Automatic modulation of therapeutic agents

Abstract

The present invention relates to a therapeutic agent comprising an automodulating element, preferably wherein the therapeutic agent is an antigen binding protein. Also disclosed are several methods of treatment comprising the use of the medicament.

Description

Technical field

[0001] The present invention relates to a therapeutic agent, preferably wherein the therapeutic agent is an antigen binding protein. Also disclosed are methods of treatment comprising the use of the therapeutic agent. [Background Technology]

[0002] Homeostasis and sensitivity to feedback regulatory signals are naturally occurring mechanisms that balance nearly all biological functions in the body. However, most drugs lack such regulatory mechanisms, and technologies that modulate the therapeutic effects of drugs in response to physiological or pathological signals are highly sought but rarely achieved.

[0003] Over the past decade, advances in molecular and cellular engineering have seen the development of complex and powerful medicines, including therapeutic monoclonal and bispecific antibodies, chimeric antigen receptor (CAR) T cells, and engineered therapeutic peptides, that achieve unique functionalities and bring major clinical benefits, particularly to patients with significant unmet needs. However, the translational potential of these novel therapies is hampered by the occurrence of serious adverse events related to their mechanism of action (MoA), which may include cytokine-mediated toxicities in adoptive immunotherapy; cytokine release syndrome; and thromboembolic and prothrombotic risks in hemophilia A. Due to the inherently long half-lives of some of these therapies (e.g., antibodies, engineered cells), such adverse events pose a serious life-threatening risk to patients. [Summary of the invention]

[0004] The present invention demonstrates how to exploit endogenous signals or factors that can be activated as a direct response to therapeutic treatment under abnormal conditions to create a new generation of therapeutic agents with built-in "on-off" mechanisms for modulating their therapeutic activity at the risk of severe MoA-associated toxicities.

[0005] The following invention describes a method for designing and developing a new generation of therapeutic agents with embedded self-regulatory elements (autoregulatory elements) that have the ability to inactivate the therapeutic agent in the event of excessive or undesirable treatment, thereby reducing the risk of adverse events and improving the safety of these therapeutic agents. Autoregulatory elements are small cleavable peptides inserted into the structure of the therapeutic agent, and their mode of action is that cleavage of the small peptide inactivates the therapeutic agent by breaking it down into non-functional or rapidly cleared fragments ( Figure 1 ).

[0006] Autoregulatory peptides are susceptible to proteolytic cleavage mediated by specifically selected endogenous factors whose concentration, activity, or bioavailability is altered in response to treatment-induced adverse events. Thus, while the therapeutic activity of the drug remains within a defined range in the absence of adverse events, the drug maintains its full therapeutic potential, whereas when the therapeutic activity is above the threshold separating normal biological activity from MoA-related adverse events, alterations in the activity of the endogenous factors will lead to cleavage of the autoregulatory element and ultimately inactivation of the therapeutic agent, reducing the risk of progression to severe toxicity ( Figure 1 ).

[0007] Proof of concept was established using two therapeutic bispecific antibodies. Emicizumab is a marketed humanized bispecific IgG4 used to treat patients with hemophilia A with or without inhibitors, while NVG-111 is a bispecific tandem-single-chain fragment variable region (scFv) investigational new drug currently in Phase I trials for chronic leukemic lymphoma (CLL) and mantle cell lymphoma (MCL). The application of autoregulation technology has also been demonstrated in CAR-T cell models. The present invention defines a universal platform that is independent of target and form, which can be applied to any therapeutic agent, including therapeutic bispecific and monoclonal antigen-binding proteins and antibodies (including non-classical forms), therapeutic non-antibody-derived peptides, and engineered cells (including but not limited to CART cells) to expand the therapeutic index of such therapies, with the potential to improve efficacy and safety, thereby achieving better clinical outcomes in patients.

[0008] Therefore, the present invention provides:

[0009] A therapeutic agent comprising an autoregulatory element, wherein the autoregulatory element is a small peptide that can be cleaved by an endogenous factor whose activity is altered by the activity of the therapeutic agent, and wherein cleavage of the autoregulatory element results in the separation of the therapeutic agent into non-functional fragments.

[0010] The present invention also provides:

[0011] - A composition comprising a therapeutic agent as defined herein and a pharmaceutically acceptable carrier.

[0012] The present invention also provides:

[0013] - An isolated nucleic acid molecule encoding an antigen binding protein as defined herein.

[0014] The present invention also provides:

[0015] - An isolated host cell transformed with a nucleic acid molecule as defined herein.

[0016] The present invention also provides:

[0017] A method of treating hemophilia A comprising administering to an individual in need thereof an antigen binding domain comprising an autoregulatory element comprising a thrombin cleavage site, or administering a composition comprising the autoregulatory element.

[0018] The present invention also provides:

[0019] A method for preventing prothrombotic risk comprising administering to an individual in need thereof an antigen binding protein comprising an autoregulatory element comprising a thrombin cleavage site, or administering a composition comprising said autoregulatory element.

[0020] The present invention also provides:

[0021] A method of treating cancer comprising administering to an individual in need thereof an antigen binding protein comprising an autoregulatory element comprising a granzyme B cleavage site, or administering a composition comprising the autoregulatory element.

[0022] The present invention also provides:

[0023] A method for preventing mechanism of action (MoA)-related toxicity associated with immunotherapy comprising administering to an individual in need thereof an antigen binding protein comprising an autoregulatory element comprising a granzyme B cleavage site, or administering a composition comprising the autoregulatory element.

[0024]

Explanation of Sequence Listing

[0025] SEQ ID NO: 1: Thrombin cleavage recognition sequence consensus sequence

[0026] SEQ ID NO: 2: Thrombin cleavage recognition sequence P1'G to L

[0027] SEQ ID NO: 3: Thrombin cleavage recognition sequence P1'G to D

[0028] SEQ ID NO: 4: Thrombin cleavage recognition sequence from human coagulation factor V (FV)

[0029] SEQ ID NO: 5: Granzyme B cleavage recognition sequence GZMB-1 from mouse BID protein

[0030] SEQ ID NO: 6: Granzyme B cleavage recognition sequence GZMB-2 from mouse BID protein

[0031] SEQ ID NO: 7: Granzyme B cleavage recognition sequence GZMB-3, consensus sequence

[0032] SEQ ID NO: 8: Granzyme B cleavage recognition sequence GZMB-4, P10 to P10' sequence of GZMB-2

[0033] SEQ ID NO: 9: Granzyme B cleavage recognition sequence GZMB-5, P4 I to V

[0034] SEQ ID NO: 10: Granzyme B cleavage recognition sequence GZMB-6, P4 I to L

[0035] SEQ ID NO: 11: Granzyme B cleavage recognition sequence GZMB-7, P2 P to G

[0036] SEQ ID NO: 12: Granzyme B cleavage recognition sequence GZMB-8, P2 P to A

[0037] SEQ ID NO: 13: Granzyme B cleavage recognition sequence GZMB-9, P1'S to A

[0038] SEQ ID NO: 14: Granzyme B cleavage recognition sequence GZMB-10, P2'L to E and P4'E to Q

[0039] SEQ ID NO: 15: Granzyme B cleavage recognition sequence GZMB-11, P2'L to E and P4'E to V

[0040] SEQ ID NO: 16: Granzyme B cleavage recognition sequence GZMB-12, P1 D to N

[0041] SEQ ID NO: 17: Granzyme B cleavage recognition sequence GZMB-13, P1 D to E

[0042] SEQ ID NO: 18: Granzyme B cleavage recognition sequence GZMB-14, P1 D to Q

[0043] SEQ ID NO: 19: ROR1 binding antigen binding domain LCDR1

[0044] SEQ ID NO: 20: ROR1 binds to antigen binding domain LCDR2

[0045] SEQ ID NO: 21: ROR1 binding antigen binding domain LCDR3

[0046] SEQ ID NO: 22: ROR1 binding antigen binding domain HCDR1

[0047] SEQ ID NO: 23: ROR1 binding antigen binding domain HCDR2

[0048] SEQ ID NO: 24: ROR1 binding antigen binding domain consensus HCDR3

[0049] SEQ ID NO: 25: ROR1 binding antigen binding domain HCDR3, clone F heavy chain CDR3

[0050] SEQ ID NO: 26: ROR1 binding antigen binding domain HCDR3, humanized 1 heavy chain CDR3

[0051] SEQ ID NO: 27: ROR1 binding antigen binding domain HCDR3, humanized 2 and 5 heavy chain CDR3

[0052] SEQ ID NO: 28: ROR1 binding antigen binding domain HCDR3, humanized 3 and 4 heavy chain CDR3

[0053] SEQ ID NO: 29: ROR1 binding antigen binding domain, clone F light chain variable region

[0054] SEQ ID NO: 30: ROR1 binding antigen binding domain, humanized 1 light chain variable region

[0055] SEQ ID NO: 31: ROR1 binding antigen binding domain, humanized 2 light chain variable region

[0056] SEQ ID NO: 32: ROR1 binding antigen binding domain, humanized 3 light chain variable region

[0057] SEQ ID NO: 33: ROR1 binding antigen binding domain, humanized 4 light chain variable region

[0058] SEQ ID NO: 34: ROR1 binding antigen binding domain, humanized 5 light chain variable region

[0059] SEQ ID NO: 35: ROR1 binding antigen binding domain, clone F heavy chain variable region

[0060] SEQ ID NO: 36: ROR1 binding antigen binding domain, humanized 1 heavy chain variable region

[0061] SEQ ID NO: 37: ROR1 binding antigen binding domain, humanized 2 heavy chain variable region

[0062] SEQ ID NO: 38: ROR1 binding antigen binding domain, humanized 3 heavy chain variable region

[0063] SEQ ID NO: 39: ROR1 binding antigen binding domain, humanized 4 heavy chain variable region

[0064] SEQ ID NO: 40: ROR1 binding antigen binding domain, humanized 5 heavy chain variable region

[0065] SEQ ID NO: 41: CD3 binding antigen binding domain, mouse light chain variable region

[0066] SEQ ID NO: 42: CD3 binding antigen binding domain, humanized 1 light chain variable region

[0067] SEQ ID NO: 43: CD3 binding antigen binding domain, humanized 2 light chain variable region

[0068] SEQ ID NO: 44: CD3 binding antigen binding domain, humanized 3 light chain variable region

[0069] SEQ ID NO: 45: CD3 binding antigen binding domain, humanized 4 light chain variable region

[0070] SEQ ID NO: 46: CD3 binding antigen binding domain, humanized 5 light chain variable region

[0071] SEQ ID NO: 47: CD3 binding antigen binding domain, mouse heavy chain variable region

[0072] SEQ ID NO: 48: CD3 binding antigen binding domain, humanized 1 heavy chain variable region

[0073] SEQ ID NO: 49: CD3 binding antigen binding domain, humanized 2 heavy chain variable region

[0074] SEQ ID NO: 50: CD3 binding antigen binding domain, humanized 3 heavy chain variable region

[0075] SEQ ID NO: 51: CD3 binding antigen binding domain, humanized 4 heavy chain variable region

[0076] SEQ ID NO: 52: CD3 binding antigen binding domain, humanized 5 heavy chain variable region

[0077] SEQ ID NO: 53: CD3 binding antigen binding domain LCDR1

[0078] SEQ ID NO: 54: CD3 binding antigen binding domain LCDR2

[0079] SEQ ID NO: 55: CD3 binding antigen binding domain LCDR3

[0080] SEQ ID NO: 56: CD3 binding antigen binding domain HCDR1

[0081] SEQ ID NO: 57: CD3 binding antigen binding domain HCDR2

[0082] SEQ ID NO: 58: CD3 binding antigen binding domain HCDR3

[0083] SEQ ID NO: 59: Linker sequence

[0084] SEQ ID NO: 60: Linker sequence

[0085] SEQ ID NO: 61: ROR1×CD3 bispecific antibody amino acid sequence

[0086] SEQ ID NO: 62: Amino acid sequence of ROR1×CD3 bispecific antibody with N-terminal hexahistidine tag (NVG-111)

[0087] SEQ ID NO: 63: Common LCDR1 of FIX-binding and FX-binding antigen-binding domains

[0088] SEQ ID NO: 64: Common LCDR2 of FIX-binding and FX-binding antigen-binding domains

[0089] SEQ ID NO: 65: Common LCDR3 of FIX-binding and FX-binding antigen-binding domains

[0090] SEQ ID NO: 66: FIX binding antigen binding domain HCDR1

[0091] SEQ ID NO: 67: FIX binding antigen binding domain HCDR2

[0092] SEQ ID NO: 68: FIX binding antigen binding domain HCDR3

[0093] SEQ ID NO: 69: FX binding antigen binding domain HCDR1

[0094] SEQ ID NO: 70: FX binding antigen binding domain HCDR2

[0095] SEQ ID NO: 71: FX binding antigen binding domain HCDR3

[0096] SEQ ID NO: 72: Common light chain variable region of FIX-binding and FX-binding antigen-binding domains

[0097] SEQ ID NO: 73: Heavy chain variable region of FIX binding antigen binding domain

[0098] SEQ ID NO: 74: Heavy chain variable region of FX binding antigen binding domain

[0099] SEQ ID NO: 75: Hinge region

[0100] SEQ ID NO: 76: Hinge region plus SEQ ID NO: 1

[0101] SEQ ID NO: 77: Linker sequence

[0102] SEQ ID NO: 78: Linker sequence

[0103] SEQ ID NO: 79: Linker sequence

[0104] SEQ ID NO: 80: Linker sequence

[0105] SEQ ID NO: 81: Linker sequence

[0106] SEQ ID NO: 82: Hinge region of CAR embodiments

[0107] SEQ ID NO: 83: Partially deleted hinge region of CAR embodiment plus SEQ ID NO: 7

[0108] SEQ ID NO: 84: Hinge region of CAR embodiment plus SEQ ID NO: 7

Brief Description of the Drawings

[0109]

【 Figure 1 : Schematic diagram of the mechanism of action of autoregulation (AR), taking therapeutic antibodies as an example]

[0110] The initial pathological state is characterized by a lack of biological activity. In the case of therapeutic intervention, AR antibodies (or therapeutic agents) produce the biological activity required for effective therapeutic effects. In the case of adverse events caused by excessive biological activity, products with proteolytic activity produced by the mechanism of action of AR antibodies are used to trigger automatic regulation. Increased activity of the enzyme product cuts the specific sensitive peptide inserted into the structure of the AR antibody (or therapeutic agent), resulting in gradual degradation of the molecule. A reduction in the concentration of AR antibodies will therefore reduce its biological activity and regulate the therapeutic effect to prevent the occurrence of adverse events.

[0111]

【 Figure 2 : Design of an autoregulatory bispecific antibody with FVIII-mimetic activity for hemophilia A]

[0112] AR_Ab8, a prototype autoregulatory bispecific therapeutic antibody for the treatment of hemophilia A, was designed using the core sequence of emicizumab, a humanized IgG4 full-length antibody targeting coagulation factor IX (FIX) and factor X (FX). A thrombin-sensitive cleavable peptide consisting of 8 residues (P4 to P4' sequence) was inserted into the hinge region of the two heavy chains of the parent antibody.

[0113]

Figure 3 : In vitro characterization of FVIII mimetic potential

[0114] (A) Coomassie blue-stained SDS-Page gel showing the migration profiles of purified emicizumab and AR_Ab8 under non-reducing conditions, indicating no premature degradation. (B) FVIII mimetic activity of emicizumab and AR_Ab8 (2 nM) was detected using a chromogenic substrate method that detects the conversion of FX to FXa mediated by FIXa. Points represent the mean OD values ​​± SD of three replicates. Control conditions were performed without bsAb. (C) In vitro procoagulant potential was measured in human plasma. 200 BU of anti-FVIII antibody was added to normal human citrated mixed plasma to neutralize endogenous FVIII. This induced hemophilia A plasma was then treated with 350 nM of emicizumab or AR_Ab8, and the activated partial thromboplastin time (aPTT) was measured. Symbols represent individual clotting time values, and lines represent the mean ± SD values ​​of three replicates. ns indicates not significant in one-way analysis of variance (ANOVA) test with Sidak's multiple comparisons.

[0115]

Figure 4 : Unimpaired therapeutic effect of AR_Ab8 in hemophilia A mice

[0116] (A) The procoagulant efficacy of AR_Ab8 was tested in vivo. FVIIIKO mice received an intravenous injection of 3 mg / kg of emicizumab or AR_Ab8. 24 h later, mice were injected with a bolus dose of human FIX and FX, followed by a tail clip assay 5 minutes after the second injection. Control animals received 2 U of human FVIII per mouse. (B) Total blood loss results are presented as box plots, showing the median (line), 25th and 75th percentiles (upper and lower boxes of the box), and minimum / maximum values ​​(error bars) from 6 to 11 mice. Symbols represent individual values. ns and * indicate not significant and p < 0.05, respectively, in a one-way ANOVA test using Sidak's multiple comparisons.

[0117]

Figure 5 :Thrombin-mediated degradation of AR_Ab8 in vitro

[0118] 2 nM AR_Ab8 or emicizumab were incubated with 2 U / mL α-thrombin to assess the cleavage kinetics of thrombin-sensitive peptides. 500 nM PPACK was added at various time points to terminate the enzymatic cleavage reaction for up to 180 minutes. (A) Reaction samples were migrated on SDS-Page gels under non-reducing conditions and then immunoblotted using an anti-human Fc antibody to visualize AR_Ab8 degradation. (B) The residual FVIII mimetic activity of the two antibodies was then measured using the FXa chromogenic substrate method. For each time point, the results were normalized and expressed as a percentage of emicizumab. Bar heights represent the mean ± SD of three replicates. ns, *, and ** indicate not significant, p < 0.05, and p < 0.01, respectively, in a two-way ANOVA test using Sidak's multiple comparisons. (C) The sensitivity of various thrombin-cleavable peptides was tested in the form of bispecific tandem scFvs. 2nM of different tandem scFv was exposed to 2U / mL α-thrombin for 20 minutes, and residual FVIII simulation activity was then measured by FXa chromogenic substrate method. Results were normalized to control conditions (no thrombin exposure), and column height represents the percentage of residual activity. Negative control group (non-cleav. (non-cleaved)) was experimented with non-autoregulatory constructs.

[0119]

【 Figure 6 Thrombin-mediated AR reduces abnormal thrombin generation in vitro

[0120] The in vitro thrombin generation curve of AR_Ab8 was studied using a thrombin generation model in the presence of an activated prothrombin complex concentrate (aPCC). APCC promoted abnormal thrombin generation together with a FVIII mimetic antibody. 200BU of anti-FVIII antibody was added to normal human citrated mixed plasma to neutralize endogenous FVIII, followed by further addition of 0.5U / mL of aPCC and 600nM. Thrombin generation was subsequently triggered using a low tissue factor solution, and thrombin generation was detected using a calibrated automated thrombogram (CAT) method. The dotted line shows the average value of the maximum value of thrombin generated in normal plasma, and the portion of the curve above the dotted line shows abnormal thrombin generation, with the average value being from three repetitions. Peak height (maximum thrombin generation) was extracted from the thrombin generation curve and plotted as three repeated individual values ​​(symbols) and average values ​​(B). ns and * indicate not significant and p < 0.05, respectively, in one-way ANOVA test with Sidak's multiple comparisons.

[0121]

【 Figure 7 Thrombin-mediated AR reduces the thrombotic characteristics of a FVIII-mimicking antibody in mice

[0122] The in vivo thrombotic profile of bispecific antibodies with FVIII mimetic activity was evaluated using a mouse model co-treated with aPCC, which enhances the thrombotic potential of emicizumab. (A) C57Bl / 6 male mice initially received a bolus of aPCC (2.5 U) and either emicizumab or AR_Ab8 (250 μg) intravenously, followed by three booster injections of aPCC at 24-h intervals. Thrombotic outcomes were assessed at t+96 h. (B) Plasma samples were collected in EDTA anticoagulant at the end of the experiment (96 h) and platelet counts were measured. Symbols correspond to individual animals, and horizontal lines represent the median for each experimental condition. ns indicates not significant, and *** indicates p<0.001, derived from an unpaired t-test.

[0123]

Figure 8 :AR_Ab8 reduces the formation of pulmonary thrombosis in mice

[0124] Lungs of animals treated with aPCC and emicizumab or AR_Ab8 were dissected, fixed with formalin, and slices were then made for histological evaluation. (A) Lung slices were subjected to immunofluorescence staining for PECAM (green) and platelet-specific integrin αIIb (purple). PECAM staining was used to delineate the vascular surface (dashed line), and αIIb staining was converted using a binary occulsion mask. 20x objective lens, scale bar represents 50 μm. (B) The degree of vascular occlusion was quantified in a blinded manner and calculated as the percentage of the occlusion mask in the total vascular surface. Symbols represent the occlusion values ​​of each blood vessel measured from each group of 8 different animals in two independent experiments. Horizontal lines represent the median of each experimental condition, and p values ​​are calculated using an unpaired t-test.

[0125]

Figure 9 :Design of an autoregulatory bispecific T cell engager targeting ROR1

[0126] An autoregulatory bispecific T cell engager (AR_TCE) was designed using the core sequence of NVG-111, a CD3×ROR1 bispecific tandem scFv antibody indicated for the treatment of ROR1-positive solid and hematological malignancies. (A) The linker region connecting the two scFvs of the parental antibody was replaced with an eight-residue granzyme B-sensitive cleavable peptide (P4 to P4' sequence) flanked by 2×G4S motifs. (B) Three AR_TCE variants were developed using different granzyme B-cleavable P4 to P4' sequences. (C) All variants were expressed in Expi-293 cells and exhibited similar migration profiles to the parental antibody NVG-111 after SDS-PAGE and Coomassie blue staining under reducing conditions.

[0127]

Figure 10 :T cell engagement ability of AR_TCE

[0128] The potential of T cell engagement of AR_TCE variants was tested using a short-term killing test model. Increasing concentrations of NVG-111 or AR_TCE were incubated for 48 hours in a co-culture model of ROR1-positive Jeko-1 cells and purified human T cells from healthy donors, using an effector-target cell ratio of 5:1. (A) T cell activation levels were detected by direct staining of CD69 using flow cytometry, expressed as the percentage of positive cells in the T cell population. (B) In parallel, the percentage of cell death in the target cell population was measured by incorporating a live cell dye. Symbols correspond to the mean ± SD of 3 repetitions. The dotted line shows baseline cell death and CD69 expression in the absence of antibody.

[0129]

Figure 11 :Granzyme B dose-dependently mediated AR_TCE inactivation in vitro

[0130] The ability of AR_TCE variants to be cleaved and inactivated by granzyme B was tested in vitro. Recombinant granzyme B was first activated using cathepsin C. The activated granzyme B was then incubated with 1 μg / mL of NVG-111 or AR_TCE variants at a final concentration of 100 nM for 2 h at 37°C. Control groups used buffer alone or cathepsin C alone. (A) Samples were migrated on SDS-Page gels under reducing conditions, followed by immunoblotting using protein L to detect intact and fragment antibodies. Subsequently, NVG-111 or AR_TCE variants (1 μg / mL) were incubated at 37°C with increasing concentrations of activated granzyme B. The reaction was then terminated after 2 h by the addition of a broad-spectrum protease inhibitor. (B) Residual intact antibodies were detected using a specialized ELISA method. Symbols represent the mean ± SD of two replicates, and curves were fit to a 4-parameter variable slope model using Prism software.

[0131]

Figure 12 :In vitro characterization of the sensitivity and specificity of AR_TCE

[0132] (A) AR_TCEs with an extended range of different AR peptide sequences were incubated with a single concentration of granzyme B (50 nM) for 2 h, and the reaction was terminated by adding a broad-spectrum protease inhibitor. (B) AR_TCE-3 was then exposed to similar concentrations of various proteases for 2 h. Residual intact antibodies were detected using ELISA. Residual antibodies were normalized to untreated counterparts and expressed as a percentage of residual intact antibodies. In (B), the results are sorted from high to low by percentage. Symbols correspond to the mean ± SD of 3 replicates. *** represents p < 0.001 in a one-way ANOVA test using Sidak's multiple comparisons.

