol Biol. 178:379-87 (2002). Single chain antibodies derived from the combinations provided herein include, but are not limited to, scFvs that contain one or more variable domain sequences disclosed herein, or one or more CDR sequences derived from one or more variable domain sequences.

[0067] C. Chimeric Antigen Receptors (CARs) and TCR Fusion Proteins (TFPs) Chimeric antigen receptors (CARs) are fusion proteins that contain an antigen recognition portion and a T cell activation domain. Exemplary CARs are provided by U.S. Patent No. 8,399,645 and U.S. Patent No. 7,638,325. Other exemplary recombinant receptors, such as CARs, recombinant T cell receptors (TCRs), TCR fusion proteins (TFPs), and methods for making and introducing them into cells, are described in International Patent Application Nos. WO2017 / 096329, WO2000 / 14257, WO2013 / 126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, and WO2013 / 071154, WO2013 / 123061, and WO / 2014055668; U.S. Patent Application Nos. US20021319 60, US2013287748, and US20130149337; U.S. Patent Nos. 6,451,995, 7,446,190, 7,638,325, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118; European Patent Application No. EP2537416; and Sadelain et al. Cancer Discov. April 3(4):388-398 (2013); Davila et al. PLoS ONE 8(4):e61338 (2013); Turtle et al. Curr. Opin. Immunol. October 24(5):633-39 (2012); and Wu et al. Cancer, March 18(2):160-75 (2012). In one embodiment, the binding agent is TFP, as described in U.S. Patent No. 15 / 419,398.

[0068] D. Amino acid substitutions As described herein, minor variations in the amino acid sequence of the binding agent are contemplated as being encompassed by the present disclosure, provided that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the variable domain of the construct disclosed herein, or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to a single CDR region, as long as the binding affinity to the PHOX2B:HLA complex is functionally maintained. For example, the binding agent may include one or more amino acid substitutions to the CDR sequences provided herein. The binding agent may include one or more amino acid substitutions in the framework region. In some embodiments, the binding agent may have no more than two amino acid substitutions in CDR-L1, no more than two amino acid substitutions in CDR-L2, no more than three amino acid substitutions in CDR-L3, no more than two amino acid substitutions in CDR-H1, no more than two amino acid substitutions in CDR-H2, or no more than four amino acid substitutions in CDR-H3 with respect to one or more of the CDR amino acid sequences provided herein. In some embodiments, the binding agent comprises amino acid substitutions in the framework regions. For example, as will be understood by those skilled in the art, routine site-directed or random mutagenesis techniques can be performed to alter the amino acid sequence in any one of the binding agents described herein, for example, to alter binding affinity (e.g., affinity maturation), to reduce susceptibility to proteolysis or oxidation, or to confer or modify other physicochemical or functional properties of the binding agent.

[0069] In some embodiments, the amino acid substitution is a conservative amino acid substitution. A conservative substitution is a substitution that occurs within a family of amino acids that have related side chains. Genetically encoded amino acids are generally classified into the following families: (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More specific families are as follows: serine and threonine are the aliphatic hydroxy family; asparagine and glutamine are the amide-containing family; alanine, valine, leucine, and isoleucine are the aliphatic family; phenylalanine, tryptophan, and tyrosine are the aromatic family. For example, it is reasonable to expect that simple substitutions of leucine for isoleucine or valine, aspartic acid for glutamic acid, threonine for serine, or similar substitutions between structurally related amino acids will not significantly affect the binding function or properties of the resulting molecule, particularly when the substitution does not involve an amino acid within a framework region. Examples of conservative amino acid substitutions are provided in Table 1 below.

[0070] Table 1. Exemplary conservative amino acid changes TIFF2024532258000003.tif136145

[0071] The present disclosure provides that the binding agent is still HLA-A * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-B * Non-conservative amino acid substitutions in the binding agents of the present disclosure are also contemplated, provided that they are capable of specifically binding to the 14:02 / PHOX2B complex. Thus, in some embodiments, the amino acid substitution is a non-conservative amino acid substitution.

[0072] Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those skilled in the art. Certain amino and carboxy termini of the fragments or analogs are near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains present in other proteins of known structure and / or function. Methods for identifying protein sequences that fold into known three-dimensional structures are known (e.g., Bowie et al., Science 253:164 (1991) or 86 ). Thus, the above examples demonstrate that one of skill in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains by the antibodies described herein.

[0073] Specific amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences (preferably in parts of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially change the structural features of the parent sequence (e.g., the replacement amino acid should not tend to disrupt helices present in the parent sequence or other types of secondary structures that characterize the parent sequence). Art-recognized examples of polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991); and Thornton et al. Nature 354:105 (1991).

[0074] For example, routine techniques can be used to introduce amino acid substitutions into CDRs to improve binding affinity. Such substitutions are made in CDR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact antigen, and the resulting variants are tested for binding affinity. Alternatively, or in addition, affinity maturation may be performed. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable region coding sequences selected for maturation by any of a variety of methods, such as error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis. A secondary library is then created. The library is then screened to identify variants with the desired affinity. Another method of introducing diversity includes a CDR-specific approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis, described below, or modeling. Specifically, CDR-H3 and CDR-L3 can be used for random mutagenesis and affinity maturation.

[0075] In certain embodiments, the substitution, insertion, or deletion is such that such alteration is associated with an HLA-A * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-B *Amino acid substitutions may occur in one or more CDRs, so long as they do not substantially reduce the ability of the binding agent to bind to the 14:02 / PHOX2B complex. In some embodiments, binding agents containing amino acid substitutions bind to HLA-A with similar affinity as binding agents that do not contain substitutions. * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-A * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-B * 14:02 / PHOX2B complex. Such substitutions may be, for example, outside of antigen contact residues in the CDRs. In some embodiments, binding agents that include amino acid substitutions bind to HLA-A with higher affinity than binding agents that do not include substitutions. * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-B * In some embodiments, the binding agents that include the amino acid substitutions bind to HLA-A 14:02 / PHOX2B complex with lower affinity than binding agents that do not include the substitutions. * 24:02 / PHOX2B complex, HLA-A * 23:01 / PHOX2B complex, or HLA-B * Binds to the 14:02 / PHOX2B complex. In certain embodiments, each CDR is unchanged or contains no more than 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the substitutions are conservative substitutions.

[0076] A useful method for identifying residues or regions of a binding agent that can be targeted for mutagenesis, described by Cunningham, Science 244:1081-1085 (1989), is called "alanine scanning mutagenesis". In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral amino acids such as alanine to determine whether the interaction of the binding agent with its antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the first substitution. Alternatively, or in addition, a crystal structure of the antigen-binding agent complex can be used to identify contact points between the binding agent and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0077] IV. Nucleic acids According to one aspect of the present disclosure, an isolated polynucleotide comprising a nucleic acid sequence encoding the binding agent described herein is also provided. An expression vector comprising a polynucleotide operably linked to a cis-acting regulatory element is also provided. The expression vector of some embodiments of the present disclosure comprises additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., as a shuttle vector). In addition, a typical cloning vector may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals. For example, such constructs typically comprise a 5'LTR, a tRNA binding site, a packaging signal, an initiation of second strand DNA synthesis, and a 3'LTR, or a portion thereof.

