Checkpoint modulators for enhancing immunotherapy

Abstract

Binder molecules that can bind to specific phosphorylated amino acids in proteins, such as signal transduction proteins, and affect the regulation of signal transduction pathways containing these proteins are disclosed. In one embodiment, the binding agent molecule can bind to intracellular phosphotyrosine in PD-1 and prevent activation of the PD-1 pathway. Intracellular expression of these binding agents, such as expression in CAR-T cells, can prevent CAR-T cell depletion.

Description

[0001] This application claims priority to U.S. Provisional Application No. 63 / 387,243, filed December 13, 2022, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art as known to those skilled in the art as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights whatsoever.

[0004] Government interests

[0005] This invention was made with government support under R00 EB030587 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0006] Sequence Listing

[0007] This application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [], is named [] and is [] bytes in size. Technical Field

[0008] The present invention relates to enhancing cell therapy with a new class of immune checkpoint modulators and methods of using the same. Background Art

[0009] Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment over the past decade. These engineered T cells are directed to cancer targets to destroy cancer cells that express the target. However, like natural T cells, CAR-T cells are easily exhausted after repeated stimulation. In this case, a variety of checkpoint inhibitory receptors (such as PD-1) on the surface of CAR-T cells are upregulated, which interact with ligand receptors on the surface of tumor cells, thereby inhibiting T cell activity. Although there are therapeutic antibodies that block signaling between PD-1 and its cognate ligand PD-L1, patient data show that this is only effective for some patients. Therefore, new checkpoint receptor modulators must be developed to improve the efficacy of CAR-T cells and other immunotherapies. Summary of the Invention

[0010] Disclosed herein are immune checkpoint modulator compositions and methods of use thereof.

[0011] One aspect of the present invention relates to an antibody system comprising: a first antibody or antigen-binding fragment thereof (B1), which binds to a phosphorylated amino acid motif in a target protein to form a first complex between B1 and the protein; and a second antibody or antigen-binding fragment thereof (B2), which binds to the first complex to form a second complex. In some embodiments, B1 can be a protein or a portion of a protein that can bind to a phosphorylated amino acid in a target protein. In some embodiments, B2 can be a scFv, a single-domain antibody, a nanobody, a DARPin, an affibody, or the like.

[0012] In some embodiments, the phosphorylated amino acid motif is embedded in a three-dimensional domain of the target protein (a tertiary folded domain of the protein). In some embodiments, the phosphorylated amino acid motif that binds to B1 comprises a three-dimensional (folded) epitope of the target protein. In some embodiments, the phosphorylated amino acid motif that binds to B1 comprises a linear epitope of the target protein.

[0013] In some embodiments, B2 bound to the first complex binds to B1. In some embodiments, B2 bound to the first complex binds to the target protein. In some embodiments, B2 bound to the first complex binds to B1 and the target protein. In some embodiments, B2 bound to the first complex recognizes an epitope that includes B1 and the target protein.

[0014] In some embodiments, if the amino acid motif in the target protein is not phosphorylated, B1 does not bind to the amino acid motif. In some embodiments, if B1 does not bind to the phosphorylated amino acid motif in the target protein, B2 does not bind to the first complex.

[0015] In certain embodiments, the phosphorylated amino acid motif includes phosphohistidine, phosphoserine, phosphothreonine or phosphotyrosine. In certain embodiments, the phosphorylated amino acid motif can be combined with other proteins, including proteins with enzymatic activity, proteins that promote the formation of signal transduction complexes, etc. In certain embodiments, the phosphorylated amino acid motif includes phosphotyrosine, and other proteins (such as proteins with phosphotyrosine binding domains or Src homology 2 (SH2) domains) can be combined with them. In certain embodiments, other proteins can promote the formation of signal transduction complexes including other proteins.

[0016] In some embodiments, the phosphorylated amino acid motif is present in a signal transduction protein. In some embodiments, the signal transduction protein regulates an immune checkpoint pathway. In some embodiments, formation of the second complex prevents activation of an immune checkpoint pathway.

[0017] In some embodiments, B1 and B2 are covalently linked.

[0018] One aspect of the present invention relates to the B1 antibody or antigen-binding fragment thereof described herein. In some embodiments, B1 is covalently linked to B2.

[0019] One aspect of the present invention relates to the B2 antibody or antigen-binding fragment thereof described herein. In some embodiments, B2 is covalently linked to B1.

[0020] One aspect of the present invention relates to a bispecific antibody comprising: a first antigen-binding domain (B1) that binds to a phosphorylated amino acid in a signal transduction protein; and a second antigen-binding domain (B2) that binds to B1 and / or the signal transduction protein when B1 binds to a phosphorylated amino acid in the signal transduction protein.

[0021] In some embodiments, when B1 binds to a phosphorylated amino acid in a signal transduction protein, B2 binds to B1 and the signal transduction protein. In some embodiments, the signal transduction protein includes an intracellular signal transduction protein.

[0022] In some embodiments, the phosphorylated amino acid is selected from the group consisting of serine, threonine, tyrosine, and histidine. In some embodiments, tyrosine is present in a motif that binds to a protein having a Src homology 2 (SH2) domain.

[0023] In some embodiments, the signal transduction protein includes an immune checkpoint pathway. In some embodiments, the immune checkpoint pathway protein includes PD-1, CTLA-4, LAG-3, TIGIT, 2B4, BTLA, CD57, TIM-3, KLRG-1, or a combination thereof. In some embodiments, the signal transduction protein includes PD-1 protein. In some embodiments, the phosphorylated amino acid in the PD-1 protein includes phosphotyrosine 223 or 248.

[0024] In some embodiments, when B1 binds to a phosphorylated amino acid in a signaling protein, and when B2 binds to B1 and / or the signaling protein, the immune checkpoint pathway is not activated.

[0025] In certain embodiments, the phosphorylated amino acid in the signal transduction protein is phosphorylated by a protein kinase. In certain embodiments, the protein kinase includes a serine / threonine protein kinase, a tyrosine kinase, or a histidine kinase. In certain embodiments, the tyrosine kinase includes a receptor tyrosine kinase.

[0026] In some embodiments, if B1 does not bind to a phosphorylated amino acid in a signaling protein, B2 does not bind to B1 and / or the signaling protein.

[0027] In some embodiments, the bispecific antibody comprises a peptide linker connecting B1 and B2. In some embodiments, the peptide linker is between about 3 amino acids and about 50 amino acids in length. In some embodiments, B1 is located in the first bispecific antibody, and B2, which binds to B1, is located in the second bispecific antibody.

[0028] One aspect of the present invention relates to the first antigen binding domain (B1) of the bispecific antibodies described herein. In some embodiments, B1 is not covalently linked to B2.

[0029] One aspect of the present invention relates to the second antigen binding domain (B2) of the bispecific antibodies described herein. In some embodiments, B2 is not covalently linked to B1.

[0030] One aspect of the present invention relates to nucleic acids encoding the isolated bispecific antibodies described herein. One aspect of the present invention relates to cells comprising the nucleic acids described herein. Cells comprising the nucleic acids can express the bispecific antibodies described herein. In some embodiments, cells comprising the nucleic acids include T cells. In some embodiments, T cells include chimeric antigen receptor (CAR)-T cells.

[0031] One aspect of the present invention relates to introducing B1-B2 molecules (e.g., bispecific antibodies) into cells. In some embodiments, B1-B2 molecules can be introduced using lipid nanoparticles, can be fused with cell penetrating peptides, etc.

[0032] One aspect of the present invention relates to a method for treating cancer, comprising administering to a patient a CAR-T cell that intracellularly expresses a bispecific antibody described herein. In some embodiments, the bispecific antibody further comprises a phosphatase. In some embodiments, when the bispecific antibody is expressed in a CAR-T cell, it can inhibit the activity of the CAR-T cell. In some embodiments, the bispecific antibody further comprises a kinase. In some embodiments, when the bispecific antibody is expressed in a CAR-T cell, it can stimulate the activity of the CAR-T cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0034] Figure 1 Schematic diagram of the pY-mediated signaling mechanism in CAR-T cells.

[0035] Figure 2Figures A to 2B are workflow diagrams for generating synthetic binders modified against pY by targeting pY with a recombinant antibody pair (pY-TRAP). Panel A provides a schematic diagram illustrating the design and affinity characterization of pY-TRAP. Panel B provides a schematic diagram illustrating that the binding of pY-TRAP is highly specific for its target. 248 TRAP, II:hUb-pY 59 TRAP, III:hRIPK2-pY 474 TRAP.

[0036] Figure 3 is a schematic diagram showing synthetic pY-mediated pathway rewiring of cellular signaling responses.

[0037] Figure 4 This is a schematic diagram comparing natural T cells and CAR-T cells.

[0038] Figure 5 It shows that engineered PD-1 + Schematic diagram of Jurkat cells.

[0039] Figure 6 A to 6C are schematic diagrams illustrating existing methods for disrupting PD-1 signaling. Panel A provides a schematic diagram of a method comprising administering a PD-1 / PD-L1 blocking antibody or using engineered CAR-T cells that secrete a PD-1 / PD-L1 blocking antibody. Panel B provides a schematic diagram of a method comprising genomic disruption of PD-1, which can specifically block the endogenous PD-1 pathway in CAR-T cells. Panel C provides a schematic diagram of a method comprising a chimeric PD-1 / CD28 receptor, which can reverse the immunosuppressive function of the PD-1 pathway to immunostimulation.

[0040] Figure 7 A to 7B, Panel A provides a schematic diagram illustrating B2-SH2 binders against linear pY antigens. Panel B provides a schematic diagram illustrating that the immune checkpoint receptors PD-1 and TIGIT both have two pY sites within the cytoplasmic signaling motif.

[0041] Figure 8 Schematic diagrams showing the consequences of reprogramming PD-1 signaling using synthetic pY binders fused to effectors are provided.

[0042] Figure 9 A schematic diagram of the native PD-1 pathway is shown on the left, and a schematic diagram of an exemplary model of a synthetic PD-1 pathway is shown on the right, in which phosphorylated amino acids in PD-1 can be targeted by the molecules described herein (e.g., cpSH2-sdAb linked to a synthetic kinase) to affect the regulation of PD-1.

[0043] Figures 10A to 10B (A) Schematic diagram of an exemplary pY-TRAP molecule having a B1 domain that binds to a phosphorylated amino acid to form a first complex, and a B2 domain that binds to the first complex to form a second complex. (B, top) Schematic diagram of an example pY-TRAP molecule having a pY binding motif from the SHP2 (B1) protein linked to an antibody (B2). (B, bottom) Schematic diagram showing an example of a region of an IgG antibody that functions as B2 in a pY-TRAP molecule.

[0044] Figures 11A to 11C A schematic diagram of a PD-1 molecule spanning the cell membrane and containing two intracellular phosphotyrosines (pY223 and pY248) is shown (A). (B) A schematic diagram of the SHP2 phosphatase (an effector protein that binds to PD-1 in its native environment) is shown, showing the dual SH2 domains in SHP2 that bind to phosphotyrosines in the PD-1 molecule, as shown in (A). (C) is a schematic diagram of a ring-shaped arrangement of C-SH2 domains as disclosed herein.

[0045] Figure 11D Similar to FIG. 11A , binding of SHP2 to phosphorylated intracellular tyrosines in PD-1 is additionally shown.

[0046] Figure 12 Shown are exemplary data from experiments using bio-layer interferometry to measure the binding of various SH2 domains and loop-arranged SH2 variants to phosphorylated tyrosines in target molecules (e.g., pY248 in PD-1).

[0047] Figures 13A to 13B Shown is a schematic diagram illustrating a method for identifying B2 molecules with enhanced binding ability to the pY-B1 complex using yeast display technology.

[0048] Figure 14 Shown Figures 13A to 13B Exemplary data from the experiments presented in .

[0049] Figure 15 Exemplary data from successive rounds of enrichment of improved B2 binders are shown.

[0050] Figures 16A to 16B (A) Exemplary data showing binding of a single B1-B2 clone (upper panel) to a baseline control binder (lower panel). (B) Exemplary data showing binding of multiple B1-B2 clones to pY248.

[0051] Figure 17 Binding data for a single B1-B2 clone are shown.

[0052] Figure 18 Exemplary data are shown for dose-response binding of two B1-B2 clones and a double SH2 control.

[0053] Figures 19A to 19B Shown are (A) exemplary data for binding of three B1-B2 clones and an SH2 control to pY248 in PD-1, and (B) exemplary data for binding of three B1-B2 clones to pY614 in Grb1-associated binding protein 2 (Gab2). The dual SH2 domains of SHP2 bind nonspecifically to both PD-1 and GAB2. The B1-B2 clones bind only to PD-1.

[0054] Figure 20 Shown are exemplary micrographs of cells expressing various B1-B2 molecules in culture.

[0055] Figure 21 Shown are data from T cell activation studies performed using a plate-based activation assay.

[0056] Figure 22 Presented are data from studies on the effects of molecules containing a CD3z domain linked to the pY-TRAP molecules disclosed herein on T cell activation.

[0057] Figure 23 Exemplary genetic pathways for stimulating or inhibiting CAR-T cells are shown.

[0058] Figure 24 An example of a role for SHP2 in T cell function is shown.

[0059] Figure 25 Examples of various pathways regulated by SHP2 in T cells are shown.

[0060] Figure 26 Figure 2 is an illustrative schematic diagram of immune checkpoint signaling as a factor of CAR-T cell exhaustion.

[0061] Figure 27 An example of immune cell circuit reconstitution is demonstrated.

[0062] Figure 28 Additional data showing the dose-dependent binding of yeast clones B#1 to B#10 to (A) pY248 in PD-1 and (B) pY614 in GAB2. Figure 28 A Similar data. Figure 28 Similar data as in FIG19B are shown in FIG19B .

[0063] Figure 29Figure 2 shows the research data of the test to reconstruct PD-1 phosphorylation in HEK293 stable cells. In order to characterize the binding agent in the model cell line system, PD1-TagBFP-HAtag and LCK-EGFP-v5tag were stably expressed in HEK293 cells (A, B). LCK is a kinase that phosphorylates PD-1. A PD-1-TagBFP-HAtag cell line alone was constructed as a control. Since HEK cells lack endogenous PD-1 and LCK expression, PD-1 is only phosphorylated in PD-1 / LCK co-expressing cells. The membrane localization of PD-1 and LCK was confirmed by fluorescence microscopy (C). LCK-dependent PD-1 phosphorylation was confirmed by immunoprecipitation experiments (D, E).

[0064] Figure 30 Example fluorescence images are provided showing co-expression of binding agents with PD-1 and LCK in stable HEK cells.Binding agents fused to mCherry were expressed in stable HEK cells.

[0065] Figure 31 Immunoprecipitation data for binders #3 and 5 in cells expressing PD-1 and in cells expressing PD-1 and LCK are shown. Binders fused to mCherry were expressed in HEK cells overexpressing PD-1 or PD-1 / LCK. Expression of binders, PD-1, and LCK was confirmed from whole cell lysates using anti-mCherry, anti-PD1, and anti-V5 antibodies, respectively. Immunoprecipitation (IP) experiments were performed using anti-HA beads that bound to HA-tagged PD-1-TagBFP fusion protein.

[0066] Figure 32 Fluorescence microscopy images showing colocalization of binder #5 with PD-1 on the cell membrane in the presence of LCK are provided. High-magnification fluorescence images of binder #5 were collected in low and high pPD-1 cells.

[0067] Figure 33 Provided are screenshots of confocal images showing that binder #5 (red) is recruited to the cell membrane and colocalizes with PD-1 (blue) in the presence of LCK.

[0068] Figures 34A to 34B (A) shows the construction of pY-TRAP by combining the circularly permuted SH2 domain (cpSH2) of SHP2 phosphatase with a synthetic nanobody library with randomized CDR sequences. (B) depicts an example of screening of pY-TRAP labeled with an HA tag on the surface of yeast cells against a biotinylated peptide with a phosphotyrosine modification (pY peptide).

[0069] Figure 34CShown are exemplary diagrams of the screening process using magnetic-activated and fluorescence-activated cell sorting.

[0070] Figure 34D An exemplary series of clone enrichment is shown. Each graph shows the binding of pY peptide visualized by streptavidin-647 signal and HA-488 signal. The cell population in the upper quadrant of the flow cytometry graph is enriched. The titer determination after the final screening round shows EC 50 In the nanomolar range.

[0071] Figures 35A to 35B In (A), titration of some enriched binders from yeast selections is shown. 50 In the nanomolar range. These binders generally bind better than cpSH2 and tandem SH2 from SHP2. (B) Shows an exemplary study in which the specificity of selected binders was tested against a panel of peptides (including non-phosphorylated forms of the peptides and pY peptides with sequence similarity to PD-pY248 and the endogenous binding site of the SHP2 SH2 domain).

[0072] Figures 36A to 36B (A) shows a depiction of a split-luciferase system in which wild-type PD1 (YY) or two tyrosine positions with alanine mutations (Y→A) are fused to LgBit from luciferase. The pY-TRAP binder is fused to SmBit from luciferase. The interactions of binder-SmBit and PD1-LgBit reconstitute the complete luciferase. (B) shows a heat map of luciferase assays using cpSH2, tandem SH2 domains of SHP2, SHP2, and selected binders in LentiX cells expressing WT PD1 (YY) and mutant PD1 (AA). The fold change in luciferase signal is shown.

[0073] Figure 37 Data from an exemplary study are shown, in which cell lysates from LentiX cells co-expressing Lck kinase, PD1 WT (YY) or Y223F / Y248F (FF), and selected binders tagged with mCherry and HA were immunoprecipitated with anti-HA beads and then blotted with anti-pTyr antibodies. Bands indicate tyrosine phosphorylation corresponding to the size of PD1 and are only present in YY cells. Lck expression was detected with the V5 antibody, and PD1 levels in whole-cell lysates were confirmed with an anti-PD1 antibody.