[0133]

【 Figure 13 Granzyme B-mediated AR reduces cytokine release in vitro

[0134] The efficacy and safety of two AR_TCE variants with low (AR_TCE-1) and high (AR_TCE-3) cleavage rates were evaluated in a long-term co-culture experiment. NVG-111 or AR_TCE variants were incubated with ROR1-positive Jeko-1 and purified human T cells from healthy donors at a concentration of 1 μg / mL, using an excess of target cells (1:10 effector-target cell ratio) until 120h. (A) The percentage of target cell death was measured every 24h using flow cytometry by incorporating a live cell dye. (B) T cell activation levels were measured by direct staining of CD25 and expressed as the mean fluorescence intensity (MFI) of the CD3-positive population. The symbols represent the mean ± SD of 3 replicates. (C) The level of human interferon gamma (IFNγ) released by activated T cells was measured in the culture supernatant at 120h. The symbols represent individual values, and the horizontal lines represent the mean ± SD of 3 replicates. * and *** indicate p<0.05 and p<0.001, respectively, in one-way ANOVA test with Sidak's multiple comparisons. (D) Supernatant samples were migrated on SDS-Page gels and then immunoblotted using anti-His tag antibodies to detect cleavage and inactivation of AR_TCE constructs.

[0135]

【 Figure 14 Granzyme B-mediated AR improves survival in a TNBC mouse xenograft model

[0136] The safety of AR was evaluated in a mouse model of TCE-induced cytokine-mediated toxicity. (A) Immunocompromised NOD.SCIDγ mice were first induced or not (vehicle) to stably express NVG-111 or AR_TCE-3. On day 0, all animals were injected subcutaneously with 2.10 6 Human ROR1 + Triple negative breast cancer (TNBC) MDA-MB-231 cells were transplanted with tumors and allowed to grow for 25 days. Mice then received 6 cycles of purified human T cells. Animals were carefully monitored during the treatment phase and were sacrificed if ethically necessary or on day 46 until the study was completed. (B) The total weight of each animal was recorded every 24 hours and displayed as a percentage of the weight measured on the day of the first T cell injection (day 25). The symbols and connecting curves represent the mean ± SD of 6 animals in each group. The arrows mark the days of T cell injection. *, **, and *** represent p < 0.05, p < 0.01, and p < 0.001 in a one-way analysis of variance test using Sidak's multiple comparisons, respectively. (C) Animals that met one of the study endpoints (severe weight loss, acute toxicity, or tumor ulceration) were humanely eliminated or removed from the study (T cell treatment was stopped). The results are displayed as the percentage of surviving animals and are plotted as Kaplan-Meier curves.

[0137]

【 Figure 15 Granzyme B-mediated AR reduces TCE-induced toxicity and cytokine release in a TNBC model

[0138] Plasma samples were prepared from mice transplanted with triple-negative breast cancer solid tumors and treated with NVG-111 or AR_TCE-3 in combination with human T cells at the time of their elimination. Circulating levels of T cell-derived cytokines, including human IFNγ and granzyme B, were measured using ELISA. Symbols correspond to individual values, and horizontal lines represent the mean for each condition. The dashed area indicates the limit of detection, and ND indicates not detectable. ns and ** indicate not significant and p < 0.01, respectively, in an unpaired t-test.

[0139]

Figure 16 : AR_TCE shows intact therapeutic effect in TNBC model

[0140] (A) Evaluation of the efficacy of AR_TCE in tumor progression. Tumor size was measured along 2 different axes using a micrometer, and the volume was calculated using the sphere formula. The volumes calculated on days 15 and 18 were combined to reduce variability, and subsequent measurements were then standardized to this value. The results are presented as the multiple increase compared to the standardized baseline. The symbols and connecting curves represent the mean ± SD of 6 animals in each group. The arrows mark the days of T cell injection. *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively, in a one-way analysis of variance test using Sidak's multiple comparison correction. (B) Tumors were explanted and photographed until the animals were sacrificed, with the horizontal bar representing 10 mm. (C) Explant volume was measured from the images and displayed as the average volume. The symbols correspond to individual values, and * indicates p < 0.05 in an unpaired statistical t-test.

[0141]

【 Figure 17 AR_TCE reduces metastatic progression in a TNBC model

[0142] The effect of the therapeutic index of TCE, which was expanded in the presence of AR, on metastatic progression in a TNBC model was evaluated. (A) Livers from animals treated with vehicle and AR_TCE-3 were explanted and photographed until the end of the study. Horizontal bars represent 10 mm. (B) Liver sections were immunofluorescently stained for MDA-MB-231 cells to assess metastatic infiltration in the tissue. Images were acquired under a wide-field microscope. Horizontal bars represent 1 mm. Arrows indicate large clusters of cancer cells, and dotted squares delineate magnified areas. (C) The density of metastatic nodules was quantified from fluorescent images and then classified as nodule diameter (d) < or >100 μm. Symbols represent individual animals, and horizontal bars correspond to the mean for each condition. ns indicates not significant, and ** indicates P < 0.01, from a two-way ANOVA test using Sidak's multiple comparisons.

[0143]

【 Figure 18 Granzyme B-mediated AR reduces TCE-induced cytokine release and improves survival in an intraperitoneal model of pancreatic cancer

[0144] ROR1 was transplanted into the peritoneal cavity + The safety of AR was evaluated in a mouse model of pancreatic tumors. (A) Immunocompromised NOD.SCIDγ mice were first induced or not (vehicle) to stably express NVG-111 or AR_TCE-3. On day 0, all animals received a single dose of 2.10 6 Human pancreatic cancer PANC-1 cells were injected intraperitoneally and allowed to recover for 8 days. Mice subsequently received 4 cycles of purified human T cells. (B) Animals were carefully monitored during the treatment phase and humanely eliminated when one of the study endpoints (severe weight loss, acute toxicity) was reached. Survival results are shown as the proportion of animals and plotted as Kaplan-Meier curves. (C) Plasma samples from all animals were prepared 24 hours after the second T cell injection, and cytokine levels were measured by ELISA. Symbols correspond to individual values, and horizontal lines represent the mean for each condition. The dotted area indicates the limit of detection, and ND indicates not detectable. ns and * indicate not significant and p < 0.05 in an unpaired t-test, respectively.

[0145]

【 Figure 19 AR_TCE reduces tumor burden in an intraperitoneal pancreatic cancer model

[0146] Tumor progression was monitored by bioluminescence imaging using an IVIS imager in mice transplanted with intraperitoneal pancreatic cancer and treated with NVG-111 or AR_TCE-3 combined with human T cells. Mice received an intraperitoneal injection of D-luciferin to detect luciferase-positive PANC-1 and were imaged 15 minutes later. The color range indicates tumor bioluminescence intensity, and white crosses mark mice that were humanely culled before day 22.

[0147]

【 Figure 20 : Design of autoregulatory CAR-T cells targeting ROR1]

[0148] Autoregulatory CAR-T cells (AR_CAR-T) are designed based on ROR1 CAR-T cells as a template. (A) The upper region of the hinge connecting the scFv targeting ROR1 and the CD8a transmembrane domain of the CAR receptor was modified to introduce the P4 to P4' sequence of a granzyme B-sensitive cleavable peptide. (B) The ability of the modified AR_CAR receptor of CAR-T cells to be cleaved by granzyme B was tested in vitro. Control CAR-T or AR_CAR-T cell variants were incubated with 100nM granzyme B at 37°C for 2h. Flow cytometry was used to detect the presence of residual CAR receptors on the cell surface using fluorescent protein L. The results were normalized to untreated counterparts and displayed as the percentage of residual CAR receptors. The column height represents the average value, and the symbol corresponds to a single value.

[0149]

Figure 21 : Activation and target engagement capabilities of AR_CAR-T cell variants

[0150] The target engagement and activation levels of AR_CAR-T cells were tested in a short-term killing test model in which ROR1-positive Jeko-1 cells were co-cultured with CAR-T cells at different effector-target cell ratios for 48 h. (A) The activation levels of CAR-T cells were measured by flow cytometry by direct staining for CD69 and expressed as the percentage of positive cells. (B) In parallel, the percentage of target cell death in the Jeko-1 population was measured by incorporating a live cell dye. Symbols represent the mean ± SD of 4 replicates.

[0151]

【 Figure 22 : AR_CAR-T cells maintain unimpaired continuous killing potential in vitro]

[0152] The continuous killing potential of AR_CAR-T cell variants was evaluated in a long-term co-culture experiment with an excess of target cells. Control CAR-T or AR_CAR-T cells were co-cultured with ROR1-positive Jeko-1 at an effector-target cell ratio of 1:10 for 5 days. The percentage of target cell death was measured every 24 hours using flow cytometry by incorporating a viable cell dye. Symbols correspond to the mean ± SD of 4 replicates. ns indicates not significant in a one-way ANOVA test using Sidak's multiple comparisons. [Specific implementation method]

[0153] It should be understood that different applications of the disclosed invention can be adjusted according to the specific needs of the art. It should also be understood that the terms used herein are only used to describe specific embodiments of the present invention and are not intended to be limiting.

[0154] In addition, in this specification and the appended claims, use of the singular forms "a," "an," and "the" includes plural references unless the context clearly dictates otherwise. Thus, for example, reference to an antibody ("an antibody") includes plural "antibodies," and so on.

[0155] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0156] Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of commonly used terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Unless otherwise explained, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which this disclosure belongs.

[0157] Generally, the term "comprising" is intended to mean including but not limited to. For example, the phrase "a therapeutic agent comprising a polypeptide" should be interpreted as meaning that the therapeutic agent comprises at least one polypeptide, but the therapeutic agent may also comprise other components.

[0158] In some embodiments of the present invention, the word "comprising" is replaced with the phrase "consisting of. The term "consisting of" is intended to be limiting. For example, the phrase "a therapeutic agent consisting of a polypeptide" should be interpreted to mean that the therapeutic agent consists of the polypeptide and no other ingredients.

[0159] In some embodiments of the present invention, the word "comprising" is replaced with the phrase "consisting essentially of." The term "consisting essentially of" indicates that certain other components may be present, i.e., components that do not materially affect the essential characteristics of the subject matter. For example, the phrase "a therapeutic agent consisting essentially of a polypeptide" may include other components in addition to the polypeptide, such as a linker sequence or a label.

[0160] The terms "self-regulating" and "auto-regulating" are equivalent and are used interchangeably herein. The term "AR" is an abbreviation for "auto-regulating." "AR" is an equivalent term for "auto-regulating" and "self-regulating."

[0161]

Therapeutic agents

[0162] The therapeutic agents of the present invention are any substances that help achieve a therapeutic purpose or effect; for example, substances or combinations of substances that can be used to prevent or treat a disease or condition. Therapeutic agents include, but are not limited to, proteins, nucleic acid molecules, and other target molecules. In some embodiments, the therapeutic agent comprises a polypeptide, such as an antigen binding protein. In some embodiments, the therapeutic agent consists mainly of a polypeptide, such as an antigen binding protein. In some embodiments, the therapeutic agent is a polypeptide, such as an antigen binding protein. It will be understood by those skilled in the art that a particular therapeutic agent can help achieve more than one effect. Therapeutic agents act on metabolic pathways associated with a disease or condition to treat the disease or condition, or to prevent symptoms associated with the disease or condition. Treatment can include alleviating symptoms, as well as preventing side effects caused by adverse reactions to such treatment.

[0163] A therapeutically effective amount of a therapeutic agent can be defined as the amount of the therapeutic agent (e.g., disclosed antigen binding proteins) that is sufficient to achieve the desired effect in the subject being treated. For example, it may be the amount necessary to inhibit tumor growth or prevent heavy bleeding. In several embodiments, a therapeutically effective amount is the amount necessary to alleviate the symptoms of a disease or condition. When administered to a subject, a dosage typically used will achieve a target tissue concentration that has been shown to achieve the desired in vitro effect.

[0164]

Self-regulating / automatic regulating elements

[0165] The self-regulating or automatic regulating elements of the present invention are small peptides that can be cleaved by endogenous factors. A small peptide can be defined as an amino acid sequence that is at least 4 amino acids but no more than 15 amino acids in length, preferably at least 5, 6, or 7 amino acids in length, and more preferably at least 8 amino acids but no more than 15 amino acids in length. In a preferred embodiment, the self-regulating / automatic regulating element is 7, 8, 9, or 10 amino acids in length. In a particularly preferred embodiment, the self-regulating / automatic regulating element is 8 amino acids in length.

[0166] The self-regulating element does not significantly affect the activity of the therapeutic agent. In other words, the therapeutic agent can function within its therapeutic range in the presence of the self-regulating element. Suitable assays for therapeutic activity are known to those skilled in the art. The self-regulating element is placed in an exposed position within the therapeutic agent so that the endogenous factor can access the self-regulating element.

[0167] In the therapeutic agents of the present invention, the activity of the endogenous factor is altered by the activity of the therapeutic agent, and the cleavage of the self-regulatory element by the endogenous factor results in the separation of the therapeutic agent into non-functional fragments. Non-functional fragments can be defined as fragments that no longer possess the desired therapeutic activity, or fragments that degrade or are eliminated at a rate that renders them functionally inactive. The therapeutic activity may include binding to the target antigen. Binding kinetics can be studied by standard laboratory techniques.

[0168] The endogenous factors described herein are factors that are endogenously present in the subject to be treated. The endogenous factors described herein have proteolytic activity. In a preferred embodiment of the present invention, the endogenous factor is a protease. In a preferred embodiment of the present invention, the autoregulatory element comprises a proteolytic cleavage site.

[0169] The autoregulatory elements of the present invention can be defined according to the following proteolytic cleavage recognition sites:

[0170] P4-P3-P2-P1-cleavage site-P1'-P2'-P3'-P4',

[0171] wherein P represents a single amino acid (aa).

[0172] The intrinsic factor has activity in the same metabolic pathway as the therapeutic agent of the present invention. It is envisioned that appropriate intrinsic factors can be selected by considering the metabolic pathway of the disease in which the therapeutic agent acts.

[0173] By way of illustration and not limitation, the following proteases are involved in the hemophilia metabolic pathway (also known as the coagulation cascade): thrombin (coagulation factor IIa - Factor IIa), coagulation factor IXa (Factor IXa), coagulation factor Xa (Factor Xa), coagulation factor XIa (Factor XIa), coagulation factor XIIIa (Factor XIIIa), and activated protein C (APC).

[0174] Factor Xa protease specifically cleaves after the amino acid sequence P4, P3, P2-Gly, P1-Arg, where P4 is Ile, Leu, Pro, or Ala, and P3 is Glu, Asp, Gln, or Asn. Factor Xa preferably cleaves after the cleavage sequence Ile, Glu, Gly, Arg. The amino acids in positions P1'-P4' may not be critical for the cleavage specificity of Factor Xa.

[0175] Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade, as well as in several other areas, has been well documented. Thrombin positively regulates coagulation by cleaving prothrombin, FV, and FVIII, and negatively regulates coagulation by cleaving protein C. The thrombin cleavage recognition sites have been extensively studied (e.g., see Gallwitze et al., The Extended Cleavage Specificity of Human Thrombin, (2012) PLoS One;7, e31756). Consensus recognition sequences have been identified as: P4 - aliphatic, P3 - a non-negatively charged aa, P2 - Pro, P1 - Arg, P1' - Ser / Ala / Gly / Thr, P2' - non-acidic, P3' - Arg, and P4 - a nonspecific aa.

[0176] In a preferred embodiment of the present invention, the self-regulatory element may comprise one of the following thrombin cleavage recognition site sequences: LTPRGVRL (SEQ ID NO: 1), LTPRLVRL (SEQ ID NO: 2), LTPRDVRL (SEQ ID NO: 3), or WYLRSNNG (SEQ ID NO: 4), wherein cleavage occurs between the two amino acids shown in bold. In a particularly preferred embodiment, the self-regulatory element consists of the sequence LTPRGVRL (SEQ ID NO: 1).

[0177] As a further illustration, T cells are involved in the immune system's response to infection, as well as to diseases such as cancer or conditions such as transplant rejection. In diseases or conditions associated with metabolic pathways in which T cells are activated (e.g., adoptive immunotherapy), the following proteases may be involved: granzyme family members (granzymes A, B, K, H, M), cathepsin family members (including cathepsins B, L, W), and matrix metalloproteinase family members (including MMP-2, MMP-9, MMP-28).

[0178] Granzymes are serine proteases stored in granules and are involved in the cytotoxic defense response mediated by T cells and natural killer cells following target cell recognition. The primary function of granzymes is to induce cell death in virally infected and other potentially harmful cells. Granzyme B is a type of granzyme that, upon contact with a target cell, undergoes directed exocytosis and enters the target cell with the assistance of perforin, a cytolytic protein expressed by cytotoxic T cells and natural killer cells. Granzyme B processes and activates various pro-caspases, thereby inducing apoptosis in the target cell. According to the present invention, the term "granzyme B protease" includes enzymes that are or can be classified under the Enzyme Commission number EC 3.4.21.79 in the Enzyme Nomenclature Database (http: / / www.expasv.orq / enzvme) (34th edition, February 2004).

[0179] Granzyme B protease is known to preferentially cleave after aspartic acid residues (D), and granzyme B is the only mammalian serine protease known to possess this P1-proteolytic specificity. Therefore, according to the present invention, useful embodiments of the granzyme B cleavage site contemplate containing an aspartic acid residue at least at the P1 position located N-terminally to the cleavage site. Several known granzyme B protease recognition sites are disclosed in Harris et al (1998) Definition and redesign of the extended substrate specificity of granzyme B, J Biol Chem: 273, pp. 27364-73.

[0180] Therefore, in an embodiment of the present invention, the granzyme B cleavage recognition site has a general amino acid sequence at the N-terminus of the cleavage site: P4-P3-P2-Pl-cleavage site, wherein P4 is preferably amino acid I or V, P3 is preferably amino acid E, Q or M, P2 is X, wherein X represents any amino acid, and P1 is preferably amino acid D.

[0181] In a preferred embodiment of the present invention, the self-regulatory element may comprise one of the following granzyme B recognition site sequences: IEPDSESQ (SEQ ID NO: 5), IEADSESQ (SEQ ID NO: 6), IEPDSLEE (SEQ ID NO: 7), HSRLGRIEAD SESQEDIIRN (SEQ ID NO: 8), VEPDSLEE (SEQ ID NO: 9), LEPDSLEE (SEQ ID NO: 10), IEGDSLEE (SEQ ID NO: 11), IEADSLEE (SEQ ID NO: 12), IEPDALEE (SEQ ID NO: 13), IEPDSEEQ (SEQ ID NO: 14), IEPDSEVE (SEQ ID NO: 15), IEPNSLEE (SEQ ID NO: 16), IEPESLEE (SEQ ID NO: 17), or IEPQSLEE (SEQ ID NO: 18. In a particularly preferred embodiment, the self-regulatory element consists of the sequence IEPDSLEE (SEQ ID NO: 7).

[0182] In a preferred embodiment of the present invention, the cleavage rate of the self-regulating element by the endogenous factor is related to the activity level of the therapeutic agent. The cleavage rate of the self-regulating element can be calculated according to standard laboratory techniques, such as using protein blots to visualize the protein fragments released over time. The activity level of the therapeutic agent can be calculated using standard laboratory techniques, such as by Kd analysis of the degree of binding to the target. Examples of more applicable techniques are discussed in the Examples of the present application.

[0183] In a preferred embodiment of the present invention, the rate of cleavage of the self-regulatory element by the endogenous factor is related to the activity level of the therapeutic agent and the amino acid sequence of the self-regulatory element. The rate of cleavage of the self-regulatory element by the endogenous factor can be adjusted by changing the amino acid sequence of the self-regulatory element.

[0184]

Peptide

[0185] In a preferred embodiment of the present invention, the therapeutic agent comprises a polypeptide or protein. In one embodiment, the therapeutic agent of the present invention consists essentially of a polypeptide.

[0186] A polypeptide or protein can be defined as any chain of amino acids, regardless of its length or the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation). In a preferred embodiment of the invention, the polypeptide is an antigen binding protein. In a preferred embodiment of the invention, the polypeptide is an antibody or fragment thereof. "Residue" refers to an amino acid or amino acid analog that is incorporated into a polypeptide via an amide bond or an amide-like bond. A polypeptide has an amino terminal (N-terminal) end and a carboxyl terminal end. Conservative amino acid substitution tables that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0187] (1) Alanine (A), serine (S), threonine (T);

[0188] (2) Aspartic acid (D), glutamic acid (E);

[0189] (3) Asparagine (N), glutamine (Q);

[0190] (4) Arginine (R), Lysine (K);

[0191] (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0192] (6) Phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0193] Antigen binding protein

[0194] An antigen can be defined as a compound, composition, or substance that stimulates the production of an antibody or T cell response in an animal, including compositions that are injected or absorbed into an animal. Antigens react with products of specific humoral or cellular immunity (including those induced by heterologous antigens), such as the disclosed antigens. An "epitope" or "antigenic determinant" refers to a region of an antigen to which B cells and / or T cells respond. In one embodiment, T cells respond to an epitope when the epitope is presented with an MHC molecule. An epitope can be composed of either contiguous amino acids or non-contiguous amino acids arranged side by side by tertiary folding of a protein. Epitopes composed of contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, more commonly at least 5, about 9, or about 8-10 amino acids, arranged in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and nuclear magnetic resonance.

[0195] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants (e.g., those recognized by immune cells). Antigens may include peptides derived from target pathogens or cancer cells. Typical pathogens include bacteria, fungi, viruses, and parasites. In some embodiments, the antigen is derived from cancer cells, such as hematologic cancer cells (chronic lymphocytic leukemia-CLL, acute lymphocytic leukemia, mantle cell lymphoma) or solid malignancies (breast cancer, pancreatic cancer, melanoma). In some preferred embodiments, the antigen is a ROR1 polypeptide or an antigenic fragment thereof.

[0196] A "target epitope" is a specific epitope on an antigen that specifically binds to a target antibody (e.g., a monoclonal antibody). In some examples, the target epitope includes amino acid residues that contact the target antibody, and thus the target epitope can be selected by determining the amino acid residues that contact the antibody.

[0197] In a preferred embodiment of the present invention, the therapeutic agent comprises an antigen binding protein. In some preferred embodiments of the present invention, the therapeutic agent is an antigen binding protein. In a preferred embodiment, the antigen binding protein of the present invention comprises a first antigen binding domain, wherein the first antigen binding domain comprises a heavy chain variable domain comprising a heavy chain complementary determining region (HCDR) 1, HCDR2 and HCDR3, and wherein the autoregulatory element is located at the C-terminus of the first antigen binding domain. In a preferred embodiment, the first antigen binding domain is a VHH antibody.

[0198] In a preferred embodiment of the present invention, the antigen binding protein comprises a first antigen binding domain, wherein the first antigen binding domain comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, and wherein the self-regulatory element is located at the C-terminus of the first antigen binding domain.

[0199] In one embodiment, the self-regulating element can be adjacent to the first antigen-binding domain and at the C-terminus of the first antigen-binding domain. The term "adjacent" means that the position of the self-regulating element is such that it does not affect the function of the first antigen-binding domain, but is placed next to the first antigen-binding domain. The self-regulating element can be located within a peptide linker sequence. The linker sequence can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. Exemplary linkers include the amino acid sequences GGGGS (SEQ ID NO: 59) and GGGGSGGGGS (SEQ ID NO: 60).

[0200] In a preferred embodiment, the antigen binding protein constitutes a part of a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In a preferred embodiment, the antigen binding protein is exogenously expressed in CAR-T cells, CAR-M cells or transformed NK cells.

[0201] Antigen binding proteins of the present invention include antibodies. Antigen binding proteins of the present invention include single chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g., VH or VL or VHH), scFv, monovalent, bivalent, trivalent or tetravalent antibodies, scFv, Bis-scFv, single domain antibodies (sdAbs, also known as VHH antibodies), nanobodies (camel-derived single domain antibodies), single domain antibodies derived from shark IgNARs, diabodies, tribodies, triabodies, tetrabodies, and epitope-binding fragments of any of the above (see, for example, Holliger P and Hudson PJ, 2005, Nat. Biotechnol., 23,: 1126-1136; Adair JR and Lawson ADG, 2005, Drug Design Reviews-Online, 2, 209-217). Methods for producing and manufacturing these antigen-binding proteins are well known in the art (e.g., see Verma et al., 1998, J. Immunol. Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009 / 040562, and its disulfide-stabilized version, Fab-dsFv, was first disclosed in WO2010 / 035012. The multivalent antigen-binding proteins of the present invention may comprise multiple specificities, such as bispecific, or may be monospecific. The antigen-binding proteins of the present invention may be bi-epitopic.

