Self-regulation of therapeutic agents

Abstract

The present invention relates to therapeutic agents comprising autoregulatory elements, preferably where the therapeutic agent is an antigen binding protein. Methods of treatment comprising the use of said agents are also disclosed.

Description

[Technical Field]

[0001] The present invention relates to therapeutic agents, preferably antigen-binding proteins. Methods of treatment including the use of said agents are also disclosed. [Background technology]

[0002] Homeostasis and sensitivity to feedback regulatory signals are naturally occurring mechanisms that balance virtually all biological functions in the body. However, most pharmaceuticals lack such regulatory mechanisms, and techniques to modulate the therapeutic effects of drugs in response to physiological or pathological signals are highly sought after but largely unmet. Summary of the Invention [Problem to be solved by the invention]

[0003] Over the past decade, advances in molecular and cellular engineering have led to the development of complex and potent drugs, including therapeutic monoclonal and bispecific antibodies, chimeric antigen receptor (CAR) T cells, and engineered therapeutic peptides, that deliver unique functionalities and offer significant clinical benefit, particularly to patients with significant unmet medical needs. However, the transformative potential of these novel therapies is limited by the occurrence of serious adverse events associated with their mechanisms of action (MoA), which can include cytokine-mediated toxicity in adoptive immunotherapy, cytokine release syndrome, and thromboembolism and prothrombotic risk in hemophilia A. Due to the inherent long half-lives of some of these therapies (e.g., antibodies and engineered cells), such adverse events represent a significant, even life-threatening, risk to patients. [Means for solving the problem]

[0004] The present invention demonstrates a method for creating next-generation therapeutic agents that utilize endogenous signals or factors that can be activated in pathological conditions as a direct response to therapeutic treatment, and that have a built-in "switch" mechanism to modulate their therapeutic activity when the risk of severe MoA-associated toxicity arises. [Effects of the Invention]

[0005] The following invention describes methods for designing and developing next-generation therapeutic agents incorporating autoregulatory elements (autoregulatory elements) that have the ability to inactivate the agent in the context of excessive or undesired therapeutic effects, reducing the risk of adverse events and improving the safety profile of the agent. Autoregulatory elements are small, cleavable peptides inserted within the structure of a therapeutic agent such that their cleavage inactivates the therapeutic agent by degrading it into either non-functional or rapidly eliminated fragments (Figure 1).

[0006] Autoregulatory peptides are susceptible to proteolytic cleavage mediated by specifically selected endogenous factors whose concentration, activity, or bioavailability changes in response to treatment-induced adverse events. Thus, when a drug's therapeutic activity remains within a range defined as free of adverse events, the drug maintains full therapeutic potential. However, when therapeutic activity exceeds a threshold separating MoA-related adverse events from normal biological activity, changes in the activity of the endogenous factors trigger cleavage of the autoregulatory elements, ultimately inactivating the therapeutic agent and reducing the risk of progression to severe toxicity (Figure 1).

[0007] Proof of concept was established with two therapeutic bispecific antibodies: emicizumab, a humanized bispecific IgG4 commercially available as a treatment for hemophilia A patients with and without inhibitors, and NVG-111, a novel bispecific single-chain variable fragment (scFv) investigational drug currently undergoing Phase I trials for chronic leukemia-lymphoma (CLL) and mantle cell lymphoma (MCL). The application of autoregulatory technology was also demonstrated in a CAR-T cell model. The present invention defines a target- and format-agnostic, versatile platform that can be applied to any therapeutic agent, including therapeutic bispecific and monoclonal antigen-binding proteins, antibodies, including non-classical formats, therapeutic non-antibody-derived peptides, and engineered cells (including, but not limited to, CAR-T cells), to extend the therapeutic index of such therapeutic agents with the potential to improve efficacy and safety for better clinical outcomes in patients.

[0008] Thus, the present invention provides: A therapeutic agent comprising an autoregulatory element, wherein the autoregulatory element is a small peptide cleavable by an endogenous factor, the activity of the endogenous factor being altered as a result of the activity of the therapeutic agent, and cleavage of the autoregulatory element results in separation of the therapeutic agent into non-functional fragments.

[0009] The present invention also provides: A composition comprising a therapeutic agent as defined herein and a pharmaceutically acceptable carrier.

[0010] The present invention also provides: An isolated nucleic acid molecule encoding an antigen binding protein as defined herein.

[0011] The present invention also provides: An isolated host cell transformed with a nucleic acid molecule as defined herein.

[0012] The present invention also provides: A method for 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 a composition comprising the autoregulatory element.

[0013] The present invention also provides: 1. A method for preventing the risk of thrombosis, comprising administering to an individual in need thereof an antigen binding protein comprising an autoregulatory element that includes a thrombin cleavage site, or a composition comprising the autoregulatory element.

[0014] The present invention also provides: A method for treating cancer, comprising administering to an individual in need thereof an antigen binding protein comprising an autoregulatory element that includes a granzyme B cleavage site, or a composition comprising the autoregulatory element.

[0015] The present invention also provides: 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 a composition comprising the autoregulatory element.

[0016] Brief description of the sequence listing SEQ ID NO: 1: thrombin cleavage recognition sequence consensus sequence SEQ ID NO: 2: Thrombin cleavage recognition sequence P1' G to L SEQ ID NO: 3: Thrombin cleavage recognition sequence P1' G to D SEQ ID NO: 4: Thrombin cleavage recognition sequence of human coagulation factor V (FV) SEQ ID NO: 5: Granzyme B cleavage recognition sequence GZMB-1 of mouse BID protein SEQ ID NO: 6: Granzyme B cleavage recognition sequence GZMB-2 of mouse BID protein SEQ ID NO: 7: Granzyme B cleavage recognition sequence GZMB-3, consensus sequence SEQ ID NO: 8: Granzyme B cleavage recognition sequence GZMB-4, P10-P10' sequence of GZMB-2 SEQ ID NO: 9: Granzyme B cleavage recognition sequence GZMB-5, P4 I-V SEQ ID NO: 10: Granzyme B cleavage recognition sequence GZMB-6, P4 I to L SEQ ID NO: 11: Granzyme B cleavage recognition sequence GZMB-7, P2 P-G SEQ ID NO: 12: Granzyme B cleavage recognition sequence GZMB-8, P2 P-A SEQ ID NO: 13: Granzyme B cleavage recognition sequence GZMB-9, P1' S to A SEQ ID NO: 14: Granzyme B cleavage recognition sequence GZMB-10, P2' L to E and P4' E to Q SEQ ID NO: 15: Granzyme B cleavage recognition sequence GZMB-11, P2' L to E and P4' E to V SEQ ID NO: 16: Granzyme B cleavage recognition sequence GZMB-12, P1 D-N SEQ ID NO: 17: Granzyme B cleavage recognition sequence GZMB-13, P1 D-E SEQ ID NO: 18: Granzyme B cleavage recognition sequence GZMB-14, P1 D-Q SEQ ID NO: 19: ROR1-binding antigen-binding domain LCDR 1 SEQ ID NO: 20: ROR1-binding antigen-binding domain LCDR 2