[0074] Figure 38Figure 2 shows exemplary cytofluorescence data for the expression of GFP-tagged PD1 (one or both tyrosine residues mutated to phenylalanine) in LentiX cells co-expressing Lck and mCherry-tagged Binder-NT7. Membrane localization of the binder was observed in wild-type PD1 (Y223 / Y248) and F223 / Y248 PD1. Membrane localization of the binder was reduced in both F223 / F248 PD1 and Y223 / F248 PD1.

[0075] Figure 39A Co-culture of Raji B cells overexpressing PDL1 with Jurkat T PD1- / - cells reconstituted with wild-type PD1 (YY) or FF mutant PD1 (FF) is shown. Jurkat T cells were activated with 1000 pM anti-CD3 / anti-CD19 bispecific antibody (BiTE).

[0076] Figures 39B to 39C Shown, in Figure 39A After activation of the cells shown in Figure 3, T cell activation was quantified by surface CD69 levels (B) and IFN-γ release (C). The data show that expression of the binder NT7 rescues T cell activation in PD1-YY mutant cells and synergizes with nivolumab (anti-PD-1). In B and C, data are mean ± SD of n = 2 biological replicates. *P < 0.05; **P < 0.001.

[0077] Figure 40A Depicted is an exemplary synthetic transcription factor release system activated by a protease fused to a pY-TRAP molecule.

[0078] Figure 40B Shown are exemplary data from LentiX cells co-transfected with PD1-TEV substrate-eGFP-tTA or CD28TM-TEV substrate-eGFP-tTA (negative control) and BinderNT7-TEV-mCherry. In cells transfected with PD1-TEV substrate-eGFP-tTA, membrane recruitment of binder-fused TEV and subsequent nuclear localization of eGFP-tagged tTA were observed. No membrane recruitment of binder-fused TEV was observed in CD28TM-TEV substrate-eGFP-tTA cells, nor was tTA nuclear localization found.

[0079] Figures 41A to 41B (A) Data from biolayer interferometry (BLI) showing binding of the binder NT7 to the PD-1pY248 peptide, K D The BLI data (B) also showed that NT7 binds specifically to the PD-1pY248 peptide compared to other peptides. DETAILED DESCRIPTION

[0080] The present application discloses reagents and methods for affecting regulatory pathways in cells, and in some embodiments, for creating regulatory pathways or rewiring existing regulatory pathways in cells. Figure 27 ). In some embodiments, the reagents and methods can target pathways regulated by phosphorylation and / or dephosphorylation of amino acids in proteins. In some embodiments, the reagents and methods can locate molecules that can affect cell regulation near phosphorylated amino acids in cells. The reagents and methods can be used to affect the regulation of many different types of cells. In some embodiments, the targeted cells can be T cells expressing chimeric antigen receptors (CAR-T cells).

[0081] In some embodiments, disclosed herein are binding agents and systems thereof that can bind to specific phosphorylated amino acids in specific proteins. In some embodiments, binding agents and systems disclosed herein can distinguish the differences between specific phosphorylated amino acids in proteins (e.g., binding to pY248 in PD-1, but not binding to pY223 in PD-1). For example, this is in contrast to existing antibodies or naturally occurring protein binding domains, which can bind to specific phosphorylated amino acids (e.g., phosphotyrosine), but are not only bound to the phosphorylated amino acids in specific proteins (e.g., binding only to the phosphotyrosine in PD-1, but not to the phosphotyrosine in other proteins). Although not wishing to be bound by theory, these non-protein-specific universal binding agents can recognize specific phosphorylated amino acids, but may not recognize the amino acid sequences flanking the phosphorylated amino acids in proteins. Although not wishing to be bound by theory, binding agents disclosed herein may be able to recognize amino acid sequences flanking or near the phosphorylated amino acids, thereby providing protein specificity of binding agents. In some embodiments, compared to known phosphorylated amino acid binding agents, binding agents disclosed herein have a higher level of specificity for the molecules they bind to.

[0082] definition

[0083] A detailed description of one or more embodiments is provided herein. However, it should be understood that the present invention can be implemented in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching those skilled in the art to use the present invention in any appropriate manner.

[0084] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. In the claims and / or the specification, when used in conjunction with the term "comprising," the use of the words "a" and "an" may mean "one," but is also consistent with "one or more," "atleast one," and "one or more than one."

[0085] Wherever the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "and not limited to." Similarly, "example," "exemplary," etc. should be understood to be non-limiting.

[0086] The term "substantially" allows deviations from the descriptive word without adversely affecting the intended purpose. Even if the word "substantially" is not explicitly mentioned, the descriptive word should be understood as being modified by the term "substantially".

[0087] The terms "comprising" and "including," as well as "having" and "involving" (and similarly "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general definition of "comprising" in U.S. patent law and, therefore, should be interpreted as open-ended terms meaning "at least the following," and should be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Wherever the terms "a" or "an" are used, they should be understood to mean "one or more" unless such an interpretation makes no sense within the context.

[0088] The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the numerical values ​​set forth. Generally, the term "about" is used herein to modify a numerical value above and below by a variance of 20 percent above or below (higher or lower).

[0089] Chimeric antigen receptor (CAR) T cell therapy

[0090] In some embodiments, cells expressing chimeric antigen receptors (CARs) can be targeted using the reagents and methods disclosed herein. For example, CAR T cell therapy redirects the patient's T cells to kill tumor cells by exogenously expressing CAR on T cells. Other cells that can express CAR include NK cells (CAR NK cells) and macrophages (CAR macrophages). CAR can be a transmembrane fusion protein that connects the antigen recognition domain of an antibody to the intracellular signaling domain of a T cell receptor and a co-receptor. CAR-T cells are commonly used to treat patient cancers.

[0091] CAR-T cells may become "exhausted." Characteristics of CAR-T exhaustion can include decreased expression of checkpoint molecules and cell effector function ( Figure 23 and 26 Without intending to be limited by theory, it is believed that CAR-T cell exhaustion results from excessive or persistent antigen signaling. An immunosuppressive tumor microenvironment may contribute to this.

[0092] Solid tumors present unique challenges for CAR-T therapy. Several barriers to CAR-T effectiveness in solid tumors include heterogeneous antigen expression, inadequate tissue homing, inadequate activation, insufficient persistence, and an immunosuppressive tumor microenvironment. Unlike blood cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, leading to healthy tissue killing by on-target and off-tumor T cells. Furthermore, immunosuppression within the tumor microenvironment (TME) limits the activation of CAR-T cells toward tumor killing.

[0093] In some embodiments, disclosed herein are methods for preventing or reversing CAR-T cell exhaustion. In some embodiments, the disclosed methods can be reversible (e.g., can be turned on and off).

[0094] For example, using the disclosed methods, the molecules disclosed herein can be expressed intracellularly. In some embodiments, the molecule, i.e., the engineered binding agent system (e.g., B1 and B2 disclosed herein), can block the function of certain cell regulatory pathways, including immune checkpoint pathways. In some embodiments, the molecules disclosed herein can be used to regulate any regulatory pathway involving molecule phosphorylation (e.g., phosphorylation of specific amino acids in a protein). In some embodiments, the disclosed molecules can bind to phosphorylated amino acids and prevent the binding of other regulatory molecules. In some embodiments, the disclosed molecules can bind to phosphorylated amino acids in a protein and affect the phosphorylation or dephosphorylation of nearby molecules (e.g., by recruiting kinases or phosphatases to the region). In some instances, the disclosed molecules (e.g., B1 and B2) can be combined with other molecules (such as kinases or phosphatases) to target these other molecules to a specific cell location. In some examples, the disclosed molecules, when linked or anchored to proteases, inteins, or fragments of these molecules, can bring these molecules to the vicinity of phosphorylated amino acids bound by the disclosed B1 / B2 molecules and catalyze the release of other molecules expressed nearby (e.g., transcription factors, epigenetic regulators, enzymes, etc.). In some embodiments, the disclosed molecules can be minibodies, scFvs, IgG molecules, DARPins, affibodies, bispecific fusion molecules, and other antibody fragments described herein.

[0095] Cells expressing these molecules (e.g., CAR-T cells) can be introduced into patients in need of treatment by infusion therapy known to those skilled in the art. In some embodiments, the patient may suffer from PD-1 related diseases or conditions as described herein, such as chronic infection or cancer. Cells (e.g., T cells) can be, for example, T lymphocytes, CD4+T cells, CD8+T cells, or a combination thereof, but are not limited thereto. In some embodiments, the cells can be CAR-NK cells or CAR-macrophages.

[0096] In some embodiments, the molecules discussed herein can be used to construct multispecific antibodies. In some embodiments, CAR-T cells can express bispecific or multispecific antibodies discussed herein within the cell.

[0097] CAR-T cells can be generated using lentiviral systems (by transduction), retroviral systems (by transfection (electroporation)), and transposon systems (by PiggyBac) according to methods known in the art. The CAR-T cells used in the methods disclosed herein can target different antigens. In some embodiments, the antigen can be a tumor-associated antigen. Non-limiting examples of tumor-associated antigens that CAR-T cells can target include ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, MN-CAIX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7.

[0098] Immune checkpoint pathways, signaling proteins, and PD-1

[0099] Immune checkpoints or immune checkpoint pathways regulate various aspects of the immune system ( Figure 23 ). These pathways can affect self-tolerance, anti-cancer immunity, etc. In some instances, these pathways can affect T cell activity, including CAR-T cell activity. In some embodiments, the stimulatory molecules or co-stimulatory molecules of CAR-T cells may include ICOS, CD28, CD40L, 4-1BB, CD27, OX40, CD2, GITR, CD226, etc. In some embodiments, the inhibitors or co-inhibitors of CAR-T cells may include PD-1, CTLA-4, LAG-3, TIGIT, 2B4, BTLA, CD57, TIM-3, KLRG-1, etc. These proteins may be referred to as signal transduction proteins. In some embodiments, these proteins may be receptors (e.g., immune checkpoint proteins, cytokine receptors, etc.). In some embodiments, these proteins may have enzymatic activity. These proteins may bind to other proteins involved in signal transduction in the cell to form so-called signal transduction pathways.

[0100] Some signal transduction proteins may contain amino acids that can be phosphorylated. In certain embodiments, the phosphorylated amino acid may be located in the intracellular domain of the signal transduction protein. In certain embodiments, the phosphorylated amino acid may be phosphohistidine, phosphoserine, phosphothreonine, or phosphotyrosine. In some signal transduction proteins, amino acid phosphorylation may play a role in regulating signal transduction pathways. Phosphorylation may affect the binding of various other proteins.

[0101] In some instances, multiple of these proteins can act as part of inhibitory / co-inhibitory pathways in cells, and some of these pathways can lead to CAR-T cell exhaustion. In some embodiments, the reagents and methods disclosed herein are intended to detect or affect the regulation of these pathways.

[0102] Programmed cell death-1 (PD-1) is a cell surface membrane protein of the immunoglobulin superfamily ( Figure 9 and 11A). This protein is a signal transduction protein. It is expressed in pre-B cells and is thought to play a role in their differentiation. As a member of the CD28 family, PD-1 is upregulated in activated T cells, B cells, and monocytes. PD-1, as well as other immune checkpoint pathway receptors, is also upregulated in exhausted T cells. PD-1 has two identified ligands within the B7 family: PD-L1 (programmed cell death protein-1 ligand 1; also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) and PD-L2. PD-L1 is a 40 kDa type I transmembrane protein. Binding of PD-L1 to PD-1 or B7.1 transmits an inhibitory signal that reduces the proliferation of CD8+ T cells in lymph nodes. PD-1 also controls the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis, which is further mediated by downregulation of the gene Bcl-2. While PD-L2 expression tends to be more restricted, primarily found on activated antigen-presenting cells (APCs), PD-L1 is more ubiquitously expressed, encompassing cells of the hematopoietic lineage (including activated T cells, B cells, monocytes, dendritic cells, and macrophages) and peripheral non-lymphoid tissues (including heart, bone, muscle, placenta, lung, kidney, and liver). The widespread expression of PD-L1 suggests a role in regulating PD-1 / PD-L1-mediated peripheral immune tolerance. PD-1 and PD-L1 are immune downregulators or "off switches" for immune checkpoints. Examples of PD-1 inhibitors include, but are not limited to, nivolumab (Opdivo) (BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680 (AMP-514), PDR001, MPDL3280A, MEDI4736, BMS-936559, and MSB0010718C.

[0103] Recent studies have shown that some chronic viral infections and cancers have developed immune evasion strategies that specifically utilize the PD-1 / PD-L1 axis by causing PD-1 / PD-L1-mediated T cell exhaustion. Many human tumor cells and tumor-associated antigen-presenting cells express PD-L1 at high levels, indicating that tumors induce T cell exhaustion to escape anti-tumor immune responses. For example, during chronic HIV infection, HIV-specific CD8+ T cell function is impaired, showing a decreased ability to produce cytokines and effector molecules, as well as a weakened ability to proliferate. Studies have shown that PD-1 is highly expressed on HIV-specific CD8+ T cells in HIV-infected patients, suggesting that blocking the PD-1 / PD-L1 pathway may have therapeutic potential for HIV infection and AIDS patients. In summary, agents that block the PD-1 / PD-L1 pathway (directly or indirectly) can provide new treatments for a variety of cancers, HIV infections, and / or other diseases and conditions associated with T cell exhaustion. Such agents are described herein.

[0104] Proteins that bind to signal transduction proteins, including SHP2 binders

[0105] In certain embodiments, protein can be combined with phosphorylated amino acids, including phosphorylated amino acids (e.g., phosphotyrosine) present in signal transduction proteins. In certain embodiments, proteins combined with phosphorylated amino acids can have enzymatic activity. In certain embodiments, proteins combined with phosphorylated amino acids can promote the formation of complexes of other proteins (including proteins that form signal transduction complexes). In certain embodiments, these proteins and their complexes can participate in signal transduction pathways. In certain embodiments, proteins combined with phosphorylated amino acids themselves can contain phosphorylated amino acids. In certain embodiments, proteins combined with phosphorylated amino acids can contain enzymatic activity, or can recruit proteins with enzymatic activity. In certain embodiments, enzymatic activity can be kinase activity or phosphatase activity.

[0106] In some embodiments, the protein that binds to the phosphorylated amino acid may have a Src homology domain or a modified variant thereof (e.g., having an amino acid substitution or a cyclic arrangement). The Src homology domain may be a Src homology 2 (SH2). In some embodiments, the protein that binds to the phosphorylated amino acid may contain a phosphotyrosine binding (PTB) domain or a modified variant thereof (e.g., having an amino acid substitution or a cyclic arrangement). Other domains capable of binding to the phosphorylated amino acid may include, for example, a HYB domain, a PH domain (including GEP100 PH), a pleckstrin homology (PH)-like domain (e.g., GEP100 PH), a PKCδ domain, a PKCθC2 domain, an RKIP domain, and the like.

[0107] SHP2 is an important signaling effector molecule for multiple receptor tyrosine kinases (RTKs), including platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and epidermal growth factor receptor (EGFR). SHP2 is also an important signaling molecule that regulates the activation of the mitogen-activated protein (MAP kinase) pathway, which can lead to cell transformation, a prerequisite for cancer development. For example, SHP2 is involved in signaling through the Ras-mitogen-activated protein kinase, JAK-STAT, and / or phosphoinositide 3-kinase-AKT pathways. SHP2 mediates the activation of Erk1 and Erk2 (Erk1 / 2, Erk) MAP kinases by receptor tyrosine kinases such as ErbB1, ErbB2, and c-Met by regulating RAS activation.

[0108] SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2; Figure 9 SHP2 consists of three SH2 domains: 11B, 11D, a catalytic domain (PTP), and a C-terminal tail. These two SH2 domains control the subcellular localization and functional regulation of SHP2. The molecule exists in an inactive conformation, where its activity is inhibited through a binding network involving residues in the N-SH2 and PTP domains. In response to growth factor stimulation, SHP2 binds to the RTK signaling machinery, inducing conformational changes and leading to SHP2 activation.

[0109] SHP2 is an immunosuppressive regulatory factor in T cells and plays a complex role in T cell function ( Figure 24 and 25 In some embodiments, SHP2 can play a role in CAR-T cell exhaustion. In some examples of the PD-1 regulatory pathway, the binding of PD-1 to PDL-1 can lead to the phosphorylation of PD-1 at pY223 and pY248 (Figure 11A). Once phosphorylated, SHP2 can bind to PD-1 at these phosphorylated amino acid residues ( Figure 11D SHP2 has phosphatase activity and can dephosphorylate nearby signaling components. This may result in negative regulation of T cells (including CAR-T cells).

[0110] binder

[0111] In the broadest sense, the term "binding agent" should be understood as a molecule that binds to a target molecule. Herein, a binding agent molecule can be represented by the letter "B", which can be followed by a number indicating a specific binding agent molecule (e.g., B1, B2, etc.). The target molecule can be present in a specific target cell population. The term "binding agent" should be understood in its broadest sense and also includes, for example, lectins, proteins that can bind to specific molecules, and phospholipid binding proteins. Such binding agents include, for example, high molecular weight proteins (binding proteins), polypeptides or peptides (binding peptides), non-peptides (e.g., aptamers (U.S. Patent No. 5,270,163) refer to Keefe AD et al., Nat. Rev. Drug Discov. 2010; 9: 537-550), or vitamins and other cell binding molecules or substances. A binding agent can be a region or domain in a molecule that can bind to another molecule (e.g., a fragment of a first molecule that can bind to a second molecule). In an embodiment, the binding agent can be from a signal transduction protein or a protein that binds to a signal transduction protein. In some embodiments, the binding agent can bind to a phosphorylated protein or a phosphorylated amino acid motif. If the amino acid motif is not phosphorylated, the binding agent may not be able to bind to the amino acid motif.