[0202] An scFv protein is a fusion protein in which the variable region of an immunoglobulin light chain and the variable region of an immunoglobulin heavy chain are joined by a linker, whereas in a dsFv, the chains are mutated to introduce disulfide bonds to stabilize the binding of the chains. The term also includes genetically engineered forms such as heterologous conjugate antibodies such as chimeric antibodies and bispecific antibodies. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0203] The antigen-binding proteins of the present invention include, but are not limited to, the following: (1) Fab, a fragment comprising a monovalent antigen-binding fragment of an antibody molecule, produced by digesting an intact antibody with papain to produce a complete light chain and a portion of a heavy chain; (2) Fab', a fragment of an antibody molecule obtained by treating an intact antibody with pepsin followed by reduction to produce a complete light chain and a portion of a heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, an antibody fragment obtained by treating an intact antibody with pepsin without subsequent reduction; (4) F(ab')2, a dimer formed by two Fab' fragments bound together by two disulfide bonds; (5) Fv, a genetically engineered fragment comprising the variable region of a light chain and the variable region of a heavy chain as its two chains; and (6) single-chain antibody (SCA), a genetically engineered molecule comprising the variable region of a light chain and the variable region of a heavy chain, the two of which are connected by a suitable polypeptide linker to form a genetically fused single-chain molecule.

[0204] In embodiments of the present invention, the antigen-binding protein may comprise a heavy chain and a light chain, each of which contains a constant region and a variable region (these regions are also referred to as "domains"). In several embodiments of the present invention, the heavy chain and light chain variable domains are combined to specifically bind to the antigen. In other embodiments of the present invention, only the heavy chain variable domain is required. For example, naturally occurring camel-derived antibodies consist only of heavy chains, which are functional and stable in the absence of light chains (see, for example, Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The light and heavy chain variable domains contain a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved across species. The framework region of an antibody, i.e., the combined framework regions of the light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0205] CDRs are primarily responsible for antigen binding. The sequences of the framework regions of different light or heavy chains are relatively conserved within the same species. The framework regions of an antibody, the combined framework regions of the light and heavy chains, serve to position and align the CDRs in three-dimensional space.

[0206] The CDRs on each chain are referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also typically identified by the chain in which the specific CDR is located. H CDR3 is located in the heavy chain variable domain of the antibody in which it is located, while V L CDR1 is the CDR1 of the light chain variable domain of the antibody in which it is located. Light chain CDRs may also be referred to as CDR L1, CDR L2, and CDR L3, or LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as CDR H1, CDR H2, and CDR H3, or HCDR1, HCDR2, and HCDR3.

[0207] The residues in the variable domains of antibodies are numbered conventionally according to IMGT (http: / / www.imgt.org). This system is described in Lefranc MP (1997, J, Immunol. Today, 18, 509). Unless otherwise specified, this numbering system is used in this specification.

[0208] The nomenclature of IMGT residues does not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or more amino acids than the strict IMGT numbering, corresponding to shortening or insertion of structural components (whether framework regions or CDRs) of the basic variable domain structure. For a given antibody, the correct IMGT numbering of the residues can be determined by aligning the homologous residues in the antibody sequence with the sequence numbered by "standard" IMGT.

[0209] Suitable antigen binding proteins, antibodies or binding fragments thereof can be disclosed herein by the CDRs of their heavy and light chains, the variable regions of their heavy and light chains and / or the full-length primary amino acid sequences of their heavy and light chains.

[0210] The antigen-binding protein, antibody, or binding fragment thereof may comprise one or more VH CDR sequences of the specific antigen-binding protein or antibody, and / or one or more VL CDR sequences other than VL CDR1. The antigen-binding protein, antibody, or binding fragment thereof may comprise one, two, or all three VH CDR sequences of the specific antigen-binding protein, antibody, or binding fragment thereof described above, and / or one, two, or all three VL chain CDR sequences of the specific antigen-binding protein, antibody, or binding fragment thereof, including VL CDR1. The antigen-binding protein, antibody, or binding fragment thereof may comprise all six CDR sequences of the specific antigen-binding protein, antibody, or binding fragment thereof described above.

[0211] Variant antigen-binding proteins or antibodies can, while maintaining antigen-binding proteins described herein or antibody activity, comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid replacements and / or disappearances compared to the specific sequences and fragments discussed above. A "deletion" variant can comprise 1, 2, 3, 4 or 5 single amino acids, or the disappearance of one or more small groups of amino acids (such as 2, 3, 4 or 5 amino acids). A "small group of amino acids" can be defined as continuous, or very close to each other but not continuous amino acids. A "replacement" variant preferably relates to the use of one or more amino acid replacements of the same number of amino acids, and carries out conservative amino acid replacements. For example, a kind of amino acid can be replaced by other amino acids with similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, another aliphatic amino acid, another tiny amino acid, another small amino acid or another large amino acid. Some properties of 20 kinds of main amino acids are as follows, and these properties can be used to select suitable substituents:

[0212] Ala Aliphatic, hydrophobic, neutral Met Hydrophobic, neutral Cys Polar, hydrophobic, neutral Asn Polar, hydrophilic, neutral Asp Polar, hydrophilic, charged (-) Pro Hydrophobic, neutral Glu Polar, hydrophilic, charged (-) Gln Polar, hydrophilic, neutral Phe Aromatic, hydrophobic, neutral Arg Polar, hydrophilic, charged (+) Gly Aliphatic, neutral Ser Polar, hydrophilic, neutral His Aromatic, polar, hydrophilic, charged (+) Thr Polar, hydrophilic, neutral Ile Aliphatic, hydrophobic, neutral Val Aliphatic, hydrophobic, neutral Lys Polar, hydrophilic, charged (+) Trp Aromatic, hydrophobic, neutral Leu Aliphatic, hydrophobic, neutral Tyr Aromatic, polar, hydrophobic

[0213] Preferred "derivatives" or "variants" include those in which the amino acids appearing in the sequence are not naturally occurring amino acids but are structural analogs thereof. The amino acids used in the sequence may also be derivatized or modified, such as labeled, provided that the function of the antibody is not significantly adversely affected.

[0214] The above-mentioned derivatives and variants can be prepared during the synthesis of the antigen-binding protein or antibody or by post-production modification, or when the antigen-binding protein or antibody is in recombinant form, using known nucleic acid site-directed mutagenesis, random mutagenesis or enzymatic cleavage and / or ligation techniques.

[0215] Preferably, the amino acid sequence of the variant antigen binding protein or antibody has more than 60%, or more than 70%, such as 75% or 80%, preferably more than 85%, such as more than 90%, 95%, 96%, 97%, 98% or 99% amino acid identity compared to the VL and / or VH of the antigen binding protein or antibody disclosed herein, or its fragment. This amino acid identity level can be seen over the entire length of the relevant SEQ ID NO sequence or a portion of the sequence, for example, in 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0216] Preferably, the variant antigen binding protein or antibody comprises one or more of the CDR sequences described herein.

[0217] "Sequence identity" with respect to amino acid sequences refers to sequences having a specified value, as assessed using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) and the following parameters:

[0218] Pairwise alignment parameters - Method: slow / accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

[0219] Multiple alignment parameters - Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: G, P, S, N, D, Q, E, K, R. Sequence identity to a specific residue is intended to include identical residues where only the residue has been derivatized.

[0220] The methods of the present invention can use antibodies having specific VH and VL amino acid sequences and variants and fragments thereof that maintain the function or activity of these VH and VL.

[0221] Mention "V H ” or “VH” refers to the variable region of an immunoglobulin heavy chain, including the variable region of an antibody fragment (such as Fv, scFv, dsFv or Fab). L ” or “VL” refers to the variable region of an immunoglobulin light chain, including the variable region of Fv, scFv, dsFv or Fab.

[0222] In some embodiments of the invention, the antigen-binding protein can be an antibody, which comprises a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. There are two types of light chains, lambda and kappa. There are five major heavy chain classes (or subclasses) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0223] IgG1 (e.g., IgG1 / κ) antibodies having an IgG1 heavy chain and one light chain can be advantageously used in the present invention. However, other human antibody subclasses are also encompassed by the present invention, including combinations of IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE with κ or λ light chains. In addition, antibodies of all animal origin of various subclasses can be used in the present invention. The antibody can be a full-length antibody or an antigen-binding fragment of an antibody, including Fab, F(ab')2, single-chain Fv fragments, or single-domain VHH, VH, or VL single domains.

[0224] The Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge at the N-terminus of these domains. For IgA and IgM, the Fc region may or may not include a tail segment and may or may not be associated with a J chain. For IgG, the Fc region includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lower hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of the Fc region can vary, the definition of the human IgG heavy chain Fc region generally includes residues C226 or P230 at its carboxyl terminus, where the numbering follows the EU index. For IgA, the Fc region includes the immunoglobulin domains Calpha2 and Calpha3 (Cα2 and Cα3) and the lower hinge between Calpha1 (Cα1) and Cα2. The definition of an Fc region encompasses functionally equivalent analogs and variants of the Fc region. A functionally equivalent analog of an Fc region can be a variant Fc region comprising one or more amino acid alterations relative to a wild-type or naturally occurring Fc region. A variant Fc region has at least 50% homology to a naturally occurring Fc region, for example, about 80%, about 90%, or at least about 95% homology. A functionally equivalent analog of an Fc region can comprise the addition or deletion of one or more amino acid residues from the N-terminus or C-terminus of the protein, for example, no more than 30 or no more than 10 additions and / or deletions. A functionally equivalent analog of an Fc region includes an Fc region operably linked to a fusion partner. A functionally equivalent analog of an Fc region must comprise the major portions of all Ig domains comprising the above-described Fc region; for example, an IgG and IgA Fc region as defined herein must comprise the major sequence encoding CH2 and the major sequence encoding CH3. Thus, the CH2 domain or the CH3 domain alone are not considered an Fc region. The Fc region can refer to this region in isolation or in the context of an Fc fusion polypeptide.

[0225] When referring to the antigen binding proteins of the present invention, the binding of the protein to the antigen refers to a binding reaction that determines the presence of a target protein, peptide or polysaccharide in the presence of a heterogeneous population of proteins and other biological products. Thus, under specified conditions, the antigen binding protein preferentially binds to a specific target protein, peptide or polysaccharide (e.g., an antigen presented on the surface of a tumor, such as ROR1), and does not bind to a significant amount of other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art. With reference to an antibody-antigen complex, the specific binding of the antigen and the antibody has a specific binding affinity of less than about 10 -5 M, 10 -6 M, 10 -7 M, for example, less than about 10 -7 M, 10 -8 M, 10 -9 Even less than about 10 -10 M's K d .

[0226] The terms "binding activity" and "binding affinity" are intended to refer to the tendency of an antigen binding protein to bind or not bind to a target. Binding affinity can be quantified by determining the dissociation constant (Kd) of the antigen binding protein to its target. Similarly, the specificity of binding of an antigen binding protein to its target can be defined by the comparative dissociation constant (Kd) of the antibody for the target, compared to the dissociation constants relative to the antigen binding protein and other non-target molecules.

[0227] Typically, the Kd of an antibody relative to a target will be less than 2-fold, preferably 5-fold, more preferably 10-fold, compared to its Kd relative to other non-target molecules (e.g., unrelated substances or accompanying substances in the environment). More preferably, the Kd will be reduced by 50-fold, even more preferably by 100-fold, and even more preferably by 200-fold.

[0228] The value of the dissociation constant can be determined directly by known methods, and even for complex mixtures it can be calculated using methods such as those described in Catechis MS and Catechis WP (1984, Byte, 9, 340-362). For example, Kd can be established using a double-filtration nitrocellulose filter binding method, such as that disclosed by Wong I and Lohman TM (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or using methods such as Octet surface plasmon resonance.

[0229] One method for assessing binding affinity is ELISA. Other standard assays for assessing the binding ability of ligands (e.g., antibodies, etc.) to targets are known in the art and include, for example, Western blotting, RIA, and flow cytometry. The binding kinetics (e.g., binding affinity) of antibodies can also be determined by standard methods known in the art, such as surface plasmon resonance, for example, by Biacore. TM System analysis.

[0230] In one embodiment, the antigen binding protein is a monoclonal antibody. Monoclonal antibodies are immunoglobulin molecules that are identical to each other and have a single binding specificity and affinity for a specific epitope. The monoclonal antibodies (mAbs) of the present invention can be produced by various techniques, including conventional monoclonal antibody methods, such as those disclosed in "Monoclonal Antibodies: a manual of techniques" (Zola H, 1987, CRC Press) and "Monoclonal Hybridoma Antibodies: techniques and applications" (Hurrell JGR, 1982 CRC Press).

[0231] In a preferred embodiment of the present invention, the therapeutic agent is an antigen binding protein comprising a first antigen binding domain and a second antigen binding domain, wherein the self-regulatory element is located between the first and second antigen binding domains. The term "between" can be interpreted as meaning that the self-regulatory element is located at the C-terminus of the first antigen binding domain and at the N-terminus of the second antigen binding domain. In an embodiment of the present invention, the antigen binding protein contains more than one self-regulatory element. In a preferred embodiment of the present invention, the antigen binding protein is a bispecific antibody comprising a self-regulatory element located at the C-terminus of the first antigen binding domain and another self-regulatory element located at the N-terminus of the second antigen binding domain.

[0232] In embodiments of the invention, the antigen binding protein comprises an scFv, and the autoregulatory element may be located within the scFv within a peptide linker sequence between the heavy and light chain variable regions, thereby rendering the scFv nonfunctional when the autoregulatory element is cleaved.

[0233] In a preferred embodiment of the present invention, the first and second antigen binding domains of the antigen binding protein are scFv proteins, covalently linked via a peptide linker. In some preferred embodiments, the autoregulatory element is present within the peptide linker.

[0234] As shown below, the sequence of each of the light chain variable domains and heavy chain variable domains mentioned above may differ from the given sequence. For example, the light / heavy chain variable domain may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in the sequence listing. Alternatively, the light / heavy chain variable domain sequence may differ at up to 10 amino acid positions, but preferably there are fewer than 10 amino acid substitutions, and thus there may be no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.

[0235] As mentioned above, in some embodiments, the light chain variable domain and the heavy chain variable domain comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. For example, the light chain framework region, the heavy chain framework region, the light chain variable domain and the heavy chain variable domain may include up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to two or up to one amino acid substitution in the amino acid sequence described above. When there is a variation in the sequence of the light chain variable domain and the heavy chain variable domain, preferably no amino acid substitution occurs in the CDR. Specifically, the light chain framework region and / or the heavy chain framework region of the antibody described above may comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described above. In addition, the light chain framework region and / or the heavy chain framework region may include up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to two, or up to one amino acid substitution in the amino acid sequence described above. Preferably, the amino acid substitutions are conservative substitutions as described above. For example, the framework regions may be replaced in such a way as to humanize the sequence. Preferably, the framework regions are humanized.

[0236] The sequence of each light chain variable domain and heavy chain variable domain of the second antigen-binding domain mentioned above may be different from the given sequence. For example, the light / heavy chain variable domain may comprise a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence described in the sequence listing. Alternatively, the light / heavy chain variable domain sequence may differ in up to 10 amino acid positions, but preferably there are fewer than 10 amino acid substitutions, and thus there may be no more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. Preferably, there are no amino acid substitutions in the CDRs of the heavy / light chain.

[0237] Antigen binding proteins comprising antigen binding domains can be prepared by proteolysis of antibodies or by expression of DNA encoding the fragments in mammalian cells. Antibody fragments can be obtained by digesting whole antibodies with pepsin or papain using conventional methods. For example, antibody fragments can be produced by enzymatic cleavage with pepsin to provide a 5S fragment of F(ab')2. This fragment can be further cleaved using a sulfhydryl reducing agent and, optionally, a blocking group for the sulfhydryl groups generated by disulfide bond cleavage to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and one Fc fragment (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al., Sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0238] Other methods of cleaving antigen binding proteins and antibodies can also be used, such as separating heavy chains to form monovalent light and heavy chain fragments, further cleaving the fragments, or using other enzymatic, chemical, or genetic techniques, so long as the fragments bind to the antigen recognized by the intact antibody or antigen binding protein.

[0239] In an embodiment of the present invention, the antigen binding protein is exogenously expressed in CAR-T cells, CAR-M cells or modified NK cells.

[0240] Location of cleavage sites within antigen-binding proteins

[0241] In an embodiment of the invention, the autoregulatory element is located at an exposed position within the antigen binding protein such that the autoregulatory element is accessible to endogenous factors.

[0242] In an embodiment of the invention, the antigen binding protein comprises an autoregulatory element, wherein the autoregulatory element is located within a peptide linker sequence.

[0243] In an embodiment of the invention, the antigen binding protein comprises an autoregulatory element, wherein the autoregulatory element is located within the peptide linker sequence of the first and / or second antigen binding domain.

[0244] In one embodiment, the self-regulatory element can be adjacent to the first antigen binding domain and at the C-terminus of the first antigen binding domain. The term "adjacent" means that the position of the self-regulatory element is such that it does not affect the function of the first antigen binding domain, but it is located within about 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the first antigen binding domain. The self-regulatory element can be located within a peptide linker sequence. The peptide linker sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. Exemplary linkers include the amino acid sequences GGGGS (SEQ ID NO: 59) and GGGGSGGGGS (SEQ ID NO: 60). In an embodiment of the present invention, the self-regulatory element is placed directly at the C-terminus of a peptide linker having the sequence GGGGS (SEQ ID NO: 59).

[0245] In an embodiment of the present invention, the self-regulatory element may be located between the first and second antigen binding domains of the antigen binding protein. In one embodiment, the self-regulatory element may be located in the hinge region between the first and second antigen binding domains.

[0246] In an embodiment of the invention, the antigen binding protein comprises a constant region, and the self-regulatory element is located in the hinge region of the constant region, between CH1 and CH2. In a preferred embodiment, there are two self-regulatory elements, each located in the hinge region of the constant region, between CH1 and CH2.

[0247] Antigen binding protein that binds to ROR1

[0248] In a preferred embodiment, the antigen binding protein of the present invention comprises an autoregulatory element comprising a cleavage site for granzyme B. In a preferred embodiment of the present invention, the antigen binding protein comprises a first antigen binding domain that selectively binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1) (also known as neurotrophic tyrosine kinase, receptor-related 1, NTRKR1), wherein the first antigen binding domain binds to a ROR1 epitope comprising amino acid Gln-261, and an autoregulatory element comprising a cleavage site for granzyme B.

[0249] In a preferred embodiment of the present invention, the antigen binding protein comprises a first antigen binding domain that selectively binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1) (also known as neurotrophic tyrosine kinase, receptor-related 1, NTRKR1), wherein the first antigen binding domain binds to a ROR1 epitope comprising amino acid Gln-261; and a second antigen binding domain that selectively binds to the CD3 subunit of the T cell receptor (TCR), and an autoregulatory element comprising a granzyme B cleavage site. In a more preferred embodiment, the autoregulatory element is as shown in SEQ ID NO: 7.

[0250] The antigen binding proteins disclosed herein that specifically bind to ROR1 and CD3 are disclosed in WO2019 / 008379, which is incorporated herein by reference.

[0251] In a first embodiment, the first antigen binding domain comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; and wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; wherein the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0252] The antigen binding protein specifically binds to the ROR1 polypeptide and specifically binds to CD3. The first and / or second antigen binding domains can be monoclonal antibodies or antigen-binding fragments thereof. In specific embodiments, the first and second antigen binding domains are both monoclonal antibodies or antigen-binding fragments thereof.

[0253] As previously mentioned, the sequence of each CDR can differ from the given sequence at up to two amino acid positions. This means that a CDR can contain one or two amino acid substitutions compared to the given sequence. However, if one or more CDRs do contain an amino acid substitution, the antibody will still be able to selectively bind to ROR1. Preferably, the amino acid substitutions are conservative substitutions.

[0254] Preferably, the sequence of each CDR may differ from the given sequence at one amino acid position. This means that the CDR may contain one amino acid substitution compared to the given sequence. Preferably, the amino acid substitution is a conservative substitution.

[0255] In some embodiments, the amino acid sequence comprised by the heavy chain complementarity determining region 3 (HCDR3) is selected from the sequences set forth in SEQ ID NOs: 25, 26, 27, and 28. Preferably, the amino acid sequence comprised by HCDR3 is selected from the sequences set forth in SEQ ID NOs: 26, 27, and 28.

[0256] Preferably, the light chain variable domain comprises an amino acid sequence as set forth in one of SEQ ID NOs: 29, 30, 31, 32, 33 and 34. More preferably, the light chain variable domain comprises an amino acid sequence as set forth in one of SEQ ID NOs: 30, 31, 32, 33 and 34.

[0257] Preferably, the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 35, 36, 37, 38, 39 and 40. More preferably, the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39 and 40.

[0258] SEQ ID NOs: 30, 31, 32, 33, and 34 are humanized light chain variable regions. SEQ ID NOs: 36, 37, 38, 39, and 40 are humanized light chain variable regions. The inventors of WO2019 / 008379 tried all possible combinations of these light and heavy chain regions, generating 25 different constructs.

[0259] Thus, in some embodiments, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39, and 40. In a specific embodiment, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 38, and 39.

[0260] In other embodiments, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 31, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0261] In another embodiment, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 32, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0262] In an alternative embodiment, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 33, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0263] In various embodiments, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 34, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0264] Similarly, in some embodiments, the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 36, and the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0265] In other embodiments, the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 37, and the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0266] In another embodiment, the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 38, and the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0267] In an alternative embodiment, the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:39, and the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs:30, 31, 32, 33, and 34.

[0268] In various embodiments, the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:40, and the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs:30, 31, 32, 33, and 34.

[0269] In a specific embodiment,

[0270] (a) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 29, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 35;

[0271] (b) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 36;

[0272] (c) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 31, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 37;

[0273] (d) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 32, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 38;

[0274] (e) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 33, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 39; or

[0275] (f) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 34, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0276] The second antigen-binding domain that selectively binds to the CD3 subunit of the T cell receptor (TCR) can be any suitable antigen-binding domain, and such binding domains are well known to those skilled in the art. For example, CD3 monoclonal antibodies are available from ThermoFisher Scientific. In addition, bispecific antibodies that bind to tumor antigens and CD3 are also known in the art, for example, as described by Baeuerle and Reinhardt (Cancer Res (2009); 69(12): 4941-4944), Chames and Baty (MAbs. (2009); 1(6): 539-547), and Hoffman et al. (Int. J. Cancer (2005) 115, 98-104).

[0277] The second antigen-binding domain that selectively binds to the CD3 subunit of the T cell receptor (TCR) can comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 41, 42, 43, 44, 45, and 46, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 47, 48, 49, 50, 51, and 52. Preferably, the light chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 42, 43, 44, 45, and 46, and the heavy chain variable domain comprises the amino acid sequence set forth in one of SEQ ID NOs: 48, 49, 50, 51, and 52.

[0278] In one embodiment of the second antigen binding domain,

[0279] (a) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 41, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 47;

[0280] (b) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 42, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 48;

[0281] (c) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 43, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 49;

[0282] (d) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 44, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 50;

[0283] (e) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 45, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 51; or

[0284] (f) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 46, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 52.