[0017] SEQ ID NO: 21: ROR1-binding antigen-binding domain LCDR 3 SEQ ID NO: 22: ROR1-binding antigen-binding domain HCDR 1 SEQ ID NO: 23: ROR1-binding antigen-binding domain HCDR 2 SEQ ID NO: 24: ROR1-binding antigen-binding domain consensus HCDR 3 SEQ ID NO: 25: ROR1-binding antigen-binding domain HCDR 3, clone F heavy chain CDR3 SEQ ID NO: 26: ROR1-binding antigen-binding domain HCDR 3, humanized 1 heavy chain CDR3 SEQ ID NO: 27: ROR1-binding antigen binding domain HCDR 3, humanized 2 and 5 heavy chain CDR3 SEQ ID NO: 28: ROR1-binding antigen-binding domain HCDR 3, humanized 3 and 4 heavy chain CDR3 SEQ ID NO: 29: ROR1-binding antigen binding domain, clone F light chain variable region SEQ ID NO: 30: ROR1-binding antigen binding domain, humanized 1 light chain variable region SEQ ID NO: 31: ROR1-binding antigen binding domain, humanized light chain variable region 2 SEQ ID NO: 32: ROR1-binding antigen-binding domain, humanized 3 light chain variable region SEQ ID NO: 33: ROR1-binding antigen-binding domain, humanized 4 light chain variable region SEQ ID NO: 34: ROR1-binding antigen-binding domain, humanized 5 light chain variable region SEQ ID NO: 35: ROR1-binding antigen-binding domain, clone F heavy chain variable region SEQ ID NO: 36: ROR1-binding antigen-binding domain, humanized heavy chain variable region 1 SEQ ID NO: 37: ROR1-binding antigen-binding domain, humanized dual heavy chain variable region SEQ ID NO: 38: ROR1-binding antigen-binding domain, humanized triple heavy chain variable region SEQ ID NO: 39: ROR1-binding antigen-binding domain, humanized 4 heavy chain variable region SEQ ID NO: 40: ROR1-binding antigen-binding domain, humanized 5 heavy chain variable region

[0018] SEQ ID NO: 41: CD3-binding antigen-binding domain, murine light chain variable region SEQ ID NO: 42: CD3-binding antigen-binding domain, humanized 1 light chain variable region SEQ ID NO: 43: CD3-binding antigen-binding domain, humanized 2 light chain variable region SEQ ID NO: 44: CD3-binding antigen-binding domain, humanized 3 light chain variable region SEQ ID NO: 45: CD3-binding antigen-binding domain, humanized 4 light chain variable region SEQ ID NO: 46: CD3-binding antigen-binding domain, humanized 5 light chain variable region SEQ ID NO: 47: CD3-binding antigen-binding domain, murine heavy chain variable region SEQ ID NO: 48: CD3-binding antigen-binding domain, humanized heavy chain variable region SEQ ID NO: 49: CD3-binding antigen-binding domain, humanized dual heavy chain variable region SEQ ID NO: 50: CD3-binding antigen-binding domain, humanized 3 heavy chain variable region SEQ ID NO: 51: CD3-binding antigen-binding domain, humanized 4 heavy chain variable region SEQ ID NO: 52: CD3-binding antigen-binding domain, humanized 5 heavy chain variable region SEQ ID NO: 53: CD3-binding antigen-binding domain LCDR 1 SEQ ID NO: 54: CD3-binding antigen-binding domain LCDR 2 SEQ ID NO: 55: CD3-binding antigen-binding domain LCDR 3 SEQ ID NO: 56: CD3-binding antigen-binding domain HCDR 1 SEQ ID NO: 57: CD3-binding antigen-binding domain HCDR 2 SEQ ID NO: 58: CD3-binding antigen-binding domain HCDR 3 SEQ ID NO: 59: linker sequence SEQ ID NO: 60: linker sequence

[0019] SEQ ID NO: 61: ROR1xCD3 bispecific antibody amino acid sequence SEQ ID NO: 62: ROR1xCD3 bispecific antibody amino acid sequence with N-terminal hexahistidine tag (NVG-111) SEQ ID NO: 63: Common LCDR 1 of FIX-binding and FX-binding antigen-binding domains SEQ ID NO: 64: Common LCDR 2 of FIX-binding and FX-binding antigen-binding domains SEQ ID NO: 65: Common LCDR 3 of FIX-binding and FX-binding antigen-binding domains SEQ ID NO: 66: FIX-binding antigen-binding domain HCDR 1 SEQ ID NO: 67: FIX-binding antigen-binding domain HCDR 2 SEQ ID NO: 68: FIX-binding antigen-binding domain HCDR 3 SEQ ID NO: 69: FX-binding antigen-binding domain HCDR 1 SEQ ID NO: 70: FX-binding antigen-binding domain HCDR 2 SEQ ID NO: 71: FX-binding antigen-binding domain HCDR 3 SEQ ID NO: 72: Common light chain variable region of FIX-binding and FX-binding antigen-binding domains SEQ ID NO: 73: Heavy chain variable region of FIX-binding antigen-binding domain SEQ ID NO: 74: FX-binding antigen-binding domain heavy chain variable region SEQ ID NO: 75: hinge region SEQ ID NO: 76: hinge region + SEQ ID NO: 1 SEQ ID NO: 77: linker sequence SEQ ID NO: 78: linker sequence SEQ ID NO: 79: linker sequence SEQ ID NO: 80: linker sequence

[0020] SEQ ID NO: 81: Linker sequence SEQ ID NO: 82: Hinge region of CAR embodiment SEQ ID NO: 83: CAR embodiment partially deleted hinge region + SEQ ID NO: 7 SEQ ID NO: 84: hinge region of CAR embodiment + SEQ ID NO: 7 [Brief explanation of the drawings]