[0078] The nucleic acid constructs of some embodiments of the present disclosure include a signal sequence for secretion or display of the binding agent from a host cell in which it is placed. Preferably, the signal sequence for this purpose is a mammalian signal sequence.

[0079] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box, located 25-30 base pairs upstream of the transcription start site, is thought to be involved in directing the initiation of RNA synthesis by RNA polymerase. Other upstream promoter elements determine the rate at which transcription is initiated. Preferably, the promoter utilized by the expression vector is active in the particular cell population transformed. Examples of cell type-specific and / or tissue-specific promoters include promoters such as liver-specific albumin (Pinkert et al. Genes Dev. 1:268-277 (1987)), lymphoid-specific promoters (Calame et al. Adv. Immunol. 43:235-275 (1988)); specifically, promoters of the T cell receptor (Winoto et al. EMBO J. 8:729-733 (1989)) and immunoglobulins (Banerji et al. Cell 33:729-740 (1983)); neuron-specific promoters, e.g., the neurofilament promoter (Byrne et al. Proc. Natl. Acad. Sci. USA 86:5473-5477 (1989)), pancreatic-specific promoters (Edlunch et al. Science 230:912-916 (1985)), or mammary gland specific promoters such as the whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. EP 0264166). In constructing an expression vector, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural context. However, as is known in the art, some variation in this distance can be accommodated without loss of promoter function.

[0080] Enhancer elements can stimulate transcription up to 1,000-fold from linked homologous or heterologous promoters. Enhancers are active when located downstream or upstream of the transcription start site. Many enhancer elements from viruses have a wide host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the present disclosure include those from polyoma virus, human or mouse cytomegalovirus (CMV), terminal repeat sequences from various retroviruses, such as murine leukemia virus, mouse or Rous sarcoma virus, and HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, NY 1983.

[0081] Polyadenylation sequences can also be added to expression vectors to increase the efficiency of TCRL mRNA translation. Accurate and efficient polyadenylation requires two distinct sequence elements: a GU-rich or U-rich sequence located downstream of the polyadenylation site, and a highly conserved 6-nucleotide sequence, AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation signals suitable for some embodiments of the present disclosure include those derived from SV40.

[0082] In addition to the elements already described, the expression vectors of some embodiments of the present disclosure may contain other special elements that are intended to increase the level of expression of cloned nucleic acid or to facilitate the identification of cells that carry recombinant DNA.For example, many animal viruses contain DNA sequences that promote extrachromosomal replication of viral genomes in permissive cell types.Plasmids carrying these viral replicons replicate episomally as long as the appropriate factors are provided by genes carried on either the plasmid or the genome of the host cell.

[0083] Vector may or may not contain eukaryotic replicon.If eukaryotic replicon exists, vector can be amplified in eukaryotic cell by using suitable selectable marker.If vector does not contain eukaryotic replicon, it cannot be amplified as episome.Instead, recombinant DNA is integrated into genome of modified cell, where promoter directs expression of desired nucleic acid.

[0084] Also provided is a cell that comprises the polynucleotide / expression vector described herein.Such a cell is typically selected for high expression of recombinant protein (e.g., bacterial cell, plant cell, or eukaryotic cell, e.g., CHO cell, HEK-293 cell), but can also be a host cell (e.g., T cell or NK cell) with specific immune effector activity, for example, when the CDR of TCRL is grafted onto the T cell receptor or CAR that is transduced into the cell used in adoptive cell therapy.

[0085] V. Diagnostic applications Due to its high specificity, the binding agent is particularly suitable for diagnostic and therapeutic applications. According to one aspect of the present disclosure, a method for detecting cells that present HLA-restricted peptide antigens of interest is provided. The method comprises contacting cells with the binding agent (e.g., antibody) of the present disclosure that has specificity for the HLA-restricted peptide antigens of interest. The contact is carried out under conditions that allow the formation of immune complexes, and the presence or level of immune complexes is indicative of cells that present HLA-restricted peptide antigens of interest.

[0086] The term "detection," as used herein, refers to the act of detecting, perceiving, finding, revealing, visualizing, or identifying a cell. The exact detection method depends on the detectable moiety to which the antibody is attached.

[0087] A single cell may be used for detection, or multiple cells may be used.For example, cells may be derived from any biological sample, such as cell line, primary cell (e.g., tumor culture), and cell sample (e.g., surgical biopsy, e.g., incision or excision biopsy, fine needle aspirate, etc.). Methods for biopsy collection are well known in the art.The above detection methods may be utilized for the diagnosis of diseases (e.g., cancer) characterized by above-normal presentation or different tissue distribution of HLA-peptide complexes.

[0088] As used herein, the term "diagnosis" refers to classifying a disease, determining the severity (grade or stage) of a disease, monitoring the progression, and predicting the outcome and / or chances of recovery from a disease.

[0089] The subject may be a healthy subject (e.g., a human) undergoing a routine medical check-up. Alternatively, the subject may be at risk for a disease. This method may be used to monitor treatment efficacy. The binding agent may include a detectable moiety, i.e., the binding agent may be attached to a detectable moiety. Alternatively, or in addition, the binding agent (or a complex containing it) may be indirectly identified, for example, by using a secondary antibody. The contacting may be performed in vitro (i.e., cell lines, primary cells), ex vivo, or in vivo.

[0090] VI. Pharmaceutical Compositions, Formulations, and Dosages Pharmaceutical compositions according to the present disclosure and for use in accordance with the present disclosure may contain, in addition to the active ingredient (i.e., binder), pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials will depend on the route of administration, which may be oral or by injection, e.g., cutaneous, subcutaneous, or intravenous injection.

[0091] For intravenous, cutaneous or subcutaneous injection, or injection into affected area, active ingredient is in the form of parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art can fully prepare appropriate solution, for example, using isotonic medium such as sodium chloride injection, Ringer's injection, lactated Ringer's injection.Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0092] In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a binding agent described herein and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0093] In one embodiment, the composition is a pharmaceutical composition comprising at least one binder as described herein, together with one or more other pharma- ceutically acceptable ingredients known to those of skill in the art, including, but not limited to, pharma- ceutically acceptable carriers, diluents, excipients, adjuvants, bulking agents, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, colorants, flavoring agents, and sweetening agents.

[0094] In one embodiment, the composition further comprises other active agents, eg, other therapeutic or prophylactic agents.

[0095] Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical reference books, see, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash) Synapse Information Resources, Inc., Endicott, New York, USA (2001), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, (2000); and Handbook of Pharmaceutical Excipients, 2nd edition (1994).

[0096] The term "pharmacologically acceptable," as used herein, refers to compounds, ingredients, materials, compositions, dosage forms, etc., that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject of interest (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0097] Formulation can be prepared by any method well known in the pharmaceutical field.Such method includes the step of associating active compound with carrier that constitutes one or more accessory ingredients.Generally, formulation is prepared by associating active compound with carrier (for example, liquid carrier, finely divided solid carrier, etc.) uniformly and homogeneously, and then, if necessary, shaping product.

[0098] The formulations may be prepared to provide quick or sustained release; immediate, delayed, extended, or sustained release; or combinations thereof.