[0285] In a first embodiment, the second antigen-binding domain comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a light chain complementarity determining region (LCDR) 1, LCDR2 and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 53; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 54; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 55; and the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 56; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 57; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 58; wherein the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0286] The first antigen-binding domain can have an antibody fragment structure, such as Fab, F(ab')2 and Fv, including heavy chain and light chain variable regions, and can bind to the epitope determinant on ROR1. Similarly, the second antigen-binding domain can have an antibody fragment structure, such as Fab, F(ab')2 and Fv, including heavy chain and light chain variable regions, and can bind to the epitope determinant on CD3. These antibody fragments retain the ability to selectively bind to the antigens described above. Methods for preparing these fragments are known in the art (e.g., see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0287] In another embodiment, the antigen binding domain can have the structure of an Fv antibody, which is typically about 25 kDa and contains a complete antigen binding site with three CDRs for each heavy chain and each light chain. To produce these antibodies, V and V can be expressed in a host cell from two separate nucleic acid constructs. H and V L If V H and V L If the antibody is expressed discontinuously, the chains of the Fv antibody are usually bound together by non-covalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to cross-link the chains using glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one example, the Fv can be a disulfide-stabilized Fv (dsFv), in which the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

[0288] In another example, an Fv fragment comprises a V H and V L These single-chain antigen-binding proteins (scFv) are prepared by constructing a structural gene containing a V encoding oligonucleotide linked to the H and V LThe structural gene is inserted into an expression vector, which is then introduced into a host cell, such as a mammalian cell or E. coli. The recombinant host cell synthesizes a polypeptide chain with a linker peptide that bridges the two V domains. Exemplary linkers include the amino acid sequences GGGGS (SEQ ID NO: 59) and GGGGSGGGGS (SEQ ID NO: 60). Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242: 423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio / Technology 11: 1271, 1993; and Sandhu, supra). Dimers of single-chain antibodies (scFV2) are also contemplated.

[0289] The antigen binding proteins disclosed herein can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the derivatization of the antigen binding protein satisfies that its binding to the ROR1 polypeptide and CD3 subunit is not adversely affected by derivatization or labeling. For example, the antigen binding protein can be functionally linked to one or more other molecular entities, such as another antibody, a detection reagent, a pharmaceutical preparation, and / or a protein or peptide that can mediate binding of the antibody to another molecule (e.g., a streptavidin core region or a polyhistidine tag), by chemical coupling, genetic fusion, non-covalent binding, or other means.

[0290] Bispecific antibodies can be produced by cross-linking two or more antibodies of the same or different types. Suitable cross-linking agents include those having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Company (Rockford, IL).

[0291] In certain embodiments, the ROR1 antigen-binding domain can be a scFv antibody. In some embodiments, the CD3 antigen-binding domain is a scFv antibody. In various embodiments, both the ROR1 antigen-binding domain and the CD3 antigen-binding domain are scFv antibodies. The two scFv antibodies can be covalently linked using a short peptide linker of 5 to 20 amino acids.

[0292] In a preferred embodiment, the autoregulatory element is located within this short peptide linker.

[0293] In some embodiments, the antigen binding protein comprises the sequence of SEQ ID NO: 61 or 62, or a sequence having at least 90% sequence identity thereto. The sequence may have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the bispecific antibody comprises the sequence of SEQ ID NO: 61 or 62.

[0294] The antibody can be labeled with a detectable moiety or marker as described above. The antibody can also be labeled with a radiolabeled amino acid. Examples of radiolabels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I. Radiolabels can be used for diagnostic and therapeutic purposes.

[0295] Methods for detecting such labels are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted illumination. Enzyme labels are typically detected by providing a substrate for the enzyme and detecting the reaction product resulting from the action of the enzyme on the substrate, while colorimetric labels are detected by simply visualizing the colored label.

[0296] Antibodies can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate-containing groups. These groups may help improve the biological properties of antibodies, such as extending serum half-life or increasing tissue binding.

[0297] In a specific embodiment, the ROR1 antigen binding domain can be part of a chimeric antigen receptor (CAR). The CAR can include a spacer sequence or hinge region to connect the ROR1 binding domain and the transmembrane domain and spatially separate the ROR1 binding domain from the internal domain of the CAR. The flexible spacer / hinge region allows the ROR1 binding domain to face different directions to achieve binding to ROR1. Herein, "flexible spacer", "spacer sequence" and "hinge region" are used interchangeably as equivalent terms. It will be understood by those skilled in the art that any suitable spacer sequence can be used. In a preferred embodiment, the self-regulatory element is located within the flexible spacer / hinge region. In a preferred embodiment, the hinge region is as described in SEQ ID NO: 82. In a preferred embodiment, the self-regulatory element is placed directly at the N-terminus of the hinge region. In a preferred embodiment, the self-regulatory element replaces the first 1, 2, 3, 4 or 5 amino acids in the hinge region. In a preferred embodiment, the self-regulatory element is a granzyme B cleavage site. In a preferred embodiment, the combined self-regulatory element and hinge region are as described in SEQ ID NO: 83 or 84.

[0298] Factor IXa and X antigen binding protein

[0299] In a preferred embodiment, the antigen binding protein of the present invention comprises an autoregulatory element comprising a thrombin cleavage site. In a preferred embodiment, the antigen binding protein comprises a first antigen binding domain that selectively binds to FIX / FIXa and a second antigen binding domain that selectively binds to FX / FXa, and an autoregulatory element comprising a thrombin cleavage site. In a preferred embodiment, the antigen binding protein comprises a first antigen binding domain that selectively binds to FIXa and a second antigen binding domain that selectively binds to FX, and an autoregulatory element comprising a thrombin cleavage site. In a preferred embodiment, the autoregulatory element comprises SEQ ID NO: 1, 2, 3 or 4. In a preferred embodiment, the autoregulatory element comprises SEQ ID NO: 1.

[0300] In a preferred embodiment, the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a light chain complementary determining region (LCDR) 1, LCDR2, and LCDR3, and the heavy chain variable domain comprises a heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3, and for the first antigen-binding domain, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; and wherein the heavy chain variable domain comprises a heavy chain complementary determining region (HCDR) 1, HCDR2, and HCDR3, and HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 66; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 67; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68; and for the second antigen-binding domain, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and NO: 64; LCDR3 comprises the amino acid sequence recorded in SEQ ID NO: 65; and the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, said HCDR1 comprising the amino acid sequence recorded in SEQ ID NO: 69; HCDR2 comprises the amino acid sequence recorded in SEQ ID NO: 70; and HCDR3 comprises the amino acid sequence recorded in SEQ ID NO: 71; wherein the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0301] In a specific embodiment, for the first antigen-binding domain, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 72, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 73, and for the second antigen-binding domain, the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 72, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 74.

[0302] In a preferred embodiment, the antigen binding protein comprises a constant region. In a preferred embodiment, the autoregulatory element is located within the constant region. In a preferred embodiment, the autoregulatory element is located within the hinge region of the constant region. In a preferred embodiment, the autoregulatory element is located within the hinge region of the constant region, between CH1 and CH2. In a preferred embodiment, the autoregulatory element and the hinge region comprise SEQ ID NO: 76. In a preferred embodiment, there are two autoregulatory elements, each located within the hinge region of the constant region, between CH1 and CH2. In a preferred embodiment, the antigen binding protein is emicizumab, wherein the antigen binding protein further comprises an autoregulatory element comprising a thrombin cleavage site.

[0303] Antigen binding proteins can be produced by cross-linking two or more antibodies of the same or different types. Suitable cross-linking agents include those having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimidyl suberate). Such linkers are available, for example, from Pierce Chemical Company (Rockford, IL).

[0304]

Polynucleotides, gene vectors and host cells

[0305] The present invention also encompasses polynucleotides, gene vectors, and expression vectors encoding the antigen binding proteins, antibodies, or binding fragments thereof described herein.

[0306] The present invention also relates to polynucleotides encoding any antigen-binding proteins or fragments described herein. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, which can be deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, genomic DNA, recombinant polynucleotides, plasmids, gene vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can be provided in an isolated or purified form.

[0307] A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. For the purposes of this disclosure, such nucleic acid sequences may include, but are not limited to, cDNA from viruses, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located at the 3' end of the coding sequence. In one embodiment, the polynucleotide comprises a sequence encoding the VH or VL amino acid sequence described above. The polynucleotide may encode a VH or VL sequence of a specific antigen binding protein, antibody, or binding fragment thereof disclosed herein.

[0308] Antigen binding proteins, antibodies, or binding fragments thereof can therefore be produced or delivered in the form of polynucleotides that encode and can express the antigen binding proteins, antibodies, or binding fragments thereof. When the antigen binding protein or antibody comprises two or more chains, a polynucleotide can encode one or more antibody chains. For example, a polynucleotide can encode an antibody light chain, an antibody heavy chain, or both. Two polynucleotides can also be provided, one of which encodes an antibody light chain and the other encodes the corresponding antibody heavy chain. Such polynucleotides or polynucleotide pairs can be expressed together to produce antibodies.

[0309] Polynucleotides can be synthesized according to methods known in the art, for example, as described by Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0310] Nucleic acid molecules of the present invention can be provided in the form of expression cassettes, which include control sequences operably linked to insertion sequences, thereby allowing the in vivo expression of antibodies of the present invention. These expression cassettes are generally provided in the form of vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be used directly to the host subject. Alternatively, the vector comprising the polynucleotide can be used to the host subject. Preferably, genetic vectors are used to prepare and / or administer the polynucleotide. Suitable gene vectors can be any vector that can carry sufficient genetic information and allow expression of polypeptides (e.g., the above-mentioned antibodies or their binding fragments).

[0311] Also disclosed is an expression vector comprising such a polynucleotide sequence. Such an expression vector is a conventional construct in the field of molecular biology and can involve the use of plasmid DNA and suitable initiators, promoters, enhancers and other elements (such as polyadenylation signals), which can be necessary and placed in the correct direction to allow expression of the peptide of the present invention. Other suitable vectors should be apparent to those skilled in the art. In this regard, as a further example, see Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0312] One skilled in the art can use the sequences described herein to clone or generate cDNA or genomic sequences, for example, as described in the examples below. The sequences are cloned into a suitable eukaryotic expression vector (e.g., pcDNA3 (Invitrogen), or a derivative thereof), and then transfected into mammalian cells (e.g., CHO cells) using a vector combination containing the appropriate light and heavy chains to allow expression and secretion of the antibodies described herein.

[0313] The skilled artisan can also make analogs of the antigen binding proteins, antibodies, or binding fragments thereof described herein by using the specific binding domains of the antigen binding proteins or antibody sequences and expressing them in different situations, such as polypeptides, such as fusion proteins. This is well known in the art.

[0314] Also disclosed are cells engineered to express antigen-binding proteins or antibodies. Such cells include transient or preferably stable higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast), or prokaryotic cells (such as bacterial cells). Specific examples of cells include mammalian HEK293, CHO, HeLa, NS0, and COS cells, which can be engineered by inserting a gene vector or expression cassette encoding an antigen-binding protein or antibody of the present invention. Preferably, the selected cell line is not only stable but also allows for mature glycosylation.

[0315] The cell lines can be cultured using conventional methods to produce antigen-binding proteins, antibodies, or binding fragments thereof, or can be used to deliver antigen-binding proteins, antibodies, or binding fragments thereof therapeutically or prophylactically to a subject. Alternatively, the polynucleotides, expression cassettes, or vectors of the present invention can be administered in vitro to cells from a subject, and the cells can then be returned to the subject.

[0316]

Drug composition

[0317] When used in the methods of the present invention, the therapeutic agent, preferably the antigen binding protein described above, can be provided in the form of a pharmaceutical composition comprising the therapeutic agent or antigen binding protein. Thus, the present invention encompasses pharmaceutical compositions comprising a therapeutic agent or antigen binding protein and a pharmaceutically acceptable carrier for use in the methods of the present invention.

[0318] As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for parenteral administration, such as intravenous, intraocular, intramuscular, subcutaneous, intradermal, or intraperitoneal administration (e.g., by injection or infusion). In certain embodiments, the pharmaceutically acceptable carrier comprises at least one carrier selected from the group consisting of a cosolvent solution, a liposome, a micelle, a crystal, a nanocrystal, a nanoparticle, an emulsion, a microparticle, a microsphere, a nanosphere, a nanocapsule, a polymer or polymeric carrier, a surfactant, a suspending agent, a complexing agent such as a cyclodextrin or an adsorbed molecule such as albumin, a surface active particle, and a chelating agent. In other embodiments, the polysaccharide comprises hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives (e.g., methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, cellulose phthalate, cellulose acetate succinate, cellulose acetate butyrate, hydroxypropyl methylcellulose phthalate), chitosan and its derivatives, [β]-glucan, arabinoxylan, carrageenan, pectin, glycogen, fucoidan, chondroitin, dermatan, heparan, heparan, pentosan, keratan, alginates, cyclodextrins, and salts and derivatives thereof, including esters and sulfates.

[0319] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. The example of suitable aqueous carriers that can be adopted in the pharmaceutical composition of the present invention includes water, buffered water and saline. The example of other carriers includes ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). For example, coating materials such as lecithin can be used, by keeping required particle size under dispersion, and by using a surfactant to keep suitable fluidity. In many cases, preferably, isotonic agents are included in the composition, such as sugar, polyols, such as mannitol, sorbitol or sodium chloride.

[0320] Pharmaceutical compositions can include pharmaceutically acceptable antioxidants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Sterilization procedures (see above) and the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.) can ensure protection against the presence of microorganisms. It may also be necessary to add isotonic agents such as sugars, sodium chloride, etc. to the composition. In addition, the absorption time of injectable pharmaceutical dosage forms can be extended by adding agents that delay absorption, such as aluminum monostearate and gelatin.

[0321] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0322] Sterile injectable solutions can be prepared by adding the desired amount of the active agent (e.g., antibody) to a suitable solvent having a combination of one or more of the ingredients listed above as desired, followed by sterilization microfiltration. Typically, dispersions are prepared by adding the active agent to a sterile vehicle containing a basic dispersion medium and the other desired ingredients listed above. Preferred methods of preparation for sterile powders for the preparation of sterile injectable solutions are vacuum drying and freeze drying (lyophilization), which produce a powder of the active agent and any other desired ingredients from a previously sterile filtered solution.

[0323] The pharmaceutical composition may comprise additional active ingredients as well as the therapeutic agent as defined above. As mentioned above, the compositions of the present invention may comprise one or more antigen binding proteins. These compositions may also comprise additional therapeutic or prophylactic agents.

[0324] Depending on the route of administration, the antigen binding protein, antibody, or binding fragment thereof may be coated in a material to protect the antigen binding protein or antibody from acid and other natural conditions that may inactivate or denature the antigen binding protein or antibody.

[0325] In a preferred embodiment, the form of the pharmaceutical composition according to the present invention is selected from one of the following: an aqueous solution, a gel, a hydrogel, a film, a paste, a cream, a spray, an ointment or a wrap.

[0326] In other embodiments, the pharmaceutical compositions described herein can be administered by routes such as intravenous, subcutaneous, intraocular, intramuscular, intraarticular, intradermal, intraperitoneal, spinal or other parenteral administration, for example by injection or infusion. Administration can be rectal, oral, ocular, topical, epidermal or via a mucosal route. Topical administration can include inhalation. In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously. In one embodiment, the pharmaceutical composition can be administered by inhalation. In one embodiment, a metered dose device containing the pharmaceutical composition is used.

[0327] Also disclosed are kits comprising the therapeutic agents or other compositions of the invention and instructions for their use. The kits may further contain one or more additional agents, such as the additional therapeutic or prophylactic agents discussed herein.

[0328]

Methods for preventing and treating diseases

[0329] The methods of the present invention can be used to prevent or treat a disease or disorder in a subject, in which case the method comprises administering to the subject a prophylactically or therapeutically effective amount of the therapeutic agent, such as the antigen binding protein.

[0330] In therapeutic applications, a therapeutic agent (e.g., an antigen binding protein) is administered to a subject who has already suffered from a disease or condition in an amount sufficient to cure, alleviate, or partially prevent one or more symptoms of the condition or condition. This treatment can result in a decrease in the severity of the disease symptoms, or an increase in the frequency or duration of the asymptomatic period. An amount sufficient to achieve this goal is defined as a "therapeutically effective amount." The effective amount for a particular purpose will depend on the severity of the disease or injury and the weight and general state of the subject. In preventive applications, a polypeptide or composition is administered to a subject who has not yet exhibited symptoms of a disease or condition in an amount sufficient to prevent or delay the development of symptoms. This amount is defined as a "prophylactic effective amount." As used herein, the term "subject" includes any vertebrate, typically any mammal, such as a human or horse. The subject is preferably a human.

[0331] In certain embodiments, a therapeutic agent, such as an antigen binding protein, can be linked (directly or indirectly) to another moiety. The other moiety can be another therapeutic agent, such as a drug. The other moiety can be a detectable label. The other moiety can be a binding moiety, such as an antibody or polypeptide binding domain specific for a therapeutic target. The antigen binding proteins of the present invention can be bispecific antibodies.

[0332] Additional therapeutic agents or detectable labels can be directly, for example, by chemical coupling, attached to the antigen-binding proteins of the present invention. Methods for coupling therapeutic agents or labels to antigen-binding proteins are well known in the art. For example, carbodiimide coupling (Bauminger S and Wilchek M, 1980, Methods Enzymol., 70, 151-159) can be used to couple various therapeutic agents including doxorubicin to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), is particularly suitable for coupling the functional moiety to the binding moiety.

[0333] Other methods can also be used to couple the moiety to the therapeutic agent. For example, sodium periodate oxidation followed by reductive alkylation using a suitable reagent can be used, or glutaraldehyde cross-linking can be used. However, it should be recognized that regardless of the method chosen to produce the conjugate of the present invention, it must be ensured that the antigen binding protein or antibody retains its targeting ability and that the functional moiety retains its relevant function.

[0334] The additional therapeutic agent linked to the antigen binding protein may comprise a polypeptide having therapeutic benefit, or a polynucleotide encoding a polypeptide. Examples of such polypeptides include anti-proliferative or anti-inflammatory cytokines.

[0335] The antigen binding protein may be linked to a detectable label. "Detectable label" refers to an antigen binding protein linked to a moiety that can be detected when the antigen binding protein is administered to a patient and localizes to a target site, typically non-invasively from outside the body and outside the target site.

[0336] Typically, the label is or comprises a radioactive atom that can be used for imaging. Suitable radioactive atoms include those used in scintigraphic studies. 99m Tc and 123 I. Other labels include, for example, spin labels for magnetic resonance imaging (MRI), such as those used in 123 I. 131 I. 111 In, 19 F. 13 C. 15 N. 17 O, gadolinium, manganese or iron. Of course, in order for the molecule to be detectable, a sufficient amount of the appropriate atomic isotope must be attached to the antibody.

[0337] Radioactive or other labels can be incorporated using known methods. For example, antigen binding proteins, antibodies, or fragments thereof can be biosynthesized, or can be synthesized by chemical amino acid synthesis using appropriate amino acid precursors (e.g., using fluorine-19 instead of hydrogen). For example, 99m Tc, 123I. 186 Rh, 188 Rh and 111 Labels such as In can be attached via cysteine ​​residues in the polypeptide. Yttrium-90 can be attached via lysine residues. Preferably, the detectable label comprises a radioactive atom, such as technetium-99m or iodine-123. Alternatively, the detectable label can be selected from the group consisting of iodine-123; iodine-131; indium-111; fluorine-19; carbon-13; nitrogen-15; oxygen-17; gadolinium; manganese; and iron.

[0338] Therapeutic agents of the present invention, preferably antigen-binding proteins of the present invention, can be administered by one or more approaches, using one or more methods known in the art. It will be understood by those skilled in the art that the route of administration and / or mode of administration will vary depending on the desired results. Preferred routes of administration for antigen-binding proteins, antibodies or compositions of the present invention include intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal or other parenteral routes, such as by injection or infusion. The phrase "parenteral administration" as used herein refers to a mode of administration other than enteral and topical administration, typically by injection. Administration can be rectal, oral, ocular, topical, epidermal or by mucosal routes. Administration can be local to the lesion, including peritumoral, paratumoral, intratumoral, tumor resection margin, intralesional, perilesional, by intracavitary infusion, intravesicular administration or inhalation. In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously.

[0339] The appropriate dosage of the therapeutic agent can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the specific therapeutic agent employed, the route of administration, the time of administration, the rate of excretion of the therapeutic agent, the duration of treatment, other drugs, compounds and / or materials employed in combination with the specific composition, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0340] A suitable dosage of the antigen binding protein can be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg relative to the body weight of the patient to be treated. For example, a suitable dosage can be about 1 μg / kg to about 50 mg / kg body weight per week, about 100 μg / kg to about 25 mg / kg body weight per week, or about 10 μg / kg to about 12.5 mg / kg body weight per week.

[0341] Suitable dosages may be about 1 μg / kg to about 50 mg / kg body weight per day, about 100 μg / kg to about 25 mg / kg body weight per day, or about 10 μg / kg to about 12.5 mg / kg body weight per day.

[0342] The dosage regimen can be adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, the dosage may be administered as a single bolus or divided over time, or the dosage may be proportionally reduced or increased according to the exigencies of the therapeutic situation. Formulating parenteral compositions in dosage unit form is particularly advantageous for simplifying administration and providing uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a single dosage for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier.

[0343] Antigen binding proteins can be administered in single doses or multiple doses. Multiple doses can be administered to the same or different locations through the same or different approaches. Alternatively, when the frequency of administration needs to be reduced, the antigen binding proteins can be administered as a sustained release formulation. Dosage and frequency can be changed according to the half-life of the antigen binding proteins in the patient's body and the expected treatment duration. The dosage and frequency of administration can also be changed according to whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages can be administered at relatively infrequent intervals over a long period of time. In therapeutic applications, relatively high dosages can be administered, for example, until the patient shows partial or complete improvement of disease symptoms.

[0344] The combined administration of two or more drugs can be achieved in a variety of different ways. In one embodiment, the therapeutic agent of the present invention and the other drug can be administered together in a single composition. In another embodiment, the therapeutic agent and the other drug can be administered in different compositions as part of a combination therapy. For example, the therapeutic agent can be administered before, after, or simultaneously with the other drug.

[0345]

Diseases that need to be diagnosed, treated or prevented

[0346] The therapeutically effective amount of the antigen binding protein (or nucleic acid encoding the antigen binding protein) will depend on the severity of the disease and the patient's general health. A therapeutically effective amount of the antigen binding protein can provide subjective relief of symptoms or objectively recognizable improvement, the latter as recorded by a clinician or other qualified observer. As previously mentioned, these compositions can be administered simultaneously or sequentially with another therapeutic agent.

[0347] When the therapeutic activity of a therapeutic agent (such as an antigen binding protein) remains within the prescribed range, the therapeutic agent will retain its full therapeutic potential. However, when the therapeutic activity is such that adverse events may occur, changes in the activity of endogenous factors will lead to the cleavage of the autoregulatory element and ultimately inactivate the therapeutic agent to reduce the risk of adverse events.

[0348] Single or multiple administrations of the compositions disclosed herein, including antigen-binding proteins, depend on the dosage and frequency required and tolerated by the patient. In any case, the composition should provide a sufficient amount of at least one antigen-binding protein disclosed herein to effectively treat the patient. The dose can be administered once, but it can also be administered periodically until a therapeutic effect is achieved or until side effects occur that require cessation of treatment. In one example, the antigen-binding protein is infused for 30 minutes every other day. In this example, about 1 to 10 doses can be administered, such as 3 or 6 doses can be administered every other day. In another example, continuous infusion is administered for about 5 to 10 days. The subject can be treated regularly, for example, once a month, until the desired therapeutic effect is achieved. Typically, the dose is sufficient to treat or improve the symptoms or signs of the disease without causing unacceptable toxicity to the patient.

[0349] Further disclosed are compositions comprising antigen-binding proteins or nucleic acids encoding antigen-binding proteins in a carrier. The compositions can be prepared in the form of unit dosage forms for administration to a subject. The amount and time of administration are determined by the attending physician to achieve the desired purpose. The antigen-binding proteins and / or nucleic acids can be formulated into dosage forms for systemic or local administration. In one example, the antigen-binding proteins or nucleic acids encoding antigen-binding proteins are formulated into dosage forms for parenteral administration, such as intravenous administration. In some embodiments, the mode of administration is intramuscular injection.

[0350] The active ingredient can also be embedded in microcapsules prepared, for example, by coacervation techniques, such as hydroxymethylcellulose or gelatin microcapsules, or by interfacial polymerization techniques, such as polymethyl methacrylate microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Specifically, methods for preparing liposomes containing antibodies are described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. No. 4,485,045 and U.S. Pat. No. 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Reverse phase evaporation can be used with lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes of a desired diameter. The polypeptides of the present invention can be coupled to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0351] The composition for administration can include a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers can be used, such as buffered saline, etc. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition can contain pharmaceutically acceptable auxiliary substances required for mimicking physiological conditions, such as pH adjusting agents and buffers, toxicity regulators, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding protein in these preparations can vary greatly and will be selected primarily based on liquid volume, viscosity, body weight, etc., as well as the specific mode of administration selected and the needs of the experimenter. In some embodiments, it is administered intravenously.