[0021] [Figure 1] This diagram shows a schematic of the mechanism of action of autoregulation (AR) exemplified by therapeutic antibodies. Initial pathological conditions are characterized by a lack of biological activity. In the context of therapeutic intervention, AR antibodies (or therapeutic agents) produce the biological activity necessary for efficient therapeutic action. In situations of excessive biological activity leading to adverse events, the proteolytic product of the AR antibody mechanism of action is used to trigger autoregulation. Increased activity of this enzymatic product cleaves a specific sensitive peptide inserted into the structure of the AR antibody (or therapeutic agent), leading to progressive molecular degradation. Consequently, a decrease in AR antibody concentration reduces biological activity and modulates the therapeutic effect to prevent adverse events. [Figure 2] Design of an autoregulatory bispecific antibody with FVIII-mimetic activity for hemophilia A. AR_Ab8, a prototype autoregulatory bispecific antibody for the treatment of hemophilia A, was designed using the core sequence of emicizumab, a full-size humanized IgG4 antibody targeting coagulation factor IX (FIX) and factor X (FX). An eight-residue thrombin-sensitive cleavable peptide (P4 to P4' sequence) was inserted into the hinge region of both heavy chains of the parent antibody. [Figure 3A-B]In vitro characterization of FVIII mimetic activity. (A) Coomassie blue stained SDS-Page gel shows the migration profiles of purified emicizumab and AR_Ab8 under non-reducing conditions, demonstrating the lack of premature degradation. (B) The FVIII mimetic activity of both emicizumab and AR_Ab8 (2 nM) was tested by a chromogenic assay measuring the conversion of FX to FXa by FIXa. Dots represent the mean OD values ​​± SD of triplicates. Control conditions were performed in the absence of bsAb. [Figure 3C] (C) In vitro procoagulant activity in human plasma was measured. 200 BU of anti-FVIII antibody was added to a pool of citrated normal human plasma to neutralize endogenous FVIII. This induced hemophilia A plasma was treated with 350 nM emicizumab or AR_Ab8, and the activated partial thromboplastin time (aPTT) was measured. Symbols represent individual clotting time values, and lines indicate the mean ± SD of triplicate experiments. ns indicates not significant by one-way analysis of variance with Sidak's multiple comparison test. [Figure 4] The loss-free therapeutic effect of AR_Ab8 in hemophilia A mice is shown. (A) The procoagulant effect of AR_Ab8 was tested in vivo. FVIIIKO mice were intravenously (IV) injected with 3 mg / kg of emicizumab or AR_Ab8. Twenty-four hours later, mice received a bolus injection of human FIX and FX, and a tail clip assay was performed 5 minutes after the second injection. Control animals received 2 U / mouse of human FVIII. (B) Total blood loss results are shown as a Wisker plot with median (line), 25th and 75th percentiles (top and bottom boxes), and minimum and maximum values ​​(bars) for 6–11 mice. Symbols indicate individual values. ns and * indicate not significant and p<0.05, respectively, in a one-way ANOVA test with Sidak's multiple comparisons. [Figure 5A-B]Thrombin-mediated in vitro degradation of AR_Ab8 is shown. 2 nM AR_Ab8 or emicizumab was incubated with 2 U / mL α-thrombin to assess the cleavage kinetics of the thrombin-sensitive peptide. The enzymatic cleavage reaction was terminated by the addition of 500 nM PPACK at different time points up to 180 min. (A) Reaction samples were run on an SDS-Page gel under non-reducing conditions and immunoblotted with an anti-human Fc antibody to visualize AR_Ab8 degradation. (B) The residual FVIII mimetic activity of both antibodies was measured using a FXa chromogenic assay. Results were normalized and expressed as a percentage relative to emicizumab at each time point. Bars represent the mean ± SD of triplicates. ns, *, and ** indicate non-significant, p<0.05, and p<0.01, respectively, in a two-way ANOVA test with Sidak's multiple comparisons. [Figure 5C] (C) The sensitivity of various thrombin-cleavable peptides was tested using a bispecific tandem scFv format. 2 nM of different tandem scFvs were exposed to 2 U / mL α-thrombin for 20 min, and the residual FVIII mimics were measured by FXa chromogenic assay. Results were normalized to the control condition (no thrombin exposure), and the bars represent the percentage of residual activity. A negative control (non-cleaved) was performed using a non-autoregulated construct. [Figure 6]Thrombin-mediated AR reduces abnormal thrombin generation in vitro. The in vitro thrombogenic profile of AR_Ab8 was investigated using a thrombin generation model in the presence of activated prothrombin complex concentrate (aPCC). aPCC potentiates abnormal thrombin generation using an FVIII-mimetic antibody. A citrated normal human plasma pool was supplemented with 200 BU of anti-FVIII antibody to neutralize endogenous FVIII, and 0.5 U / mL of aPCC combined with 600 nM was added. Thrombin generation was induced with a low-tissue factor solution and measured by a calibrated automated thrombogram (CAT) method. (A) The dotted line indicates the mean maximum thrombin generated by normal plasma, and the curve above the dotted line indicates abnormal thrombin generation as an average of triplicates. (B) Peak heights (maximum thrombin generation) were extracted from the thrombogram and plotted as individual values ​​(symbols) and the average of triplicates. ns and * mean not significant and p<0.05, respectively, in one-way ANOVA test with Sidak's multiple comparisons. [Figure 7] Thrombin-mediated AR reduces the thrombotic profile of FVIII-mimetic antibodies in mice. The in vivo thrombogenic profile of a bispecific antibody with FVIII-mimetic activity was evaluated in a mouse model co-treated with aPCC, which mitigates the thrombogenic potential of emicizumab. (A) C57Bl / 6 male mice received a bolus intravenous injection of aPCC (2.5 U) and emicizumab or AR_Ab8 (250 μg), followed by three booster injections of aPCC at 24-hour intervals. Thrombotic outcomes were assessed at t+96 h. (B) At the end of the experiment (96 h), plasma samples were collected in EDTA anticoagulant, and platelet counts were measured. Symbols represent individual animals, and horizontal bars represent the median distribution for each experimental condition. In an unpaired t-test, ns indicates no significance, and *** indicates p<0.001. [Figure 8]AR_Ab8 reduces pulmonary thrombus formation in mice. Lungs from animals treated with aPCC and emicizumab or AR_Ab8 were dissected, fixed in formalin, and sections mounted for histological evaluation. (A) Immunofluorescent staining for PECAM (green) and platelet-specific