[0112] In some embodiments, the binding agent can be a protein domain that can bind to a signal transduction protein. In some embodiments, the binding agent can be an SH2 domain from the SHP2 protein. In some embodiments, the SH2 domain can be the N-SH2 domain or the C-SH2 domain of SHP2 ( FIG. 11B ). In some embodiments, these SH2 domains can bind to pY223 or pY248 of PD-1 ( Figure 11D In some embodiments, the SH2 domain does not bind to amino acids 223 or 248 in PD-1 unless the amino acid (e.g., tyrosine) is phosphorylated.

[0113] The binding agent can be, for example, an antibody and antibody fragment or antibody mimetic, such as, for example, an affibody, an adnectin, anticalins, DARPin, avimer or nanobody (see Gebauer M. et al., Curr. Opinion in Chem. Biol. 2009; 13: 245-255; Nuttall SD et al., Curr. Opinion in Pharmacology 2008; 8: 608-617). The binding peptide is, for example, a ligand of a ligand / receptor pair, such as VEGF of, for example, a ligand / receptor pair VEGF / KDR, transferrin such as a ligand / receptor pair transferrin / transferrin receptor, or a cytokine / cytokine receptor such as TNFalpha of a ligand / receptor pair TNFalpha / TNFalpha receptor. In some embodiments, the binding peptide or polypeptide can comprise a protein that binds to a signal transduction protein. In some embodiments, the binding peptide / polypeptide can comprise a Src homology 2 (SH2) domain.

[0114] In some embodiments, a binding agent that is an antibody can bind to a phosphorylated amino acid or phosphorylated amino acid motif and may not bind to it if the amino acid motif is not phosphorylated. In some embodiments, the phosphorylated amino acid motif may be located in a signal transduction protein or a protein that binds to a signal transduction protein. In some embodiments, the antibody can bind to an amino acid motif comprising pY223 or pY248 of PD-1. In some embodiments, the antibody will not bind to the amino acid unless it is phosphorylated.

[0115] The phosphorylated amino acid motif to which the antibody binds can be an amino acid epitope. The epitope can be a linear amino acid epitope. The epitope can be embedded in a three-dimensional domain of the target protein. The epitope can be a non-linear amino acid epitope of the target protein. The epitope can be a three-dimensional (folded) amino acid epitope of the target protein.

[0116] The epitope can be a phosphorylated serine or phosphorylated threonine to which a WW domain (also referred to as an rsp5 domain or WWP domain) can bind. The epitope can be a phosphohistidine motif to which an SH2 domain can bind. The epitope can be a sulfotyrosine to which a protein with a modified SH2 domain can bind.

[0117] In some embodiments, a binding agent can bind to another binding agent that is already bound to its target. In some embodiments, a first binding agent B1 can target a phosphorylated amino acid motif and form a first complex between B1 and the phosphorylated amino acid motif. A second binding agent B2 can target the first complex and form a second complex. Herein, such an antibody system can target a regulatory pathway in a cell and alter its regulation or prevent its regulation from being altered. For example, the formation of a second complex can prevent a molecule from binding to and / or regulating a phosphorylated amino acid motif in a signal transduction molecule. The formation of a second complex can prevent the activation of an immune checkpoint pathway. In some embodiments, B1 can be an antibody, ScFv, single domain antibody, nanobody, DARPin, affibody, etc. that binds to a phosphorylated amino acid. In some embodiments, B1 can be part of a protein that can bind to a phosphorylated amino acid. In some embodiments, when B1 binds to a specific phosphorylated amino acid, B2 can be an antibody, ScFv, single domain antibody, nanobody, DARPin, affibody, etc. that can bind to at least B1.

[0118] In some of the above embodiments, the SH2 domain that has bound to pY248 in PD-1 (the SH2 domain is the first binding agent or B1) forms a first complex between the SH2 domain and pY248. In some embodiments, this complex can bind to a second binding agent (B2) to form a second complex. In some embodiments, B2 can be an antibody. In some embodiments, B2 can bind to B1, to a target bound by B1, to B1 and B1's target, or to other configurations of the epitope. In some embodiments, B2 does not bind to the first complex unless B1 is bound to the B1 target protein.

[0119] In some embodiments, the B1 binding agent can bind to phosphorylated amino acids in different proteins. B1 binding agents with this property can include phosphorylated amino acid binding domains from proteins that can bind to phosphorylated amino acids in different proteins. In some embodiments, such amino acid binding domains are SH2 domains from SHP2 (e.g., Figure 25 , right panel, showing that the SH2 domain in SHP2 can bind to multiple proteins in addition to PD-1). In cases where the B1 binder can bind to different phosphorylated amino acids within a protein, or can bind to phosphorylated amino acids in different proteins, the B2 counterpart of B1 can provide additional specificity. For example, the data herein show that a B1 binder comprising the SH2 domain from SHP2, when used with its cognate B2 binder, can bind to phosphotyrosine in PD-1, but not to phosphotyrosine in GAB2 ( Figures 19A to 19B ).

[0120] In such antibody systems, such as the B1 / B2 system described above, B1 and B2 can be separate molecules (i.e., non-covalently linked), or B1 and B2 can be covalently linked. In some embodiments, B1 and B2 can be connected by a peptide linker (e.g., having a length of between about 8 and about 50 amino acids). In some embodiments, the B1 and B2 binding agents can be part of a multifunctional antibody (e.g., a bispecific antibody). The B1 / B2 antibody system can include additional functions. For example, the B1 / B2 antibody system can include a motif to which another molecule can bind. In some embodiments, the B1 / B2 antibody system can include an immunoreceptor tyrosine-based activation motif (ITAM), an immunoreceptor tyrosine-based inhibition motif (ITIM), an immunoreceptor tyrosine-based switch motif (ITSM), etc. When this system is expressed intracellularly, it binds to a specific phosphorylated amino acid in the target and recruits a kinase or phosphatase to the vicinity of the phosphorylated amino acid. The recruited kinase or phosphatase will act on a substrate near the phosphorylated amino acid.

[0121] In some embodiments, the B1 / B2 system can be integrated into an enzyme, such as a kinase or phosphatase. In this embodiment, the kinase or phosphatase will bind to a specific phosphorylated amino acid in the target, and the kinase or phosphatase as part of the system will act on the nearby substrate. In some embodiments, a B1 / B2 antibody system containing kinase or phosphatase activity can bind to the phosphorylated amino acid motif, thereby activating the kinase or phosphatase.

[0122] In some embodiments, the B1 / B2 binder system can be fused to another enzyme. In some embodiments, the another enzyme can be a protease. In some embodiments, the B1 / B2 system with a protease is expressed in a cell. A membrane-bound effector protein (e.g., a transcription factor, an epigenetic regulator, an enzyme, etc.) containing a protease substrate sequence can also be expressed in a cell. For example, in an embodiment where B1 of the B1 / B2 / protease system targets phosphorylated amino acids in PD-1, the protease can be co-localized with the membrane anchor protein, resulting in the cleavage of the protease substrate sequence and the release of the effector protein to initiate and drive the reaction.

[0123] In the broadest sense, "target molecule" is understood to be a molecule present in the target cell population, and it can be a protein (e.g., a growth factor receptor) or a non-peptide molecule (e.g., a sugar or phospholipid). It can be a receptor or an antigen. In some embodiments, the target protein can include a signal transduction protein. In some embodiments, the target protein can include molecules such as PD-1, CTLA-4, LAG-3, TIGIT, 2B4, BTLA, CD57, TIM-3, KLRG-1. In some embodiments, these molecules can be signal transduction molecules. In some embodiments, the protein can bind to a signal transduction molecule and affect the regulation of the immune system. In some embodiments, the target protein can have one or more phosphorylated amino acids.

[0124] The term "extracellular" target molecule describes a target molecule that is attached to a cell, is located outside the cell, or is located at a portion of the target molecule outside the cell, i.e., a binding agent can bind to its extracellular target molecule on a complete cell. The extracellular target molecule can be anchored to the cell membrane or is a component of the cell membrane. Those skilled in the art know methods for identifying extracellular target molecules. For proteins, this can be achieved by determining the transmembrane domain and the orientation of the protein in the membrane. These data are typically stored in protein databases (e.g., SwissProt). In some instances, the extracellular target molecule can comprise a receptor or an extracellular domain of a receptor.

[0125] A binding agent can be attached to another molecule. In some embodiments, a first binding agent can be attached to a second binding agent. In some embodiments, a first binding agent can be attached or linked to a second binding agent via a linker (which can include an amino acid).

[0126] The attachment of the binding agent can be performed via heteroatoms of the binding agent. According to the present invention, heteroatoms of the binding agent that can be used for attachment are sulfur (in one embodiment, via sulfhydryl groups of the binding agent), oxygen (in one embodiment, via carboxyl or hydroxyl groups of the binding agent), and nitrogen (in one embodiment, via primary or secondary amine groups or amide groups of the binding agent). These heteroatoms can be present in natural binding agents or introduced by chemical methods or molecular biological methods. According to the present invention, the attachment of the binding agent to the toxic group has only a slight effect on the binding activity of the binding agent for the target molecule. In a preferred embodiment, the attachment has no effect on the binding activity of the binding agent for the target molecule.

[0127] An "isolated" antibody is a binding agent that has been purified to remove other cellular components. Contaminating cellular components that may interfere with diagnostic or therapeutic use are, for example, enzymes, hormones, or other peptide or non-peptide components of the cell. Preferred antibodies or binding agents are those that are purified to a degree greater than 95% by weight relative to the antibody or binding agent (e.g., as determined by the Lowry method, UV-visible spectroscopy, or by SDS capillary gel electrophoresis). In addition, the antibody should be purified to a degree that allows determination of at least 15 amino acids of amino-terminal or internal amino acid sequence, or to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions (detection can be determined by Coomassie brilliant blue staining or silver staining). However, antibodies are typically prepared by one or more purification steps.

[0128] The term "specific binding" or "binds specifically" refers to an antibody or binding agent that binds to a predetermined antigen / target molecule. Specific binding of an antibody or binding agent is typically described as an antibody or binding agent having an affinity of at least 10-7 M (as a Kd value; i.e., an antibody or binding agent having a Kd value of less than 10-7 M), and the affinity of the antibody or binding agent for the predetermined antigen / target molecule is at least twice that for a nonspecific antigen / target molecule (e.g., bovine serum albumin or casein), which is not a predetermined antigen / target molecule or a closely related antigen / target molecule. The antibody can have an affinity of at least 10-7 M (as a Kd value; in other words, an antibody having a Kd value of less than 10-7 M), at least 10-8 M, and more preferably in the range of 10-9 M to 10-11 M. The Kd value can be determined, for example, by surface plasmon resonance spectroscopy.

[0129] Binders and antibody systems targeting PD-1

[0130] Described herein are systems for targeting regulatory systems, including immune checkpoint pathways. In some embodiments, the binding agents used in these systems are antibodies.

[0131] Unique recombinant monoclonal antibody clones are described herein. "Recombinant" when referring to a polypeptide (such as an antibody) or polynucleotide, refers to a non-naturally occurring form of the polypeptide or polynucleotide, which, by way of non-limiting example, can be created by combining polynucleotides or polypeptides that do not normally occur together.

[0132] In some embodiments, the binding agent can be a domain from a protein capable of binding to another molecule. In some embodiments, such a binding agent can be an SH2 domain. The SH2 domain can be from the SHP2 protein. In some embodiments, the SH2 domain from the SHP2 protein can be a C-SH2 domain or an N-SH2 domain that binds to pY248 in PD-1 ( Figures 11A , 11B, and 11D ). In some embodiments, the SH2 domain can be a ring-arranged SH2 domain ( Figure 11C ). In some embodiments, the amino acid sequence of the ring-arranged SH2 domain is as shown in Tables 1 and 2.

[0133]

[0134]

[0135] In some embodiments, the B2 antibody used in the B1 / B2 binding agent system described herein can be an IgG heavy chain variable region. In some embodiments, the nucleic acid sequence of the scaffold region of this region is as shown in Table 3. Provided herein is a nucleic acid sequence scaffold for a VH domain. In some embodiments, the VH domain can be located between bases 1 and 378. In some embodiments, CDR1 can be located between bases 82 and 108. In some embodiments, CDR2 can be located between bases 151 and 183. In some embodiments, CDR3 can be located between bases 298 and 339. In some embodiments, the CDRs of the IgG heavy chain variable region of the antibodies disclosed herein are as shown in Table 4 (see also Examples 4 and 5). Figure 17 ).

[0136]

[0137]

[0138]

[0139] In some embodiments, the B2 antibody scaffold used in the B1 / B2 binding agent system described herein can be an IgG heavy chain variable region (e.g., a nanobody). In some embodiments, the amino acid sequence of the scaffold region of this region is as shown in Table 5.

[0140]

[0141] In some embodiments, exemplary sequences of pY-TRAP molecules can be as shown in Table 6.

[0142]

[0143] In some embodiments, the CDRs of the IgG heavy chain variable regions of the antibodies disclosed herein are as shown in Table 7.

[0144]

[0145]

[0146] The antibodies described herein include a first antibody or an antigen-binding fragment thereof (B1), which binds to a phosphorylated amino acid motif in a target protein to form a first complex between B1 and the protein. In another embodiment, the antibodies described herein include a second antibody or an antigen-binding fragment thereof (B2), which binds to the first complex to form a second complex.

[0147] In one embodiment, B1 is a mutant of a universal anti-pY antibody. In another embodiment, B2 binds to a complex of B1 and a specific pY protein. In one embodiment, the B1 and B2 antibodies have high affinity and specificity for phosphorylated amino acid motifs in the target protein.

[0148] Some embodiments also relate to antibodies having a specified percentage of identity or similarity to the amino acid or nucleotide sequences of the anti-pY antibodies described herein. For example, the terms "homologous" or "identity" or "similarity" refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity compared to a specific region or the entire length of any of the anti-pY antibodies described herein. For example, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity compared to a specific region or the entire length of any anti-pY antibody described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al., eds. (2007) Current Protocols in Molecular Biology. For example, percent sequence identity or similarity to the nucleic acids and proteins of the invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0149] "Protein" or "peptide" includes polymers of amino acid residues linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein is at least three amino acids long. A protein can refer to a single protein or a collection of proteins. The proteins of the present invention can contain only natural amino acids, but non-natural amino acids (i.e., compounds that do not occur in nature but can be incorporated into polypeptide chains) and / or amino acid analogs known in the art can also be used. In addition, one or more amino acids in the proteins of the present invention can be modified, for example, by adding chemical entities (such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications, etc.). Proteins can also be single molecules or can be multimolecular complexes. Proteins can be simply fragments of naturally occurring proteins or peptides. Proteins can be naturally occurring, recombinant, or synthetic, or any combination thereof.

[0150] As used herein, "polypeptide" may encompass the singular "polypeptide" as well as the plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" refers to any chain of two or more amino acids, rather than a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other terms used to refer to a chain of two or more amino acids are included within the definition of "polypeptide," and the term "polypeptide" may replace any of these terms or be used interchangeably therewith. "Polypeptide" may also refer to a product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. With respect to amino acid sequences, it will be readily understood by those skilled in the art that individual substitutions, deletions, or additions made to nucleic acid, peptide, polypeptide, or protein sequences (which alter, add, delete, or replace a single amino acid or a small proportion of amino acids in the encoded sequence) are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the replacement of an amino acid with an amino acid that has similar chemical properties. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0151] For example, a "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and the side chain comprises a basic side chain (e.g., lysine, arginine, histidine), an acidic side chain (e.g., aspartic acid, glutamic acid), an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), a non-polar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), a beta-branched side chain (e.g., threonine, valine, isoleucine) and an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is replaced by another amino acid residue from the same side chain family. In another embodiment, an amino acid string can be replaced with a structurally similar string that differs in the order and / or composition of the side chain family members.

[0152] As used herein, "kinase" refers to a large class of enzymes that catalyze the transfer of gamma-phosphate from ATP to the hydroxyl groups on Ser / Thr or Tyr side chains in proteins and peptides and are intimately involved in the regulation of a variety of important cellular functions, perhaps most notably signal transduction, differentiation, and proliferation. It is estimated that there are approximately 2,000 different protein kinases in the human body. Although each kinase phosphorylates a specific protein / peptide substrate, they all bind the same secondary substrate, ATP, in a highly conserved pocket. Approximately 50% of known oncogene products are protein tyrosine kinases (PTKs), whose kinase activity has been shown to lead to cellular transformation.

[0153] In some embodiments, the kinase is a tyrosine kinase. As used herein, "tyrosine kinase" refers to an enzyme that uses ATP as a substrate to phosphorylate tyrosine residues on proteins. In some embodiments, the tyrosine kinase is a non-receptor tyrosine kinase. Mammalian non-receptor tyrosine kinases (NRTKs) are divided into ten families: Src, Abl, Jak, Ack, Csk, Fak, Fes, Frk, Tec, and Syk.

[0154] Antibody

[0155] As used herein, "antibody" or "antigen-binding polypeptide" may refer to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a complete antibody and any antigen-binding fragment or single chain thereof. For example, an "antibody" may comprise any protein- or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementary determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region (FR) or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" may refer to an immunoglobulin molecule and an immunoactive portion of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen-binding site that specifically binds (immunoreacts). "Specific binding" or "immunoreacts" refers to a reaction of an antibody with one or more antigenic determinants of the desired antigen, without reacting with other polypeptides.