[0352] Controlled-release parenteral dosage forms can be prepared as implants, oily injections, or particle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or a drug, as a central core. In the microspheres, the therapeutic drug is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are typically referred to as nanoparticles, nanospheres, and nanocapsules, respectively. The capillary diameter is about 5 μm, so only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0353] Polymers can be used for controlled ion release of the antigen-binding proteins disclosed herein. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, the block copolymer, polaxamer 407 exists as a viscous but flowable liquid at low temperatures, but forms a semisolid gel at body temperature. Polaxamer 407 has been shown to be an effective medium for the formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9: 425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44 (2): 58-65, 1990). In addition, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). On the other hand, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)).

[0354] Common pharmaceutical compositions for intravenous administration include about 0.1 to 10 mg / kg of antigen-binding protein per day, or 0.5 to 15 mg / kg of antigen-binding protein per day. A dose of 0.1 to about 100 mg / kg can be used per subject per day, particularly when the drug is administered to a closed site and does not enter the circulation or lymphatic system (e.g., into the lumen of a body cavity or organ). Exemplary dosages include 1 to 10 mg / kg, such as 2 to 8 mg / kg, such as 3 to 6 mg / kg. The actual method of preparing the composition to be administered should be known or apparent to those skilled in the art and is described in more detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0355] Antigen binding proteins can be provided in lyophilized form and rehydrated with sterile water before administration, although they can also be provided as sterile solutions of known concentrations. The antigen binding protein solution is then added to an infusion bag containing 0.9% sodium chloride (USP standard), USP, and is typically administered at a dose of 0.1 to 10 mg / kg body weight or 0.5 to 15 mg / kg body weight. Example doses include 1 to 10 mg / kg, such as 2 to 8 mg / kg, such as 3 to 6 mg / kg. This area has extensive experience in the administration of antibody drugs, and since 1997, Since its approval, antibody drugs have been on the market in the United States. Antigen binding proteins can be administered by slow infusion rather than intravenous push or single rapid injection (bolus). In one example, a higher loading dose is first administered, followed by a maintenance dose at a lower level. For example, an initial loading dose of 4 mg / kg can be infused over about 90 minutes, and if the previous dose is well tolerated, a maintenance dose of 2 mg / kg is given weekly for the next 4 to 8 weeks, infused over 30 minutes.

[0356] A therapeutically effective amount of a nucleic acid encoding an antigen-binding protein can be administered to a subject in need. One method of administering the nucleic acid is to directly immunize using plasmid DNA, for example, using a mammalian expression plasmid. The nucleotide sequence encoding the antigen-binding protein can be placed under the control of a promoter to increase the expression level of the molecule. Immunization via nucleic acid constructs is well known in the art and is taught, for example, in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972, and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids to organisms. These methods include liposome delivery of nucleic acids.

[0357] In another method using nucleic acid, the antigen-binding proteins can also be expressed by an attenuated viral host or viral vector or bacterial vector that can be applied to a subject. Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytomegalovirus, pox virus or other viral vectors can be used to express antibodies. For example, vaccinia vectors are described in U.S. Patent No. 4,722,848. Bacillus Calmette-Guérin (BCG) provides another vector for the expression of publicly available antigen-binding proteins (see Stover, Nature 351:456-460, 1991).

[0358] In one embodiment, nucleic acid encoding an antigen binding protein is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microparticles by standard methods and expressed as a protein ... The nucleic acid can be introduced into the skin using a device such as a Gene Gun. The nucleic acid can be "naked" or consist of a plasmid under the control of a strong promoter.

[0359] Typically, DNA is injected into muscle, although it can also be injected directly into other sites. The injection dose is generally about 0.5 mg / kg to about 50 mg / kg, with a typical dose being about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0360] In some examples, DNA encoding antigen-binding proteins is administered to a subject for in vivo production of antigen-binding proteins, for example, utilizing the subject's cellular machinery. Immunization by nucleic acid constructs is well known in the art, for example, as taught in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972, and U.S. Patent No. 5,817,637. Several methods for delivering encoding nucleic acids to organisms are described in U.S. Patent No. 5,880,103. These methods include liposome delivery of nucleic acids. One of ordinary skill in the art can use such methods for the production of antigen-binding proteins.

[0361] One method of nucleic acid administration is direct administration of plasmid DNA, such as a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibodies can be placed under the control of a promoter to increase expression levels.

[0362] Treatment of hemophilia A

[0363] In an embodiment of the present invention, when the autoregulatory element comprises a thrombin, factor IXa, factor Xa, factor XIa, factor XIIIa, or APC cleavage site, it can be used to treat hemophilia A. Embodiments of the present invention in which the autoregulatory element comprises a thrombin cleavage site can be used to treat hemophilia A. In a preferred embodiment of the present invention, the antigen-binding protein of the present invention that can be used to treat hemophilia A comprises a thrombin cleavage site. In a preferred embodiment of the present invention, the antigen-binding protein of the present invention that can be used to treat hemophilia A comprises an autoregulatory element as described in SEQ ID NO: 1, 2, 3, or 4. In a preferred embodiment of the present invention, the antigen-binding protein of the invention that can be used to treat hemophilia A comprises an autoregulatory element as described in SEQ ID NO: 1.

[0364] In a preferred embodiment of the invention, the antigen binding protein comprising a thrombin cleavage site comprises a first antigen binding domain that specifically binds to FIXa / FIX (as defined herein) and a second antigen binding domain that specifically binds to FX / FXa (as defined herein), and a thrombin cleavage site (as defined herein).

[0365] Thus, also disclosed is a method of treating hemophilia A in a subject, comprising administering to the subject a therapeutically effective amount of a disclosed antigen binding protein and / or a nucleic acid encoding the antigen binding protein, thereby treating hemophilia.

[0366] The present invention also relates to the disclosed antigen binding protein for treating hemophilia A. In addition, the present invention also relates to the use of the disclosed antigen binding protein in the manufacture of a medicament for treating hemophilia A.

[0367] In an embodiment of the present invention, when the self-regulatory element comprises a thrombin, factor IXa, factor Xa, factor XIa, factor XIIIa or APC cleavage site, it can be used to prevent the risk of prothrombosis. The embodiment of the present invention in which the self-regulatory element comprises a thrombin cleavage site can be used to prevent the risk of prothrombosis. In a preferred embodiment of the present invention, the antigen-binding protein of the invention that can be used to prevent the risk of prothrombosis comprises a thrombin cleavage site. In a preferred embodiment of the present invention, the antigen-binding protein of the invention that can be used to prevent the risk of prothrombosis comprises a self-regulatory element as described in SEQ ID NO: 1, 2, 3 or 4. In some preferred embodiments of the present invention, the antigen-binding protein of the invention that can be used to prevent the risk of prothrombosis comprises the self-regulatory element described in SEQ ID NO: 1.

[0368] Therefore, in another embodiment of the present invention, a method for preventing the risk of prothrombosis in a subject is also disclosed, which comprises administering to the subject a therapeutically effective amount of the disclosed antigen-binding protein and / or nucleic acid encoding the antigen-binding protein, thereby preventing the risk of prothrombosis.

[0369] The present invention also relates to the disclosed antigen binding protein for preventing the risk of thrombosis. In addition, the present invention also relates to the use of the disclosed antigen binding protein in the manufacture of a medicament for preventing the risk of thrombosis.

[0370] Cancer Treatment

[0371] In some embodiments of the present invention, when the self-regulatory element comprises a cleavage site for granzyme A, granzyme B, granzyme K, granzyme H, granzyme M, cathepsin B, cathepsin L, cathepsin W, or matrix metalloproteinases (such as MMP-2, MMP-9, and MMP-28), it is suitable for treating cancer. The embodiment of the present invention in which the self-regulatory element comprises a granzyme B cleavage site is suitable for treating cancer. In a preferred embodiment of the present invention, the antigen binding protein comprises a self-regulatory element as described in any one of SEQ ID NOs: 5 to 18. In a preferred embodiment of the present invention, the antigen binding protein comprises a self-regulatory element as described in SEQ ID NO: 7. In a preferred embodiment of the present invention, the antigen binding protein comprising a granzyme B cleavage site comprises a first antigen binding domain (as defined herein) that specifically binds to ROR1. In a preferred embodiment of the present invention, the antigen binding protein comprising a granzyme B cleavage site comprises a first antigen binding domain (as defined herein) that specifically binds to ROR1 and a second antigen binding domain (as defined herein) that specifically binds to CD3.

[0372] Therefore, also disclosed is a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a disclosed antigen binding protein and / or a nucleic acid encoding the antigen binding protein, thereby treating the cancer.

[0373] The disclosed antigen binding proteins can be cytotoxic to cancer cells.

[0374] Preferably, the cancer is leukemia (such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell leukemia or hairy cell leukemia), pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma, renal cancer. In addition, ROR1 is expressed in some cancer stem cell subpopulations.

[0375] The present invention also relates to the disclosed antigen-binding protein for treating cancer. In addition, the present invention also relates to the use of the disclosed antigen-binding protein in the manufacture of a drug for treating cancer.

[0376] Preferably, the cancer is leukemia (such as chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cell leukemia or hairy cell leukemia), pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma, renal cancer. In addition, ROR1 is expressed in some cancer stem cell subpopulations.

[0377] The composition need not completely eliminate the cancer or tumor for it to be effective. For example, the antibody can reduce the size of the tumor by a desired amount, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%, compared to the absence of the composition.

[0378] Administration of an antigen binding protein of the invention may result in 5, 10, 20, 50, 75, 90, 95 or 99% depletion (ie, reduction) of malignant cells.

[0379] In another example, an effective amount of an additional drug, such as a chemotherapeutic drug, can also be administered to the subject. The method can include administering one or more additional drugs known in the art.

[0380] Severe toxicities associated with the mechanism of action (MoA) of immunotherapy include, but are not limited to, cytokine release syndrome (CRS) and neurotoxicity, and lead to acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction. Acute systemic inflammatory syndrome may be associated with (i) chimeric antigen receptor (CAR)-T cell therapy (ii) therapeutic antibodies and (iii) haploidentical allogeneic stem cell transplantation (haplo-allo-HSCT). Granzyme B levels are elevated in subjects who progress to severe MoA-related toxicity. In some embodiments of the present invention, when the self-regulatory element includes a cleavage site for granzyme A, granzyme B, granzyme K, granzyme H, granzyme M, cathepsin B, cathepsin L, cathepsin W, or matrix metalloproteinases (such as MMP-2, MMP-9, and MMP-28), it is suitable for preventing MoA-related toxicities associated with immunotherapy, such as cytokine release syndrome. The embodiment in which the self-regulatory element of the present invention comprises a granzyme B cleavage site can be used to prevent MoA-related toxicities associated with immunotherapy. In a preferred embodiment of the present invention, the antigen binding protein comprises an autoregulatory element as described in any one of SEQ ID NOs: 5 to 18. In a preferred embodiment of the present invention, the antigen binding protein comprises an autoregulatory element as described in SEQ ID NO:7.

[0381] In other embodiments of the present invention, a method for preventing MoA-related toxicity associated with immunotherapy in a subject is also disclosed, the method comprising administering to the subject a therapeutically effective amount of the disclosed antigen binding protein and / or nucleic acid encoding the antigen binding protein, thereby preventing MoA-related toxicity.

[0382] The present invention also relates to the disclosed antigen binding proteins for preventing MoA-related toxicity associated with immunotherapy. In addition, the present invention also relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for preventing MoA-related toxicity.

[0383] Other antigen-binding proteins that may benefit from the introduction of granzyme B autoregulatory elements to prevent MoA-associated toxicity include moruomab, basiliximab, daclizumab, blinatumumab, and alemtuzumab.

[0384] In another embodiment of the present invention, a method for preventing cytokine release syndrome in a subject is also disclosed, which comprises administering to the subject a therapeutically effective amount of the disclosed antigen binding protein and / or nucleic acid encoding the antigen binding protein, thereby preventing cytokine release syndrome.

[0385] The present invention also relates to the disclosed antigen-binding protein for preventing cytokine release syndrome. In addition, the present invention also relates to the use of the disclosed antigen-binding protein in preparing a medicament for preventing cytokine release syndrome.

[0386] Other antigen-binding proteins that may benefit from the introduction of granzyme B autoregulatory elements to prevent cytokine release syndrome include moruomab, basiliximab, daclizumab, blinatumumab, and alemtuzumab.

[0387] [Example]

[0388] [Example 1: Theoretical basis]

[0389] FVIII-mimicking antibodies in hemophilia A

[0390] Coagulation factor VIII (FVIII) is a key cofactor in the coagulation cascade, facilitating the catalytic conversion of factor X (FX) to activated FX (FXa) by forming a complex with activated factor IX (FIXa) and FX. In hemophilia A (an X-linked monogenic disorder with an incidence of 1 in 5,000 male newborns), FVIII deficiency impairs coagulation, leading to a life-threatening spontaneous bleeding diathesis. FVIII replacement therapy, as standard of care treatment, requires frequent intravenous infusions of FVIII. However, approximately 30% of treated patients develop anti-FVIII neutralizing antibodies, making FVIII replacement therapy inefficient and significantly shortening life expectancy.

[0391] Emicizumab, a humanized IgG4 bispecific antibody approved in late 2017, is a FVIII mimetic molecule that simultaneously binds to FIXa and FX, promoting the catalytic conversion of FX to activated FXa via FIXa, leading to the restoration of thrombin generation and clot formation. Emicizumab has redefined the treatment of hemophilia A by reducing bleeding events by 80% and achieving a preventive effect even in patients with neutralizing antibodies. However, an unusually high incidence of life-threatening thrombotic microangiopathy and thromboembolism (3 cases per 1,000 treated patients) and unexplained deaths have been observed in patients treated with emicizumab, leading the FDA to issue a black box warning regarding the use of this treatment.

[0392] FVIII and emicizumab maintain the coagulation cascade through distinct mechanisms (Lenting, Denise et al., 2017). FVIII circulates in an inactive form and requires activation to FVIIIa to promote amplified thrombin generation. Thrombin further activates protein C (PC), inhibiting FVIIIa through a negative feedback loop, thereby preventing excessive procoagulant activity. In contrast, emicizumab is permanently active and cannot be "turned off" by activated PC (aPC) after clot formation. Therefore, as long as it remains in circulation, it promotes a persistent prothrombotic state. This dangerous state could be addressed by developing autoregulatory FVIII-mimetic antibodies. Incorporating a thrombin-cleavable peptide as a built-in "off" mechanism would allow the autoregulatory antibody to be cleaved into a nonfunctional component when sufficient thrombin is generated to form a clot, thereby restoring the negative feedback loop and preventing the risk of thrombosis.

[0393] Bispecific T cell engagers in adoptive immunotherapy

[0394] Bispecific T cell engagers (TCEs) are bispecific antibodies that redirect the cytotoxic activity of T cells toward malignant tumor cells. This is a highly effective mechanism of action, but it is also associated with the development of serious adverse events, leading to life-threatening immune hyperactivation in a significant number of patients, including cytokine release syndrome (CRS) and neurotoxicity. In fact, a 100% adverse event rate has been observed with the therapeutic use of TCEs, with grade 3 and 4 life-threatening toxicities observed in over 50% of patients treated with currently approved TCEs (blinatumomab, mosenozumab, and teclistamab). Palliative measures such as tumor burden reduction, corticosteroid administration, and dose escalation can help mitigate but not eliminate these toxicities. These limitations limit optimal dosing, shorten treatment duration, and increase the burden of monitoring, particularly in complex or fragile patients. Therefore, developing an auto-regulating TCE (AR_TCE) that can respond to biological signals in a threshold-limited manner by inactivation before the onset of grade 3 / 4 life-threatening toxicity will effectively reduce the occurrence of adverse events and broaden the therapeutic index of TCE, thereby improving the treatment effect of cancer patients.

[0395] When simultaneously engaging both targets, TCEs aggregate CD3 on the T cell surface, leading to their activation and the formation of an immune synapse. This stimulates the local release of cytotoxic enzymes, including granzymes, triggering apoptosis and clearance of target cells. Effective cancer cell killing relies on this T cell-mediated release of cytokines and proteolytic enzymes, which, at low levels, are associated with the mechanism of action of TCEs. However, experience gained from studies of TCEs and CAR-T cells indicates that life-threatening MoA-associated toxicity is a hallmark of T cell hyperactivation, in which granzyme B levels can be upregulated as much as 40-fold (Kochenderfer, Somerville et al., 2017). Therefore, inserting a granzyme B-sensitive peptide between the targeting and effector arms of a TCE could render the drug susceptible to degradation and inactivation upon exposure to granzyme B levels that rise above the threshold associated with the development of MoA-associated toxicity, thereby reducing the risk of serious adverse events. [Chimeric Antigen Receptor (CAR)-T Cell Therapy]

[0396] Chimeric antigen receptor (CAR)-T cells are a type of cell therapy that are genetically engineered to express a chimeric cell surface receptor that targets a target tumor-associated antigen (TAA). They have been clinically demonstrated to effectively redirect T cell cytotoxicity to eliminate cancer cells. The mechanism of action of CAR-T cells is similar to that of bispecific TCEs and, therefore, is associated with a higher risk of severe MoA-related toxicities, which are characteristic of T cell redirection technologies, including cytokine release syndrome and neurotoxicity.

[0397] The hallmark features of CAR-T cell toxicity are similar to those of TCEs, including the release of granzyme B due to overactivation of CAR-T cells. Therefore, an autoregulatory AR_CAR-T cell was developed by inserting a granzyme B-sensitive peptide into the structure of the CAR receptor, enabling it to respond to signals associated with the progression of MoA-related toxicity, cleave and remove the receptor from the cell surface, thereby significantly improving the safety of this type of therapy without compromising its efficacy.

[0398] [Example 2: Materials and Methods]

[0399] Preparation and purification of bispecific antibodies

[0400] Emicizumab was obtained from the commercial Hemlibra provided by the Katharine Dormandy Haemophilia and Thrombosis Centre (Royal Free Hospital, London). TM The production and purification of the auto-regulated prototype AR_Ab8 was outsourced to Absolute Antibody (now Absolute Biotech). The antibody was expressed in the human embryonic kidney (HEK)-293 cell line and purified using Absolute Antibody's proprietary method by size exclusion chromatography to a final product purity of >99%.

[0401] NVG-111 and autoregulatory AR_TCE variants were produced in-house using transient transfection in the mammalian Expi293 expression system (Gibco). TM Cells were routinely subcultured in Expi293 expression medium at 37°C, 8% CO2, and 130 rpm. For transfection, 1 × 10 6 The cells were seeded in Expi293 expression medium at a density of viable cells. The next day, the cells were expressed using ExpiFectamine TMFollowing the manufacturer's instructions, the required volume of cell suspension was transfected with plasmids containing different antibody coding sequences cloned from the pcDNA3.1 hygro(+) expression vector. 16-18 hours after transfection, production enhancers were added to the cultured cells as recommended to increase protein production, and the cultures were incubated for another 5 days. Cells were removed by centrifugation (4000 g, 20 minutes) and the production supernatant was clarified and collected by filtration using a 0.22 μm filter. Affinity chromatography was performed using a 5-well plate filled with MabSelect TM 5 mL HiTrap of PrismA resin (Cytiva) TM Chromatographic column, Different variants were purified on a Cytiva Avant 25 chromatography system. The purified protein was eluted in 50 mM glycine, 150 mM NaCl, pH 3 and immediately neutralized with 2% v / v 2M Tris-Base solution (pH 8.3). After purification, the antibody buffer was exchanged into a formulation solution containing 10 mM histidine, 150 mM NaCl, 0.02% Tween 80, pH 6.0, and stored at -80°C.

[0402]

Chromogenic substrate method

[0403] The FVIII mimetic activity of emicizumab and AR_Ab8 was evaluated by measuring FIX-mediated activation of FX to FXa using the FXa-specific S-2765 chromogenic substrate. In a polystyrene 96-well flat-bottom half-well plate (Greiner Bio-one), 50 μL of coagulation factor solution containing 40 μM phospholipid-TGT (Rossix AB), 280 nM plasma-derived human FX ( Bioproducts Laboratories), 6 nM plasma-derived human FIXa (Haematologic Technologies), and 10 mM CaCl2 were dissolved in running buffer (50 mM Tris-Base, 150 nM NaCl, 0.1% BSA (protease-free), pH 7.8). Then, 25 μL of S-2765 chromogenic substrate (Chromogenix) diluted to 1.2 mM in running buffer was added to each well, and the chromogenic conversion kinetics were immediately recorded using a microplate reader (SpectraMax m3, Molecular Devices) at a wavelength of 405 nm and 37°C for 20 minutes (read once every minute, with 2 seconds of shaking before each reading).

[0404] Activated Partial Thromboplastin Time (aPTT)

[0405] 200 Bethesda units of FVIII neutralizing polyclonal antibodies (PAHFVIII-S, Haematologica Technologies) were added to human citrated pooled plasma prepared from at least 20 healthy volunteers to remove FVIII activity. 350 nM of emicizumab or AR-Ab8 were then added to induced hemophilia A plasma and incubated at room temperature for 15 minutes. Coagulation time was measured using aPTT synthaSil reagent (Werfen Limited) on an ACL Top 700 coagulation analyzer (Werfen Limited).

[0406] Thrombin cleavage test

[0407] To evaluate thrombin-mediated inactivation of the bispecific antibodies, 2 nM emicizumab or AR-Ab8 solutions were mixed with 2 U / mL human α-thrombin (Enzyme Research Laboratories) in running buffer (50 mM Tris-Base, 150 nM NaCl, 0.1% BSA (protease-free), pH 7.8) and incubated at 37° C. The cleavage reaction was terminated at different time points (15 min to 3 h) by adding an equal volume of 500 nM PPACK inhibitor (Merck-Millipore) solution, and the samples were frozen for subsequent analysis.

[0408] Antibody cleavage was detected using SDS-PAGE and Western blotting. Samples were loaded directly onto NuPAGE 4-12% bis-Tris precast gels (Invitrogen) without dilution and electrophoretically migrated at 120-180 V in MOPS-SDS running buffer (Invitrogen). Five microliters of prestained PageRuler protein molecular weight standards (Thermofisher) were added to another well for molecular weight comparison. Proteins were further transferred to nitrocellulose blotting membranes using a wet transfer method in NuPAGE transfer buffer (Invitrogen). The membrane was blocked for 1 hour in TBS.T buffer (50 mM Tris-Base, 150 mM NaCl, 0.1% Tween 20, pH 7.8) containing 5% skim milk on an orbital shaker. A horseradish peroxidase (HRP)-labeled polyclonal goat anti-human Fc antibody (#31413, Invitrogen) was then added (diluted 1:5000 in TBS.T + 5% skim milk) and incubated overnight at 4°C. The membrane was washed three times in TBS.T and incubated with SuperSignal WestPico PLUS chemiluminescent substrate (Thermofisher) for 5 minutes at room temperature. Images were acquired using the GelDoc system (Bio-Rad).

[0409] The residual FVIII mimetic activity of the samples was measured using a chromogenic substrate method as described above. The samples were diluted 1:2 to a bispecific antibody concentration of 1 nM.

[0410] Thrombin generation assay (TGA)

[0411] Abnormal thrombin generation was detected using a calibrated automated thrombogram (CAT) method on a Thrombinoscope (Diagnostica stago). As previously described, 200BU of anti-FVIII antibodies were added to human mixed plasma to neutralize FVIII activity. 600nM emicizumab or a combination of AR_Ab8 and 0.5U / mL aPCC (FEIBA, Takeda Pharmaceutical) were then added to the neutralized plasma and incubated at room temperature for 15 minutes. For each experimental condition, 80 μL of plasma was mixed with 20 μL of tissue factor PPPlow trigger reagent (Diagnostica stago) in an Immulon 2HB U-bottom microplate (Diagnostica Stago) and incubated at 37°C for 10 minutes. The thrombin generation reaction was initiated by automatically injecting 20 μL of preheated FluCa reagent and recording data for 60 minutes. Endogenous thrombin potential (ETP) was calculated as the area under the thrombin generation curve, representing the total thrombin generation amount.