integrin αIIb (purple) was performed on lung sections. PECAM staining was used to define the vascular surface (dotted line), and αIIb staining was converted to a binary occlusion mask. The observation magnification was 20x, and the scale bar measures 50 μm. (B) Vascular occlusion was quantified blinded and calculated as the percentage of the occluded mask relative to the total vascular surface. Symbols represent the occlusion values ​​for each vessel measured from eight different animals per group in two independent experiments. Horizontal bars represent the median distribution for each experimental condition, and p values ​​were calculated using an unpaired t-test. [Figure 9A] Design of an autoregulatory bispecific T cell engager targeting ROR1. The autoregulatory bispecific T cell engager (AR_TCE) was designed using the core sequence of NVG-111, a CD3xROR1 bispecific tandem scFv antibody applied to the treatment of ROR1-positive solid and hematological malignancies. (A) The linker region connecting the two scFvs of the parent antibody consists of eight residues (P4 to P4' sequence) and is substituted with a granzyme B-sensitive cleavable peptide flanked by two G4S motifs. [Figure 9B-C] (B) Three variants of AR_TCE were developed, each differing in the P4-P4' sequence cleavable by granzyme B. (C) All variants were expressed in Expi-293 cells and showed migration profiles similar to those of the parent antibody NVG-111 after Coomassie blue staining on SDS-Page under reducing conditions. [Figure 10]Figure 1 shows the T cell engagement ability of AR_TCE. The potential of AR_TCE variants for T cell engagement was tested in a short-term killing assay model. Increasing concentrations of NVG-111 or AR_TCE were incubated with purified human T cells from ROR1-positive Jeko-1 and healthy donors at an effector-to-target ratio of 5:1 in a coculture model for 48 hours. (A) T cell activation levels were measured by direct staining for CD69 in flow cytometry and 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 using viability dye uptake. Symbols represent the mean ± SD of triplicate experiments. The dotted line indicates basal cell death and CD69 expression in the absence of antibody. [Figure 11] Dose-dependent granzyme B-mediated inactivation of AR_TCE in vitro is shown. The ability of AR_TCE variants to be cleaved and inactivated by granzyme B was tested in vitro. Recombinant granzyme B was first activated with cathepsin C. Activated granzyme B was incubated with 1 μg / mL NVG-111 or AR_TCE variants at a final concentration of 100 nM for 2 hours at 37°C. Controls included buffer alone or cathepsin C alone. (A) Samples were run on an SDS-Page gel under reducing conditions and immunoblotted with protein L to detect intact and fragment antibodies. NVG-111 or AR_TCE variants (1 μg / mL) were incubated with increasing concentrations of activated granzyme B at 37°C. The reaction was then stopped by the addition of a broad-spectrum protease inhibitor for 2 hours. (B) Residual intact antibodies were detected using a dedicated ELISA assay. Symbols are the mean ± SD of duplicate experiments and curves were fitted to a four-parameter variable slope model using Prism software. [Figure 12]In vitro characterization of sensitivity and specificity for AR_TCE is shown. (A) An expanded range of AR_TCEs bearing various AR peptide sequences was incubated with a single concentration of granzyme B (50 nM) for 2 hours, and the reaction was stopped by adding a broad-spectrum protease inhibitor. (B) AR_TCE-3 was then exposed to a broad panel of proteases at similar concentrations for 2 hours. Residual intact antibodies were detected by ELISA. Residual antibodies were normalized to untreated and expressed as a percentage of remaining intact antibodies. In (B), results were ranked from highest to lowest percentage. Symbols represent the mean ± SD of triplicates. *** indicates p<0.001 by one-way ANOVA with Sidak's multiple comparisons. [Figure 13A] Granzyme B-mediated AR reduces cytokine release in vitro. The efficacy and safety profiles of two AR_TCE variants with low (AR_TCE-1) and high (AR_TCE-3) cleavage rates were evaluated in a long-term coculture assay. NVG-111 or AR_TCE variants were incubated at 1 μg / mL with purified human T cells from ROR1-positive Jeko-1 and healthy donors and excess target cells (effector-to-target ratio 1:10) for up to 120 hours. (A) The percentage of cell death was measured every 24 hours by flow cytometry of target cells using viability dye uptake. [Figure 13B-C] (B) T cell activation levels were measured by direct staining for CD25 and expressed as mean fluorescence intensity (MFI) of the CD3+ population. Symbols represent the mean ± SD of triplicate experiments. (C) The level of human interferon-γ (IFNγ) release by activated T cells was measured in the culture supernatant at 120 hours. Symbols represent individual values, and horizontal bars represent the mean ± SD of triplicate experiments. * and *** indicate p<0.05 and p<0.001, respectively, by one-way ANOVA with Sidak's multiple comparisons. [Figure 13D](D) Supernatant samples were run on an SDS-Page gel and immunoblotted with anti-His-Tag antibody to detect cleavage and inactivation of the AR_TCE construct. [Figure 14A] Granzyme B-mediated AR increases survival in a mouse xenograft model of TNBC. The safety profile of AR was evaluated in a mouse model of TCE-induced cytokine-mediated toxicity. (A) Immunocompromised NOD.SCID gamma mice were first induced with stable expression of either NVG-111 or AR_TCE-3, or not (vehicle). On day 0, all animals were implanted with 2 x 106 human ROR1+ triple-negative breast cancer (TNBC) MDA-MB-231 cells via subcutaneous injection, and tumors were allowed to engraft for 25 days. Mice were then administered six cycles of purified human T cells. Animals were carefully monitored throughout the treatment period and culled on day 46, if ethically required, or at the end of the study. [Figure 14B] (B) Total body weight of each individual animal was recorded every 24 hours and expressed as a percentage of the body weight measured on the day of the first T cell injection (day 25). Symbols and connected curves represent the mean ± SD of six animals per group. Arrows indicate the day of T cell injection. *, **, and *** indicate p<0.05, p<0.01, and p<0.001, respectively, by one-way ANOVA with Sidak's multiple comparisons. [Figure 14C] (C) Animals that met any of the study endpoints (severe weight loss, acute toxicity, or tumor ulceration) were culled or removed from the study (T cell treatment was discontinued). Results are presented as the proportion of surviving animals and plotted as Kaplan-Meier curves. [Figure 15]Granzyme B-mediated AR reduces TCE-induced toxicity and cytokine release in