[0156] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as an F (ab′)2 、F (ab)2 、F ab ′、F ab , Fv, scFv, VH, etc. Regardless of the structure, antibody fragments will bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (such as mirror image aptamers), miniantibodies and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex. The antibodies, antigen-binding polypeptides, variants or derivatives described herein include but are not limited to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), miniantibodies, disulfide-linked Fv (sdFv), fragments comprising VL or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies.

[0157] "Single-chain variable fragment" or "scFv" refers to the heavy chain (V H ) and light chain (V L) variable region. Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed by a gene fusion comprising VH and VL encoding genes connected by a peptide encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected by a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, and rich in serine or threonine for solubility, and can connect V H N-terminal and V L The C-terminus of the antibody is retained, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker. Various methods have been described to identify chemical structures that convert the naturally aggregated but chemically separated light and heavy chain polypeptide chains in the antibody V region into scFv molecules, which will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site. (See, for example, U.S. Patent Nos. 5,091,513; 5,892,019; 5,132,405; and 4,946,778, each of which is incorporated herein by reference in its entirety.)

[0158] Very large natural human scFv libraries have been and can be constructed to provide a large source of rearranged antibody genes for a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0159] Antibody molecules obtained from the human body are divided into five classes of immunoglobulins: IgG, IgM, IgA, IgE, and IgD, which differ in the nature of the heavy chains present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with several subclasses within these (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, are well characterized and are known to confer functional specificity. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. These four chains are typically connected by disulfide bonds in a "Y" configuration, with the light chains beginning at the mouth of the "Y," sandwiching the heavy chains, and continuing through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0160] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be combined with either a κ or λ light chain. Typically, the light and heavy chains are covalently bonded to each other, and when the immunoglobulin is produced by a hybridoma, B cell, or genetically engineered host cell, the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds or non-covalent disulfide bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the fork end of the Y configuration to the C-terminus at the base of each chain.

[0161] Both light and heavy chains are divided into structural and functional homologous regions. The terms "constant" and "variable" are used functionally. The variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement fixation, etc. The term "antigen binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and light chain ("L") chains. Three highly variable segments within the heavy and light chain V regions, called "hypervariable regions," are located between more conserved flanking segments, called "framework regions," or "FRs." Therefore, the term "FR" can refer to amino acid sequences naturally occurring between or adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen. The three hypervariable regions of each heavy and light chain are called "complementarity determining regions," or "CDRs."

[0162] The six CDRs present in each antigen-binding domain are short, discontinuous amino acid sequences, which are specifically positioned to form antigen-binding domains when the antibody presents its three-dimensional configuration in an aqueous environment. The remaining amino acids (FR regions) in the antigen-binding domains show less intermolecular variability. The framework region mainly adopts a β-sheet conformation, and CDR forms a loop that connects the β-sheet structure and forms a part of the β-sheet structure in some cases. The framework region is used to form a support, which positions the CDR in the correct orientation by interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDR provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody and its cognate epitope. One of ordinary skill in the art can readily identify the amino acids in the heavy or light chain variable region that comprise the CDRs and framework regions, respectively, as they have been previously defined (see, “Sequences of Proteins of Immunological Interest,” Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0163] When the term used and / or accepted in the art has two or more definitions, unless otherwise expressly stated, the definition of the term used herein is intended to include all of these meanings. A specific example is to use the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites found in the variable region of heavy and light chain polypeptides. This specific region has been described by Kabat et al., US Pat. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which is incorporated herein by reference in its entirety. When compared to each other, according to the definition of Kabat and Chothia, CDR comprises overlapping or subsets of amino acid residues. However, the application of any definition to refer to the CDR of an antibody or its variant is intended to fall within the scope of the term as defined and used herein. The corresponding amino acid residues comprising the CDR defined by each of the above-mentioned references are shown in the table below as a comparison. The exact number of residues comprising a specific CDR will vary according to the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR given the amino acid sequence of an antibody variable region.

[0164] CDR Kabat numbering Chothia Number VH CDR1 31-35 26-32 VH CDR2 50-65 52-58 VH CDR3 95-102 95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VL CDR3 89-97 91-96

[0165] Kabat et al. defined a variable domain sequence numbering system applicable to any antibody. Those skilled in the art can clearly assign this "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth in Kabat et al., U.S. Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0166] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine ​​residue), contains approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at approximately the 15th residue after the end of CDR-H1, contains approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at approximately the 33rd amino acid residue after the end of CDR-H2; contains 3-25 amino acids; and ends with the sequence WGXG, where X is any amino acid. CDR-L1 starts at approximately residue 24 (i.e., after the cysteine ​​residue); contains approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 starts at approximately the 16th residue after the end of CDR-L1 and contains approximately 7 residues. CDR-L3 begins approximately thirty-three residues after the end of CDR-L2 (ie, after the cysteine ​​residue); comprises approximately 7-11 residues, and terminates with the sequence F or WGXG, where X is any amino acid.

[0167] As used herein, the terms "nanoantibody" and "isolated VHH domain" are used interchangeably and refer to camelid single-domain antibody fragments. "Nanoantibody" refers to the smallest antigen-binding fragment or single variable domain ("VHH") derived from natural heavy-chain antibodies. Nanoantibodies are derived from heavy-chain-only antibodies present in camelids. In the "Camelid" family, immunoglobulins lacking light polypeptide chains were found. "Camelids" include Old World camelids (Bactrian camels and dromedaries) and New World camelids (e.g., alpacas, llamas, guanacos, and vicuñas). Low-specificity nanoantibodies bind to multiple different epitopes (or polypeptide regions) through a single antigen-binding site or binding domain, while high-specificity nanoantibodies bind to one or several epitopes (or polypeptide regions) through a single antigen-binding site or binding domain.

[0168] It should be noted that the term "Nanobody" as used herein in its broadest sense is not restricted to a specific biological source or to a specific method of preparation. For example, the Nanobodies herein can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanizing" a naturally occurring VHH domain or by expressing a nucleic acid encoding such a humanized VHH domain; (4) by "camelizing" a naturally occurring VH domain from any animal species (in particular a mammalian species, such as a human), or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelizing" a "domain antibody" or "Dab" as described in the art, or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic techniques known per se for the preparation of proteins, polypeptides or other amino acid sequences; (7) by preparing a nucleic acid encoding a Nanobody using techniques known per se for nucleic acid synthesis and then expressing the nucleic acid thus obtained; and / or (8) by any combination of one or more of the aforementioned methods.

[0169] As used herein, the term "monomer" refers to an antigen-binding molecule with a heavy chain variable domain and no light chain variable domain. Monomers can bind to antigens without light chains and typically have three CDR regions, respectively named CDRH1, CDRH2, and CDRH3. A heavy chain IgG monomer has two heavy chain antigen-binding molecules connected by a disulfide bond. The heavy chain variable domain includes one or more CDR regions, preferably CDRH3 regions. "VhH" or "VHH" refers to the variable domains of a heavy chain antibody (such as a monomer). "Camelid monomer" or "Camelid VHH" refers to a monomer or its antigen-binding portion thereof obtained from a camelid source (comprising an animal with two toes and a leathery sole).

[0170] The term "DARPin" (designed ankyrin repeat protein) refers to an antibody-mimicking protein with high specificity and high binding affinity for a target protein, which is prepared by genetic engineering. DARPin is derived from a natural ankyrin and has a structure comprising at least two ankyrin repeat motifs (e.g., comprising at least three, four, or five ankyrin repeat motifs). DARPin can have any suitable molecular weight, depending on the number of repeat motifs. DARPin comprises a core portion that provides a structure and a target binding portion that is located outside the core and binds to a target. The structural core comprises a conserved amino acid sequence, and the target binding portion comprises an amino acid sequence that varies depending on the target. DARPin has target specificity similar to that of an antibody. Therefore, by connecting DARPin to an antibody or antibody fragment, such as an IgG (e.g., IgG1, IgG2, IgG3, or IgG4) antibody or a scFv-Fc antibody fragment, a new form of bispecific chimeric protein is provided.

[0171] As used herein, the term "affimer" refers to a protein engineered to mimic a monoclonal antibody with a target protein or peptide bonded to a high-affinity target protein or peptide, and is therefore a member of an antibody mimics family. Affibody is composed of a three-helix bundle domain of the IgG binding domain derived from staphylococcal protein A. This protein domain is composed of 58 amino acid sequences, of which 13 randomized amino acids provide a series of affibody variants. Although affibody molecules are significantly smaller than antibodies (affimer weights approximately 6kDa, and antibodies weigh approximately 150kDa conventionally), affibody molecules act like antibodies because their binding sites are roughly equal to those of antibodies in surface area.

[0172] As used herein, the term "epitope" can include any protein determinant that can be specifically bound to an immunoglobulin, scFv or T cell receptor. The variable region allows antibodies to selectively recognize and specifically bind to the epitope on the antigen. For example, the VL domain and VH domain or a subset of complementary determining regions (CDRs) of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site present at the end of each arm of the Y. The epitope determinant can be made up of the chemically active surface groups (such as amino acids or sugar side chains) of the molecule, and generally has specific three-dimensional structural characteristics and specific charge characteristics. For example, antibodies can be produced against the N-terminal or C-terminal peptide of a polypeptide. More specifically, the antigen binding site is defined by three CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) on each of the VH and VL chains.

[0173] As used herein, the terms "immunobinding" and "immunobinding properties" may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunobinding interaction may be measured by the dissociation constant (K) of the interaction. d ) indicates that the smaller K d =A greater affinity indicates greater affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complexing partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions. Thus, an "association rate constant" (K) can be determined by calculating the concentration and the actual association and dissociation rates. on ) and the “dissociation rate constant” (K off (See Nature 361: 186-87 (1993)). off / K on The ratio allows the elimination of all parameters not related to affinity and is equal to the equilibrium binding constant K D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).

[0174] For example, in some embodiments, K D Between approximately 1E-12M and K D In some embodiments, K D Between approximately 1E-11M and K D In some embodiments, K D Between approximately 1E-10M and K D In some embodiments, K D Between approximately 1E-9M and K D In some embodiments, K D Between approximately 1E-8M and K D In some embodiments, K D Between approximately 1E-7M and K D For example, in some embodiments, K D is about 1E-12M, while in other embodiments, K D In some embodiments, K D is about 1E-10M, while in other embodiments, K D is about 1E-9M. In some embodiments, K Dis about 1E-8M, while in other embodiments, K D In some embodiments, K D is about 1E-6M, while in other embodiments, K D is about 1E-5M. For example, in some embodiments, K D is about 3E-11M, while in other embodiments, K D In some embodiments, K D The term "specifically binds" or "is specific for" may refer to an antibody that binds to an epitope via its antigen-binding domain, and such binding requires some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen-binding domain more readily than to a random, unrelated epitope.

[0175] For example, the anti-pY antibodies, B1 and B2, can be monovalent or bivalent and can comprise single or double chains. Functionally, the binding affinity of the antibodies described herein is between 10 -5 M to 10 -12 For example, the binding affinity of anti-pY antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 - 7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10-9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 - 9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M, or 10 -5 M to 10 -6 M.

[0176] The target protein or its derivatives, fragments, analogs, homologs or orthologs can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components. The target protein or its derivatives, fragments, analogs, homologs or orthologs coupled to proteoliposomes can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components.

[0177] Another method for determining whether a human monoclonal antibody has the specificity of the human monoclonal antibodies disclosed herein is to pre-incubate the human monoclonal antibodies of the present invention with their normally reactive target protein, and then add the human monoclonal antibody to be tested to determine whether the binding ability of the tested human monoclonal antibody is inhibited. If the tested monoclonal antibody is inhibited, it is likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibodies of the present invention.

[0178] Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies against the proteins of the present invention or against derivatives, fragments, analogs, homologs or orthologs thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated by reference).

[0179] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG portion of immune serum. Subsequently, or alternatively, the specific antigen or its epitope as the target of the immunoglobulin sought can be fixed on a column to purify the immunospecific antibody by immunoaffinity chromatography. For example, D.Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, volume 14, phase 8 (April 17, 2000), pages 25 to 28) has discussed the purification of immunoglobulins.

[0180] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" refers to a population of antibody molecules containing only one molecular species of antibody molecule composed of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a specific antigenic epitope and are characterized by a unique binding affinity for that specific antigenic epitope.

[0181] Monoclonal antibodies can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0182] Immunizing agent can comprise protein antigen, its fragment or its fusion protein.For example, if the cell of human origin is needed, peripheral blood lymphocyte can be used, or if the cell of non-human mammal origin is needed, spleen cell or lymph node cell can be used.Then lymphocyte is fused with immortalized cell line using suitable fusogenic agent (such as polyethylene glycol), to form hybridoma cell (referring to Goding, Monoclonal Antibodies:Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell line can be transformed mammalian cell, particularly myeloma cell of rodent, cattle and human origin.For example, rat or mouse myeloma cell line is used.Hybridoma cell can be cultured in suitable culture medium, and this culture medium contains one or more substances that suppress the growth or survival of unfused immortalized cells.

[0183] Useful immortalized cell lines are those that effectively fuse, support the stable high-level expression of antibodies by selected antibody production cells, and are sensitive to culture medium (such as HAT culture medium). For example, immortalized cell lines can be mouse myeloma cell lines, which can be obtained from, for example, the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51 to 63).

[0184] The presence of monoclonal antibodies against antigens in the culture medium of hybridoma cells can then be determined. For example, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be, for example, determined by Scatchard analysis by Munson and Pollard, Anal. Biochem., 107: 220 (1980). In addition, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

[0185] After identifying the desired hybridoma cells, clones can be subcloned by limiting dilution and cultured by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites.

[0186] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0187] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (incorporated herein by reference in its entirety). DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the present invention are used as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell, such as ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not otherwise produce immunoglobulins, thereby synthesizing monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by replacing the homologous mouse sequences with human heavy and light chain constant region coding sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0188] For example, a fully human antibody is an antibody molecule in which the complete sequence of both the light chain and the heavy chain (including CDRs) is derived from human genes. Such antibodies are referred to as "human antibodies" or "fully human antibodies". Human monoclonal antibodies (such as fully human antibodies and humanized antibodies) can be prepared by trioma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4:72); and EBV hybridoma technology to produce human monoclonal antibodies (see Cole et al., 1985 in: MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used and can be produced by using human hybridomas (see Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (see Cole et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0189] A "humanized antibody" can be an antibody from a non-human species (such as a mouse) whose amino acid sequence (e.g., in the CDR region) has been modified to increase its similarity to an antibody variant produced by humans. Antibodies can be humanized by methods known in the art, such as CDR grafting. See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29:175-86. In addition, humanized antibodies can be produced in transgenic plants as a cheap production alternative to existing mammalian systems. For example, the transgenic plant can be a tobacco plant, i.e., Nicotiana benthamiana, and Nicotiana tabaccum. The antibody is purified from the leaves of the plant. Stable transformation of plants can be achieved by using Agrobacterium tumefaciens or particle bombardment. For example, via transformation, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed in a bacterial culture (i.e., Agrobacterium tumefaciens strain BLA4404). Infiltration of the plant can be accomplished via injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and pestle and centrifuging. Isolation and purification of antibodies can be readily performed by many methods known to those skilled in the art. Other methods for producing antibodies in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5; and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Thus, the present invention further provides any cell or plant comprising a vector encoding an antibody of the present invention or producing an antibody of the present invention.

[0190] Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, incorporated herein by reference in its entirety). “Humanization” (also known as reshaping or CDR grafting) is a well-established technique understood by those skilled in the art for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (usually rodents) and for improving their ability to activate the human immune system (see, e.g., Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). “Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling”. J Biochem. 144(1): 115–20).

[0191] In addition, other techniques (including phage display libraries) can also be used to produce human antibodies (such as human antibodies). (See Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol, 222: 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). After challenge, the production of human antibodies is observed, which is very similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoires. This approach has been described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0192] In addition, transgenic non-human animals can be used to produce human antibodies, which are modified to produce fully human antibodies, rather than endogenous antibodies of animals attacked by reactive antigens. (See, PCT Publication No. WO94 / 02602 and U.S. Patent No. 6,673,986). The endogenous genes encoding heavy and light chains of immunoglobulins in non-human hosts have lost their ability, and the active loci encoding human immunoglobulin heavy and light chains are inserted into the genome of the host. For example, yeast artificial chromosomes containing necessary human DNA fragments are used to incorporate human genes. Then, by hybridizing intermediate transgenic animals containing less than the fully complementary modified ones, animals providing all the desired modifications are obtained as offspring. A non-limiting example of such non-human animals is a mouse, and is referred to as a Xenomouse. TM, as disclosed in PCT Publication Nos. WO96 / 33735 and WO96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with the immunogen of interest, such as preparations of polyclonal antibodies, or alternatively from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to directly obtain antibodies, or can be further modified to obtain antibody analogs, such as single-chain Fv (scFv) molecules.

[0193] Thus, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using this technology. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, PCT Publication No. WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be engaged to provide human antibodies against selected antigens using techniques similar to those described herein.

[0194] An example of a method for producing a non-human host (e.g., mouse) lacking endogenous immunoglobulin heavy chain expression is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being achieved by a targeting vector containing a gene encoding a selectable marker; and generating a transgenic mouse from the embryonic stem cell whose somatic and germ cells contain a gene encoding the selectable marker.

[0195] U.S. Patent No. 5,916,771 discloses a method for producing an antibody of interest (e.g., a human antibody). The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a cultured mammalian host cell, introducing an expression vector containing a nucleotide sequence encoding a light chain into another cultured mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing both heavy and light chains.

[0196] In a further refinement of this approach, methods for identifying clinically relevant epitopes on immunogens and related methods for selecting antibodies that immunospecifically bind to the relevant epitopes with high affinity are disclosed in PCT Publication No. WO 99 / 53049.