[0412] Tail clip assay

[0413] The procoagulant potential of FVIII-mimetic bispecific antibodies in FVIII-deficient mice was evaluated using a tail-chopping experiment, and the procedure was performed as previously described (Ferriere et al., blood 2020). Briefly, FVIII-deficient male mice (8-12 weeks old) were given 3 mg / kg of emicizumab or AR_Ab8 by retroorbital injection 24 h before surgery. Under ketamine (100 mg / kg) / xylazine (10 mg / kg) anesthesia, a mixture of 100 U / kg of human FIX and FX was administered by a second retroorbital injection 5 minutes before tail chopping 3 mm distal to the tail. Control animals received a single retroorbital injection of 2 U / mouse of recombinant human FVIII. The severed tail was then immersed in preheated saline (37°C), and blood was collected for 30 minutes. The hemoglobin content in the collection tube was detected using a spectrophotometer at a wavelength of 416 nm, and blood loss was calculated based on the standard curve.

[0414] In vivo thrombosis model

[0415] The thrombogenic potential of a FVIII-mimetic bispecific antibody was evaluated using an in vivo protocol designed to replicate the combined effects of emicizumab and aPCC. Eight- to 10-week-old C57Bl / 6 male mice (Charles River Laboratories) received an initial intravenous (IV) injection of 2.5 U of aPCC (FEIBA, Takeda Pharmaceuticals) and 250 μg of the bispecific antibody, followed by three booster injections of aPCC (2.5 U each IV) at 24, 48, and 72 hours. Control animals received aPCC alone without the bispecific antibody. At 96 hours, all animals were under irreversible gas anesthesia (isoflurane), and blood was collected via cardiac puncture of the posterior vena cava in EDTA (final concentration 0.05 M). All animals were euthanized humanely by cervical dislocation. Platelet counts were performed immediately after blood collection using an automated cell counter (Scil Vet ABC Plus, Horiba Medical).

[0416] Explanted lungs were used for thrombosis analysis and fixed in 10% neutral buffered formalin (CellPath, Inc.) for 24 hours at room temperature. The fixed organs were briefly rinsed in PBS, embedded in OCT matrix (CellPath, Inc.), and frozen in liquid nitrogen. Tissue sections were prepared from lung frozen blocks and then subjected to immunofluorescence (IF) staining of platelets and blood vessels. Lung tissue sections were washed in PBS for 10 minutes to remove the OCT matrix and then incubated in PBS + 0.05% Tween 20 (PBS.T) and 0.5% Triton X-100 for 10 minutes for detergent-based antigen retrieval. Sections were washed 3×5 minutes in PBS.T and then incubated with 3% (w / v) bovine serum albumin (BSA) in PBS.T for 1 hour at room temperature (RT) to block nonspecific binding sites. Sections were incubated overnight at 4°C with rat anti-mouse CD41 / integrin αIIb (clone MWreg30, BD bioscience) and goat anti-mouse CD31 / PECAM-1 (AF3628, Bio-Techne) diluted in PBS-T + 1% BSA for platelet and endothelial cell detection, respectively. Slides were washed 3 × 5 minutes in PBS and then incubated with Alexa 488 donkey anti-rat (A48269) and Alexa-555 donkey anti-goat (A32816) polyclonal antibodies diluted in PBS-T + 1% BSA for 1 hour at room temperature on an orbital shaker. After a final 3 × 5 minute wash step, slides were placed with a drop of ProLong Diamond antifade reagent containing DAPI (ThermoFisher Scientific), mounted with coverslips, and allowed to dry in the dark for 24 hours.

[0417] Images were collected using a Z1 inverted microscope (Zeiss) at a 20x objective. 4 μm 2 Individual blood vessels (identified by CD31 staining) were imaged. Vessel boundaries were manually delineated based on CD31 staining using Fiji software (open source). Using the αIIb (platelet) staining channel, signal intensity was empirically thresholded based on the signal-to-noise ratio and converted into a binary occlusion mask to define positive and negative occlusion areas. This mask was applied to all images from the same experiment. The percent occlusion for each vessel was calculated using Fiji software (open source, GNU General Public License), defined as the percentage of area within the vessel boundary occupied by the occlusion mask.

[0418] Protease-mediated TCE cleavage assay

[0419] Granzyme B-specific cleavage of autoregulatory AR_TCE variants was evaluated in vitro. Recombinant human granzyme B protein (Bio-Techne) was first activated using mouse cathepsin C (Bio-Techne) as recommended in the manufacturer's instructions. 1 μg / mL of NVG-111 or AR_TCE was then mixed with varying concentrations of activated granzyme B diluted in assay buffer (50 mM Tris-Base, 150 mM NaCl, 5 mM CaCl2, 100 μg / mL protease-free BSA, pH 7.6) and incubated at 37°C for 2 h. A broad-spectrum protease inhibitor cocktail (Tebu-Bio) was added to a final concentration of 1x (1:10 dilution) to terminate the catalytic reaction, and the samples were frozen for subsequent analysis. Undiluted samples were used to examine antibody cleavage by SDS-Page electrophoresis and Western blotting, following the above-mentioned experimental protocol. Nitrocellulose membranes were incubated with HRP-labeled protein L (Genscript) diluted 1:2000 in TBS.T + 5% emulsion at 4°C overnight.

[0420] In order to assess the sensitivity of AR-TCE to different proteases, a panel of 50 nM representative proteases was used to determine the cleavage of the protein. The panel was composed of activated FIIa, FVIIa, and FXa (Prolytix), caspase-3, -8 (Bio-Techne), and -9 (Abcam), and matrix metalloproteinases (MMPs)-3 and -9 (Abcam).

[0421] Residual intact antibodies were quantified using a dedicated ELISA method that specifically recognizes intact antibodies. Polystyrene 96-well flat-bottom half-well plates (Greiner Bio-one) were coated with 2 μg / mL ROR1 protein (Abcam) diluted in carbonate buffer (Na2CO3 12.2mM, NaHCO3 35mM, pH 9.6) at 4°C overnight. The plates were washed three times in TBS.T (50mM Tris-Base, 150mM NaCl, 0.1% Tween20, pH 7.8) and nonspecific binding sites were blocked at 37°C for 1 hour using TBS.T + 3% BSA. After washing three times, the samples were diluted with TBS.T + 1% BSA and incubated at 37°C for 2 hours. After three washes in TBS to remove unbound sample, the plate was incubated with an HRP-conjugated monoclonal anti-His tag antibody (clone J099B12, Biolegend) diluted 1:2500 in TBS-T + 1% BSA for 2 hours at 37°C. The intact antibody recognizes the histidine tag motif on the CD3 binding arm (released upon cleavage). After a final three washes, the 96-well plate was incubated with a slow-kinetic 3,3',5,5'-tetramethylbenzidine (TMB) substrate and allowed to develop for 5–15 minutes. The reaction was terminated by adding an equal volume of 2 M H2SO4, and the optical density was read at 450 nm using a SpectraMax m3 (Molecular Devices) within 30 minutes.

[0422] T cell preparation and culture

[0423] Human peripheral blood mononuclear cells (PBMCs) were obtained from leukocyte reduction chambers of healthy individuals and purified by Ficoll-Plaque density gradient. PBMCs were then cultured at 37°C and 8% CO2 at a concentration of 5 × 10 5 The cells were cultured at a density of 10 cells / ml in RPMI1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U / mL recombinant human interleukin-2 (IL-2, Miltenyi Biotec) for 5 days to achieve the gradual depletion of non-T cell related lineages.

[0424] Short-term co-culture experiment

[0425] NVG-111 and AR_TCE variants were characterized using short-term co-culture assays to assess their T cell engagement abilities and their effects on ROR1. +Cytotoxic potential of the mantle cell lymphoma Jeko-1 cell line. To track target cells in co-culture, CellTrace was used according to the manufacturer's instructions. TM Jeko-1 cells were stained with Violet (CTV; C34557; Life Technologies). Jeko-1 cells were then mixed with an excess of purified human T cells (prepared as described above) to a target to effector ratio of 1:5 and co-cultured for 48 h in the presence of increasing concentrations of NVG-111 or AR_TCE.

[0426] At the end of the assay, the cells were centrifuged at 300 g for 5 minutes and washed once in PBS. The pellet was resuspended in a PBS solution containing Live / Dead fixable green dye (1:800 dilution, L23101, Life Technology) and APC-labeled anti-human CD69 antibody (1:50 dilution, 310910, Biolegend). The cells were then incubated at 4°C for 60 minutes, washed once in PBS, and then analyzed by flow cytometry using CytoFlex (Beckman Coulter Life Sciences). CTV staining gates were set to distinguish T cells (CTV-) and Jeko-1 cells (CTV+). Cytotoxicity was measured using the percentage of dead cells (live / dead marker staining positive) in the Jeko-1 population. T cell activation levels were measured using the mean fluorescence intensity (MFI) of the T cell population. All flow cytometry data were analyzed by FlowJo software (BD Biosciences).

[0427]

Long-term co-culture experiment

[0428] The effects of autoregulation on cytokine release were assessed in vitro using a long-term co-culture assay, using an excess of target cells to simulate continuous T cell engagement. Purified human T cells were prepared as described above and mixed with an excess of Jeko-1 cells at a target-effector ratio of 10:1. Cells were co-cultured for up to 120 h with or without the addition of 1 μg / mL NVG-111 or AR_TCE. Co-culture samples were collected every 24 h, and the cells were separated from the supernatant by centrifugation at 300 g for 5 minutes. The supernatant was collected and stored at -80°C for subsequent analysis. The cell pellet was washed once in PBS and incubated in Live / Dead fixable green dye solution as described above. The cells were washed again in PBS and fixed for 20 minutes at 4°C using CytoFix / CytoPerm reagent (BD Biosciences).

[0429] In order to assess cytotoxic potential and T cell activation level, anti-human CD3 antibody (1:50 dilution, 300417, Biolegend) and PE-labeled anti-human CD25 antibody (1:50 dilution, 356134, Biolegend) labeled fixed cells were marked using the aforementioned experimental method, and flow cytometry analysis was then further performed. T cells and target cells were distinguished using a gating strategy based on CD3. Cytotoxicity was measured using the percentage of dead cells in CD3 cell mass (Jeko-1 cells), and the average MFI of CD25 expression in T cell mass (CD3+) was used to measure T cell activation degree. According to the manufacturer's recommendation, ELISA MAX Deluxe set (Biolegend) was used to detect the people's INFγ released by activated T cells from co-culture supernatant. Using undiluted supernatant samples, the cutting of AR_TCE was detected by SDS-Page electrophoresis and protein L immunoblotting, and the method was as described above.

[0430] In vivo models of severe toxicities associated with TCE mechanism of action, such as cytokine release syndrome

[0431] The potential of AR_TCE to reduce the risk of severe toxicities associated with its mode of action and excessive T cell engagement was tested in an in-house mouse adoptive immunotherapy model, in which mice were transplanted with either triple-negative breast cancer (TBNC) solid tumors or disseminated pancreatic cancer cells, both of which express ROR1. + .

[0432] First, a proprietary method was used to induce stable expression of NVG-111 or autoregulatory AR_TCE-3 variants in the circulation of 8-10 week old NOD.SCIDγ (NSG) male mice (Charles River Laboratory). For the TNBC model, 2×10 6 Cells of the MDA-MB-231 cell line were injected subcutaneously into the mammary fat pad of mice and the solid tumors formed were allowed to grow for 25 days. 6 PANC-1 cells were injected directly into the peritoneal cavity and mice were allowed to recover for 8 days. After tumors were established, all animals were treated with 6 (TNBC) or 4 (pancreatic cancer) cycles of purified human T cells. T cells were prepared as described above, resuspended in 100 μL PBS, and injected into the tail vein of the mice. Each mouse was given 10 7 T cells, the subsequent dose is halved, and the injection is carried out according to the "4 days / 3 days" interval plan.

[0433] Animal body weights were recorded daily during the T cell administration phase and normalized using the body weight measured on the day of the first injection (day 25 for TNBC and day 8 for pancreatic cancer). The study protocol had an ethical limit of 20% weight loss, so any animal weighing less than 1 gram was humanely culled. In addition, the general health of the mice and the appearance of solid tumors (in the TNBC model) were observed daily by staff at an independent animal facility; any signs of distress, inflammation, or tumor ulceration were grounds for humane culling of the animals.

[0434] In the TNBC model, the progression of solid tumors was monitored every 2 to 3 days. The average tumor diameter (d) was measured along two different axes using a micrometer and the spheroid formula V = (4 / 3) × π × (d / 2) was used. 2 Calculate volume (V). The average of the tumor volumes calculated on days 15 and 18 was used as a reference value for measuring tumor progression. In the pancreatic cancer model, bioluminescence imaging (BLI) was used to assess the tumor burden of luciferase-expressing PANC-1 cells. Mice were placed under gas anesthesia and injected intraperitoneally with 200 μg of D-luciferin (Melford Laboratories) diluted in 200 μL PBS per mouse. BLI images were collected 15 minutes after injection using the IVIS Lumina II in vivo imaging system (Perkin-Elmer). Image processing was performed using Fiji software.

[0435] At study completion or early sacrifice, mice were bled by cardiac puncture of the posterior vena cava under irreversible gas anesthesia (isoflurane) in EDTA (final concentration 0.05 M). All animals were euthanized humanely by cervical dislocation. Blood samples were centrifuged at 1500 g for 20 minutes to separate the plasma fraction and stored at −80°C. Human cytokine release was measured using ELISAs, including INFγ using the ELISA MAX Deluxe set (Biolegend) and granzyme B using the DuoSet system (DY-2906-05, Bio-Techne), according to the manufacturer's recommendations. In the TNBC model, tumors were explanted, photographed with a micrometer, and their volumes calculated as described above.

[0436] Quantification of metastatic progression

[0437] After the study was terminated, livers from mice transplanted with TNBC solid tumor models were explanted and fixed in 10% neutral buffered formalin (CellPath, Inc.) for 24 hours at room temperature before processing into paraffin blocks. Paraffin sections were prepared for immunofluorescence, dewaxed in xylene for 3 × 5 minutes, rehydrated with 100% ethanol washes for 3 × 5 minutes, and equilibrated in H2O. Sections were washed in PBS for 10 minutes and then incubated in PBS + 0.05% Tween 20 (PBS.T) and 0.5% TritonX-100 for 10 minutes for detergent-based antigen retrieval. Sections were then washed in PBS.T for 3 × 5 minutes and then incubated with 3% (w / v) bovine serum albumin (BSA) in PBS.T for 1 hour at room temperature (RT) to block nonspecific binding sites. Sections were incubated overnight at 4°C with goat anti-luciferase (NB1000-1677SS, Novus Biologicals) diluted 1:200 in PBS.T + 1% BSA to detect infiltrating MDA-MB-231 cancer cells. Slides were washed 3 × 5 minutes in PBS and then incubated with Alexa-555 donkey anti-goat (A32816, Thermofisher) polyclonal antibody diluted 1:2000 in PBS.T + 1% BSA for 1 hour at room temperature on an orbital shaker. After a final 3 × 5 minute wash step, a drop of ProLong Diamond antifade reagent with DAPI (ThermoFisher Scientific) was applied to the slides, which were then mounted with coverslips and dried in the dark for 24 hours. Images were acquired using a PhenoImager HT slide scanner (Akoya Bioscience), and the diameter of metastatic nodules in each section was measured and quantified manually using Fiji software (open source software, GNU General Public License).

[0438] Preparation of CAR-T cells

[0439] Lentivirus encoding ROR1-targeted CAR receptor or AR_CAR variants was prepared by triple transfection of human embryonic kidney (HEK)-293T cell line using a second-generation lentiviral packaging system consisting of pMD2.G for viral envelope, pCMVδR8.2 for packaging, and a transgene plasmid containing the CAR expression cassette. Cells were cultured at 1.65×10 6Cells were plated at a density of 10 cells / dish in 10 cm dishes and incubated overnight in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 10% FBS. The seeded cells were transfected using GeneJuice (Millipore) according to the manufacturer's instructions. Supernatant containing functional lentivirus was harvested 48 hours later and used immediately or stored at -80°C.

[0440] Peripheral blood mononuclear cells were obtained from healthy volunteers and purified using Ficoll-Paque Plus (GE Healthcare) density gradients as previously described. Isolated PBMCs were activated overnight using human T-activator CD3 / CD28 Dynabeads (ThermoFisher) and 100 U / mL human IL-2 (Miltenyi). Transduction was performed in RetroNectin-coated 24-well plates (Takara). 3 × 10 cells were plated per well. 5 Each activated cell was cultured in 0.5 ml of RPMI medium supplemented with 10% FBS, and then 1.5 mL of supernatant containing lentivirus and 100 U / mL human IL-2 was added to each well. The transduced cells were incubated in RetroNectin-coated plates for 5 days, and the transduction efficiency was assessed using flow cytometry. The transduced cells were centrifuged at 300 g for 5 minutes and washed once in PBS. The cells were then incubated at 4 ° C for 60 minutes with PE-labeled protein L (ACROBiosystems) diluted 1:50 in PBS to detect the expression of CAR receptors. The stained cells were washed once in PBS and analyzed by flow cytometry using CytoFlex (Beckman Coulter Life Sciences).

[0441] Granzyme B cleavage of CAR-T cells

[0442] Granzyme B-mediated cleavage of the AR_CAR receptor was evaluated in vitro. 1×10 5 Each CAR-T cell was resuspended in 200 μL of RPMI medium and incubated with 100 nM activated human granzyme B at 37°C for 2 h. The cells were centrifuged at 300 g for 5 minutes and washed once in PBS. The presence of residual CAR receptors on the cell surface was determined by flow cytometry using the protein L assay as described previously.

[0443] Short-term co-culture study with CAR-T cells

[0444] AR_CAR-T cells were tested using a short-term co-culture assay similar to that described above for TCE. + Jeko-1 cells were mixed with various amounts of CAR-T cells to achieve an effector-to-target cell ratio ranging from 0.04:1 (target cell excess) to 10:1 (CAR-T cell excess) and then co-cultured for 48 hours. Cytotoxicity was measured by the percentage of target cell death, with dead cells distinguished by positive staining for the Live / Dead marker, while the degree of CAR-T cell activation was measured by measuring the mean fluorescence intensity (MFI) of cell surface CD69 staining using flow cytometry.

[0445]

Long-term co-culture study with CAR-T cells

[0446] In long-term co-culture experiments, the continuous engagement and killing capabilities of CAR-T cells were tested using excess target cells. CAR-T cells were mixed with excess Jeko-1 cells at a 1:10 effector to target cell ratio and co-cultured for up to 5 days. Co-culture samples were harvested every 24 hours, and target cell death was measured by flow cytometry as described previously.

[0447] Example 3: Design of a prototype FVIII-mimicking bispecific antibody embedded with an autoregulatory peptide

[0448] Prothrombin (coagulation factor II) is a core member of the coagulation cascade, and it is one of the main products of the coagulation process that prothrombin activates and forms thrombin (FIIa). The abnormal rise of the level of this enzyme is also the hallmark feature of thrombosis and prothrombotic events. Therefore, utilizing the rising of thrombin enzymatic activity as having FVIII mimetic activity as trigger with autoregulation bispecific antibodies (such as emicizumab) has great potential in the generation of prevention thrombotic events.

[0449] To design AR_Ab8, a prototype FVIII-mimicking bispecific antibody with autoregulatory capacity controlled by thrombin levels, an 8-amino acid peptide (LTPRGVRL) from P4 to P4' was inserted into the sequence of emicizumab. This 8-amino acid peptide was identified by Gallwitz and his collaborators as a consensus recognition sequence for thrombin-mediated cleavage. To achieve degradation and inactivation of the autoregulatory bispecific antibody, the peptide (Gallwitz, Enoksson et al., 2012) was inserted between the K junction of the hinge region of the two heavy chains. 228 and Y 229 Between residues ( Figure 2 ). Thus, thrombin-mediated cleavage produces three non-functional fragments, comprising the Fc region and two isolated Fab domains.

[0450] After production and purification, the migration patterns of AR_Ab8 and emicizumab were compared by electrophoresis under non-reducing conditions. Both constructs showed similar characteristics, with the intact antibody dominating ( Figure 3 A). In particular, no degradation products were observed in AR_Ab8, indicating that premature nonspecific cleavage of the thrombin-sensitive peptide did not occur during its production and purification. [Example 4: In vitro procoagulant activity of a prototype FVIII-mimicking antibody with thrombin-mediated autoregulation]

[0451] To assess whether structural changes in the hinge region caused by the insertion of the thrombospondin would have a disruptive effect on therapeutic function, the FVIII mimetic potential of AR_Ab8 and emicizumab was evaluated in vitro.

[0452] In the chromogenic substrate activity assay, both antibodies successfully catalyzed the FIXa-mediated conversion of FX to FXa, and their kinetic curves overlapped ( Figure 3 B), indicating that the insertion of a cleavable peptide into AR_Ab8 does not affect its ability to bridge FIXa and FX and thus restore FVIII-like activity. In addition, compared with the pooled plasma of healthy volunteers, the clotting time of the aPTT test in FVIII-deficient human plasma was severely prolonged (mean ± SD; 95.6 ± 12.6 vs 29.6 ± 0.7 s, respectively). The addition of 350 nM of emicizumab and AR_Ab8 corrected the clotting time (22.0 ± 0.3 and 28.1 ± 2.0, respectively) to a level comparable to that of normal controls, and there was no significant difference between the two antibodies ( Figure 3 C).

[0453] Example 5: Undiminished Therapeutic Potential of Autoregulating FVIII Mimetic Antibodies in Vivo

[0454] The procoagulant potential of AR_Ab8 and its parent antibody, emicizumab, was subsequently tested in vivo using a bleeding assay in a hemophilia A mouse model (FVIIIKO) and specifically optimized for FVIII-mimicking bispecific antibodies, as described by Ferriere and his collaborators (Ferriere, Peyron et al., 2020). In this model, untreated FVIIIKO animals exhibited a strong bleeding phenotype with an average bleeding volume of 900 ± 92 μL (mean ± SD) if the tail was cut 3 mm distal to the tail and bleeding was monitored within 30 minutes. Control animals treated with 2 U of recombinant human FVIII per mouse showed complete correction of the bleeding trend ( Figure 4A). As expected, when mice were pretreated with a single intravenous injection of 3 mg / kg emicizumab 24 h before the study, partial correction of bleeding was observed, with a mean blood loss of 677 ± 198 μL. Interestingly, treatment with an equivalent dose of AR_Ab8 corrected the bleeding phenotype to the same extent as the parental antibody emicizumab, with a mean blood loss of 609 ± 182 μL, thus indicating that the insertion of the thrombospondin into the core structure of the bispecific antibody does not compromise its procoagulant potential and therapeutic efficacy ( Figure 4 B).

[0455] [Example 6: Thrombin-mediated inactivation of autoregulatory FVIII mimetic antibodies]

[0456] Insertion of a thrombin-cleavable peptide into the structure of AR_Ab8 renders this prototypical autoregulatory antibody inactivated and degraded upon exposure to high concentrations of thrombin. Therefore, the sensitivity of AR_Ab8 to thrombin-mediated cleavage was assessed in vitro.