a TNBC model. Plasma samples were prepared at cull from mice implanted with triple-negative breast cancer solid tumors and treated with either NVG-111 or AR_TCE-3 in combination with human T cells. Circulating levels of T cell-derived cytokines, including human IFNγ and granzyme B, were measured by ELISA. Symbols represent individual values, and horizontal bars represent the mean distribution for each condition. The shaded area indicates the lower limit of detection, and ND means not detectable. ns and ** mean not significant and p<0.01, respectively, in an unpaired t-test. [Figure 16A] AR_TCE demonstrated therapeutic efficacy without any loss in the TNBC model. (A) The efficacy of AR_TCE was assessed by tumor progression. Tumor size was measured biaxially using a micrometric caliper, and volume was calculated using the spherical formula. The volumes calculated on days 15 and 18 were integrated to reduce variability, and subsequent measurements were normalized to this value. Results are shown as fold increase from normalized baseline. Symbols and connected curves represent the mean ± SD of six mice per group. Arrows indicate the day of T cell injection. *, **, and *** indicate p<0.05, p<0.01, and p<0.001, respectively, by one-way ANOVA with Sidak's multiple comparisons. [Figure 16B-C] (B) Tumors were excised at the time of animal culling and photographed with a bar representing 10 mm. (C) The excision volume was measured from the images and expressed as the mean volume. Symbols represent individual values, and * indicates p<0.05 by unpaired t-test. [Figure 17]AR_TCE reduces metastatic progression in a TNBC model. The impact of the expanded therapeutic index of TCE in the presence of AR on metastatic progression in a TNBC model was evaluated. (A) Livers from vehicle- and AR_TCE-3-treated animals were removed at the end of the study and photographed, with the bar representing 10 mm. (B) To assess metastatic invasion within the tissue, immunofluorescence staining for MDA-MB-231 cells was performed on liver sections, and images were acquired under a wide-field microscope, with the bar representing 1 mm. Arrows indicate large cancer cell clusters, and dotted boxes define the magnified area. (C) Metastatic nodule density was quantified from the fluorescence images and classified as nodule diameter (d) <100 μm or >100 μm. Symbols represent individual animals, and bars represent the mean values ​​for each condition. ns indicates non-significant and **P < 0.01 by two-way ANOVA with Sidak's multiple comparisons. [Figure 18A] In an intraperitoneal model of pancreatic cancer, granzyme B-mediated AR reduced TCE-induced cytokine release and increased survival. The safety profile of AR was evaluated in a mouse model of intraperitoneally implanted ROR1+ pancreatic tumors. (A) Immunocompromised NOD.SCID gamma mice were first induced to stably express NVG-111 or AR_TCE-3, or not (vehicle). On day 0, all animals received a single intraperitoneal injection of 2 x 106 human pancreatic cancer PANC-1 cells and allowed to recover for 8 days. Mice then received four cycles of purified human T cell therapy. [Figure 18B-C] (B) Animals were carefully monitored during the treatment period and ethically terminated when one of the study endpoints (severe weight loss, acute toxicity) was met. Survival outcomes are expressed as the proportion of animals and plotted as Kaplan-Meier curves. (C) Plasma samples were prepared from all animals 24 hours after the second T cell injection, and cytokine levels were measured by ELISA. Symbols represent individual values, and horizontal bars represent the mean distribution for each condition. The shaded area indicates the lower limit of detection, and ND means not detectable. ns and * mean not significant and p<0.05, respectively, in an unpaired t-test. [Figure 19]AR_TCE reduces tumor burden in an intraperitoneal pancreatic cancer model. Mice were implanted intraperitoneally with pancreatic cancer and treated with either NVG-111 or AR_TCE-3 in combination with human T cells. Tumor progression was monitored using bioluminescence imaging on an IVIS imager. Mice were injected intraperitoneally with D-luciferin to detect luciferase-positive PANC-1 cells and imaged 15 minutes later. The color range indicates tumor bioluminescence intensity, and white crosses mark mice ethically culled before day 22. [Figure 20A] Design of autoregulatory CAR-T cells targeting ROR1. Autoregulatory CAR-T cells (AR_CAR-T) were engineered using the ROR1 CAR-T cell template. (A) The upper region of the hinge connecting the ROR1-targeting scFv of the CAR receptor and the CD8a transmembrane domain was modified to incorporate the P4-P4' sequence of a granzyme B-sensitive cleavable peptide. [Figure 20B] (B) The ability of the modified AR_CAR receptor to be cleaved from CAR-T cells by granzyme B was tested in vitro. Control CAR-T or AR_CAR-T cell variants were incubated with 100 nM granzyme B for 2 hours at 37°C. The presence of remaining CAR receptor on the cell surface was detected using fluorescent protein L in flow cytometry. Results were normalized to untreated and expressed as the percentage of remaining CAR receptor. Histograms represent mean values, and symbols represent individual values. [Figure 21] Activation and target engagement capacity of AR_CAR-T cell variants. Target engagement and activation of AR_CAR-T cells were tested in a short-term killing assay model in which ROR1-positive Jeko-1 cells and CAR-T cells were co-cultured at various effector / target ratios for 48 hours. (A) CAR-T cell activation levels were measured by direct staining for CD69 in flow cytometry and expressed as the percentage of positive cells. (B) In parallel, the percentage of target cell killing in the Jeko-1 population was measured using viability dye uptake. Symbols represent the mean ± SD of four replicates. [Figure 22]AR_CAR-T cells maintain serial killing potential in vitro without loss. The serial killing potential of AR_CAR-T cell variants was evaluated in a long-term coculture assay using excess target cells. Control CAR-T cells or AR_CAR-T cells were cocultured with ROR1-positive Jeko-1 at an effector-to-target ratio of 1:10 for 5 days. The percentage of target cell killing was measured every 24 hours by flow cytometry using viability dye uptake. Symbols represent the mean ± SD of four replicates. ns indicates not significant by one-way ANOVA with Sidak's multiple comparisons test. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention It is understood that different applications of the disclosed invention may be tailored to the particular needs of the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0023] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes "antibodies," and the like.