[0197] Antibodies can be expressed by vectors containing DNA fragments encoding single-chain antibodies as described herein. Vectors include, but are not limited to, chemical conjugates such as those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), plasmids, phages, viral vectors, etc. containing a target moiety (e.g., a target cell-specific antibody) and a nucleic acid binding moiety (e.g., protamine). Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, including Moloney murine leukemia virus. DNA viral vectors may also be used and include poxvirus vectors (such as orthopoxvirus or avipoxvirus vectors), herpesvirus vectors (such as herpes simplex virus type 1 (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F. et al., in DNA Cloning: Mammalian Systems, D. Glover, ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci USA 87:1149 (1990), adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995) and adeno-associated virus vectors (see Kaplitt, MG et al., Nat. Genet. 8:148 (1994)).

[0198] Poxvirus vectors introduce genes into the cytoplasm. Fowlpoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The selection of a specific vector will depend on the target cells and the condition being treated. Introduction can be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0199] The vector can be used to target substantially any desired target cell. For example, stereotactic injection can be used to guide the vector (e.g., adenovirus, HSV) to the desired location. In addition, a micropump infusion system (such as a synchronous infusion system) can be used to deliver particles by intracerebroventricular (icv) infusion. A method based on bulk flow, called convection, has also been shown to be effective in delivering macromolecules to extended areas of the brain and can be used to deliver vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994); Brigham et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injections, and oral or other known routes of administration.

[0200] These vectors can be used to express large numbers of antibodies that can be used in a variety of ways. For example, they can be used to detect the presence of TIGIT in a sample. Antibodies can also be used to attempt to bind to and disrupt TIGIT activity.

[0201] In one embodiment, the antibodies described herein may be full-length antibodies containing an Fc region similar to a wild-type Fc region that binds to an Fc receptor.

[0202] These techniques can be adapted to produce single chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). ab Expression libraries (see, e.g., Huse et al., 1989 Science 246: 1275-1281) are used to allow for the rapid and efficient identification of monoclonal F3kp ... ab Fragments. Antibody fragments containing idiotypes directed against protein antigens can be produced by techniques known in the art, including but not limited to: (i) F-terminal fragments produced by pepsin digestion of antibody molecules;(ab')2 Fragment; (ii) by reducing F (ab')2 F ab (iii) F fragments produced by treating the antibody molecule with papain and a reducing agent ab fragment; and (iv) F v fragment.

[0203] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. For example, such antibodies can target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980) and are used to treat HIV infection (see PCT Publication No. WO91 / 00360; WO92 / 20373). Antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolates and methyl-4-mercaptobutyrimide salts and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0204] The antibodies of the present invention can be modified according to effector functions, thereby enhancing the effectiveness of, for example, antibodies in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibodies thus generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1 191-1 195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies with dual Fc regions can be engineered to have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). In one embodiment, the antibodies of the present invention are modified to the Fc region so that the Fc region does not bind to Fc receptors. For example, the Fc receptor is an Fcγ receptor. As described herein, antibodies in which the Fc region is modified so that the Fc region does not bind to Fcγ but still binds to the neonatal Fc receptor are useful.

[0205] In certain embodiments, the antibodies of the present invention may include Fc variants that include amino acid substitutions that alter the antigen-dependent effector function of the antibody (particularly the circulating half-life of the antibody). Compared to antibodies lacking these substitutions, such antibodies exhibit increased or decreased binding to FcRn and therefore have an increased or decreased half-life in serum, respectively. The improved affinity of Fc variants for FcRn can have a longer serum half-life, and such molecules have useful applications in methods for treating mammals, where it is desirable that the half-life of the administered antibody be longer, for example, to treat chronic diseases or conditions. In contrast, Fc variants with reduced affinity for FcRn are expected to have a shorter half-life, and such molecules are also useful, for example, when administered to mammals, a shortened circulation time may be advantageous, such as for in vivo diagnostic imaging, or in cases where the initial antibody has toxic side effects when it is present in the circulation for a longer time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore also useful in treating diseases or conditions in pregnant women. In addition, other applications that may need to reduce FcRn binding affinity include those that need to be localized to the brain, kidneys and / or liver. In one embodiment, antibodies containing Fc variants can show reduced transport from the vasculature through the glomerular epithelium. In another embodiment, antibodies containing Fc variants show reduced transport from the brain through the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, antibodies with altered FcRn binding include an Fc domain that has one or more amino acid substitutions in the "FcRn binding loop" of the Fc domain. The FcRn binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No. WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, antibodies of the present invention or fragments thereof include Fc domains with one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).

[0206] In certain embodiments, mutation is introduced into the constant region of mAb so that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of mAb is changed.For example, mutation is the LALA mutation in the CH2 domain.In one embodiment, antibody (for example, human mAb or bispecific Ab) contains mutation on a scFv unit of heterodimeric mAb, which reduces ADCC activity.In another embodiment, mAb contains mutation on two chains of heterodimeric mAb, which completely eliminates ADCC activity.For example, the mutation introduced into one or two scFv units of mAb is the LALA mutation in the CH2 domain.These mAbs with variable ADCC activity can be optimized so that mAb shows maximum selective killing to the cells expressing a kind of antigen recognized by mAb, but shows minimum killing to the second antigen recognized by mAb.

[0207] In other embodiments, the antibodies of the present invention used in the diagnostic and therapeutic methods described herein have a constant region, such as an IgG1 or IgG4 heavy chain constant region, which can be altered to reduce or eliminate glycosylation. For example, the antibodies of the present invention can also include Fc variants that include amino acid substitutions that alter the glycosylation of the antibody. For example, the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In some embodiments, the Fc variant includes reduced glycosylation of N-linked glycans typically found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within the glycosylation motif, for example, an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In a specific embodiment, the antibody includes an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody includes an IgG1 or IgG4 constant region that includes S228P and T299A mutations (EU numbering).

[0208] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, pokeweed proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, toxin proteins, crotonic acid, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. A variety of radionuclides can be used to prepare radioconjugated antibodies. Non-limiting examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0209] Conjugates of antibodies and cytotoxic agents are prepared using a variety of bifunctional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumdiphenylyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described by Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see PCT Publication No. WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0210] One of ordinary skill in the art will appreciate that a wide variety of possible moieties can be conjugated to the resulting antibodies or other molecules of the invention (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and R.E. Lewis, Jr (eds.), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0211] As long as the antibody and other parts maintain their respective activity, coupling can be completed by any chemical reaction in conjunction with two molecules. The connection can include many chemical mechanisms, such as covalent binding, affinity binding, insertion, coordination binding and complexing. In one embodiment, the combination is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by introducing external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as the antibody of the present invention) to other molecules. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This enumeration is not intended to exhaust various coupling agents known in the art, but the example of more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987).) Non-limiting examples of linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719 which describes the use of haloacetic acid hydrazide derivatives coupled to antibodies via oligopeptide linkers. Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., catalog number (21558G)); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., catalog number #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propionamido]hexanoate (Pierce Chem. Co. catalog number #2165-G); and (v) sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number #24510), coupled with EDC.

[0212] The linkers described herein contain components with different properties, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS-esters have poorer solubility than sulfo-NHS esters. In addition, the linker SMPT contains sterically hindered disulfide bonds and can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in less available conjugates. In particular, Sulfo-NHS can enhance the stability of carbodiimide coupling. Carbodiimide coupling (such as EDC) when used in combination with sulfo-NHS forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0213] The antibodies disclosed herein can also be formulated into immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and described in U.S. Patent Nos. 4,485,045 and 4,544,545. U.S. Patent No. 5,013,556 discloses liposomes with extended circulation time.

[0214] Non-limiting examples of useful liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to obtain liposomes having the desired diameter. The Fab' fragments of the antibodies of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide exchange reaction.

[0215] Multispecific antibodies (bispecific and trispecific)

[0216] Multispecific antibodies are antibodies that can identify two or more different antigens. For example, bispecific antibodies (bsAb) are antibodies comprising two variable domains or scFv units so that the resulting antibody recognizes two different antigens. For example, trispecific antibodies (tsAb) are antibodies comprising two variable domains or scFv units so that the resulting antibody recognizes three different antigens. The invention provides multispecific antibodies that recognize the phosphorylated amino acid motif in the target protein and a second antigen and / or a third antigen, such as bispecific antibodies and trispecific antibodies. In one embodiment, multispecific antibodies (for example, bispecific antibodies and trispecific antibodies) can include the anti-pY specific fusion protein encompassing antibodies as described herein. Exemplary second and / or third antigens include tumor-associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2); IL-18 (protein sequence having NCBI Reference No. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_000576.1); IL-7 (protein sequence having NCBI Reference No. NP_000871.1); IL-2 (protein sequence having NCBI Reference No. NP_000577.2); and IL-21 (protein sequence having NCBI Reference No. NP_068575.1)), cytokine cognate receptors (e.g., IL-12R), and cell surface receptors. Non-limiting examples of the second and / or third antigen include CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, CD73, PD-1, 2B4, CD57, KLRG-1, or a combination thereof. In one embodiment, the bispecific antibody includes a first antigen binding domain (B1) and a second antigen binding domain (B2). In one embodiment, the first antigen binding domain (B1) is bound to a phosphorylated amino acid in a signal transduction protein. In another embodiment, when B1 is bound to a phosphorylated amino acid in a signal transduction protein, the second antigen binding domain (B2) is bound to B1 and / or a signal transduction protein.

[0217] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies) of the present invention can be constructed using methods known in the art. In some embodiments, the bispecific antibody is a single polypeptide in which two scFv fragments are connected by a long linker polypeptide, the length of which is sufficient to allow intramolecular binding between the two scFv units to form an antibody. In other embodiments, the bispecific antibody is more than one polypeptide connected by covalent or non-covalent bonds. Targeting PD1 Use of antibodies (therapeutic methods)

[0218] In various embodiments, antibodies or fragments thereof that specifically bind to phosphorylated amino acids in signal transduction proteins of the present invention can be expressed in cells, thereby enabling the antibodies to affect the regulation of cells. In some embodiments, the cells comprise CAR-T cells. In some embodiments, CAR-T cells expressing the antibody systems disclosed herein can be infused into a patient. In some embodiments, the patient may have cancer. In some embodiments, the cancer may be a blood cancer. In some embodiments, the cancer may be a solid cancer, including lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer.

[0219] As used herein, the terms "treat" and "treatment" refer to therapeutic treatments and disease prophylactic or preventative measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, alleviation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation (whether partial or complete), whether detectable or undetectable. "Treatment" can refer to prolonging survival compared to expected survival if not receiving treatment. Those in need of treatment include those already suffering from the condition or disorder, as well as those predisposed to suffering from the condition or disorder, or those in need of prevention of the condition or disorder.

[0220] The present invention provides methods for treating a subject at risk of (or susceptible to) cancer (e.g., if an early detection cancer biomarker is identified in the subject) or other cell proliferation-related diseases or conditions. Such diseases or conditions include, but are not limited to, for example, those diseases or conditions associated with abnormal expression of PD-1. For example, these methods are used to treat, prevent or alleviate cancer symptoms. In one embodiment, these methods are used to treat, prevent or alleviate solid tumor symptoms. Non-limiting examples of other tumors that can be treated by the compositions described herein include lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer or gastric cancer. In addition, the methods of the present invention can be used to treat hematological cancers, such as leukemia and lymphoma. Alternatively, these methods can be used to treat, prevent or alleviate the symptoms of metastatic cancer. For example, cancers that can be treated, prevented or whose symptoms can be alleviated include B-cell chronic lymphocytic leukemia (CLL), non-small cell lung cancer, melanoma, ovarian cancer, lymphoma or renal cell carcinoma. Cancers that can also be treated, prevented, or whose symptoms can be alleviated include those solid tumors with high mutation load and WBC in the filtrate.Cancers that can be treated or prevented, or whose symptoms can be alleviated further include cancers in which the signal in the PD-1 / TIGIT axis is regulated, including but not limited to breast cancer, lung cancer (e.g., non-small cell lung cancer or lung adenocarcinoma), gastric cancer, colorectal cancer, bladder cancer, pancreatic cancer, prostate cancer, esophageal squamous cell carcinoma, nasopharyngeal carcinoma, and liquid tumors with active PD-1 / PDL1 axis (such as diffuse large B-cell lymphoma (DLBCL) and B-cell chronic lymphocytic leukemia (B-CLL)) (see, e.g., Han et al., PD-1 / PD-L1 pathway: current researches in cancer, Am J Cancer Res 2020; 10(3): 727-742).

[0221] Therefore, in one aspect, the present invention provides a method for preventing, treating or alleviating the symptoms of a subject's cancer or cell proliferative disease or disorder by administering to the subject a monoclonal antibody, scFv antibody or bispecific antibody of the present invention. For example, cells expressing the antibody system disclosed herein can be administered in a therapeutically effective amount.

[0222] Pharmaceutical composition

[0223] In some embodiments, cells expressing the antibodies of the present invention can be administered in the form of a pharmaceutical composition for the treatment of cancer. The principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising antibodies, as well as guidance on component selection, are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th Edition (Alfonso R. Gennaro, et al., eds.) Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0224] The specific dosage and treatment regimen for any particular patient will depend on various factors, including the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex and diet, as well as the time of administration, rate of excretion, drug combination and the severity of the specific disease being treated. The judgment of the medical caregiver on these factors is within the scope of those of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease and the desired effect. The dosage can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0225] A therapeutically effective amount of the cells of the present invention can be the amount required to achieve a therapeutic goal.

[0226] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for regulating tension, such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be sealed in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0227] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In an embodiment, the composition is sterile and fluid to the extent that it is easy to inject. It can be stable under manufacturing and storage conditions and can be preserved against the contaminating effects of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant. The effect of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols (such as mannitol, sorbitol), sodium chloride, can be included in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent (such as aluminum monostearate and gelatin) that delays absorption in the composition.

[0228] Combination Methods

[0229] The compositions of the present invention described herein can be administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, cytoxan ... hydrazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorethoxyquin, and testolactone); nitrogen mustard derivatives (e.g., melphalan, chlorambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations thereof (e.g., betamethasone sodium phosphate); and others (e.g., dacarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0230] In another embodiment, the compositions of the present invention described herein can be administered in combination with cytokines. Cytokines that can be administered with the compositions include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0231] In further embodiments, the compositions described herein can be administered in combination with other therapeutic or preventive regimens, such as radiation therapy.

[0232] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivacizumab, dapoxetine ... bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, Dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotum ab), futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab,labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumab, naptumomab ), necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab omab), pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab b), taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and 3F8.

[0233] Other embodiments

[0234] While the invention has been described in conjunction with detail, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0235] Also disclosed herein are nucleic acids encoding any binding agent molecules disclosed herein. Contemplated are vectors containing nucleic acids encoding binding agent molecules and cells containing vectors. In some embodiments, cells containing nucleic acids and / or vectors can be T cells. Cells can be CAR-T cells, CAR-NK cells, CAR-macrophages, and the like.

[0236] Also disclosed herein are methods for introducing the antibodies or antibody systems disclosed herein, or genes encoding the antibodies or antibody systems, into cells. In some embodiments, the antibodies / antibody systems or encoding genes can be introduced into cells in vitro or ex vivo. After introduction, cells expressing the antibodies can be introduced into the patient's body. In some embodiments, the antibodies / antibody systems or genes encoding the antibodies / antibody systems can be introduced into the patient's body and targeted to desired cell types, such as T cells, NK cells, macrophages, etc.

[0237] In some embodiments, antibodies / antibody systems or genes encoding antibodies / antibody systems can be introduced into cells using lipid nanoparticles or viral vectors. In some embodiments, lipid nanoparticles can contain mRNA encoding the antibody / antibody system, which can be used to introduce the mRNA into cells. In some embodiments, the antibodies / antibody systems disclosed herein can be linked to toxins or toxin fragments. The toxins / toxin fragments can contact cells. The toxins / toxin fragments can bind to receptors on the surface of the cells. The toxins / toxin fragments can be internalized by the cells, and can also internalize the linked antibody / antibody system.

[0238] In some embodiments, the reagents and methods disclosed herein can be used as immunoassay reagents or biosensors to detect the presence of specific phosphorylated amino acids in cells. As disclosed elsewhere herein, binding agents can bind to specific phosphorylated amino acids (e.g., phosphotyrosine), but can also bind to these phosphorylated amino acids only within specific proteins (e.g., within PD-1, but not to phosphotyrosine in other proteins). The binding agents disclosed herein can be used to detect and visualize the presence of phosphorylated amino acids in specific proteins within cells.

[0239] In some embodiments, when a binding agent used as a biosensor binds to a specific phosphorylated amino acid in a specific protein, it can be detected in the cell. In some embodiments, this binding can be detected using imaging methods. In some embodiments where the binding agent is used as a biosensor, the binding agent molecule can be fused to a protein that is easily visualized (such as a fluorescent protein, etc.). Fluorescent proteins can be detected using, for example, a microscope or other imaging methods to confirm the presence or absence of specific phosphorylated amino acids in a specific protein. Figure 30 、 32 Examples of this are shown in and 33. Readouts other than fluorescence can be used with these binder biosensor systems.

[0240] For example, the disclosed binding agent biosensors can be used for diagnostics. In some embodiments, these biosensors can be used to screen cells or cell lines, or to determine the phenotype of cells or cell lines. In some embodiments, these biosensors can be used, for example, to detect disease, to confirm that a particular therapy for a disease will be effective, to determine the effectiveness of a treatment, etc. In some embodiments, the binding agents used as biosensors can detect functional or defective regulatory networks in cells.

[0241] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0242] Examples

[0243] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for making and implementing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are provided for illustrative purposes only, as alternative methods may be utilized to obtain similar results.