[0457] When exposed to 2 U / mL human α-thrombin for up to 180 minutes, the parental antibody emicizumab (2 nM) showed no signs of cleavage, and only bands corresponding to intact antibody were detected by Western blotting ( Figure 5 A). In contrast, AR_Ab8 was gradually cleaved, and its degradation fragments were detectable after 15 minutes of exposure. In addition, the appearance of two groups of fragments at approximately 90-100 kDa and 50-55 kDa indicated that both autoregulatory peptides present in AR_Ab8 were effectively cleaved, and the autoregulatory antibody was almost completely degraded after 180 minutes of exposure ( Figure 5 A). These results were confirmed by assessing the residual FVIII mimetic activity of each sample after exposure to α-thrombin using a chromogenic substrate assay. As expected, the activity of emicizumab remained unchanged, while AR_Ab8 showed a significant decrease in FVIII mimetic activity after 30 minutes of α-thrombin exposure, followed by a gradual decrease in residual activity. After 180 minutes of exposure, almost complete inactivation was achieved, with the average residual activity reduced to only 1.5 ± 1.3% of that of emicizumab ( Figure 5 B). The sensitivity of several cleavable peptides to thrombin exposure was then compared. The FVIII mimetic antibody constructs were designed as bispecific tandem-scFvs and contained (or did not contain) different thrombin-sensitive peptides. The peptides tested were: the aforementioned LTPRGVRL consensus sequence, as well as peptides with a single amino acid change at the P1 position (LTPRDVRL and LTPRLVRL), and the natural cleavage site of human coagulation factor V (FV) at position R1753 (WYLRSNNG). After exposure to α-thrombin for 20 minutes, these peptides showed different levels of sensitivity, as shown by the residual levels of FVIII mimetic activity ( Figure 5C). Indeed, the consensus (LTPRGVRL) cleavage site showed the highest sensitivity, with only 17.5% residual activity; in contrast, the LVRL site showed much lower sensitivity to thrombin exposure, with a residual FVIII mimetic activity of 56.7%, while the DVRL site showed no cleavage at all. Interestingly, the naturally occurring FV_R1573 cleavage site showed only minimal inactivation upon exposure to thrombin, retaining 88.7% of FVIII mimetic activity after 20 minutes of exposure ( Figure 5 C).

[0458] To determine whether thrombin-mediated inactivation could effectively reduce the thrombotic risk of FVIII mimetic antibodies, thrombin generation curves of AR_Ab8 and emicizumab were tested in the presence of activated prothrombin complex concentrate (aPCC). This assay, previously described by Hartmann and collaborators (Hartmann, Feenstra et al., 2018), mimics the prothrombotic potential of emicizumab observed clinically when co-administered with this bypassing agent. Indeed, the addition of 600 nM emicizumab and 0.5 U / mL aPCC to hemophilia A plasma resulted in abnormally high levels of thrombin generation, with a maximum peak increase of 1.79-fold compared to normal pooled plasma ( Figure 6 A~B). However, in the presence of AR_Ab8, thrombin generation was significantly reduced, and its peak value was not significantly different from that in normal plasma (1.15 times), indicating that autoregulation was effectively triggered to prevent excessive thrombin generation ( Figure 6 B).

[0459] Example 7: Thrombin-mediated autoregulation reduces the prothrombotic phenotype associated with FVIII mimetic antibodies in vivo

[0460] The potential of thrombin-mediated autoregulation in controlling the therapeutic activity of FVIII-mimicking bispecific antibodies and reducing the risk of thrombosis was then evaluated in vivo using a mouse model that replicates the prothrombotic potential of emicizumab associated with aPCC. C57Bl / 6 mice were given an initial injection of 2.5U aPCC alone or in combination with 250μg of emicizumab or AR_Ab8. All animals subsequently received three additional aPCC administrations every 24h to maintain a sustained increase in bypass agent levels and enhance the prothrombotic phenotype ( Figure 7 A).

[0461] Blood samples were collected from mice 30 minutes after the last aPCC injection to measure total platelet counts. As expected, the combination of emicizumab and aPCC induced a significant platelet-consuming coagulopathy, with the average platelet count dropping to 621×10 3 platelets / μL, while the control group using aPCC alone had 908×10 3 Platelets / μL ( Figure 7 B) However, in mice treated with aPCC and AR_Ab8, platelet counts in peripheral blood were completely corrected, with an average of 958×10 3 platelets / μL, which was not significantly different from the control group, thus indicating that thrombin-mediated autoregulation effectively controlled the risk of coagulopathy associated with FVIII mimetic antibodies ( Figure 7 B).

[0462] Thrombosis in the lungs was subsequently assessed using immunofluorescence microscopy ( Figure 8 ). Immunofluorescence staining for PECAM (blood vessels) and integrin aIIb (platelets) was then performed to further confirm that these structures were platelet-rich thrombi that partially blocked pulmonary vasculature ( Figure 8 A). These immunofluorescence-stained images were then used to define a quantitative mask and calculate the percentage of vascular occlusion, defined as the percentage of platelet-positive staining within the vessel-delineated area ( Figure 8 Blinded quantification of lung sections showed a significant increase in mean occlusion rate in mice treated with emicizumab compared to the control group that received aPCC alone, with several vessels exhibiting luminal occlusion exceeding 20% ​​( Figure 8 B). In contrast, mice treated with the autoregulatory antibody AR_Ab8 had a reduced incidence of highly obstructed vessels, with the distribution of obstruction rates closer to that of the control group ( Figure 8 B), again demonstrating that thrombin-mediated autoregulation can reduce the risk of adverse events associated with FVIII mimetic antibodies.

[0463] [Example 8: Design of a ROR1×CD3 bispecific adaptor containing a granzyme B-sensitive autoregulatory peptide]

[0464] To demonstrate that autoregulation can also be achieved using nonclassical antibody formats, in another setting, a bispecific T-cell engager (TCE) embedded with an autoregulatory element was designed to prevent the occurrence of adverse events related to its mechanism of action (MoA), which is one of the major risks associated with this class of therapeutics. Granzyme B is an enzyme unique to immune cells and, in T-cell redirecting immunotherapies such as TCE, is released by activated T cells. Interestingly, circulating levels of granzyme B are dramatically increased in patients who develop severe MoA-related adverse events, making this enzyme a promising target for triggering TCE autoregulation and reducing the risk of MoA-related toxicities associated with this class of T-cell redirecting immunotherapies.

[0465] The design of the autoregulatory bispecific T cell engager (AR_TCE) is based on NVG-111, a ROR1×CD3 bispecific tandem scFv (td_scFv). The central GSGGGGS linker was replaced by a granzyme B cleavable peptide (8 amino acids in length), flanked by a GGGGS extender ( Figure 9 A). Several granzyme B-sensitive peptides were first tested. AR_TCE-1 and AR_TCE-2 were designed with GZMB-1 and GZMB-2 peptides, respectively, derived from the P4 to P4' granzyme B cleavage sequence of the mouse or human BH3-interacting domain death agonist (BID), a well-described granzyme B target. AR_TCE-3 obtained the GZMB-3 peptide derived from the granzyme B cleavage consensus sequence previously described by Wee and coworkers (Wee, Er et al., 2011). Figure 9 B). All constructs were expressed and purified from mammalian cells, and their migration patterns were compared to those of the parent antibody NVG-111 by electrophoresis under reducing conditions. All constructs showed a discrete band at approximately 55 kDa, which corresponds to the expected size of a td-scFv antibody, indicating that the granzyme B sensitive peptide was not prematurely cleaved and the autoregulatory construct was not degraded ( Figure 9 C).

[0466] [Example 9: Autoregulation of the Cytotoxicity and T Cell Engagement Potential of AR_TCE]

[0467] The cytotoxicity and T cell engagement abilities of AR_TCE and its parent NVG-111 were evaluated in parallel using a short-term killing assay. + Jeko-1 target cells were co-incubated with excess purified human T cells at a target-effector ratio of 5:1 and in the presence of increasing concentrations of either antibody for 48 h. By detecting the expression level of cell surface CD69, both AR_TCE-1 and AR_TCE-3 showed T cell activation potential similar to that of the parent NVG-111 ( Figure 10 A). In addition, all three constructs induced comparable T cell-mediated killing of target cells ( Figure 10 B), thus demonstrating that insertion of a granzyme B-cleavable peptide into the linker of the autoregulatory AR_TCE does not affect its mechanism of action in vitro.

[0468] [Example 10: Autoregulation enables threshold-dependent inactivation of TCE by exposure to granzyme B]

[0469] To determine whether the granzyme B-cleavable peptide inserted into the AR_TCE provides efficient sensitivity to granzyme B exposure, several AR_TCE variants were tested using an in vitro cleavage assay. After incubation with 100 nM recombinant activated granzyme B for 2 h, both AR_TCE-1 and AR_TCE-2 showed partial cleavage, with faint bands between 25 and 30 kDa detected by electrophoresis and protein L immunoblotting, corresponding to the cleaved single scFv. The remaining intact body was still located at around 55 kDa ( Figure 11 A). In contrast, the AR_TCE-3 variant showed significantly higher sensitivity to granzyme B, achieving complete cleavage within 2 h, and no intact body was detected ( Figure 11 A).

[0470] Subsequent ELISA assays to detect the remaining intact antibody showed that, as expected, exposure to increasing concentrations of granzyme B had no effect on the parental antibody NVG-111 ( Figure 11 In contrast, the level of intact antibody for AR_TCE-1 decreased slightly at the highest concentration of 100 nM, consistent with the partial degradation observed by Western blotting, while AR_TCE-3 was gradually cleaved with increasing concentrations of granzyme B until it was completely inactivated ( Figure 11 B). This observation emphasizes that the autoregulatory inactivation mechanism in TCE is dose-dependent and threshold-mediated, i.e., AR_TCE-3 remains intact at low levels of granzyme B but becomes completely inactivated at high concentrations, which are associated with a higher risk of serious MoA-related adverse events.

[0471] Example 11: Specificity and sensitivity of granzyme B-cleavable peptides for autoregulation

[0472] To further explore the relationship between the sequence of the autoregulatory peptides and their sensitivity to granzyme B exposure, several additional peptides were generated from the consensus peptide GZMB-3 by adjusting the P4 to P4' sequences. These new peptides (GZMB-4 to -14) were further used to generate other AR_TCE variants as previously described ( Figure 12Interestingly, upon exposure to a single dose of granzyme B (50 nM), the different autoregulatory peptides displayed a wide range of cleavage sensitivities, ranging from constructs similar to the parental antibody NVG-111 (AR_TCE-2, -13) that showed little sign of inactivation, to highly sensitive variants at the other end of the spectrum (AR_TCE-3, -8), which retained only approximately 25% of the intact antibody after 2 h of exposure ( Figure 12 A) This diversity in granzyme B sensitivity supports the possibility of fine-tuning the dynamics of the autoregulatory element and selecting a specific sequence that inactivates the AR_TCE only at granzyme B levels relevant to MoA-associated adverse events.

[0473] In addition, the specificity of the GZMB-3 peptide as a substrate for the proteolytic activity of granzyme B was evaluated in vitro. AR_TCE-3 was exposed for 2 hours to a panel of equimolar concentrations (50 nM) of proteases, which were selected based on their reported presence in the circulation or tumor microenvironment and as representatives of different protease families. In this experiment, AR_TCE-3 showed a unique proteolytic sensitivity to granzyme B, i.e., the molecule was significantly inactivated upon exposure to granzyme B, while remaining intact upon exposure to any of the other proteases in the test panel ( Figure 12 B) This selectivity for the protease used to trigger autoregulation significantly reduces the risk of nonspecific premature inactivation of AR_TCE.

[0474] Example 12: Granzyme B-mediated autoregulation reduces TCE-induced cytokine release in vitro

[0475] Two AR_TCEs with low (AR_TCE-1) and high (AR_TCE-3) sensitivity to granzyme B exposure were characterized alongside NVG-111 in a long-term killing assay assessing continuous target cell killing and progressive T cell engagement. + Jeko-1 target cells (effector-target cell ratio 1:10) were co-cultured in the presence of bispecific antibodies. Figure 10 ) were similar to the observations in the previous study, the cytotoxic potential of all three antibodies was the same within 120 h of co-culture, with maximum target cell death rates of 60.5, 58.7, and 55.7% for NVG-111, AR_TCE-1, and AR_TCE-3, respectively ( Figure 13A). However, as measured by increased cell surface expression of CD25 (IL-2 receptor α subunit), long-term T cell activation levels in AR_TCEs stabilized after 96 hours compared to NVG-111, with the strongest effect observed in the highly sensitive AR_TCE3 molecule. In fact, at 120 hours, mean CD25 expression levels were reduced by 14.2% and 21.5% in AR_TCE-1 and AR_TCE-3, respectively ( Figure 13 B).

[0476] Furthermore, the marked reduction in T cell activation was associated with a strong and significant reduction in human interferon gamma (INFγ) release. At 120 h, the levels of INFγ released by T cells co-cultured with AR_TCE-1 and AR_TCE-3 were equivalent to 77.3% and 52.3% of those co-cultured with parental NVG-111, respectively. Figure 13 C). This reduction in cytokine release was also associated with the observed autoregulation and inactivation of AR_TCE. In fact, NVG-111 remained intact in the co-culture supernatant throughout the assay, whereas AR_TCE-1 and AR_TCE-3 showed evidence of cleavage, i.e., the presence of fragmented by-products ( Figure 13 D). As expected, for the less sensitive AR_TCE-1 construct, antibody fragments were detected only at 120 h, whereas for the more sensitive AR_TCE-3 molecule, inactivation was observed as early as 72 h ( Figure 13 D) In ​​summary, these results demonstrate that antibody engineering to incorporate granzyme B-mediated autoregulatory mechanisms into TCEs can significantly reduce cytokine release induced by T cell engagement and activation while preserving their cytotoxic potential. Furthermore, the inactivation kinetics of the antibody, and thus its effect on cytokine release, can be modified by careful selection of the autoregulatory peptide sequence.

[0477] [Example 13: Autoregulation reduces the occurrence of severe toxicities associated with the TCE mechanism of action (e.g., cytokine release syndrome) in a triple-negative breast cancer model]

[0478] The mechanism of granzyme B-mediated autoregulation was further investigated in an in vitro model that recapitulates the severe toxicity associated with TCE-associated MoA. In this model, immunocompromised NSG mice were induced to express stable levels of NVG-111 or AR_TCE-3 variants at concentrations (mean range 0.66-2.01 μg / mL) known to induce rapid toxicity in the unregulated parental antibody. Figure 14 A) ROR1 was then transplanted subcutaneously into mice +The solid tumors of the MDA-MB-231 triple-negative breast cancer (TNBC) cell line were treated with purified human T cells for 6 cycles for a total of 21 days (from day 25 to day 46).

[0479] Weight loss and animal survival are the main indicators of TCE-induced toxicity in this model. As expected, animals treated with NVG-111 showed rapid signs of toxicity 24 hours after the second T cell injection, manifested by a sudden and significant loss of total body weight on day 30 ( Figure 14 B). Subsequent increased weight loss and overall deterioration of the animals' health led to the humane culling of four of the six mice by day 36 ( Figure 14 C), the remaining animals were removed from the protocol. In contrast, animals treated with the autoregulatory AR_TCE-3 molecule showed no signs of toxicity and had stable body weight fluctuations like the vehicle group ( Figure 14 Therefore, all animals in this group were retained in the experimental protocol until the end of the study on day 46 ( Figure 14 C). Interestingly, the results indicate that autoregulation responds rapidly to granzyme B release and can control the onset of toxicity within 24 hours of T cell injection. Human cytokine release from T cells was also measured in plasma samples collected on the day of sacrifice in both groups of mice. Circulating levels of human IFNγ and granzyme B were significantly reduced in animals treated with AR_TCE-3 compared to NVG-111, thus suggesting that reduced T cell activation is associated with improved survival ( Figure 15 ).

[0480] Example 14: Autoregulation of AR_TCE in vivo exhibits intact anti-tumor efficacy

[0481] To assess whether the reduced toxicity and cytokine release observed in mice treated with autoregulatory TCEs correlated with reduced therapeutic efficacy, tumor progression was measured in each group. AR_TCE-3 demonstrated significant unimpaired efficacy, as evidenced by significantly slower tumor progression compared to the vehicle group, detectable as early as day 34 ( Figure 16 A). In fact, at the completion of the study, tumor volume in the vehicle group had increased by 18.5 ± 0.94 times (mean ± SD) compared to that on day 15, but in the AR_TCE-3 treated animals it had increased by only 8.0 ± 3.4 times. In addition, in 3 of 6 mice in the vehicle group, tumors had progressed to the ulcer stage, triggering early humane euthanasia on day 40 ( Figure 16 A). These data are supported by postmortem tumor explant volume measurements ( Figure 16 B), the average of the medium group is 910mm 3 , while in animals treated with AR_TCE-3 only 578 mm3 ( Figure 16 Interestingly, although the data are incomplete because mice treated with NVG-111 were sacrificed early, tumor progression in this group did not differ compared to mice receiving AR_TCE-3 until day 36 (4.6 ± 2.3- and 5.2 ± 1.7-fold progression, respectively), supporting the idea that autoregulatory AR_TCE efficacy was not compromised ( Figure 16 A).

[0482] [Example 15: Autoregulation achieves sustained treatment with AR_TCE and reduces TNBC metastatic proliferation in vivo]

[0483] The improved safety profile of AR_TCE-3 compared to the parental NVG-111 allowed for a longer treatment period in mice ( Figure 14 Therefore, the metastatic progression of the TNBC model was used to evaluate the long-term therapeutic effect of continuous exposure to AR_TCE-3. The liver surface of animals in the vehicle group showed obvious metastatic nodules ( Figure 17 A), which can be confirmed by the presence of clusters of cancer cells in liver sections ( Figure 17 B), whereas such nodules were not seen in mice treated with AR_TCE-3. Quantification of metastatic nodules by immunofluorescence staining further showed that the number of large cancer cell clusters (d>100 μm) infiltrating the liver tissue of animals treated with AR_TCE-3 was significantly reduced compared with that of mice in the vehicle group ( Figure 17 C), thus demonstrating that AR technology does not affect therapeutic efficacy, but the reduced toxicity compared to NVG-111 can extend the therapeutic window.

[0484] In summary, the results obtained using this severe toxicity model relevant to the TCE mechanism of action suggest that the inherent risk of serious adverse events associated with T-cell engager therapies can be effectively managed through built-in autoregulation of granzyme B-cleavable peptides, thereby enabling prolonged treatment and sustained therapeutic effects on tumor progression and metastatic dissemination.

[0485] Example 16: Autoregulation Improves Survival in Vivo While Maintaining Therapeutic Efficacy in a Pancreatic Cancer Model

[0486] The results obtained in the TNBC model were further confirmed in another cancer type using a shorter xenograft model established by intraperitoneal injection of ROR + The PANC-1 pancreatic cancer cell line was treated with four cycles of human T cells ( Figure 18A). Similar to the results in the TNBC xenograft model, NVG-111-treated mice showed rapid signs of toxicity following T cell administration, as evidenced by increased mortality, with 4 of 6 mice requiring humane culling between days 16 and 20. In contrast, only 1 of 6 animals treated with the autoregulatory AR_TCE-3 required humane culling by day 21 ( Figure 18 B).

[0487] To assess cytokine release in response to T cell engagement, a single blood draw was performed on mice in all groups 24 h after the second injection (e.g., day 13). Compared with NVG-111, mice treated with AR_TCE-3 showed a significant decrease in granzyme B levels and a significant decrease in IFNγ release, indicating that the autoregulatory mechanism of AR_TCE-3 effectively controlled T cell activation within a 24-h time window ( Figure 18 C). The therapeutic effect of AR_TCE was further verified by bioluminescent reading of tumor progression. Three of the five animals in the vehicle group had multiple tumor proliferation foci at the end of the study (day 22), while all mice treated with AR_TCE-3 had cleared tumor progression ( Figure 19 ).

[0488] [Example 17: Application of Granzyme B-Mediated Autoregulation in a ROR1-Targeted CAR-T Cell Model]

[0489] To demonstrate the applicability of autoregulation to genetically engineered cells in the context of T cell redirection therapy, an autoregulatory AR_CAR-T cell variant was designed using ROR1-targeted CAR-T cell therapy. The eight residues (sequence P4 to P4') of the granzyme B-sensitive peptide GZMB-3 used for autoregulation of AR_TCE-3 were inserted into the upper region of the chimeric receptor ( Figure 20 A). The autoregulatory peptide was inserted directly between the ROR1-targeting scFv and the hinge region of the chimeric receptor (AR_CAR-T27) or by replacing the first five residues of the hinge region (AR_CAR-T22).

[0490] Both the autoregulatory AR_CAR-T variants and the parental CAR-T cells were generated by lentiviral infection of PBMCs obtained from healthy donors. The CAR-T cells were then exposed to 100 nM activated human granzyme B for 2 h in vitro to assess the sensitivity of the AR_CAR-T variants to the proteolytic activity of granzyme B. When evaluated using flow cytometry, both AR_CAR-T22 and AR_CAR-T27 showed significant cleavage and removal of the chimeric receptor from the cell surface, with protein L detecting 39.5% and 38.0% of the residual CAR receptor ( Figure 20 B). In contrast, parental CAR-T cells were unaffected by granzyme B exposure.

[0491] [Example 18: Autoregulation maintains the cytotoxic potential and continuous engagement ability of AR_CAR-T cells]

[0492] The cytotoxicity of AR_CAR-T cells and parental ROR1-targeted CAR-T cells was compared using a short-term killing assay at different cell concentrations. + The Jeko-1 target cell line was co-incubated with CAR-T cells for 48 h using effector-target cell ratios ranging from 0.04 (target cell excess) to 10 (CAR-T cell excess). Interestingly, for all tested cell ratios, both autoregulatory AR_CAR-T cell variants showed similar activation levels to the parental CAR-T cells ( Figure 21 A). In addition, activation levels correlated with comparable target cell killing ( Figure 21 B) The function and ability of autoregulatory AR_CAR-T cells to promote continuous killing were then evaluated using a long-term killing assay, which reflects the gradual CAR-T cell engagement. + Jeko-1 target cells (effector-target cell ratio 1:10) were co-cultured for up to 5 days. Similar to previous observations, the engagement and cytotoxicity of CAR-T cells were not affected in the presence of autoregulatory elements. Progressive killing was observed, as evidenced by the highest target cell death rate observed at 120 h, with AR_CAR-T22, AR_CAR-T27, and parental CAR-T cells at 72.4, 74.8, and 79.1%, respectively ( Figure 22 ).

[0493] These results demonstrate that engineering the CAR receptor to introduce a granzyme B-sensitive peptide does not impair CAR-T cell function, thus suggesting that autoregulation can be successfully used in genetically engineered CAR-T cells to achieve granzyme B-mediated inactivation and reduce the risk of serious adverse events related to the CAR-T cell mechanism of action.