[0024] All publications, patents, and patent applications cited in this document, whether supra or infra, are hereby incorporated by reference in their entirety.

[0025] Unless otherwise specified, technical terms are used according to conventional usage. Definitions of common 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 in this document have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0026] In general, the term "comprising" is intended to include, but is not limited to, For example, the phrase "a therapeutic agent comprising a polypeptide" should be interpreted to mean that the agent comprises at least one polypeptide, although the agent may include additional components.

[0027] In some embodiments of the invention, the word "comprising" is replaced with the phrase "consisting of." The word "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 agent consists of the polypeptide and does not contain additional components.

[0028] In some embodiments of the present invention, the word "comprising" is replaced with the phrase "consisting essentially of." The term "consisting essentially of" means that certain additional components may be present, i.e., components that do not significantly affect the essential characteristics of the invention. 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.

[0029] The terms "self-regulation" and "autoregulation" are equivalent and are used interchangeably in this disclosure. The term "AR" is an abbreviation for "autoregulation." "AR" is an equivalent term to "autoregulation" and "self-regulation."

[0030] therapeutic agent A therapeutic agent of the present invention is any substance useful for achieving a therapeutic goal or result, for example, a substance or combination of substances useful for preventing or treating a disease or condition. Therapeutic agents include, but are not limited to, proteins, nucleic acid molecules, and other molecules of interest. In some embodiments, a therapeutic agent comprises a polypeptide, such as an antigen-binding protein. In some embodiments, a therapeutic agent consists essentially of a polypeptide, such as an antigen-binding protein. In some embodiments, a therapeutic agent is a polypeptide, such as an antigen-binding protein. One of skill in the art will appreciate that a particular agent may be useful for achieving more than one result. A therapeutic agent acts on a metabolic pathway 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, including preventing side effects resulting from the adverse effects of such treatment.

[0031] A therapeutically effective amount of a therapeutic agent may be defined as the amount of said agent, such as the disclosed antigen binding proteins, sufficient to achieve a desired effect in a treated subject. For example, this may be the amount necessary to inhibit tumor growth or prevent bleeding. In some 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 is generally used that achieves target tissue concentrations shown in vitro to achieve the desired effect.

[0032] Self-regulating / autoregulating elements The autoregulatory or autoregulatory elements of the present invention are small peptides that are cleavable by endogenous factors. Small peptides can be defined as amino acid sequences that are at least 4 but not more than 15 amino acids in length, preferably at least 5, 6, or 7 amino acids, and more preferably at least 8 but not more than 15 amino acids in length. In preferred embodiments, the autoregulatory / autoregulatory element is 7, 8, 9, or 10 amino acids in length. In particularly preferred embodiments, the autoregulatory / autoregulatory element is 8 amino acids in length.

[0033] The autoregulatory element does not significantly affect the activity of the therapeutic agent; i.e., the therapeutic agent can function within its therapeutic range even in the presence of the autoregulatory element. Suitable tests for therapeutic activity are known to those of skill in the art. The autoregulatory element is placed in an exposed position within the therapeutic agent so that the autoregulatory element has access to endogenous factors.

[0034] In the therapeutic agents of the present invention, the activity of the endogenous factor is altered as a result of the activity of the therapeutic agent, and cleavage of the autoregulatory element by the endogenous factor results in the separation of the therapeutic agent into non-functional fragments. Non-functional fragments may be defined as fragments that no longer have the required therapeutic activity or fragments that are subject to degradation or removal at such a rate that they become functionally inactive. Therapeutic activity includes binding to the target antigen. Binding kinetics may be examined by standard experimental techniques.

[0035] 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.

[0036] The autoregulatory elements of the present invention can be defined according to the following proteolytic cleavage recognition sites: P4-P3-P2-P1-cleavage site-P1'-P2'-P3'-P4', Here, P represents a single amino acid (aa).

[0037] The endogenous factor is one that is active in the same metabolic pathway as the therapeutic agent of the present invention acts in. It is envisioned that a suitable endogenous factor can be selected taking into consideration the metabolic pathway of the disease on which the therapeutic agent acts.

[0038] By way of example and not limitation, the following proteases are associated with the hemophilia metabolic pathway (also known as the coagulation cascade): thrombin (clotting factor IIa, IIa), clotting factor IXa (IXa), clotting factor Xa (Xa), clotting factor XIa (XIa), clotting factor XIIIa (XIIIa), and activated protein C (APC).

[0039] 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 sequences Ile, Glu, Gly, and Arg. The amino acids at positions P1'-P4' may not determine the cleavage specificity of factor Xa.

[0040] Thrombin is one of the most widely studied proteases. Its central role in the coagulation cascade as well as in several other areas has been thoroughly documented. Thrombin regulates the coagulation process both positively by cleaving prothrombin, FV, and FVIII, and negatively by cleaving protein C. The cleavage recognition sites of thrombin have been extensively studied (see, for example, Gallwitz et al. The Extended Cleavage Specificity of Human Thrombin, (2012) Plos One; 7, e31756). The consensus recognition sequences have been identified as follows: P4 - aliphatic, P3 - non-negatively charged aa, P2 - Pro, P1 - Arg, P1' - Ser / Ala / Gly / Thr, P2' - non-acidic, P3' - Arg, and P4 - non-specific aa.

[0041] In a preferred embodiment of the invention, the autoregulatory element may comprise one of the following thrombin cleavage recognition site sequences: LTP RG VRL (SEQ ID NO: 1), LTP RL VRL (SEQ ID NO: 2), LTP RD VRL (SEQ ID NO: 3), or WYL RS NNG (SEQ ID NO:4), the cleavage occurs between the two underlined amino acids. In a particularly preferred embodiment, the autoregulatory element has the sequence LTP RG VRL (SEQ ID NO: 1).

[0042] By way of further example, T cells are involved in the immune system's response to infection, to diseases such as cancer, or to 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: members of the granzyme family (including granzymes A, B, K, H, M), members of the cathepsin family (including cathepsins B, L, W), members of the matrix metalloproteinase family (including MMP-2, MMP-9, MMP-28).

[0043] Granzymes are granule-stored serine proteases that participate in T cell- and natural killer cell-mediated cytotoxic defense responses after target cell recognition. Their primary function is to induce the death of virus-infected and other potentially harmful cells. Granzyme B, a type of granzyme, undergoes directional exocytosis upon contact with target cells with the assistance of perforin (a cytolytic protein expressed by cytotoxic T cells and natural killer cells) and enters the target cell. Granzyme B processes and activates various caspase precursors, thereby inducing apoptosis of the target cell. In the present invention, the term "granzyme B protease" includes enzymes classified or potentially classified under Enzyme Commission number EC 3.4.21.79 in the Enzyme Nomenclature Database (34th edition, February 2004, http: / / www.expasv.orq / enzvme).