[0099] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may further contain other pharma- ceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is about 1 ng / ml to about 10 μg / ml, e.g., about 10 ng / ml to about 1 μg / ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0100] It will be understood by those skilled in the art that the appropriate dosage of the binding agent and the composition comprising the binding agent may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against risk or adverse side effects. The selected dosage level depends on a variety of factors, including, but not limited to, the activity of the specific compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, the race, sex, age, weight, condition, general health, and past medical history of the patient. The amount and route of administration of the binding agent are ultimately at the discretion of the physician, veterinarian, or clinician, but generally, the dosage is selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial toxic or adverse side effects.

[0101] Administration can be performed in one dose, or can be performed continuously or intermittently (e.g., in divided doses at appropriate intervals) during the course of treatment.The method of determining the most effective means and dosage of administration is well known to those skilled in the art, and varies according to the formulation used for treatment, the purpose of treatment, the target cell being treated, and the subject being treated.Single administration can be performed, or multiple administrations can be performed, and the dose level and pattern are selected by the physician, veterinarian, or clinician who performs treatment.

[0102] In some embodiments, the binding agent or the composition containing it is administered once a week for a therapeutically effective period. In some embodiments, the binding agent or the composition containing it is administered once a day for a therapeutically effective period. In some embodiments, the binding agent or the composition containing it is administered once a month for a therapeutically effective period. In some embodiments, the binding agent or the composition containing it is administered once a year for a therapeutically effective period.

[0103] In general, suitable dosages of binding agents range from about 100 ng to about 25 mg (more typically, from about 1 μg to about 10 mg) per kilogram of subject body weight per day. Where the composition includes a salt, ester, amide, prodrug, or the like, the amount administered is calculated based on the parent compound and therefore the actual weight used will be increased proportionately.

[0104] VII. Treatment The binding agents (e.g., antibodies, CARs) of the present disclosure are particularly useful for the treatment of cancer. In a specific embodiment, the cancer is characterized by the expression of PHOX2B. The types of cancers treated by the binding agents of the present disclosure include, but are not limited to, blood cancers, solid tumors, and non-solid tumors. Examples of solid tumors, such as sarcomas and cell tumors, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct Cancers include, but are not limited to, carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases). Also included are adult tumors / cancers and pediatric tumors / cancers.

[0105] The term "treatment" as used herein with respect to the treatment of a condition generally refers to treatment and therapy, whether in humans or animals (e.g., veterinary applications), in which a desired therapeutic effect is achieved, e.g., inhibition of progression of the condition, including slowing the rate of progression, halting the rate of progression, regressing the condition, ameliorating the condition, and curing the condition. Treatment as a preventative measure (i.e., prevention, prophylaxis) is also included.

[0106] The term "therapeutically effective amount," as used herein, relates to an amount of a material, such as a binding agent, or an antibody drug conjugate, composition, or dosage form that includes an active binding agent, that is effective to produce a desired therapeutic effect when administered in accordance with a desired treatment regimen.

[0107] In some embodiments, the treatment reduces or inhibits tumor growth for at least 6, 12, 24, 36, or 48 months. In some embodiments, the treatment enhances the immune response against the tumor.

[0108] The subject / patient is an animal or any mammalian species, including, but not limited to, a horse, dog, cat, pig, or primate. In a specific embodiment, the subject / patient is a human. EXAMPLES

[0109] VIII. Examples The following examples are included to illustrate specific embodiments. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in implementing the embodiments, and therefore can be considered to constitute specific implementation modes. However, in light of this disclosure, those skilled in the art should understand that many changes can be made to the disclosed specific embodiments while still obtaining the same or similar results without departing from the spirit and scope of this disclosure.

[0110] Example 1 – Materials and Methods ReD library panning. Beasley et al. 56 Using human germline IGLV1-51, IGLV3-1, and IGLV6-57 scaffolds paired with the IGHV3-23 scaffold, V L and V H Ruby scFv library (>10 11 We have constructed a system of 3D models that can represent the diversity of the data.

[0111] The Ruby scFv library and its combination and use with the Retained Display platform for antibody screening are described in WO / 2011 / 075761 (protein display), WO / 2013 / 023251 (soluble polypeptides), and WO / 2013 / 000023 (methods for protein display).

[0112] PHOX2B(43-51)A bound to MyOne Streptavidin C1 Dynabeads (ThermoFisher, Cat: 65002) * 24:02 The Ruby library was panned twice using MHC complexes. The panned library output was then displayed on the ReD cell display platform. 56 The cells were transferred to 0.5% β-d-thioglucopyranoside (Anatrace, Cat: 0314), permeabilized with 0.5% n-octyl β-d-thioglucopyranoside, and labeled with recombinant PHOX2B pMHC complexes ligated to fluorophores excitable by 405 and 488 nm lasers. Cells positive for target binding were isolated using a FACSMelody sorter (Becton-Dickinson).

[0113] PHOX2B A * 24:02 After two rounds of positive selection for binding to MHC complexes, counterlabeled A containing an irrelevant peptide * Two further rounds of FACS were performed using the 24:02 MHC complex. After four rounds of FACS, individual colonies were picked and grown in 96-well plates prior to scFv induction, cell permeabilization, and PHOX2B MHC labeling and detection by CytoFLEX (Beckman Coulter).

[0114] Clones identified as specifically binding to the PHOX2B(43-51) MHC complex were sequenced and unique scFvs were expressed in E. coli as fusions with the AviTag™ biotinylation motif. Biotinylated scFv proteins were released via permeabilization with 0.5% n-octyl β-d-thioglucopyranoside and purified to approximately 90% purity on nickel NTA agarose resin (ABT, catalog: 6BCL-NTANi).

[0115] Binding kinetics. Affinity measurements were performed using the BLItz™ system (ForteBio, USA) and analyzed using BLItz Pro™ software. A streptavidin biosensor (ForteBio, catalog: 18-5019) was loaded with AviTag™ biotinylated scFv, blocked with biotin, washed with PBS, and then associated with pMHC ligand in PBS.

[0116] Steady-state binding assay. Equilibrium binding assays with target pMHC were also established using MyOne Streptavidin C1 Dynabeads. Briefly, 50 micrograms of Streptavidin C1 Dynabeads were incubated with excess biotinylated scFv, then blocked with free biotin and washed with PBS. Fluorophore-labeled pMHC complexes were added at a concentration of 3.5 nM and incubated for 1 h at 4°C, followed by incubation at 25°C for 10 min. Binding of free MHC complexes to the beads was quantified by CytoFLEX at 488 nm (ex) / 525 nm (em). Binding was normalized to beads without scFv and to beads containing an irrelevant control MHC complex.

[0117] The bead-binding assay assessed the binding of scFv to MHC complexes containing alanine-scanning substitutions of the PHOX2B peptide and 95 unrelated 9-residue long peptides A. *24:02 It was used to quantify the binding of MHC complexes to the plate and the degree of cross-reactivity of binding of MHC complexes containing peptides identified by eXpitope 2.0 as having high homology to PHOX2B peptides.