[0244] Example 1

[0245] R&D

[0246] We observed that Jurkat cells expressing SHP2 N-SH2-C-SH2 and N-SH2-C-SH2-ITAM (which can bind to phosphorylated PD-1) showed enhanced Jurkat activation in vitro. We also successfully screened and validated protein binders that specifically recognize phosphorylated PD-1.

[0247] question

[0248] Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment over the past decade. These engineered T cells are directed to cancer targets to destroy cancer cells that express the target. However, like natural T cells, CAR-T cells are easily exhausted after repeated stimulation. In this case, a variety of checkpoint inhibitory receptors (such as PD-1) on the surface of CAR-T cells are upregulated, which interact with ligand receptors on the surface of tumor cells, thereby inhibiting T cell activity. Although there are therapeutic antibodies that block signaling between PD-1 and its cognate ligand PD-L1, patient data show that this is only effective for some patients. Therefore, new checkpoint receptor inhibitors are needed.

[0249] Our approach is to develop binding agents that can bind to tyrosine phosphorylated or "active" forms of PD-1 in CAR-T cells and use these binding agents to regulate CAR-T cell exhaustion in cells. Using the pY-TRAP method previously developed by Dr. Xin Zhou (Zhou, Xin X. et al. "Targeting phosphotyrosine in native proteins with conditional, bispecific antibody traps." Journal of the American Chemical Society 142.41 (2020): 17703-17713) and yeast surface display technology, we screened out binding agents that are fusions of the ring-arranged SH2 of SHP-2 (phosphatase that binds to PD-1) and the heavy chain of a Fab antibody. This approach has many advantages over other methods of controlling T cell exhaustion because we specifically target the source of the exhaustion signal and can reconnect this signal to multiple different outputs. In addition, we can control the expression of this binding agent to turn on / off PD-1 signaling, which studies have shown may be better than simply knocking out PD-1 or other checkpoint inhibitors. Another approach would be to knock out SHP-2, a phosphatase thought to be responsible for binding to pY PD-1 and shutting down T cell activity. However, studies have also shown that knocking out SHP-2 does not prevent T cell exhaustion. This is likely because SHP-2 binds to multiple different phospho-tyrosine proteins, and completely removing it from cells would alter T cell function in other ways.

[0250] We are using this in conjunction with genetic engineering of CAR-T cells to treat human cancers. In this case, the binder will be expressed intracellularly, and the construct will therefore be contained within a lentivirus that introduces the CAR-T receptor. Another approach would be to deliver the protein binder into the cell via lipid nanoparticles, AAV approaches, or cell-penetrating peptides.

[0251] This approach could be combined with similar binders targeting other phosphorylated tyrosine sites on other checkpoint receptors. Furthermore, combining it with other CAR-T engineering approaches (e.g., knocking out the TGFβ receptor) might also improve efficacy.

[0252] Example 2

[0253] Specific goals

[0254] Although chimeric antigen receptor (CAR)-T cell therapies have demonstrated efficacy in the treatment of hematologic cancers, numerous barriers remain to the widespread application of these powerful anticancer strategies. A major challenge is the lack of reliable in situ methods to determine how the architecture of CARs regulates T cell signaling mechanisms, hindering the rational design of superior CAR therapeutics. Another obstacle is the deleterious immunosuppressive tumor microenvironment (TME) in solid tumors, which is caused by immunosuppressive cytokines, tumor-associated macrophages, and inhibitory checkpoints that promote severe CAR-T cell exhaustion. Among the various inhibitory signals in the TME, the interaction between PD-1 on T cells and PD-L1 on tumor and non-tumor cells is the main driver of CAR-T cell exhaustion. Accordingly, strategies to attenuate PD-1 signaling can significantly enhance the efficacy of CAR-T cells. However, current approaches have considerable limitations due to safety issues, inefficiency, or compensatory signaling mechanisms.

[0255] Tyrosine phosphorylation (pY) is a core and ubiquitous component of T-cell signaling pathways, mediating both activating and inhibitory signaling responses. Given these critical functions, engineering strategies to modulate pY-specific signaling events would be a powerful approach to study, engineer, and optimize CAR-T cell signaling and function. However, this strategy has not been explored previously because there are no readily available methods to engineer specific and tight synthetic anti-pY binders.

[0256] In previous work, we developed a powerful platform for engineering synthetic anti-pY binders, called "recombinant antibody pairs targeting pY," or "pY-TRAP" (Zhou, Xin X. et al. "Targeting phosphotyrosine innative proteins with conditional, bispecific antibody traps." Journal of the American Chemical Society 142.41(2020):17703-17713). We engineered two binders, B1 and B2, where B1 is a mutant of a universal anti-pY binder and B2 binds to a complex of B1 and pY antigen. This dual-binder approach addresses the challenge of phosphate recognition and also improves affinity and specificity. This is the first in vitro method that allows engineering specific binders for pY modifications in three-dimensional protein structures. This work opens up the possibility of rewiring endogenous signaling pathways and engineering new cell signaling responses in CAR-T cells.

[0257] Reprogramming the PD-1 signaling pathway in CAR-T cells (R00 stage). When activated on T cells, PD-1 terminates phosphorylation of signaling proteins by recruiting SHP phosphatases to their pY signaling motifs. Synthetic PD-1 pathways can be engineered using synthetic effectors that bind to the pY motif on phosphorylated PD-1. We can use a derivative of the pY-TRAP approach to develop specific and tight anti-pY binders for the two cytoplasmic pY modifications on PD-1. These domains can be fused to different enzymes (phosphatases or kinases) and expressed in CAR-T cells. Their superior affinity can surpass the binding of endogenous SHP to phosphorylated PD-1, thereby achieving customized signaling output of the PD-1 pathway. Although not wishing to be limited by theory, expression of fusions with phosphatases can produce immunosuppressive functions, while expression of fusions with kinases can produce immunostimulatory functions. This signaling rewiring strategy can be tested to determine whether it can be extended to other immunosuppressive pathways, such as TIGIT, and to study the effects of co-inhibition of PD-1 and TIGIT.

[0258] Example 3

[0259] Research strategy

[0260] background:

[0261] Chimeric antigen receptor (CAR)-T cell therapy has demonstrated unprecedented success in treating hematologic malignancies, particularly B-cell leukemias and lymphomas. However, efficacy is very limited against solid malignancies, which account for 90% of all cancer deaths. Numerous obstacles remain to the widespread application of these potent anticancer strategies.

[0262] A major obstacle is the inefficient activation and limited persistence of CAR-T cells, primarily due to T-cell exhaustion caused by immunosuppressive factors in the malignant tumor microenvironment (TME) associated with solid tumors. A major mechanism of CAR-T cell inhibition is the interaction between programmed cell death protein 1 (PD-1), expressed on activated or exhausted CAR-T cells, and its ligand PD-L1, presented on various tumor and non-tumor cells within the TME. Accordingly, attenuating PD-1 signaling in CAR-T cells can significantly enhance the efficacy of CAR-T cells. More than 10 clinical trials are testing strategies to block PD-1 signaling as a means of improving CAR-T cell therapy (clinicaltrials.gov). However, current approaches have limitations, including poor safety or reliance on inefficient genome engineering of primary T cells (see below for details). A simple, safe, and effective method for engineering CAR-T cells to resist PD-1-mediated T-cell dysfunction has yet to be reported.

[0263] Tyrosine phosphorylation (pY) is a universal mode of regulating protein function and forms the basis of a set of key signal transduction pathways in CAR-T cells ( Figure 1 During CAR activation, the signaling domain in the CAR receptor (such as the CD3ζ domain) is phosphorylated in its immunoreceptor tyrosine-based activation motif (ITAM), which leads to the recruitment of kinases to the CAR signalosome and the activation of downstream signaling pathways. In the immunosuppressive pathway, a common mechanism used by many receptors (including PD-1) is to recruit phosphatases to their phosphorylated immunoreceptor tyrosine-based inhibitory motifs or switch-like motifs (ITIM or ITSM, Figure 1 ) to terminate the phosphorylation of signaling proteins. Given the important role of pY modification in CAR-T cells, methods to modulate specific pY events are powerful tools for studying and engineering CAR-T cells. However, this approach has not been exploited, primarily because natural pY binding motifs (such as Src-homology 2 (SH2) domains) are highly promiscuous (i.e., pY binding domains do not specifically and exclusively bind to specific amino acids in specific proteins), and methods for engineering synthetic anti-pY binders with specificity and tightness are not yet available.

[0264] This study used a phage display platform to engineer sequence-specific anti-pY binders. This work shows how the structure of CARs influences stimulatory and inhibitory signaling pathways. This work also involves reprogramming endogenous inhibitory pathways to generate more powerful CAR-T cells.

[0265] Specifically, we show how to engineer the PD-1 pathway in CAR-T cells to reprogram the immunosuppressive function of this pathway.

[0266] Innovation:

[0267] The main challenge in studying tyrosine phosphorylation is the development of specific detection and purification methods. Most antibodies against pY sequences are generated using animal immunization, but this method has low throughput, high cost, and usually produces low affinity, low specificity, and non-renewable reagents, and is not suitable for non-immunogenic antigens. Previously, we developed an in vitro phage display workflow called "recombinant antibody pair targeting pY" (pY-TRAP) to generate synthetic binders to pY modifications ( Figure 2 A to 2B; Zhou, Xin X. et al. "Targeting phosphotyrosine in native proteins with conditional, bispecific antibody traps." Journal of the American Chemical Society 142.41(2020):17703-17713). In this approach, we engineered two binders, B1 and B2, where B1 is a mutant of a universal anti-pY antibody and B2 binds to a complex of B1 and a specific pY protein (rather than B1 or pY protein alone). This dual-binder approach addresses the challenge of phosphate recognition and promotes improved affinity and sequence specificity. Linking B1 and B2 together yields a subnanomolar Kd binder for pY modifications, one of the tightest anti-PTM binders known to date. In this article, we describe an innovative technology based on synthetic pY binders for studying and reprogramming the effects of CAR-T cells.

[0268] Reprogramming natural cell signaling pathways with synthetic signaling outputs. Natural pY pathways exhibit significant crosstalk due to the molecular promiscuity of their binding patterns. Such interactions typically have low affinity and modest selectivity, allowing a single protein to bind to multiple partners in a dynamic manner. In contrast, leveraging the tightness and specificity of pY-TRAP, we precisely engineered linear synthetic pathways. Such synthetic pathways may allow rewiring of endogenous signaling to drive novel cell signaling responses in CAR-T cells ( Figure 3In this work, we used such a strategy to rewire the output of PD-1 signaling to enhance CAR-T cell resistance to the suppressive TME (tumor microenvironment).

[0269] Although many CAR-T engineering approaches have been developed, engineering pY-binder-based signaling pathways to improve CAR-T cell function has not yet been explored. The generalization of this approach, engineering compact synthetic signaling pathways to detect pY signals and then rewiring them to customizable reactions, will allow the construction of synthetic signaling mechanisms that perform user-defined functions in any biological system of interest.

[0270] Reprogramming the PD-1 signaling pathway in CAR-T cells.

[0271] In some embodiments, constructing a synthetic PD-1 pathway in CAR-T cells, in which phosphorylated PD-1 recruits synthetic effectors rather than endogenous SHP phosphatases, can rewire the pathway's output from immunosuppression to a customized response.

[0272] introduce:

[0273] To overcome the immunosuppressive function of PD-1, methods to disrupt PD-1 signaling in CAR-T cells are being developed and have shown good promise in preclinical and clinical trials. This approach involves administering PD-1 / PD-L1 blocking antibodies or using engineered CAR-T cells that secrete PD-1 / PD-L1 blocking antibodies ( Figure 6 A) However, these approaches may induce severe autoimmune reactions associated with systemic immune checkpoint blockade 5 Alternatively, genomic disruption of PD-1 can specifically block the endogenous PD-1 pathway in CAR-T cells, but this approach requires extremely inefficient genome engineering of primary T cells ( Figure 6 B). Finally, the chimeric PD-1 / CD28 receptor can reverse the immunosuppressive function of the PD-1 pathway to immunostimulation, but it does not prevent the endogenous PD-1 signaling pathway from performing its immunosuppressive effects ( Figure 6 C).

[0274] We can rewire the signaling output of the PD-1 pathway, turning it into a potentiator of T cell activity rather than an inhibitor ( Figure 8 This approach has several advantages over existing approaches: (1) it only modulates the endogenous PD-1 pathway of CAR-T cells and therefore does not cause toxicity caused by systemic PD-1 blockade, (2) it does not require precise genome editing of T cells, (3) it blocks the immunosuppressive function of the endogenous PD-1 pathway, and (4) the output can be flexibly programmed.

[0275] A derivative of the pY-TRAP method for engineering "superbinders" targeting pY modifications in PD-1. In order to study and modulate cell signaling pathways, a method for engineering binders that interact with linear pY antigens is needed. Not only are pY in flexible linear structural motifs (such as ITAM, ITIM, and ITSM) widespread mediators of cell signaling transduction, but commonly used detection methods (such as protein immunoblotting) also require reagents that bind to linear or denatured epitopes. Although the pY-TRAP method ( Figure 2 ) is widely useful for pY sites in folded proteins, but this approach may not be effective for linear epitopes because B1 is not optimal for peptide binding. Replacing B1 in the pY-TRAP system with the SH2 domain (which is the natural motif for binding to linear pY epitopes) can generate a new binding mode that recognizes linear pY antigens ( Figure 7 A).

[0276] The cytoplasmic domain of PD1 contains ITIM and ITSM motifs ( Figure 7 B), which is a linear pY-containing motif that is important for regulating immune cell signaling activities. In the endogenous PD-1 pathway, activated PD-1 recruits SHP phosphatase through the interaction between phosphorylated ITIM and ITAM and the SH2 domain in SHP phosphatase. In order to engineer a "super binder" targeting the pY site in PD-1, we can use the SH2 domain of SHP as B1, and then screen the domain that can recognize the SH2-pY complex from our in-house Fab or single domain VH library and use it as B2 ( Figure 7A). The library can be cpSH2 fused to a single domain antibody library (VH fragment) (see Bracken, Colton J. et al. "Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2." Nature chemical biology 17.1 (2021): 113-121). The library can be cpSH2 fused to a nanobody library (see McMahon, Conor et al. "Yeast surface display platform for rapid discovery of conformationally selective nanobodies." Nature structural & molecular biology 25.3 (2018): 289-296). The affinity of this B2-SH2 fusion for pY modification in PD-1 should be significantly enhanced than that of endogenous SHP phosphatase.

[0277] Specifically, we can use two approaches to identify B2. Based on our experience developing pY-TRAP, a stable pY antigen / B1 interaction (Kd <50 nM) is key to successful B2 identification. The interaction between the SH2 domain and the pY antigen typically has a Kd value of 100 nM to 10 μM, and therefore requires additional engineering steps to promote a stronger interaction. In the first approach, we can link the synthetic PD-1pY peptide to the SHP SH2 domain using methionine-based conjugation chemistry or subtilisin ligase-based bioconjugation methods and then use this covalent fusion to screen for B2. The high local concentration of the two moieties in a single polypeptide chain significantly stabilizes their interaction. We can also genetically fuse the SHP SH2 domain to a library of VH domains to construct a phage library displaying VH-SH2 fusions. We can then use the immobilized PD-1pY peptide to pull the strongest interacting SH2-VH variants from this new phage library. Using the resulting binders, we were able to engineer B2-SH2 fusions and characterize their binding affinity and specificity for the PD-1pY antigen using biofilm interferometry. These fusions bind to PD-1 with higher affinity than the SH2 domain alone in SHP. In the pY-TRAP approach, we found that the B1-B2 fusion had a subnanomolar Kd for the pY antigen, while the Kd for binding of B1 or B2 alone was >10 nM ( Figure 2 A). Binding to endogenous PD-1 will be verified by western blotting or immunoprecipitation.

[0278] Engineering synthetic PD-1 pathways in CAR-T cells. The B2-SH2 domain can be fused to different effector domains (including phosphatases and kinases) to create synthetic effector domains for expression in CAR-T cells ( Figure 8 ). Although not wishing to be bound by theory, B2-SH2 not fused to an effector can inhibit PD-1 signaling because it will bind to PD-1 with a higher affinity than native SHP, thereby preventing endogenous SHP from being recruited to the immune synapse and its subsequent immunosuppressive function. Although not wishing to be bound by theory, a fusion of B2-SH2 with the phosphatase domain of SHP (B2-SH2-SHP) can produce a strong immunosuppressive effect because its binding to the immune synapse will be more stable than native SHP. Although not wishing to be bound by theory, fusion of B2-SH2 with LCK (B2-SH2-LCK) or ZAP70 (B2-SH2-ZAP70), kinases that promote the assembly and function of the CAR signalosome, can convert the initial immunosuppressive result of PD-1 signaling into a stimulatory signal.

[0279] A model cell line, the PD-1+ Jurkat cell line ( Figure 5 ). Jurkat cells can be infected with a retrovirus encoding an anti-CD19-CAR and a synthetic effector and then co-cultured with CD19+PD-L1+K562 cells (as described elsewhere), which can activate CAR signaling as well as the PD-L1 / PD-1 pathway. We can determine whether overexpression of B2-SH2, B2-SH2-SHP2, or B2-SH2-LCK in the Jurkat cell line can lead to varying degrees of CAR activation by measuring the intensity of NFAT-GFP and the expression of CD69 (an early surface marker for Jurkat cell activation). Although not wishing to be bound by theory, B2-SH2-SHP2 expression can lead to the weakest CAR-T activation, while B2-SH2-LCK / ZAP70 expression can lead to the strongest activation. Different orientations of the protein fusions, linker lengths, and mutations in the effector domain can be tested to determine a construct with the desired signaling output.