[0044] Granzyme B proteases are known to have a tendency to cleave after aspartic acid residues (D), and granzyme B is the only mammalian serine protease known to have this P1 proteolytic specificity. Therefore, in accordance with the present invention, the cleavage site of granzyme B in useful embodiments is envisioned to include at least an aspartic acid residue at the P1 position located N-terminal to the cleavage site. Some currently known recognition sites for granzyme B proteases 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.

[0045] Thus, in an embodiment of the invention, the granzyme B cleavage recognition site has an amino acid sequence of the following general formula: P4-P3-P2-P1-cleavage site located at the N-terminus of the cleavage site, where P4 is preferably amino acid I or V, P3 is preferably amino acid E, Q or M, P2 is X (X represents any amino acid), and P1 is preferably amino acid D.

[0046] In a preferred embodiment of the present invention, the autoregulatory element may comprise one of the following granzyme B recognition site sequences: IEP DS ESQ (SEQ ID NO: 5), IEA DS ESQ (SEQ ID NO: 6), IEP DS LEE (SEQ ID NO: 7), HSRLGRIEA DS ESQEDIIRN (SEQ ID NO: 8), VEP DS LEE (SEQ ID NO: 9), LEP DS LEE (SEQ ID NO: 10), IEG DS LEE (SEQ ID NO: 11), IEA DS LEE (SEQ ID NO: 12), IEP DA LEE (SEQ ID NO: 13), IEP DS EEQ (SEQ ID NO: 14), IEP DS EVE (SEQ ID NO: 15), IEP NS LEE (SEQ ID NO: 16), IEP ES LEE (SEQ ID NO: 17), or IEP QS LEE (SEQ ID NO: 18). In a particularly preferred embodiment, the autoregulatory element has the sequence IEP DS LEE (SEQ ID NO: 7).

[0047] In a preferred embodiment of the present invention, the cleavage rate of the autoregulatory element by an endogenous factor correlates with the activity level of the therapeutic agent. The cleavage rate of the autoregulatory element can be calculated according to standard experimental techniques, such as using Western blotting to visualize protein fragments released over time. The activity level of the therapeutic agent can be calculated using standard experimental techniques, such as the degree of binding to the target via Kd analysis. Further examples of suitable techniques are discussed in the examples of this application.

[0048] In a preferred embodiment of the present invention, the rate of cleavage of an autoregulatory element by an endogenous factor correlates with the activity level of the therapeutic agent and the amino acid sequence of the autoregulatory element. The rate of cleavage of an autoregulatory element by an endogenous factor can be adjusted by altering the amino acid sequence of the autoregulatory element.

[0049] Polypeptides In a preferred embodiment of the invention, a therapeutic agent comprises a polypeptide or protein. In one embodiment, a therapeutic agent of the invention consists essentially of a polypeptide.

[0050] A polypeptide, or protein, can be defined as any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). In a preferred embodiment of the present invention, the polypeptide is an antigen-binding protein. In a preferred embodiment of the present invention, the polypeptide is an antibody or a fragment thereof. A "residue" refers to an amino acid or amino acid mimetic incorporated into a polypeptide by an amide bond or an amide bond mimetic. Polypeptides have an amino-terminus (N-terminus) and a carboxy-terminus. Conservative amino acid substitution tables providing functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0051] antigen-binding proteins An antigen can be defined as a compound, composition, or substance that stimulates antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens such as the disclosed antigens. "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 presented in association with an MHC molecule. Epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and nuclear magnetic resonance.

[0052] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants recognized by immune cells. Antigens can include peptides derived from pathogens or cancer cells of interest. Exemplary pathogens include bacteria, fungi, viruses, and parasites. In some embodiments, the antigen is derived from cancer cells, such as blood cancer cells (chronic lymphocytic leukemia - CLL, acute lymphoblastic leukemia, mantle cell lymphoma) or solid malignant tumors (breast, pancreatic, melanoma). In some preferred embodiments, the antigen is a ROR1 polypeptide or an antigenic fragment thereof.

[0053] A "target epitope" is a specific epitope on an antigen that specifically binds to an antibody of interest, such as a monoclonal antibody. In some instances, the target epitope includes amino acid residues that contact the antibody of interest, such that the target epitope can be selected by the amino acid residues determined to contact the antibody.

[0054] In a preferred embodiment of the present invention, the therapeutic agent comprises an antigen-binding protein. In a preferred embodiment 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, which comprises a heavy chain variable domain including heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3, and 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.

[0055] In a preferred embodiment of the present invention, the antigen-binding protein comprises a first antigen-binding domain, 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 region (LCDR)1, LCDR2, and LCDR3, 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.

[0056] In one embodiment, the autoregulatory element may be adjacent to the first antigen-binding domain and located at the C-terminus of the first antigen-binding domain. The term "adjacent to" means that the autoregulatory element is positioned so as not to affect the function of the first antigen-binding domain, but is located next to the first antigen-binding domain. The autoregulatory element may be located within a peptide linker sequence. The linker sequence may 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).

[0057] In a preferred embodiment, the antigen binding protein forms 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 a CAR-T cell, a CAR-M cell, or an engineered NK cell.

[0058] 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 (single domain antibodies derived from camels), shark IgNAR-derived single domain antibodies, 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 (see, for example, Verma R 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. Multivalent antigen-binding proteins of the invention may comprise multiple specificities, e.g., bispecific, or may be monospecific. Antigen-binding proteins of the invention may be biparatopic.

[0059] An scFv protein is a fusion protein in which the light and heavy chain variable regions of an immunoglobulin are linked by a linker, while in dsFv, the chains are mutated to introduce disulfide bonds to stabilize the association of the chains. This term also includes genetically engineered heteroconjugate antibodies such as chimeric antibodies and bispecific antibodies. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd See also Ed., W.H. Freeman & Co., New York, 1997.

[0060] Antigen-binding proteins of the present invention include, but are not limited to, (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibodies with the enzyme papain to yield an intact light chain and a portion of the heavy chain; (2) Fab', a fragment of an antibody molecule obtained by treating whole antibodies with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, a fragment of an antibody obtained by treating whole antibodies with the enzyme pepsin, without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single-chain antibodies ("SCAs"). A genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable polypeptide linker as a genetically fused single-chain molecule.