[0118] Virus production and transduction of Jurkat cells and primary T cells. Retroviruses for transduction of Jurkat cells and primary CD4 / 8 T cells were produced using Platinum-A (Plat-A) cells, a retrovirus packaging cell line. 7 × 10 cells were cultured at 4 °C for 1 h. 5 Cells / well were seeded in 6-well plates and transfected with 2.5 μg of the appropriate TCR or CAR construct in the retroviral vector pMP71 using Lipofectamine 3000 (Life Technologies, Invitrogen). After 24 hours, the medium was replaced with IMDM-10% FBS or AIM-V-10% FBS for Jurkat cells or primary cells, respectively. After 24 hours of incubation, the supernatant was collected and filtered through a 0.2 mM filter.

[0119] To generate replication-deficient lentivirus, a second-generation lentivirus system was used. 15 million HEK 293T cells were seeded in 15 cm dishes the day before transfection. On the day of transfection, 80 μL of Lipofectamine 3000 (Life Technologies, Invitrogen) was added to 3.5 mL of room temperature Opti-MEM medium (Gibco). At the same time, 80 μL of P3000 reagent (Thermo Fisher Scientific), 12 μg of psPAX2 (Gag / Pol), 6.5 μg of pMD2.6 (VSV-G envelope), and equimolar amounts of transfer plasmids were added to 3.5 mL of room temperature Opti-MEM medium. Viral supernatants were collected after 24 and 48 h, briefly centrifuged at 300 g, and passed through a 0.45 μM syringe.

[0120] Jurkat cells were cultured at 1 × 10 per well in a 6-well plate pretreated with Retronectin (20 mg / mL, Takara Bio. Inc.). 6 Cells were seeded / well and spinoculated with 2 mL of retroviral supernatant at 800×g for 30 min at RT. After 24 h, cells were harvested and grown in IMDM-10% FBS.

[0121] Primary T cells were thawed and activated by culturing for 3 days in the presence of 100 U / ml IL-2 and anti-CD3 / CD28 beads (Dynabeads, Human T-Activator CD3 / CD28, Life Technologies) at a bead:T cell ratio of 3:1. On days 4 and 5, activated cells were plated at 1 × 10 cells / mL in 6-well plates pretreated with Retronectin (20 mg / mL, Takara Bio.Inc.). 6 Cells / well were seeded and spinoculated with 2 mL of retroviral supernatant for 2 hours at 2400 rpm at 32° C. On day 6, cells were harvested, washed, beads were magnetically removed, and cells were expanded in AIM-V-10% FBS supplemented with 25 U / ml IL-2.

[0122] Primary human T cells were thawed and activated in G-Rex-based vessels (Wilson Wolf) for 1 day in the presence of 5ng / ml recombinant IL-7, 5ng / ml recombinant IL-15, and anti-CD3 / CD28 beads (Dynabeads, Human T-Activator CD3 / CD28, Life Technologies) at a bead:T cell ratio of 3:1. On day 2, thawed lentiviral vectors were added to the cultured T cells together with 10μg / mL polybrene (Millipore Sigma), and 24 hours later the vessels were filled with complete AIM-V medium supplemented with the indicated concentrations of IL-7 and IL-15. On day 10, cells were harvested and washed. Activation beads were magnetically removed and cell viability was measured before freezing.

[0123] Human neuroblastoma cell lines were seeded in 6 cm dishes and 2 mL of thawed lentiviral vector generated by transfer plasmid pLenti-CMV-eGFP-Puro (Addgene plasmid #17448) was added along with 10 μg / mL polybrene (Millipore Sigma). Cells were selected for eGFP expression using flow-assisted cell sorting (BD FACSJazz, BD Biosciences), followed by 10 μg / mL puromycin selection.

[0124] Selective cross-reactive antigen presentation ( S elective C ross- R eactive A ntigen P Tumor antigens were compared to the entire normal human proteome in matched HLA (85,915,364 total normal peptides in HLA 84 HLA). Each residue at the same position in the tumor peptide and human peptide was assigned a score for perfect match, similar amino acid classification, or different polarity, scored as 5, 2, or -2, respectively. Residues 1 and 3-8 were used to calculate similarity scores based on amino acid classification, excluding MHC anchor residues, and to determine hydrophobicity. The maximum normal tissue RPKM value was then identified from 1643 normal tissues in GTEx. The normal peptides were compared to a database of normal tissue immunopeptidomes. 67 An overall cross-reactivity score for each normal peptide was then calculated using the following formula: TIFF2024532258000004.tif14128 (where n is the peptide length, P is the score of each amino acid in the normal peptide compared to the tumor antigen, b is the pMHC binding affinity of the normal peptide, E max is maximum normal tissue expression). The algorithm is available at marisshiny.research.chop.edu / sCRAP.

[0125] Tetramer / dextramer staining and flow cytometry analysis. Surface expression and binding of CAR-transduced Jurkat cells and primary T cells was measured by staining with dextramer conjugated to PE or APC bearing NB antigen peptide-MHC (Immudex). Cells were harvested from culture, washed with 2 ml PBS at 800×g for 5 min, incubated with 1 μl dextramer for 10 min in the dark, washed again, and resuspended in 300 ul PBS for analysis. Typically, 5×10 5 Cells were used for staining and analyzed on a BD LSR II (BD Biosciences) or an Attune Acoustic Focusing Cytometer (Applied Biosystems, Life Technologies).

[0126] Cross-reactive pMHC screening. Potential cross-reactive peptides (GenScript) were suspended at a working concentration of 200 μM. For each test, 0.5 μL of peptide was added to 5 μL of HLA-A * Add 24:02 empty loadable tetramer (Tetramer Shop) and then incubate on ice for 30 min or use TABBR peptide exchange as previously described 68 Once prepared, pMHC tetramers were used to stain the cells (see above).

[0127] Incucyte cytotoxicity assay. 0.5 x 10 5 Tumor cell targets were incubated with varying ratios of transduced primary cells (5 × 10 for effector:target (E:T) ratios of 10:1, 5:1, 2:1, 1:1, and 1:2, respectively) in 96-well plates at 37 °C in the presence of 0.05 μM Caspase-3 / 7 Red. 5 , 2.5×10 5 , 1×10 5 , 0.5×10 5 , and 2.5 × 10 4) (Incucyte, Essence BioScience). Plates were run on the Incucyte for 24-72 hours and measured for apoptotic activity via caspase cleavage and comparison of relative confluency. After the assay, supernatants were collected for ELISA. Total GFP integrated intensity (total GCU × μm 2 / image) was assessed as a quantitative measure of live GFP+ tumor cells. Values ​​were normalized to the t=0 measurement.

[0128] Cytokine secretion assay. Cell supernatants collected from the cytotoxicity assay were thawed and plated in triplicate for each condition. IFN-γ and IL-2 levels were measured using ELISA kits according to the manufacturer's protocol (BioLegend).