[0494] [Sequence Listing]

[0495] [SEQ ID NO: 1: Thrombin cleavage recognition sequence consensus sequence]

[0496]

[0497] [SEQ ID NO: 2: Thrombin cleavage recognition sequence P1'G to L]

[0498]

[0499] [SEQ ID NO: 3: Thrombin cleavage recognition sequence P1'G to D]

[0500]

[0501] [SEQ ID NO: 4: Thrombin cleavage recognition sequence from human coagulation factor V (FV)]

[0502]

[0503] [SEQ ID NO: 5: Granzyme B cleavage recognition sequence GZMB-1, from mouse BID protein]

[0504]

[0505] [SEQ ID NO: 6: Granzyme B cleavage recognition sequence GZMB-2, from mouse BID protein]

[0506]

[0507] [SEQ ID NO: 7: Granzyme B cleavage recognition sequence GZMB-3, consensus sequence]

[0508]

[0509] [SEQ ID NO: 8: Granzyme B cleavage recognition sequence P10 to P10' sequence of GZMB-4 and GZMB-2]

[0510]

[0511] [SEQ ID NO: 9: Granzyme B cleavage recognition sequence GZMB-5, P4 position I changed to V]

[0512]

[0513] [SEQ ID NO: 10: Granzyme B cleavage recognition sequence GZMB-6, P4 position I changed to L]

[0514]

[0515] [SEQ ID NO: 11: Granzyme B cleavage recognition sequence GZMB-7, P2 position P changed to G]

[0516]

[0517] [SEQ ID NO: 12: Granzyme B cleavage recognition sequence GZMB-8, P at position P2 changed to A]

[0518]

[0519] [SEQ ID NO: 13: Granzyme B cleavage recognition sequence GZMB-9, S at P1' changed to A]

[0520]

[0521] [SEQ ID NO: 14: Granzyme B cleavage recognition sequence GZMB-10, L at P2' changed to E and E at P4' changed to Q]

[0522]

[0523] [SEQ ID NO: 15: Granzyme B cleavage recognition sequence GZMB-11, L at P2' changed to E and E at P4' changed to V]

[0524]

[0525] [SEQ ID NO: 16: Granzyme B cleavage recognition sequence GZMB-12, D at P1 changed to N]

[0526]

[0527] [SEQ ID NO: 17: Granzyme B cleavage recognition sequence GZMB-13, P1 position D changed to E]

[0528]

[0529] [SEQ ID NO: 18: Granzyme B cleavage recognition sequence GZMB-14, D at P1 changed to Q]

[0530]

[0531] [SEQ ID NO: 19: ROR1 binding antigen binding domain LCDR1]

[0532]

[0533] [SEQ ID NO: 20: ROR1 binding antigen binding domain LCDR2]

[0534]

[0535] [SEQ ID NO: 21: ROR1 binding antigen binding domain LCDR3]

[0536]

[0537] [SEQ ID NO: 22: ROR1 binding antigen binding domain HCDR1]

[0538]

[0539] [SEQ ID NO: 23: ROR1 binding antigen binding domain HCDR2]

[0540]

[0541] [SEQ ID NO: 24: ROR1 binding antigen binding domain consensus HCDR3]

[0542] XXHRYNLFDS (where X1 is A or T and X2 is S, K, or R)

[0543] [SEQ ID NO: 25: ROR1 binding antigen binding domain HCDR3, clone F heavy chain CDR3]

[0544]

[0545] [SEQ ID NO: 26: ROR1 binding antigen binding domain HCDR3, humanized 1 heavy chain CDR3]

[0546]

[0547] [SEQ ID NO: 27: ROR1 binding antigen binding domain HCDR3, humanized 2 and 5 heavy chain CDR3]

[0548]

[0549] [SEQ ID NO: 28: ROR1 binding antigen binding domain HCDR3, humanized 3 and 4 heavy chain CDR3]

[0550]

[0551] [SEQ ID NO: 29: ROR1 binding antigen binding domain, clone F light chain variable region (CDRs underlined)]

[0552]

[0553] [SEQ ID NO: 30: ROR1 antigen binding domain, humanized 1 light chain variable region (CDRs underlined)]

[0554]

[0555] [SEQ ID NO: 31: ROR1 binding antigen binding domain, humanized 2 light chain variable region (CDRs underlined)]

[0556]

[0557] [SEQ ID NO: 32: ROR1 binding antigen binding domain, humanized 3 light chain variable region (CDRs underlined)]

[0558]

[0559] [SEQ ID NO: 33: ROR1 binding antigen binding domain, humanized 4 light chain variable region (CDRs underlined)]

[0560]

[0561] [SEQ ID NO: 34: ROR1 binding antigen binding domain, humanized 5 light chain variable region (CDRs underlined)]

[0562]

[0563] [SEQ ID NO: 35: ROR1 binding antigen binding domain, clone F heavy chain variable region (CDRs underlined)]

[0564]

[0565] [SEQ ID NO: 36: ROR1 antigen binding domain, humanized 1 heavy chain variable region (CDRs underlined)]

[0566]

[0567] [SEQ ID NO: 37: ROR1 binding antigen binding domain, humanized 2 heavy chain variable region (CDRs underlined)]

[0568]

[0569] [SEQ ID NO: 38: ROR1 binding antigen binding domain, humanized triple heavy chain variable region (CDRs underlined)]

[0570]

[0571] [SEQ ID NO: 39: ROR1 binding antigen binding domain, humanized 4 heavy chain variable regions (CDRs are underlined)]

[0572]

[0573] [SEQ ID NO: 40: ROR1 binding antigen binding domain, humanized 5 heavy chain variable region (CDRs underlined)]

[0574]

[0575]

[0576] [SEQ ID NO: 41: CD3 binding antigen binding domain, mouse light chain variable region (CDRs are underlined)]

[0577]

[0578] [SEQ ID NO: 42: CD3 binding antigen binding domain, humanized 1 light chain variable region (CDRs are underlined)]

[0579]

[0580] [SEQ ID NO: 43: CD3 binding antigen binding domain, humanized 2 light chain variable region (CDRs underlined)]

[0581]

[0582] [SEQ ID NO: 44: CD3 binding antigen binding domain, humanized 3 light chain variable region (CDRs are underlined)]

[0583]

[0584] [SEQ ID NO: 45: CD3 binding antigen binding domain, humanized 4 light chain variable region (CDRs are underlined)]

[0585]

[0586] [SEQ ID NO: 46: CD3 binding antigen binding domain, humanized 5 light chain variable region (CDRs are underlined)]

[0587]

[0588] [SEQ ID NO: 47: CD3 binding antigen binding domain, mouse heavy chain variable region (CDRs are underlined)]

[0589]

[0590] [SEQ ID NO: 48: CD3 binding antigen binding domain, humanized 1 heavy chain variable region (CDRs underlined)]

[0591]

[0592] [SEQ ID NO: 49: CD3 binding antigen binding domain, humanized 2 heavy chain variable region (CDRs are underlined)]

[0593]

[0594] [SEQ ID NO: 50: CD3 binding antigen binding domain, humanized 3 heavy chain variable region (CDRs are underlined)]

[0595]

[0596] [SEQ ID NO: 51: CD3 binding antigen binding domain, humanized 4 heavy chain variable regions (CDRs are underlined)]

[0597]

[0598] [SEQ ID NO: 52: CD3 binding antigen binding domain, humanized 5 heavy chain variable region (CDRs are underlined)]

[0599]

[0600] [SEQ ID NO: 53: CD3 binding antigen binding domain LCDR1]

[0601]

[0602] [SEQ ID NO: 54: CD3 binding antigen binding domain LCDR2]

[0603]

[0604] [SEQ ID NO: 55: CD3 binding antigen binding domain LCDR3]

[0605]

[0606] [SEQ ID NO: 56: CD3 binding antigen binding domain HCDR1]

[0607]

[0608] [SEQ ID NO: 57: CD3 binding antigen binding domain HCDR2]

[0609]

[0610] [SEQ ID NO: 58: CD3 binding antigen binding domain HCDR3]

[0611]

[0612] [SEQ ID NO: 59: linker sequence]

[0613]

[0614] [SEQ ID NO: 60: linker sequence]

[0615]

[0616] [SEQ ID NO: 61: ROR1×CD3 bispecific antibody amino acid sequence]

[0617]

[0618] [SEQ ID NO: 62: Amino acid sequence of ROR1×CD3 bispecific antibody with N-terminal hexahistidine tag (NVG-111)]

[0619]

[0620] [SEQ ID NO: 63: Common LCDR1 of FIX-binding and FX-binding antigen-binding domains]

[0621]

[0622] [SEQ ID NO: 64: Common LCDR2 of FIX-binding and FX-binding antigen-binding domains]

[0623]

[0624] [SEQ ID NO: 65: Common LCDR3 of FIX-binding and FX-binding antigen-binding domains]

[0625]

[0626] [SEQ ID NO: 66: FIX binding antigen binding domain HCDR1]

[0627]

[0628] [SEQ ID NO: 67: FIX binding antigen binding domain HCDR2]

[0629]

[0630] [SEQ ID NO: 68: FIX binding antigen binding domain HCDR3]

[0631]

[0632] [SEQ ID NO: 69: FX binding antigen binding domain HCDR1]

[0633]

[0634] [SEQ ID NO: 70: FX binding antigen binding domain HCDR2]

[0635]

[0636] [SEQ ID NO: 71: FX binding antigen binding domain HCDR3]

[0637]

[0638] [SEQ ID NO: 72: Common light chain variable region of the FIX-binding and FX-binding antigen-binding domains (CDRs shown in bold)]

[0639]

[0640] [SEQ ID NO: 73: Heavy chain variable region of the FIX-binding antigen-binding domain (CDRs shown in bold)]

[0641]

[0642] [SEQ ID NO: 74: Heavy chain variable region of FX binding antigen-binding domain (CDRs shown in bold)]

[0643]

[0644] [SEQ ID NO: 75: hinge region]

[0645]

[0646] [SEQ ID NO: 76: hinge region plus SEQ ID NO: 1 (SEQ ID NO: 1 is underlined)]

[0647]

[0648] [SEQ ID NO: 77: linker sequence]

[0649]

[0650] [SEQ ID NO: 78: linker sequence]

[0651]

[0652] [SEQ ID NO: 79: linker sequence]

[0653]

[0654] [SEQ ID NO: 80: linker sequence]

[0655]

[0656] [SEQ ID NO: 81: linker sequence]

[0657]

[0658] [SEQ ID NO: 82: Hinge region of CAR embodiments]

[0659]

[0660] [SEQ ID NO: 83: Partially deleted hinge region of a CAR embodiment plus SEQ ID NO: 7]

[0661]

[0662] [SEQ ID NO: 84: Hinge region of a CAR embodiment plus SEQ ID NO: 7]

[0663]

[0664] [Implementation Method]

[0665] 1. A therapeutic agent comprising a self-regulatory element,

[0666] The self-regulatory element is a small peptide that can be cleaved by endogenous factors,

[0667] wherein the activity of the intrinsic factor is altered due to the activity of the therapeutic agent, and

[0668] wherein cleavage of the autoregulatory element results in separation of the therapeutic agent into non-functional fragments.

[0669] 2. The therapeutic agent of embodiment 1, wherein the intrinsic factor is active in the same metabolic pathway as the therapeutic agent.

[0670] 3. The therapeutic agent of embodiment 1 or 2, wherein the intrinsic factor is a protease.

[0671] 4. The therapeutic agent of any one of the above embodiments, wherein the autoregulatory element comprises a proteolytic cleavage site.

[0672] 5. The therapeutic agent of any one of the above embodiments, wherein the rate at which the intrinsic factor is cleaved from the regulatory element is correlated with the activity level of the therapeutic agent.

[0673] 6. The therapeutic agent of embodiment 5, wherein the rate at which the endogenous factor cleaves the autoregulatory element is related to the activity level of the therapeutic agent and the peptide sequence of the autoregulatory element.

[0674] 7. The therapeutic agent of any one of embodiments 1 to 6, wherein the autoregulatory element comprises a thrombin or granzyme B cleavage recognition site.

[0675] 8. The therapeutic agent of any one of the above embodiments, wherein the therapeutic agent comprises a polypeptide.

[0676] 9. The therapeutic agent of embodiment 8,

[0677] wherein the therapeutic agent is an antigen binding protein comprising a first antigen binding domain,

[0678] wherein the first antigen-binding domain comprises a heavy chain variable domain comprising a heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and

[0679] The self-regulatory element is located at the C-terminus of the first antigen-binding domain.

[0680] 10. The antigen binding protein of embodiment 9, wherein the first antigen binding domain is a VHH antibody.

[0681] 11. The antigen binding protein according to embodiment 9,

[0682] wherein the antigen binding protein comprises a first antigen binding domain, wherein the first antigen binding domain comprises a light chain variable domain and a heavy chain variable domain,

[0683] wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and

[0684] wherein the heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, and

[0685] The self-regulatory element is located at the C-terminus of the first antigen-binding domain.

[0686] 12. The antigen binding protein of embodiments 9, 10 or 11, wherein the antigen binding protein forms part of a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0687] 13. The antigen-binding protein according to any one of embodiments 9 to 12,

[0688] wherein the antigen binding protein comprises a first antigen binding domain and a second antigen binding domain,

[0689] wherein the autoregulatory element is located between the first and second antigen binding domains.

[0690] 14. The antigen binding protein of embodiment 13,

[0691] wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain,

[0692] wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and

[0693] The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3.

[0694] 15. The antigen binding protein of embodiment 13 or 14, wherein the first antigen binding domain and the second antigen binding domain comprise the same CDRs, comprise bi-epitope CDRs, or comprise bispecific CDRs.

[0695] 16. The antigen binding protein of any one of embodiments 13 to 15, wherein the antigen binding protein is an antibody or a fragment thereof.

[0696] 17. The antigen-binding protein of any one of embodiments 9 to 16, wherein the antigen-binding protein comprises a single domain fragment, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain Fab (scFab) fragment, a single-chain Fv protein (scFv), a tandem scFv protein, a disulfide-stabilized Fv protein (dsFv) or a scFv-Fc protein.

[0697] 18. The antigen binding protein of any one of embodiments 9 to 17, wherein the antigen binding protein is comprised in a monoclonal antibody, a bispecific antibody, or a bispecific T cell engager.

[0698] 19. The antigen binding protein of any one of embodiments 9 to 18, wherein the first and second antigen binding domains are scFv proteins covalently linked via a peptide linker.

[0699] 20. The antigen binding protein of any one of embodiments 9 to 19, wherein the autoregulatory element is present within a peptide linker.

[0700] 21. The antigen binding protein of any one of embodiments 9 to 20, wherein the autoregulatory element comprises a granzyme B cleavage site and the endogenous factor is granzyme B.

[0701] 22. The antigen binding protein of any one of embodiments 9 to 21, wherein the first antigen binding domain selectively binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1).

[0702] 23. The antigen binding protein of embodiment 22,

[0703] wherein the first antigen-binding domain comprises a light chain variable domain and a heavy chain variable domain,

[0704] The light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3, wherein the

[0705] LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19;

[0706] LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and

[0707] LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; and

[0708] The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the

[0709] HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22;

[0710] HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23; and

[0711] HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24;

[0712] The sequence of each complementarity determining region may differ from the given sequence at a maximum of two amino acid positions.

[0713] 24. The antigen binding protein of embodiment 23, wherein:

[0714] (a) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 29, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 35;

[0715] (b) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 36;

[0716] (c) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 31, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 37;

[0717] (d) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 32, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 38; or

[0718] (e) the light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 33, and the heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 39,

[0719] Each of the light chain variable domain and heavy chain variable domain may have at least 90% identity with the amino acid sequence described above.

[0720] 25. The antigen binding protein of any one of embodiments 22 to 24, wherein the second antigen binding domain selectively binds to the CD3 subunit of the T cell receptor (TCR).

[0721] 26. The antigen binding protein of any one of embodiments 22 to 25, wherein the granzyme B cleavage site comprises SEQ ID NO: 7.

[0722] 27. The antigen binding protein of any one of embodiments 9 to 20, wherein the autoregulatory element comprises a thrombin cleavage site and the endogenous factor is thrombin.

[0723] 28. The antigen binding protein of any one of embodiments 9 to 20 and 27, wherein the first antigen binding domain selectively binds to FIXa / FIX, and the second antigen binding domain selectively binds to FX / FXa.

[0724] 29. The antigen-binding protein according to any one of embodiments 9 to 20 and 27 or 28,

[0725] wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain,

[0726] wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and

[0727] wherein the heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3;

[0728] wherein the first antigen-binding domain

[0729] LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63;

[0730] LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and

[0731] LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; and

[0732] The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the

[0733] HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 66;

[0734] HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 67; and

[0735] HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68; and

[0736] wherein the second antigen-binding domain

[0737] LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63;

[0738] LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and

[0739] LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; and

[0740] The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the

[0741] HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 69;

[0742] HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 70; and

[0743] HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:71.

[0744] 30. The antigen binding protein of embodiment 29, wherein

[0745] For the first antigen binding domain,

[0746] The light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 72, and

[0747] The heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 73, and

[0748] For the second antigen-binding domain,

[0749] The light chain variable domain comprises the amino acid sequence set forth in SEQ ID NO: 72, and

[0750] The heavy chain variable domain comprises the amino acid sequence set forth in SEQ ID NO:74.

[0751] 31. The antigen binding protein of any one of embodiments 9 to 20 and 27 to 30, wherein the thrombin cleavage site comprises SEQ ID NO: 1.

[0752] 32. The antigen binding protein of any one of embodiments 9 to 31, wherein the antigen binding protein is labeled.

[0753] 33. The antigen binding protein of embodiment 32, wherein the label is a fluorescent label, an enzyme label or a radioactive label.

[0754] 34. A composition comprising the therapeutic agent of any one of embodiments 1 to 33 and a pharmaceutically acceptable carrier.

[0755] 35. An isolated nucleic acid molecule encoding the antigen binding protein of any one of embodiments 8 to 31.

[0756] 36. The isolated nucleic acid molecule of embodiment 35, which is operably linked to a promoter.

[0757] 37. An expression vector comprising the isolated nucleic acid molecule of embodiment 35 or embodiment 36.

[0758] 38. An isolated host cell transformed with the nucleic acid molecule of embodiment 35 or 36, or the gene vector of embodiment 37.

[0759] 39. The antigen binding protein of any one of embodiments 9 to 31, wherein the antigen binding protein is exogenously expressed in CAR-T cells, CAR-M cells or modified NK cells.

[0760] 40. A method of treating hemophilia A, comprising administering the antigen binding protein of any one of embodiments 27 to 31, or the composition of embodiment 34, to an individual in need thereof.

[0761] 41. A method for preventing prothrombotic risk, comprising administering to an individual in need thereof the antigen binding protein of any one of embodiments 27 to 31, or the composition of embodiment 34.

[0762] 42. A method of treating cancer, comprising administering the antigen binding protein of any one of embodiments 20 to 26, or the composition of embodiment 34, to an individual in need thereof.

[0763] 43. The method of embodiment 42, wherein the cancer is leukemia, pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma, or kidney cancer.

[0764] 44. A method for preventing toxicity associated with a mechanism of action (MoA) associated with immunotherapy, comprising administering to a subject in need thereof the antigen binding protein of any one of embodiments 20 to 26, or the composition of embodiment 34.

[0765] 45. A method for preventing cytokine release syndrome, comprising administering the antigen binding protein of any one of embodiments 20 to 26, or the composition of embodiment 34, to an individual in need thereof.

[0766] References

[0767] Ferriere, S., I. Peyron, OD Christophe, C. Kawecki, C. Casari, V. Muczynski, ACNathwani, A. Kauskot, PJ Lenting and CVDenis (2020). "A hemophilia A mousemodel. for the in vivo assessment of Emicizumab function." Blood.

[0768] Gallwitz, M., M. Enoksson, M. Thorpe and L. Hellman (2012). "The extended cleavage specificity of human thrombin." PLoS One 7(2):e31756.

[0769] Harris et al.,Definition and redesign of the extendedsubstratespecificity of granzyme B,(1998)J Biol Chem:273 pp.27364-73.

[0770] Hartmann,R.,T.Feenstra,L.Valentino,M.Dockal and F.Scheiflinger(2018).“In vitro studies show synergistic effects of aprocoagulant bispecificantibody and bypassing agents.”J Thromb Haemost.

[0771] Kochenderfer,J.N.,R.P.T.Somerville,T.Lu,V.Shi,A.Bot,J.Rossi,A.Xue,S.L.Goff,J.C.Yang,R.M.Sherry,C.A.Klebanoff,U.S.Kammula,M.Sherman,A.Perez,C.M.Yuan,T.Feldman,J.W.Friedberg,M.J.Roschewski,S.A.Feldman,L.McIntyre,M.A.Toomey and S.A.Rosenberg(2017).“Lymphoma Remissions Caused by Anti-CD19ChimericAntigen Receptor T Cells Are Associated With High Serum Interleukin-15Levels.”J Clin Oncol 35(16):1803-1813.

[0772] Lenting,P.J.,C.V.Denis and O.D.Christophe(2017).“Emicizumab,abispecific antibody recognizing coagulation factors IX and X:how doesitactually compare to factor VIII?”Blood 130(23):2463-2468.

[0773] Wee,L.J.,E.P.Er,L.F.Ng and J.C.Tong(2011).“In silicoprediction of thegranzyme B degradome.”BMC Genomics 12 Suppl 3:S11.

Claims

1. Therapeutic agents comprising an autoregulatory element, The autoregulatory element is a small peptide that can be cleaved by endogenous factors. wherein the activity of the intrinsic factor is altered due to the activity of the therapeutic agent, and wherein cleavage of the autoregulatory element results in separation of the therapeutic agent into non-functional fragments.

2. The therapeutic agent according to claim 1, wherein the intrinsic factor is active in the same metabolic pathway as the therapeutic agent, Optionally, wherein the endogenous factor is a protease.

3. The therapeutic agent of claim 1 or claim 2, wherein the autoregulatory element comprises a proteolytic cleavage site.

4. The therapeutic agent according to any one of the preceding claims, wherein the rate at which the intrinsic factor cleaves the autoregulatory element is correlated with the activity level of the therapeutic agent, Optionally, wherein the rate at which the endogenous factor cleaves the autoregulatory element is related to the activity level of the therapeutic agent and the peptide sequence of the autoregulatory element. 5 . The therapeutic agent according to claim 1 , wherein the autoregulatory element comprises a cleavage recognition site for thrombin or granzyme B.

6. The therapeutic agent according to any one of claims 1 to 5, wherein the therapeutic agent is an antigen binding protein comprising a first antigen binding domain, wherein the first antigen-binding domain comprises a heavy chain variable domain comprising a heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, and The autoregulatory element is located at the C-terminus of the first antigen binding domain.

7. The antigen-binding protein according to claim 6, wherein the antigen binding protein comprises a first antigen binding domain, wherein the first antigen-binding domain comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and wherein the heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, and The autoregulatory element is located at the C-terminus of the first antigen binding domain.

8. The antigen-binding protein according to any one of the preceding claims, wherein the antigen binding protein comprises a first antigen binding domain and a second antigen binding domain, wherein the autoregulatory element is located between the first and second antigen binding domains.

9. The antigen-binding protein according to claim 8, wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3.

10. The antigen binding protein of claim 8 or claim 9, wherein the antigen binding protein is an antibody or a fragment thereof, optionally wherein the antigen binding protein comprises a single domain fragment, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain Fab (scFab) fragment, a single-chain Fv protein (scFv), a tandem scFv protein, a disulfide-stabilized Fv protein (dsFv) or a scFv-Fc protein.

11. The antigen binding protein of any one of claims 6 to 10, wherein the autoregulatory element is present within a peptide linker.

12. The antigen-binding protein according to any one of claims 6 to 11, wherein the autoregulatory element comprises a granzyme B cleavage site and the endogenous factor is granzyme B, optionally The first antigen-binding domain selectively binds to receptor tyrosine kinase-like orphan receptor 1 (ROR1).

13. The antigen-binding protein according to claim 12, wherein the first antigen-binding domain comprises a light chain variable domain and a heavy chain variable domain, The light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 19; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 20; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; and The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; The sequence of each complementarity determining region may differ from the given sequence at a maximum of two amino acid positions.

14. The antigen binding protein of claim 13, wherein the first antigen binding domain is comprised in a chimeric antigen receptor (CAR).

15. The antigen binding protein of claim 13, wherein the second antigen binding domain selectively binds to the CD3 subunit of the T cell receptor (TCR).

16. The antigen binding protein of any one of claims 13 to 15, wherein the granzyme B cleavage site comprises SEQ ID NO:

7.

17. The antigen-binding protein according to any one of claims 6 to 11, wherein the autoregulatory element comprises a thrombin cleavage site and the intrinsic factor is thrombin, Optionally, wherein The first antigen binding domain selectively binds to FIXa / FIX, and The second antigen-binding domain selectively binds to FX / FXa.

18. The antigen-binding protein according to any one of claims 6 to 11 and 17, wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3, and wherein the heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3; wherein the first antigen-binding domain LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; and The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 66; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 67; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68; and wherein the second antigen-binding domain LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 63; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 64; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; and The heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 69; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 70; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

71.

19. The antigen binding protein of any one of claims 6 to 11 and 17 or 18, wherein the thrombin cleavage site comprises SEQ ID NO:

1.

20. A composition comprising the therapeutic agent of any one of claims 1 to 19 and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid molecule encoding the antigen binding protein of any one of claims 6 to 19; optionally, the nucleic acid molecule is operably linked to a promoter.

22. An isolated host cell transformed with the nucleic acid molecule of claim 21.

23. A method for treating hemophilia A, comprising administering the antigen-binding protein according to any one of claims 17 to 19, or the composition according to claim 20, to an individual in need thereof.

24. A method for preventing the risk of prothrombotic events, comprising administering the antigen binding protein of any one of claims 17 to 19, or the composition of claim 20, to an individual in need thereof.

25. A method for treating cancer, comprising administering the antigen binding protein of any one of claims 11 to 16 or the composition of claim 20 to an individual in need thereof.

26. A method for preventing mechanism of action (MoA)-related toxicity associated with immunotherapy, comprising administering to an individual in need thereof the antigen binding protein of any one of claims 11 to 16, or the composition of claim 20.

27. A method for preventing cytokine release syndrome, comprising administering the antigen binding protein of any one of claims 11 to 16 or the composition of claim 20 to an individual in need thereof.

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