[0280] The optimal construct can be expressed in primary CAR-T cells. To induce an exhausted / hypofunctional phenotype, transduced T cells can be cultured for three days in the presence of 10 ng / ml Staphylococcal enterotoxin B (Charles River), which significantly increases PD-1 expression on T cells. PD-1 levels can be measured using flow cytometry and anti-PD-1 antibodies. These PD-1+ exhausted primary CAR-T cells can be co-cultured with CD19+PD-L1+ K562 cells to activate the CAR and PD-1 signaling pathways. Surface markers (CD69 / CD25 / CD137), protein phosphorylation (PLC-γ1-pY783, CD3ζ-pY142, ZAP70-pY319), cytokine release (IL-2, IFN-γ, TNF-α), and cytotoxicity can be characterized to determine whether cells transduced with B2-SH2 and B2-SH2-LCK elicit stronger activation / cytotoxicity than cells expressing mock constructs or B2-SH2-SHP29. Together, these experiments can determine whether the synthetic pathway has successfully rewired the endogenous function of PD-1 and has an impact on CAR-T cell function.

[0281] Strategies for other immunosuppressive pathways. Comparative analysis of gene expression in patients showed that 156 genes related to immune function were differentially expressed. Although PD-1 has been studied in many aspects as a promising candidate, these data emphasize the need to consider other immunomodulatory factors. One of the similar immunosuppressive receptors is T cell immunoglobulin with ITIM domain (TIGIT). Recent studies have shown that overexpression of TIGIT-CD28 chimeric receptors in CAR-T cells can offset the immunosuppressive function of this pathway. TIGIT has a cytoplasmic tail ( Figure 7 B). In the TIGIT pathway, binding to its ligands (such as CD155 and CD112) induces phosphorylation of the ITIM and ITT motifs of TIGIT, leading to binding to the cytoplasmic adaptor protein Grb2, which then recruits SHIP1 to terminate CAR signaling. To engineer a synthetic TIGIT pathway, we can follow the same strategy as above. In short, we can develop B2-SH2 binders targeting the ITIM or ITT domains of TIGIT phosphorylation, fuse them with SHIP1 or LCK, and express them in primary CAR-T cells. To test the effect, we can prepare CD155+K562 and CD155-K562 cells and co-culture them with engineered CAR-T cells. Although not wishing to be bound by theory, expression of the B2-SH2-SHIP1 fusion can produce a powerful immunosuppressive function, while expression of the B2-SH2-LCK fusion can be immunostimulatory.

[0282] In some embodiments, a combined effect involving co-inhibition of the PD-1 and TIGIT pathways is described. While not wishing to be bound by theory, in CAR-T cells, co-inhibition of TIGIT and PD-1 can produce a CAR-T cell phenotype that is more resistant to exhaustion.

[0283] Additional methods:

[0284] Additional approaches are to perform SH2 affinity maturation to generate SHP (or Grb2) SH2 mutants with stronger affinity for the pY sequence in PD-1 (or TIGIT) by incorporating mutations known to increase affinity for the pY antigen in the SH2 domain or by developing SH2 libraries to screen for tighter binders.

[0285] Influence

[0286] This work focuses on the ex vivo study and engineering of primary CAR-T cells to determine the performance of these engineered cells in animal models.

[0287] Example 4

[0288] We have selected binders that are fusions of the looped SH2 of SHP-2 (a phosphatase that binds PD-1) and the heavy chain variable domain (VH) of a Fab antibody to target PD-1. In this example, experiments demonstrating these binders are described.

[0289] Figure 10A shows a pY-Trap molecule. Figure 10B shows a schematic diagram of an embodiment of a molecule disclosed herein. The top of Figure 10B shows an intracellular phosphorylated tyrosine in a PD-1 molecule. SH2 represents a binding agent (B1) that binds to phosphotyrosine. The binding agent comprises an SH2 region that binds to phosphotyrosine in PD-1. The SH2 region is connected to the VH domain of the antibody (B2; as shown in the bottom of Figure 10B) via a peptide linker. The antibody domain binds to SH2, binds to PD-1 (e.g., phosphotyrosine), or binds to both SH2 and PD-1.

[0290] Figure 11A shows a PD-1 molecule, comprising an extracellular domain and an intracellular domain. Intracellular phosphorylated tyrosines pY223 and pY248 are shown. pY248 is targeted in our exemplary system. Figure 11B shows the various domains of a protein (e.g., SHP2) that binds to PD-1. The N-SH2 domain binds to pY223. The C-SH2 domain binds to pY248. The modified C-SH2 domain used herein is used for the B1 binder in our molecule. Figure 11C shows a ring-shaped version of the C-SH2 domain of the SHP2 phosphatase used in our molecule. This configuration of the SH2 domain facilitates its fusion with other polypeptides. Figure 11D Figure 2 shows that SHP2 binds to PD-1 via its N-terminal and C-terminal SH2 domains. In addition to PD-1, the SH2 domain in SHP2 also binds to other phosphorylated molecules, such as GAB2 pY614 ( Figure 25 , right small picture).

[0291] Figure 12 The results show that a ring-shaped version of the C-SH2 domain of SHP2 phosphatase still retains weak binding to pY248 in the PD-1 molecule.

[0292] Figures 13A to 13B A strategy for isolating the molecules disclosed herein is presented. The target molecules are those containing a ring-arranged SH2 domain connected to a library of VH antibodies (see also FIG10B ). We used the ring-shaped SH2 domain to bind to pY248 of PD-1, but the screening was aimed at finding antibodies from the library that bind more strongly to pY248 after being connected to the ring-shaped SH2 domain (e.g., a better B2 antibody). As shown in FIG13B , yeast display was used to express cpC-SH2 connected to various VH antibodies from the library.

[0293] Figure 14 Screening results from Figure 13B are shown for yeast cells exposed to a peptide containing the PD-1 pY248 motif, stained, and analyzed by flow cytometry. The x-axis represents yeast cells expressing cpC-SH2 / VH antibody molecules on their cell surface. The y-axis represents pY248 binding in the library of molecules expressed on yeast cells. Yeast clones that both express cpC-SH2 / VH antibody molecules and bind to pY248 are indicated within the oval.

[0294] Figure 15 Show the Figure 14 The cpC-SH2 / VH antibody molecules shown within the oval (B1-B2 molecules) were sorted in successive rounds (R2, R3, R4) to obtain molecules that bind better to pY248.

[0295] Figures 16A to 16B Figure 16A shows a single clone obtained from screening of yeast display cells. Figure 16A shows flow cytometric analysis of negative controls (bottom) and high binding clones (top). Figure 16B shows the PD-1 binding profiles of each clone obtained from the cell sorting experiment. On the left, the dual SH2 domain from SHP2 is used as a positive control for high binding. On the right is the low binding cpSH2 (see Figure 12 )

[0296] Figure 17Binding data for 10 clones obtained from cell sorting experiments are shown. DNA sequences for some of these molecules were obtained. Next generation sequencing was also used to obtain the DNA sequences of the most abundant sequences in the mixed sample. The CDRs contained in the VH domains from some of these molecules are shown in Table 2. The nucleic acid sequence of one of the VH regions is shown in Table 1.

[0297] Figure 18 Shown are dose-response binding curves for A4 and F10 clones compared to a double SH2 positive control (high binder).

[0298] Figure 19A shows the dose-response binding curves of A3, A4 and F10 clones compared to a double SH2 positive control (high binder) and a cpSH2 low binder to pY248 in PD-1. Additional data of this type are shown in Figure 19A. Figure 28 As shown in Figure 19B, the dose-response binding curves of clones A3, A4, and F10 to pY614 in Grb2-associated binding protein-2 (Gab2) are shown. Gab2 contains an SH2 domain. Gab2 is involved in granulocyte colony stimulating factor (G-CSF) signaling. These data show the specificity of our molecules for pY248 in PD-1. Additional data of this type are shown in Figure 19B. Figure 28 As shown in B.

[0299] Figure 20 It was shown that the A4, F10 and A3 VH domains in a fusion protein with looped SH2 domains can be expressed in mammalian cells.

[0300] Example 5

[0301] Cells expressing the antibody system described herein (e.g., comprising the B1 domain of a portion of SHP2 that binds to pY248 in PD-1 and the B2 domain that binds to the complex formed between B1 and pY248 in PD-1) and having a functional PD-1 pathway were used. Control cells having a functional PD-1 pathway but not expressing the antibody system described herein were also used.

[0302] Recombinant PDL-1 or PDL-1-expressing cancer cells were added to cells expressing the antibody system and control cells not expressing the antibody system. The response of the PD-1 pathway after the addition of PDL-1 was measured.

[0303] The data will show that in control cells that do not express the antibody system, the PD-1 pathway activated by PDL-1 recruits the native SHP2 effector protein, and the recruited SHP2 dephosphorylates T cell effector proteins (such as CD3 and ZAP70). The data will show that the PD-1 pathway activated by PDL-1 in cells expressing the antibody system does not recruit SHP2, or recruits less SHP2, compared to cells that do not express the antibody system. As a result, T cell effector proteins (such as CD3 and ZAP70) are not dephosphorylated, or are dephosphorylated to a lesser extent.

[0304] Example 6

[0305] Figure 21 An example of a plate-based activation assay is shown. In this assay, cells on a plate are treated with increasing concentrations of an anti-CD3 antibody, which activates T cells. The activation response of cells to the anti-CD3 antibody is measured by flow cytometry using CD69. CD69 is a marker for activated T cells. The data in the figure show that activation of both Jurkat cells and Jurkat cells expressing PD-1 is enhanced with increasing doses of the anti-CD3 antibody. However, due to the expression of the PD-1 pathway in Jurkat PD-1 cells, activation of Jurkat PD-1 cells is lower than that of Jurkat cells.

[0306] Figure 22 The expression of pY-TRAP binders A4, F10 and A3 fused to the CD3z motif in Jurkat-PD1 cells is shown. Figure 21 Plate-based activation assay presented in . CD3z represents the signaling domain of the CAR receptor that contains an ITAM (immunoreceptor tyrosine-based activation motif). The data suggest that binders fused to the CD3z motif can enhance CAR-T activity. These data suggest that pY-TRAP binders fused to the CD3z motif can convert immune checkpoint pathways (e.g., PD-1) into immune activation pathways.

[0307] Example 7

[0308] Binding molecules to specific phosphorylated amino acids were tested in a model cell line system. To this end, cell lines stably expressing non-phosphorylated PD-1 and phosphorylated PD-1 were constructed.

[0309] To construct a cell line expressing PD-1, PD1-TagBFP-HAtag was stably expressed in HEK293 cell line (schematic diagram as shown in Figure 29As shown in Figure A). PD1-TagBFP-HAtag has PD1 molecules, BFP fluorescent tags (blue) and HA tags. Separately, PD1-TagBFP-HAtag and LCK-EGFP-v5tag were expressed in another HEK293 cell line (schematic diagram as shown in Figure A). Figure 29 (Shown in B). LCK-EGFP-v5tag contains LCK kinase, an EGFP fluorescent tag (green), and a V5 tag. LCK is a kinase that phosphorylates PD-1. Because HEK cells lack endogenous PD-1 and LCK expression, we anticipate that PD-1 will only be expressed in HEK293 cell lines that also express LCK kinase.

[0310] The expression and membrane localization of PD-1 and LCK in these cell lines were confirmed using fluorescence microscopy ( Figure 29 C). LCK-dependent PD-1 phosphorylation was confirmed in immunoprecipitation experiments ( Figure 29 D to 29E).

[0311] Next, we investigated whether the binder molecules could be expressed in cells and whether these molecules were fully functional within the cells. This question was raised because some single-domain antibodies may not be naturally stable in the reducing intracellular environment (e.g., due to the presence of intramolecular disulfide bonds).

[0312] Multiple binder molecules were fused to the mCherry fluorescent tag (red). Among the binders tested, binders #1, #2, #3 (F10), and #5 had moderate intracellular expression in cells (Binders #1 and #2 were Figure 30 Other binding agents showed little intracellular expression (e.g., Figure 30 A3 shown in ).

[0313] We next performed experiments to determine whether the intracellularly expressed binders recognized PD-1. Various binder molecules (Binders #1, #2, #3 (F10), and #5) were expressed in the above-mentioned HEK293 cells expressing PD-1 (unphosphorylated PD-1) and HEK293 cells overexpressing PD-1 and LCK (phosphorylated PD-1). The expression of PD-1 and LCK was confirmed by western blotting using anti-PD-1 and anti-V5 tag antibodies, respectively. Figure 31 The expression of the binder was confirmed by Western immunoblotting using an anti-mCherry antibody ( Figure 31 Whole-cell lysates were analyzed by western blot.

[0314] To test the binding of the binder molecules to PD-1, immunoprecipitation experiments were performed using anti-HA beads that bind to the HA-tagged PD-1-TagBFP fusion protein expressed in cells. First, the immunoprecipitates were subjected to protein immunoblotting with anti-pTyr1000 antibody to identify phosphorylated PD-1. Phosphorylated PD-1 was only recognized in cells expressing LCK ( Figure 31 ). Second, the immunoprecipitates were immunoblotted with anti-mCherry antibodies to identify the binder molecules. These data show that binders #3 and #5 co-immunoprecipitated with PD-1 in LCK-expressing cell lysates, indicating that these binders do bind to phosphorylated PD-1 in cells ( Figure 31 (see the fourth small picture in the figure).

[0315] To observe the localization of the binding agent molecules within the cells, high-magnification fluorescence images were collected for cells expressing binding agent #5 and PD-1 or PD-1 and LCK. These images are shown in Figure 32 The images show that the binder colocalizes with PD-1 and LCK at the cell membrane of cells expressing PD-1 and LCK, but not in cells lacking LCK. Figure 33 Higher magnifications of the figures are shown.

[0316] Example 8

[0317] Another library was used to search for binders. The molecules in this library have a circularly arranged SH2 domain (cpSH2) from the SHP2 phosphatase fused to a library of nanobodies (i.e., heavy chains) with randomized CDR sequences ( FIG34A ; Table 6 ). FIG34B shows an example of yeast surface display screening of pY-TRAP from the library, which was labeled with an HA tag to a biotinylated peptide (pY peptide) with a phosphotyrosine modification. Figure 34C A multi-step enrichment process using magnetic enrichment and fluorescence-activated cell sorting is shown. Figure 34D Flow cytometry analysis of different enrichment steps is shown (cell populations in the upper quadrants of the graph are enriched). Titration after the final round of enrichment shows EC 50 These binders generally bound better than cpSH2 and tandem SH2 from SHP2 in the nanomolar range (Figure 35A, Table 7).

[0318] Biolayer interferometry (BLI) data showed that NT7 binder bound to PD-1pY248 peptide with a K D is about 17 nm ( FIG. 41A ).

[0319] Example 9

[0320] Additional studies were performed on the molecules described in Example 8 to examine their function.

[0321] To investigate the binding specificity of the molecule, binding studies were performed with a panel of peptides, including non-phosphorylated forms of the peptides and pY peptides with amino acid sequences similar to the endogenous binding sites of PD-1 pY248 and the SHP2 SH2 domain. The data ( FIG35B ) demonstrate the binding specificity of these molecules. Biolayer interferometry of NT7 binding to the PD-1 pY248 peptide also demonstrated specific binding of the molecule compared to other phosphorylated and non-phosphorylated amino acid motifs ( FIG41B ).

[0322] Other studies were conducted to test the binding specificity of the molecules. Figure 36A shows a schematic diagram of a split luciferase system, in which LgBit and SmBit polypeptides / peptides from luciferase are combined to reconstruct a functional luciferase molecule. As shown, wild-type PD1 (YY) or alanine mutations (Y→A) at two tyrosine sites are fused to LgBit from luciferase. The pY-TRAP binding agent is fused to SmBit from luciferase. The interaction between the binding agent-SmBit and PD1-LgBit reconstructs a complete luciferase, which can be measured. The research data (Figure 36B) shows a heat map of luciferase assays performed using the tandem SH2 domains of cpSH2, SHP2, SHP2, and selected binding agents in LentiX cells expressing WT PD1 (YY) and mutant PD1 (AA). The fold change of the luciferase signal is shown. The data show that the tested molecules are specific for binding to phosphorylated tyrosine residues (YY).

[0323] Figure 37 Data are shown in which cell lysates from LentiX cells co-expressing Lck kinase, PD1 WT (YY) or PD1 Y223F / Y248F (FF), and selected binders tagged with mCherry and HA were immunoprecipitated with anti-HA beads (to precipitate the binders) and then blotted with anti-pTyr antibody. Figure 37 The band in the middle shows tyrosine phosphorylation corresponding to the size of PD1 and is only present in YY cells. The expression of Lck was detected with V5 antibody, and the PD1 level in whole cell lysates was confirmed with anti-PD1 antibody. These data also show the binding specificity of the test molecules.

[0324] Figure 38Cell fluorescence data for expression of GFP-tagged PD1 (green) are shown. One or both tyrosine residues in the PD1 molecule were mutated to phenylalanine. The cells used were LentiX cells co-expressing Lck and mCherry-tagged Binder-NT7 (red). Membrane localization of the binder was observed in wild-type PD1 (Y223 / Y248) and F223 / Y248 PD1. Membrane localization of the binder was reduced in both F223 / F248 PD1 and Y223 / F248 PD1.

[0325] Example 10

[0326] Studies were performed to test whether expression of binder molecules in T cells could inhibit the suppression of T cell activation by PDL-1.

[0327] Figure 39A Schematic diagram showing the co-culture of Raji B cells overexpressing PDL1 (right) and Jurkat T PD1- / - cells reconstituted with wild-type PD1 (YY) or FF mutant PD1 (FF) (left). Jurkat T cells were activated with 1000 pM anti-CD3 / anti-CD19 bispecific antibody (BiTE).