[0129] Expression, refolding, and purification of recombinant peptide / HLA molecules. HLA-A * 02:01 and HLA-A * The 24:02 construct was cloned into the pET24a+ plasmid for expression in bacteria. * 02:01 (heavy chain), HLA-A * DNA plasmids encoding 24:02 (heavy chain) and human β2M (light chain) were prepared as previously described. 71 The plasmid p53 was transformed into E. coli BL21-DE3 (Novagen) using the plasmid p53-100001 and expressed as inclusion bodies and refolded. E. coli cells were grown in autoinduction medium (16-18 h). 72The E. coli cells were then harvested by centrifugation and resuspended in 25 mL of BugBuster (Milipore Sigma) per liter of culture. The cell lysate was sonicated and then pelleted by centrifugation (5,180×g, 4° C., 20 min) to collect the inclusion bodies. The inclusion bodies were washed with 25 mL of wash buffer (100 mM Tris pH 8.0, 2 mM EDTA, and 0.01% v / v deoxycholate), sonicated, and pelleted by centrifugation. A second wash was performed using 25 mL of Tris-EDTA buffer (100 mM Tris pH 8.0 and 2 mM EDTA). Again, the solution was resuspended by sonication and then centrifuged. The inclusion bodies were then solubilized by resuspension in 6 mL of resuspension buffer (100 mM Tris pH 8.0, 2 mM EDTA, 0.1 mM DTT, and 6 M guanidine-HCl). The solubilized inclusion bodies of heavy and light chains were mixed in a 1:3 molar ratio and then added dropwise over 2 days to 1 L of refolding buffer (100 mM Tris pH 8.0, 2 mM EDTA, 0.4 M arginine-HCl, 4.9 mM L-glutathione reduced, 0.57 mM L-glutathione oxidized) containing 10 mg of synthetic peptide (Genscript) of >98% purity as confirmed by mass spectrometry. Refolding was allowed to proceed for 4 days at 4° C. without stirring. After this incubation period, the refolding mixture was dialyzed against size exclusion buffer (25 mM Tris pH 8.0 and 150 mM NaCl). After dialysis, the sample was first concentrated using a Labscale Tangential Flow Filtration system and then concentrated to a final volume of 5 mL using Amicon Ultra-15 Centrifugal 10 kDa MWCO filter units (Millipore Sigma). Purification was performed using size exclusion chromatography on a HiLoad 16 / 600 Superdex 75 column.After size exclusion, the sample was further purified by anion exchange chromatography using a MonoQ 5 / 50 GL column and a 0-100% gradient of Buffer A (25 mM Tris pH 8.0 and 50 mM NaCl) and Buffer B (25 mM Tris pH 8.0 and 1 M NaCl). The purified protein was exhaustively exchanged into 20 mM sodium phosphate (pH 7.2) and 50 mM NaCl. The final sample was validated using SDS-PAGE to confirm the formation of pMHC complexes containing both heavy and light chains.

[0130] Immunohistochemistry. CD3 (Dako A0452), PHOX2B (Abcam ab183741), and HLA-ABC (Abcam ab70328) antibodies were used to stain formalin-fixed paraffin-embedded tissue slides. Staining was performed with a Bond Max automated staining system (Leica Biosystems). The Bond Refine polymer staining kit (Leica Biosystems, DS9800) was used. Standard protocols were followed, except that primary antibody incubation was extended to 1 h at room temperature. CD3, PHOX2B, and HLA-ABC antibodies were at dilutions of 1:100, 1:500, and 1:1200, respectively. Antigen retrieval was performed in E1 (Leica Biosystems) retrieval solution for 20 min (E2 for PHOX2B). Slides were rinsed and dehydrated in a series of ascending concentrations of ethanol and xylene, then coverslipped. Stained slides were then digitally scanned at 20x magnification on an Aperio CS-O slide scanner (Leica Biosystems).

[0131] Mouse PC-CAR T cell preclinical study. NOD SCID gamma (NSG) female (6-8 weeks old) mice (stock no. 005557) from Jackson Laboratories were used to propagate subcutaneous xenografts. All mice were maintained in barrier conditions and experiments were performed using the IACUC protocols and conditions of the Children's Hospital of Philadelphia. Treatment was initiated via lateral tail vein injection. The dose administered was 100ul of vehicle or CAR T cells per mouse as a single treatment. Tumor volumes of 150mm 3 ~250mm 3 Treatment was administered when tumors reached 2.0 cm 3 Tumor volume and survival were monitored by twice weekly measurements until the tumor reached a size of 100 μg / kg or until the mice showed signs of graft-versus-host disease. After the onset of GVHD, when an animal showed circling, tachypnea, urine staining, weight loss, and a body condition score of 2 as determined by visual inspection, the animal was removed from the study and the study was terminated. Onset of GVHD was defined as urine staining and a 20% body weight loss, or a 10-15% weight loss accompanied by circling, tachypnea, or poor body condition.

[0132] Example 2 - Results Generation of PC-CAR T cells against PHOX2B. Because multiple screenings did not identify high affinity TCRs due to the lack of immunogenicity of self-antigens, the inventors pursued the development of scFv-based CARs rather than modified T cell receptors (TCRs) against PHOX2B. It was reasoned that a synthetic peptide-centered receptor could be used to induce immunogenicity in pMHC that is not naturally immunogenic.

[0133] To screen for PHOX2B peptide-specific clones, we developed a protein display platform, Retained Display, which allows flow cytometric selection of pMHC-binding scFvs in permeabilized bacterial cells. 56 (ReD) system, >10 11The scFv library was used with 10 members of the PHOX2B(43-51) scFv library. From there, two clones, 10 (SEQ ID NO:3) and 302 (SEQ ID NO:12), were isolated and further characterized. Figures 1A and 1B show the binding kinetics of the scFv proteins of clones 10 and 302 to the target PHOX2B(43-51) MHC complex compared to binding to an irrelevant complex, with an extremely slow kinetic off-rate (k d =7.6×10 -4 The HLA-A complex was also stained with PHOX2B(43-51) as a target. * A panel of 95 unrelated peptides and four highly similar peptides assembled into the 24:02 complex Clones were further characterized by binding to TIFF2024532258000005.tif11128. Figures 2A-B show that both clones 10 (Figure 2A) and 302 (Figure 2B) show no detectable binding to a panel of 95 unrelated pMHC complexes. As a further indication of specificity for the target complex, Figures 3A-B show that both clones 10 (Figure 3A) and 302 (Figure 3B) show highly selective binding to the PHOX2B target pMHC and no detectable binding to four peptides identified from the human proteome with homology to the target. Two of these peptides, TIFF2024532258000006.tif4128 was also identified using mass spectrometry to be presented in vivo by the immunopeptidome (www.iedb.org). To elucidate the interaction between the scFv and the PHOX2B MHC complex and also to clarify the sensitivity of the clones' specific binding to the substitution of different amino acids at each position of the target peptide, we performed "X-scan" mutagenesis of a PHOX2B target peptide that was sequentially mutated to 18 natural amino acids (other than cysteine) at positions 1, 3, 4, 5, 6, 7, and 8 (i.e., non-anchor positions). Figure 4 shows that clone 10 tolerates Phe / His / Lys / Trp / Tyr at position 3 (Asn for PHOX2B), but has a strong preference for PHOX2B target identity at positions 4, 5, and 7, and an absolute constraint for Arg at position 5. Figure 5 shows that clone 302 shows a highly specific footprint at positions 5, 6, 7, and 8, with a constraint at position 4. This strong interaction of the binder of the invention with 4+ residues of the PHOX2B targeting peptide highlights the superior selectivity of PC-CAR compared to the typical 3-4 residues that interact with the TCR. 58 .

[0134] To address cross-reactivity with pMHC in normal tissues, we performed alanine scanning or receptor 57 We developed an algorithm to predict potential selective cross-reactive antigen presentation (sCRAP; marisshiny.research.chop.edu / sCRAP) in the same HLA allotype, which allows preemptive selectivity filtering in the early stages of scFv screening without the need for HLA-A (Figure 25A-C). * The sCRAP algorithm was evaluated by testing its ability to predict cross-reactivity of the MAGE-A3 peptides presented on 01:01. * Targeting of the MAGE-A3 peptide presented above has previously been shown to mediate HLA-A expression in myocardial tissue. *01:01 resulted in lethal cross-reaction with another peptide derived from the TITIN protein presented above. 15 We predicted MAGE-A3 cross-reactivity with TITIN peptide as the fourth most predictive out of 1,143,861 possible self-peptides presented in cardiac tissue (FIGS. 26A-B).

[0135] We then screened a panel of PHOX2B-specific CARs against the top seven pMHC predicted by sCRAP (Figure 6), thereby eliminating cross-reactive CARs and prioritizing those with the highest target selectivity. We identified clone 10LH CAR (SEQ ID NO:21) and clone 302LH CAR (SEQ ID NO:22) as possessing the highest specificity profile for further development.

[0136] To test the functional significance of binding to potential off-target pMHC predicted by sCRAP, we pulsed HLA-matched / PHOX2B-negative SW620 colon adenocarcinoma cells with PHOX2B peptide and potential cross-reactive peptides at a range of concentrations (Figure 7). Pulsing of PHOX2B peptide resulted in complete cytotoxicity when co-cultured with 10LH at the lowest tested concentration of 0.1 μM. 10LH CAR T cells were not induced to cytotoxicity by the most cross-reactive predicted peptide, ABCA8, at 10 μM and only at a supraphysiological concentration of 50 μM. The second most cross-reactive peptide (MYO7B) showed no CAR-induced cytotoxicity with 10LH at concentrations up to 50 μM. Neither ABCA8 nor MYO7B have been detected in normal tissue immunopeptidomes. 11None of the peptides previously detected in normal tissue immunopeptidomes show cross-reactivity with PC-CAR 10LH. These screens demonstrate the potential of sCRAP to preemptively identify off-target effects, efficiently screen for their functional significance, and identify binders with highly selective binding to tumor targets.

[0137] PC-CAR T cells break the HLA restriction imposed by conventional TCRs. Because antigen processing and presentation are prerequisites for the detection of a given MHC peptide by immunopeptidomics, we hypothesized that the same peptide could be presented on additional HLA allotypes that could bind to the peptide's anchor residues, and that some of these peptides could be presented in a similar enough conformation to be recognized by peptide-centric scFv binders. To identify additional HLA allotypes that could present the same PHOX2B peptide, we used the population-scale antigen presentation tool ShinyNAP. 7 Using pMHC structural modeling software RosettaMHC, we identified eight additional HLAs predicted to bind to the nine-residue stretch of PHOX2B. We then used the pMHC structural modeling software RosettaMHC to model the 3D conformations and binding free energies of peptides presented by the additional HLA alleles. 59 Using HLA-A * HLA-A was the highest scoring candidate for PC-CAR recognition of the first PHOX2B peptide discovered at 24:02. * 23:01 and HLA-B * After confirming the binding of QYNPIRTTF (SEQ ID NO:1) to these alternative allotypes, we measured the ability of 10LH to recognize these pMHC and found that 10LH binds to HLA-A * In addition to 24:02, HLA-A * 23:01 and HLA-B *We also found that the 9-residue long QYNPIRTTF (SEQ ID NO:1) of PHOX2B presented by 14:02 binds with high affinity (FIGS. 8A-C). * 07:02, but 10LH binds to HLA-C * It was also found to have 17.4-fold lower binding to 07:02. * To demonstrate functionally relevant recognition of the predictive value of PHOX2B presentation in HLA-A 23:01, we * 23:01 / PHOX2B - Melanoma cell line WM873 was pulsed with QYNPIRTTF (SEQ ID NO:1) peptide, and antigen-specific killing was induced in cells pulsed with the peptide, but cytotoxicity was not induced in cells pulsed with a mismatched peptide (Figure 9A-B). * 23:01 is the most common non-A2 allele in people of African descent, highlighting the potential of PC-CAR to extend clinical application to underserved populations. These findings demonstrate the potential to significantly expand the eligible patient population for peptide-centric scFv-based immunotherapy.

[0138] PC-CAR T cells against PHOX2B have potent antitumor activity in preclinical models of neuroblastoma. * 24:02 and HLA-A * The on-target killing potential of 10LH was tested using 23:01 neuroblastoma cell lines (SKNAS, NBSD, and SKNFI), showing complete tumor cell killing and robust IFNγ release after 24 hours at an effector-target ratio (E:T) of 5:1 (Figure 10). Functional cross-reactivity of PC-CAR to the peptide milieu presented by off-target tissues was tested, demonstrating that 10LH inhibited the expression of PHOX2B in three HLA-A-associated tumors that do not express PHOX2B. *No activity was observed in 24:02 cell lines (SW620; colorectal adenocarcinoma, KATO III gastric adenocarcinoma, and HEPG2 hepatocellular carcinoma). To confirm the specificity of killing by PC-CAR, we pulsed HLA-matched PHOX2B-negative cancer cell lines with PHOX2B peptides to force overexpression of PHOX2B. Specific killing was only observed in cells pulsed with PHOX2B peptides and cells transduced with full-length PHOX2B mRNA, but not in cells pulsed with non-specific CHRNA3, ABCA8, and MYO7B peptides presented on the same HLA, or in cells transduced with full-length PRAME mRNA, indicating that native PHOX2B is processed and presented on MHC, where it is specifically recognized by PC-CAR. To detect PHOX2B pMHC on the cell surface, tetramerized 10LH scFv was generated and stained on-target and off-target cell lines, showing significant surface PHOX2B pMHC on neuroblastoma cells and not on HLA-matched controls (Figure 11), suggesting that these reagents have the potential to be used to assess the presence of antigens in biopsy tissue samples. CARs marked as cross-reactive by sCRAP were also found to show significant cross-reactivity, confirming the functional significance of the algorithmic cross-reactivity (data not shown).

[0139] The present inventors then investigated HLA-A * 24:02 (SKNAS and COG-564x) and HLA-A * Immunodeficient mice bearing xenografts of 23:01 (NBSD) were cultured at 100 mm 3 ~250mm 3 After reaching 10, 10LH and 302LH transduced 6 Mice treated with 10LH and 302LH PC-CARs showed both HLA-A and HLA-B phenotypes. *In 24:02 xenografts, complete tumor responses were observed (Figure 12), whereas HLA-A * In 23:01 NBSD xenografts, only mice treated with 10LH showed this, which is consistent with HLA-A * The relative affinity of these two constructs for the PHOX2B peptide presented on 23:01 (Figure 9A) directly correlated with the relative affinity of these two constructs for the PHOX2B peptide presented on 23:01, suggesting that threshold affinity or different binding modes by different scFvs may contribute to their ability to recognize peptides with slightly altered conformations when presented by different HLA alleles. We also observed that CAR treatment induced a significant upregulation of MHC in the tumor. The COG-564x PDX model was generated from postmortem blood draws from a patient with high-risk MYCN-amplified neuroblastoma who had experienced multiple relapses and shows a very rapid tumor growth rate in mice. In this experiment, one mouse treated with the 10LH construct grew to 2 cm just one week after PC-CAR T cell treatment. 3 The tumor reached endpoint size of 1000 and was available for analysis, while all other tumors in this arm just missed the endpoint size and then all regressed. The only COG-564x tumors and NBSD tumors that reached the endpoint showed significant PC-CAR T cell infiltration and dramatic upregulation of MHC expression compared to endpoint tumors treated with non-transduced CAR-T cells (Figure 13). This upregulation was likely due to the strong IFNγ release measured in vitro, suggesting that these treatments may activate T cell expansion at low antigen density to initiate a feed-forward cascade that increases MHC and antigen presentation.