[0328] exist Figures 39B to 39C In the data shown in , the activation of T cells treated with anti-CE3 / anti-CD19 antibodies was quantified by measuring surface CD69 levels (Figure 39B) and IFN-γ release (Figure 39C), both of which are indicators of T cell activation. The data show that the tested binding agent molecule (NT7) reversed the PDL-1 inhibition of T cell activation, as did the positive control nivolumab (anti-PD1). The data show that the reversal of PDL-1 inhibition by NT7 binding agent combined with nivolumab is stronger than the reversal of PDL-1 inhibition by NT7 or nivolumab alone.

[0329] Example 11

[0330] A study was conducted to demonstrate one way in which the binder molecules described herein can be used to regulate gene expression in cells. The study showed that synthetic transcription factors can be released by a protease fused to a pY-TRAP molecule.

[0331] Figure 40A Figure 2 is a schematic diagram of a cell membrane containing a PD-1 molecule with a tetracycline transactivator (tTA) fused to a nuclear localization signal (NLS). This molecule is referred to as PD1-TEV substrate-eGFP-tTA. Not shown are negative control cells with a CD28 transmembrane protein also with tTA fused to an NLS (referred to as CD28TM-TEV substrate-eGFP-tTA0).

[0332] Figure 40B Data from this study are shown. LentiX cells were co-transfected with PD1-TEV substrate-eGFP-tTA (bottom row) or CD28TM-TEV substrate-eGFP-tTA (top row) and BinderNT7-TEV-mCherry. In cells transfected with PD1-TEV substrate-eGFP-tTA, membrane recruitment of binder-fused TEV (lower left) and nuclear localization of eGFP-tagged tTA (lower right) were observed. No membrane recruitment of binder-fused TEV was found in CD28TM-TEV substrate-eGFP-tTA cells (upper left), and no nuclear localization of tTA was found (upper right).

[0333] The data showed that when the binding agent molecule specifically binds to phosphorylated PD-1, the protease can cleave the transactivator from PD-1, and due to the fused nuclear localization signal, the cleaved transactivator can be localized to the cell nucleus.

[0334] *****

[0335] Equivalent

[0336] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. An antibody system comprising: a first molecule (B1) that binds to a phosphorylated amino acid motif in a target protein to form a first complex between B1 and the protein; and A second molecule comprises an antibody or antigen-binding fragment thereof (B2), which binds to the first complex to form a second complex.

2. The antibody system of claim 1, wherein B1 comprises an antibody or an antibody-binding fragment thereof. 3 . The antibody system of claim 1 , wherein B1 comprises a portion of a protein capable of binding to the phosphorylated amino acid motif in the target protein. The antibody system according to claim 3 , wherein B1 comprises a protein having enzymatic activity or capable of promoting the formation of a protein signaling complex.

5. The antibody system of claim 4, wherein the protein having enzymatic activity comprises an SH2 domain. The antibody system according to claim 5 , wherein the SH2 domain is from SHP2 protein.

7. The antibody system of claim 5, wherein the SH2 domain comprises a looped arrangement of SH2 domains.

8. The antibody system of claim 6, wherein the SH2 domain comprises a looped C-terminal SH2 domain of SHP2.

9. The antibody system of claim 8, wherein the circularly arranged C-terminal SH2 domain of SHP2 has an amino acid sequence as shown in Tables 1 and 2, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

10. The antibody system according to claim 1, wherein the phosphorylated amino acid motif is embedded in the three-dimensional domain of the target protein (the tertiary folding domain of the protein).

11. The antibody system of claim 1, wherein the phosphorylated amino acid motif comprises a non-linear epitope.

12. The antibody system of claim 11, wherein the phosphorylated amino acid motif comprises a three-dimensional (folded) epitope of the target protein.

13. The antibody system of claim 1, wherein the phosphorylated amino acid motif comprises a linear epitope of the target protein. The antibody system of claim 1 , wherein B2 bound to the first complex is bound to B1. The antibody system of claim 1 , wherein B2 bound to the first complex is bound to the target protein. The antibody system of claim 1 , wherein B2 bound to the first complex is bound to B1 and the target protein. The antibody system of claim 1 , wherein B2 bound to the first complex recognizes an epitope comprising B1.

18. The antibody system of claim 1, wherein B2 bound to the first complex recognizes an epitope comprising the target protein. The antibody system of claim 1 , wherein B2 bound to the first complex recognizes an epitope comprising B1 and the target protein.

20. The antibody system of claim 1, wherein if the amino acid motif in the target protein is not phosphorylated, B1 does not bind to the amino acid motif.

21. The antibody system of claim 1, wherein if B1 does not bind to the phosphorylated amino acid motif in the target protein, B2 does not bind to the first complex.

22. The antibody system of claim 1, wherein the phosphorylated amino acid motif comprises phosphohistidine, phosphoserine, phosphothreonine, or phosphotyrosine.

23. The antibody system of claim 1, wherein the phosphorylated amino acid motif is present in a signal transduction protein.

24. The antibody system of claim 23, wherein the signal transduction protein comprises a receptor.

25. The antibody system of claim 23, wherein the signal transduction protein has enzymatic activity.

26. The antibody system of claim 23, wherein the signaling protein modulates an immune checkpoint pathway.

27. The antibody system of claim 26, wherein the signal transduction protein comprises PD-1, CTLA-4, LAG-3, TIGIT, 2B4, BTLA, CD57, TIM-3, A2AR, VISTA, KIR, IDO, B7-H3, B7-H4, or KLRG-1.

28. The antibody system of claim 27, wherein the signal transduction protein comprises PD-1.

29. The antibody system of claim 23, wherein the signal transduction protein comprises phosphothisidine, phosphoserine, phosphothreonine, or phosphotyrosine.

30. The antibody system according to claim 23, wherein a protein having enzymatic activity or capable of promoting the formation of a signaling complex is capable of binding to the phosphorylated amino acid motif of the signal transduction protein.

31. The antibody system according to claim 30, wherein the protein having enzymatic or complex-forming ability comprises a phosphothisidine binding domain, a phosphoserine binding domain, a phosphothreonine binding domain or a phosphotyrosine binding domain.

32. The antibody system of claim 30, wherein the protein having enzymatic activity comprises a Src homology 2 (SH2) domain or a phosphotyrosine binding (PTB) domain.

33. The antibody system of claim 32, wherein the protein having enzymatic activity comprises SHP2.

34. The antibody system of claim 23, wherein the phosphorylated amino acid motif comprises a phosphotyrosine to which a protein having a Src homology 2 (SH2) domain can bind.

35. The antibody system of claim 23, wherein the phosphorylated amino acid motif comprises phosphoserine or phosphothreonine to which a protein having a WW domain can bind.

36. The antibody system of claim 23, wherein the phosphorylated amino acid motif comprises phosphohistidine to which a protein having an SH2 domain can bind.

37. The antibody system of claim 23, wherein the phosphorylated amino acid motif comprises sulfotyrosine to which a protein having a modified SH2 domain can bind.

38. The antibody system of claim 30, wherein formation of the second complex prevents a protein from binding to and / or modulating the phosphorylated amino acid motif in the signal transduction protein.

39. The antibody system of claim 26, wherein formation of the second complex prevents activation of the immune checkpoint pathway.

40. The antibody system of claim 1, wherein said B1 and said B2 are covalently linked.

41. The antibody system of claim 40, wherein said B1 and / or said B2 comprises a scFv, a single domain antibody, a nanobody, a monomer, a DARPin, or an affibody.

42. The antibody system of claim 40, wherein said B1 and said B2 are linked by a peptide linker to form linked B1-B2.

43. The antibody system of claim 42, wherein the linked B1-B2 further comprises a phosphatase or kinase bindable motif.

44. The antibody system of claim 42, wherein the linked B1-B2 further comprises an immunoreceptor tyrosine-based activation motif (ITAM), an immunoreceptor tyrosine-based inhibitory motif (ITIM), or an immunoreceptor tyrosine-based switch motif (ITSM).

45. The antibody system according to any one of claims 1 to 44, which is expressed in a cell or introduced into a cell.

46. ​​The antibody system of claim 45, wherein the cells comprise T cells.

47. The antibody system of claim 46, wherein the cell comprises a CAR-T cell, a CAR-NK cell, or a CAR-macrophage.

48. A kinase or phosphatase comprising the linked B1-B2 of claim 42.

49. A protease comprising the linked B1-B2 of claim 42.

50. The antibody system of claim 6, wherein the B2 comprises an antibody or antibody binding fragment thereof comprising a heavy chain, wherein the heavy chain comprises the CDR1, CDR2 and CDR3 of 10.1 / E4, 10.2, 10.3 / F10, 10.4, 10.5, 10.6, 10.7, 30.3, 30.5, 30.7, C6, A4, B5, C8, F9, E10 or D9 as shown in Table 4.

51. The antibody system of claim 50, wherein the scaffold of the heavy chain is encoded by a nucleic acid sequence as shown in Table 3, or a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

52. B1 according to claim 1.

53. The B1 of claim 52, comprising a scFv, a single domain antibody, a nanobody, a monomer, a DARPin, or an affibody.

54. The B1 of claim 52, comprising a ring-arranged SH2 domain from a SHP2 protein.

55. The B1 of claim 52, having an amino acid sequence as shown in Table 1 or Table 2, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

56. The B1 of claim 52, wherein the B1 is not covalently linked to B2.

57. The B2 antibody or antigen-binding fragment thereof according to claim 1.

58. B2 according to claim 57, which comprises a scFv, a single domain antibody, a nanobody, a monomer, a DARPin or an affibody.

59. The B2 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain, wherein the heavy chain comprises the CDR1, CDR2 and CDR3 of 10.1 / E4, 10.2, 10.3 / F10, 10.4, 10.5, 10.6, 10.7, 30.3, 30.5, 30.7, C6, A4, B5, C8, F9, E10 or D9 as shown in Table 4.

60. The B2 antibody or antigen-binding fragment thereof according to claim 59, wherein the scaffold of the heavy chain is encoded by a nucleic acid sequence as shown in Table 3, or a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. The B2 antibody or antigen-binding fragment thereof of claim 57 , wherein the B2 is not covalently linked to B1.

62. A bispecific antibody comprising: a first antigen-binding domain (B1) that binds to a phosphorylated amino acid in a signal transduction protein; and The second antigen-binding domain (B2) binds to the B1 and / or the signal transduction protein when the B1 binds to the phosphorylated amino acid in the signal transduction protein. The bispecific antibody according to claim 62 , wherein when the B1 binds to the phosphorylated amino acid in the signal transduction protein, the B2 binds to the B1 and the signal transduction protein.

64. The bispecific antibody of claim 62, wherein the signal transduction protein comprises a cellular receptor.

65. The bispecific antibody of claim 64, wherein the cellular receptor comprises an intracellular domain comprising the phosphorylated amino acid.

66. The bispecific antibody of claim 64, wherein the cellular receptor comprises a cytokine receptor.

67. The bispecific antibody of claim 64, wherein the cellular receptor modulates cancer in a cell or in an individual.

68. The bispecific antibody of claim 62, wherein the phosphorylated amino acid is selected from the group consisting of serine, threonine, tyrosine, and histidine.

69. The bispecific antibody of claim 68, wherein the tyrosine is present in a motif to which a protein having a Src homology 2 (SH2) domain can bind.

70. The bispecific antibody of claim 62, wherein the signaling protein comprises an immune checkpoint pathway.

71. The bispecific antibody of claim 70, wherein the regulatory proteins in the immune checkpoint pathway comprise PD-1, CTLA-4, LAG-3, TIGIT, 2B4, BTLA, CD57, TIM-3, KLRG-1, or a combination thereof. The bispecific antibody of claim 71 , wherein the signal transduction protein comprises a PD-1 protein. The bispecific antibody of claim 72 , wherein the phosphorylated amino acid in the PD-1 protein comprises phosphotyrosine 223 or 248.

74. The bispecific antibody of claim 70, wherein the immune checkpoint pathway is not activated or is minimally activated when B1 binds to the phosphorylated amino acid in the signaling protein and wherein B2 binds to B1 and / or the signaling protein.

75. The bispecific antibody of claim 62, wherein the phosphorylated amino acid in the signal transduction protein is phosphorylated by a protein kinase.

76. The bispecific antibody of claim 75, wherein the protein kinase comprises a serine / threonine protein kinase, a tyrosine kinase, or a histidine kinase.

77. The bispecific antibody of claim 76, wherein the tyrosine kinase comprises a receptor tyrosine kinase. The bispecific antibody according to claim 62 , wherein if the B1 does not bind to the phosphorylated amino acid in the signal transduction protein, the B2 does not bind to the B1 and / or the signal transduction protein. The bispecific antibody of claim 62 , wherein the bispecific antibody comprises a peptide linker connecting B1 and B2.

80. The bispecific antibody of claim 79, wherein the peptide linker is between about 3 amino acids and about 50 amino acids in length. The bispecific antibody of claim 62 , wherein the B1 is located in a first bispecific antibody and the B2 to which the B1 binds is located in a second bispecific antibody.

82. The bispecific antibody of any one of claims 62 to 81, which is expressed in a cell or introduced into a cell.

83. The bispecific antibody of claim 82, wherein the cell comprises a T cell.

84. The bispecific antibody of claim 83, wherein the cell comprises a CAR-T cell, a CAR-NK cell, or a CAR-macrophage.

85. A first antigen-binding domain (B1) of the bispecific antibody according to claim 57.

86. The B1 of claim 85, comprising a scFv, a single domain antibody, a nanobody, a monomer, a DARPin, or an affibody.

87. A second antigen binding domain (B2) of the bispecific antibody according to claim 57.

88. B2 according to claim 87, which comprises a scFv, a single domain antibody, a nanobody, a monomer, a DARPin or an affibody.

89. A nucleic acid encoding the isolated bispecific antibody of claim 62.

90. A vector comprising the nucleic acid of claim 89.

91. A cell comprising the nucleic acid of claim 89 or the vector of claim 90.

92. The cell of claim 91, wherein the cell comprises a T cell.

93. The cell of claim 92, wherein the T cell comprises a chimeric antigen receptor (CAR)-T cell, a CAR-NK cell, or a CAR-macrophage.

94. A method for treating cancer, comprising administering to a patient CAR-T cells that express the bispecific antibody according to any one of claims 62 to 81 within the cells.

95. A method for treating cancer, comprising administering CAR-NK cells or CAR-macrophages to a patient, wherein the cells express the bispecific antibody according to any one of claims 62 to 81 intracellularly.

96. A method for introducing a bispecific antibody according to any one of claims 62 to 81 or a nucleic acid encoding a bispecific antibody according to any one of claims 62 to 81 into a cell or a cell in an individual, the method comprising introducing a lipid nanoparticle comprising the bispecific antibody or nucleic acid or a viral vector containing the nucleic acid into the cell.

97. The method of claim 96, wherein the lipid nanoparticles contain a nucleic acid comprising mRNA.

98. A method for introducing B1 and / or B2 according to claim 1 into a cell or a cell in an individual, the method comprising linking the B1 and / or B2 to a toxin or a fragment thereof, or to a cell-penetrating peptide to produce toxin-linked or penetrating peptide-linked B1 and / or B2, and introducing the toxin-linked or penetrating peptide-linked B1 and / or B2 into the cell.

99. The method of claim 98, wherein the toxin or fragment thereof can bind to a receptor on the surface of the cell.

100. The method of claim 98, wherein the toxin or fragment thereof that binds to the receptor can be internalized by the cell together with the B1 and / or B2.

101. The method of claim 98, wherein the penetrating peptide can be internalized by the cell together with the B1 and / or B2.

102. The bispecific antibody according to any one of claims 62 to 81, further comprising a phosphatase or kinase bindable motif.

103. The bispecific antibody of claim 102, wherein the motif comprises an immunoreceptor tyrosine-based activation motif (ITAM), an immunoreceptor tyrosine-based inhibitory motif (ITIM), or an immunoreceptor tyrosine-based switch motif (ITSM).

104. The bispecific antibody of any one of claims 62 to 81, further comprising a phosphatase or a kinase.

105. The bispecific antibody of any one of claims 62 to 81, further comprising a protease.

106. The bispecific antibody according to claim 105, wherein When the bispecific antibody is expressed in a cell, the protease can cleave the substrate sequence of the membrane-tethered effector protein and release the effector protein from the membrane.

107. The bispecific antibody of claim 106, wherein the effector protein comprises a transcription factor, an epigenetic regulator, or an enzyme.

108. The bispecific antibody of claim 107, wherein the epigenetic regulator comprises a DNA methylase, an enzyme that modifies histones, or a non-coding RNA.

109. A method for modulating the activation of T cells, the method comprising expressing in a cell the antibody system of any one of claims 1 to 47 or 50 to 51, the kinase or phosphatase of claim 48, the protease of claim 49, or the bispecific antibody of any one of claims 62 to 84.

110. The antibody system according to any one of claims 1 to 47 or 50 to 51 or the bispecific antibody according to any one of claims 62 to 84, comprising a detectable marker.

111. The antibody system or bispecific antibody of claim 110, wherein the detectable marker comprises a fluorescent protein or moiety.

112. A method for detecting a specific phosphorylated amino acid in a specific protein, the method comprising: contacting the antibody system according to any one of claims 1 to 47 or 50 to 51 or the bispecific antibody according to any one of claims 62 to 84 with a cellular protein in a permeabilized cell or a living cell, or with a cell extract; and The binding of the antibody system or the bispecific antibody to the specific phosphorylated amino acid in the specific protein is detected.

113. The method according to claim 112, wherein said detecting comprises visualizing a marker of said antibody system or bispecific antibody.

114. The method of claim 113, wherein the marker comprises a fluorescent protein or moiety.

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