[0140] In vitro characterization of clones 9, 1113, and 1114. scFv clones 9 (SEQ ID NO:23), 1113 (SEQ ID NO:32), and 1114 (SEQ ID NO:41) also bind to PHOX2B(43-51)A. *These were isolated from the ReD library as binding to the 24:02 MHC complex. They were characterized for their binding kinetics to the target complex by Biolayer Interferometry and showed no binding kinetics to the non-target complex (Figures 16A-B and 17) and to 95 unrelated A * The scFvs were shown to specifically bind to the PHOX2B target complex compared to the 24:02 MHC complex. They were further analyzed for binding to homologous peptides, two of which, RYVIIPTTF (SEQ ID NO:61) and KYNIFRSTF (SEQ ID NO:62), were also identified using mass spectrometry to be presented in vivo by the immunopeptidome (www.iedb.org) (Figures 20A-B and 21). To clarify the interaction between each scFv and the PHOX2B MHC complex and also to clarify the sensitivity of the specific binding of the clones to the substitution of different amino acids at each position of the target peptide, we performed an "X-scan" mutagenesis of the PHOX2B target peptide sequentially mutated to 18 natural amino acids (other than cysteine) at positions 1, 3, 4, 5, 6, 7, and 8 (i.e., non-anchor positions). Figure 22 shows that clone 9 stringently interacts with positions 3, 4, 5, and 6 of the PHOX2B target peptide, and also constrains amino acid identity at positions 1, 7, and 8. Figure 23 shows that clone 1113 stringently interacts with positions 3, 4, 5, 6, 7, and 8 of the target peptide. Figure 24 shows that clone 1114 stringently interacts with positions 3, 4, 5, and 6 of the target peptide, and also constrains binding identity at positions 7 and 8.

[0141] The sequence homology of the CDR3 of clones 9 and 1114 establishes the binding motif. Figures 22 and 24 show that clones 9 and 1114 show close pairwise preferences for MHC target peptide amino acid identity in the X-scan through each position. Examination of the sequences of these clones shows that the light chain variable domain CDR3 loops have the same length and consensus QAWDS[L / I]G[V / N][N / M]TVV (SEQ ID NO:50). Similarly, the heavy chain variable domain CDR3 loops have the same length and consensus ASE[A / Y][Y / T][S / N]AFDI (SEQ ID NO:51). This conservation and identity of CDR3 length, combined with the close similarity of the X-scan, indicates that these clones represent two related solutions for binding to the PHOX2B target with high specificity. Other binding solutions with close CDR identity were identified by methods known in the art, such as V and V, and V and V, by the above methods. L Domains and V H Mutational scanning of both CDR domains or V domains of clones 9 (SEQ ID NO:25), 10 (SEQ ID NO:5), 302 (SEQ ID NO:14), 1113 (SEQ ID NO:34), and 1114 (SEQ ID NO:43) H Diversified V by Domain L It can be found by swapping domains.

[0142] In vitro characterization of bispecific antibodies. Figure 28 shows the in vitro characterization of human HLA-A treated with PHOX2B targeting peptide (114) at a concentration of 1 μM. *We show that K562 cells stably transfected with the 24:02 expression construct were cytotoxic by clone 10, but not by an equal concentration of a closely related unrelated peptide (693XR, RYVIIPTTF (SEQ ID NO:61)) or an equal volume of dimethylsulfoxide (DMSO, negative control). Cytotoxicity was measured by incubating K562 cells with peptide for 3 hours at 28°C, and the K562 cells were incubated with activated human primary CD3+ cells in the presence or absence of purified bispecific antibody of clone 10 (RU141-10) at concentrations of 100 ng / ml, 50 ng / ml, 20 ng / ml, 10 ng / ml, and 5 ng / ml, or an unrelated (RU68-615) bispecific control antibody at 100 ng / ml. + T cells (effector). Effector and target cells were incubated at a ratio of 3:1, respectively. After 24 h of co-incubation at 37° C., surviving K562 target cells were quantified by flow cytometry, and the relative percentage of surviving cells was calculated with reference to the experiment lacking bispecific antibodies. No cytotoxicity was observed for the conditions without bispecific control or with RU86-615 unrelated bispecific control, either when incubated with PHOX2B target peptide (114), when incubated with unrelated peptide (693XR, RYVIIPTTF (SEQ ID NO:61)), or when incubated with DMSO (negative control).

[0143] FIG. 29 shows human HLA-A receptor agonists treated with either a closely related unrelated peptide (693XR, RYVIIPTTF (SEQ ID NO:61)) or a PHOX2B targeting peptide (114) at a concentration of 1 μM, or an equal volume of dimethyl sulfoxide (DMSO, negative control). * 24:02 (Figure 29A) or human HLA-A *Figure 29B compares the cytotoxicity of clones 10 and 302 against K562 cells stably transfected with an expression construct for 23:01 (Figure 29B). After incubating K562 cells with peptides for 3 hours at 28°C, the K562 cells were incubated with purified bispecific antibody clone 10 (RU141-10) at a concentration of 100 ng / ml or 10 ng / ml, clone 302 (RU141-302) at a concentration of 100 ng / ml or 10 ng / ml, and with or without an irrelevant (RU68-615) bispecific control antibody at a concentration of 100 ng / ml, activated human primary CD3 + T cells (effector). Effector and target cells were incubated at a ratio of 3:1, respectively. After 24 hours of co-incubation at 37°C, surviving K562 target cells were quantified by flow cytometry, and the relative percentage of surviving cells was calculated with reference to the experiment lacking bispecific antibody.

[0144] Referring to FIG. 29, bispecific clone 10 (RU141-10) at concentrations of 100 ng / ml and 10 ng / ml and bispecific clone 302 (RU141-302) at a concentration of 100 ng / ml were pulsed with PHOX2B targeting peptide (114) and human HLA-A * In contrast, cytotoxicity was clearly observed in cells incubated with stably transfected K562 cells expressing 24:02. * When incubated with stably transfected K562 cells expressing 23:01, cytotoxicity was observed only for bispecific clone 10 (RU141-10) at concentrations of 100 ng / ml and 10 ng / ml, but not for bispecific clone 302 (RU141-302) at concentrations of 100 ng / ml and 10 ng / ml. Conditions without bispecific control or with the RU86-615 unrelated bispecific control did not show any significant cytotoxicity against human HLA-A. * Stably transfected K562 cells expressing 23:01 or human HLA-A* When incubated with stably transfected K562 cells expressing 24:02, no cytotoxicity was observed with the PHOX2B targeting peptide (114), an unrelated peptide (693XR, RYVIIPTTF (SEQ ID NO:61)), or DMSO (negative control).

[0145] Table 1. Exemplary antigen binding protein sequences TIFF2024532258000007.tif220150TIFF2024532258000008.tif226150TIFF2024532258000009.tif232150 TIFF2024532258000010.tif226150TIFF2024532258000011.tif228150TIFF2024532258000012.tif111150

[0146] Table 2: Sequence of PHOX2B 10LH PC-CAR 4-1bb / CD3z TIFF2024532258000013.tif219149

[0147] (Table 3) PHOX2B 302LH PC-CAR 4-1bb / CD3z TIFF2024532258000014.tif210150

[0148] All compositions and methods disclosed and claimed herein can be manufactured and carried out without undue experimentation in light of this disclosure.Although the compositions and methods of this disclosure are described with reference to specific embodiments, it is clear to those skilled in the art that variations can be applied to the compositions and methods described herein, and to the steps or sequence of steps of the methods, without departing from the concept, spirit and scope of this disclosure.More specifically, it is clear that the agents described herein can be substituted with certain agents that are both chemically and physiologically related, and still achieve the same or similar results.All such similar substitutions and modifications that are clear to those skilled in the art are deemed to be within the spirit, scope and concept of this disclosure, as defined by the appended claims.

[0149] IX. References The following references, and other references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2024532258000015.tif189150TIFF2024532258000016.tif210150TIFF2024532258000017.tif217150TIFF2024532258000018.tif217150 TIFF2024532258000019.tif210150TIFF2024532258000020.tif217150TIFF2024532258000021.tif224150TIFF2024532258000022.tif190150

Claims

1. a peptide having the sequence QYNPIRTTF (SEQ ID NO:1), an HLA α chain polypeptide, and a β 2 HLA PHOX2B containing microglobulin polypeptides QYNPIRTTF A binding agent comprising an antigen binding site that specifically binds to the complex.

2. The antigen-binding site has a dissociation constant (K) of about 500 nM or less, about 200 nM or less, or about 13 nM or less. D ) and HLA PHOX2B QYNPIRTTF 10. The binding agent of claim 1, which binds to the complex.

3. 10. The binding agent of claim 1, which is not MHC restricted.

4. The antigen-binding site is HLA-A * 24:02, HLA-A * 23:01, HLA-B * 14:02, HLA-C * 07:01, HLA-C * 06:02, HLA-A * 29:02, and HLA-A * HLA PHOX2B presented by two or more, three or more, or four or more of 32:01 QYNPIRTTF 10. The binding agent of claim 1, which binds to the complex.

5. The antigen-binding site is (a) a CDR-L1 region of SEQ ID NO: 6, a CDR-L2 region of SEQ ID NO: 7, and a CDR-L3 region of SEQ ID NO: 8; L and / or (b) V comprising a CDR-H1 region set forth in SEQ ID NO: 9, a CDR-H2 region set forth in SEQ ID NO: 10, and a CDR-H3 region set forth in SEQ ID NO: 11; H ; or (a) a CDR-L1 region of SEQ ID NO: 15, a CDR-L2 region of SEQ ID NO: 16, and a CDR-L3 region of SEQ ID NO: 17; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 18, a CDR-H2 region as set forth in SEQ ID NO: 19, and a CDR-H3 region as set forth in SEQ ID NO: 20; H ; or (a) a CDR-L1 region of SEQ ID NO: 26, a CDR-L2 region of SEQ ID NO: 27, and a CDR-L3 region of SEQ ID NO: 28; L and / or (b) a CDR-H1 region set forth in SEQ ID NO: 29, a CDR-H2 region set forth in SEQ ID NO: 30, and a CDR-H3 region set forth in SEQ ID NO: 31; H ; or (a) a CDR-L1 region of SEQ ID NO: 35, a CDR-L2 region of SEQ ID NO: 36, and a CDR-L3 region of SEQ ID NO: 37; L and / or (b) a CDR-H1 region as set forth in SEQ ID NO: 38, a CDR-H2 region as set forth in SEQ ID NO: 39, and a CDR-H3 region as set forth in SEQ ID NO: 40; H ; or (a) a CDR-L1 region of SEQ ID NO: 44, a CDR-L2 region of SEQ ID NO: 45, and a CDR-L3 region of SEQ ID NO: 46; L and / or (b) a CDR-H1 region set forth in SEQ ID NO: 47, a CDR-H2 region set forth in SEQ ID NO: 48, and a CDR-H3 region set forth in SEQ ID NO: 49; H 2. The binder of claim 1, comprising:

6. The binder is V H and V L and where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:5, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:4; or The binder is V H and V L and where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:14, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:13; or The binder is V H and V L and where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:25, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:24; or The binder is V H and V L and where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:34, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:33; or The binder is V H and V L and where V H has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:43, and / or V L has at least 75%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO:42; The binder of claim 1.

7. 10. The binding agent of claim 1, which is an antibody.

8. V H and V L and V H V L 10. The binding agent of claim 1, wherein the binding agent is fused to

9. mAb, Fab, Fab', F(ab') 2 2. The binding agent of claim 1, wherein the binding agent is selected from the group consisting of: Fv, Dab single chain antibody, scFv, CAR, ADC, KIR, BiTE, BsMAb, and TFP.

10. 10. The binding agent of claim 9, which is a single-chain variable fragment (scFv).

11. 2. The binding agent of claim 1, which is a chimeric antigen receptor (CAR).

12. 2. The binding agent of claim 1, which is a killer Ig-like receptor (KIR).

13. 2. The binding agent of claim 1, which is a modular bispecific T cell-like inducer (BiTE).

14. 12. The binding agent of claim 11, wherein the CAR comprises the intracellular signaling domain of CD3ε, CD3γ, CD3δ, TCRα, or TCRβ.

15. 10. An isolated polynucleotide comprising a nucleic acid sequence encoding the binding agent of claim 1.

16. 16. An expression vector comprising the polynucleotide of claim 15 operably linked to a cis-acting regulatory element.

17. A cell comprising the polynucleotide of claim 15.

18. A cell comprising the expression vector described in claim 16.

19. 19. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 14, the isolated polynucleotide of claim 15, the expression vector of claim 16, or the cell of claim 17 or 18.

20. An in vitro method for detecting cancer cells, comprising: QYNPIRTTF contacting a cell with a binding agent according to any one of claims 1 to 10 under conditions that allow binding of the binding agent to a complex, QYNPIRTTF The method, wherein the binding to or level of the complex is indicative of cancer cells.

21. The method described in claim 20, wherein the cancer cells are neuroblastoma cells.

22. 20. The pharmaceutical composition of claim 19, for use in a method for diagnosing and treating cancer in a subject in need thereof, the method comprising: (a) detecting the presence of cancer cells in said subject by the method of claim 20; (b) diagnosing the subject as having cancer if cancer cells are detected; and (c) treating the subject with an anti-cancer therapy 10. A pharmaceutical composition comprising:

23. 23. The pharmaceutical composition of claim 22, wherein the cancer cells are neuroblastoma cells.

24. 20. The pharmaceutical composition of claim 19 for use in a method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the binding agent of any one of claims 1 to 14, the isolated polynucleotide of claim 15, the vector of claim 16, or the cell of claim 17 or 18, thereby treating cancer.

10. A pharmaceutical composition comprising:

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is neuroblastoma.

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