[0061] In one embodiment of the invention, an antigen-binding protein may comprise a heavy chain and a light chain comprising a constant region and a variable region (regions are also known as "domains"). In some embodiments of the invention, heavy and light chain variable domains combine to specifically bind to an antigen. In further embodiments of the invention, only the heavy chain variable domain is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Light and heavy chain variable domains contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., 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 within a species. The framework region of an antibody, collectively the framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0062] CDRs are primarily responsible for antigen binding. The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, consisting of the framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0063] The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus) and are typically identified by the chain in which the particular CDR is located. H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while V LCDR1 is the CDR1 of the antibody light chain variable domain in which it is found. Light chain CDRs may also be referred to as CDR L1, CDR L2, CDR L3, or LCDR1, LCDR2, LCDR3. Heavy chain CDRs may also be referred to as CDR H1, CDR H2, CDR H3, or HCDR1, HCDR2, HCDR3.

[0064] Residues in antibody variable domains are conventionally numbered according to IMGT (http: / / www.imgt.org). This system is described in Lefranc MP (1997, J. Immunol. Today, 18, 509). This numbering system is used herein unless otherwise indicated.

[0065] The IMGT residue designations do not necessarily correspond directly to the linear numbering of the amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict IMGT numbering, corresponding to shortening of, or insertion into, structural elements of the basic variable domain structure, whether framework or CDR. The correct IMGT numbering of residues may be determined for a given antibody by alignment of homologous residues in the antibody sequence with the "standard" IMGT numbering sequence.

[0066] Suitable antigen binding proteins, antibodies or binding fragments thereof may be disclosed herein by the primary amino acid sequences of the heavy and light chain CDRs, the heavy and light chain variable regions, and / or the full-length heavy and light chains.

[0067] An antigen-binding protein, antibody, or binding fragment thereof may comprise, in addition to a VL CDR1, one or more VH CDR sequences of said specific antigen-binding protein or antibody, and alternatively or additionally, one or more VL CDR sequences. An antigen-binding protein, antibody, or binding fragment thereof may comprise one, two, or all three of the VH CDR sequences of said specific antigen-binding protein, antibody, or binding fragment thereof as described above, and alternatively, or additionally, one, two, or all three of the VL chain CDR sequences of said specific antigen-binding protein, antibody, or binding fragment thereof, including a VL CDR1. An antigen-binding protein, antibody, or binding fragment thereof may comprise all six CDR sequences of said specific antigen-binding protein, antibody, or binding fragment thereof as described above.

[0068] Variants of antigen-binding proteins or antibodies may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions from the specific sequences and fragments described above while retaining the activity of the antigen-binding proteins or antibodies described herein. "Deletion" variants include, for example, the deletion of 1, 2, 3, 4, or 5 individual amino acids, or the deletion of one or more small groups of amino acids, such as 2, 3, 4, or 5 amino acids. "Small groups of amino acids" may be defined as those that are contiguous or closely adjacent to each other but not contiguous. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids, making conservative amino acid substitutions. For example, an amino acid can be substituted with an alternative amino acid having similar properties, such as 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 minute amino acid, another small amino acid, or another large amino acid. Some properties of the 20 main amino acids that can be used to select appropriate replacements are as follows:

[0069] [Table 1]

[0070] Preferred "derivatives" or "variants" include those in which an amino acid that appears in the sequence instead of a naturally occurring amino acid is its structural analog. The amino acids used in the sequence may be derivatized or modified, e.g., labeled, as long as this does not significantly adversely affect the function of the antibody.

[0071] Such derivatives and variants may be prepared by modification during synthesis or after production of the antigen binding protein or antibody, or when the antigen binding protein or antibody is in recombinant form, using known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0072] Preferably, the antigen binding protein or antibody variant has an amino acid sequence that has more than 60%, or more than 70%, such as 75% or 80%, preferably more than 85%, for example more than 90%, 95%, 96%, 97%, 98% or 99% amino acid identity to the VL and / or VH of an antigen binding protein or antibody disclosed herein, or a fragment thereof. This level of amino acid identity may be found over the entire length of the relevant SEQ ID NO sequence, or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full-length polypeptide.

[0073] Preferably, the antigen binding protein or antibody variant comprises one or more of the CDR sequences as described herein.

[0074] In the context of amino acid sequences, "sequence identity" refers to sequences having the indicated values ​​when assessed using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680) with the following parameters: Pairwise alignment parameters - Method: Slow / Accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10; Multiple alignment parameters - matrix: PAM, gap opening 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 penalty: on, hydrophilic gap penalty: on, hydrophilic residues: G, P, S, N, D, Q, E, K, R. Sequence identity at specific residues is intended to include only derivatized identical residues.

[0075] The methods of the present invention may use antibodies having specific VH and VL amino acid sequences, as well as variants and fragments thereof that maintain the function or activity of these VH and VL.

[0076] "V H References to "V" or "VH" refer to an immunoglobulin heavy chain variable region, including the heavy chain variable region of an antibody fragment such as an Fv, scFv, dsFv or Fab. L" or "VL" refers to an immunoglobulin light chain variable region, including the light chain variable region of an Fv, scFv, dsFv or Fab.

[0077] In some embodiments of the invention, the antigen-binding protein may be an antibody comprising heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0078] In the present invention, IgG1 (e.g., IgG1 / kappa) antibodies having IgG1 heavy and light chains may be advantageously used. However, other human antibody isotypes, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE in combination with kappa or lambda light chains, are also encompassed by the present invention. Animal-derived antibodies of all different isotypes may also be used in the present invention. The antibody may be a full-sized antibody or an antigen-binding fragment of an antibody, including Fab, F(ab')2, a single-chain Fv fragment, or a single VHH, VH, or VL domain.

[0079] 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 the tailpiece and may or may not be connected by a J chain. For IgG, the Fc region includes immunoglobulin domains C gamma 2 and C gamma 3 (Cγ2 and Cγ3) and the lower portion of the hinge between C gamma 1 (Cγ1) and Cγ2. Although the boundaries of the Fc region vary, the human IgG heavy chain Fc region is usually defined as including residues C226 or P230 through the carboxyl terminus, according to the EU index. In the case of IgA, the Fc region comprises immunoglobulin domains C alpha 2 and C alpha 3 (C alpha 2 and C alpha 3) and the lower hinge region between C alpha 1 (C alpha 1) and C alpha 2. Included in the definition of an Fc region are functionally equivalent analogs and variants of an Fc region. A functionally equivalent analog of an Fc region may be a mutant Fc region containing one or more amino acid modifications relative to a wild-type or naturally occurring Fc region. A mutant Fc region has at least 50% homology to a naturally occurring Fc region, e.g., about 80%, about 90%, or at least about 95% homology. A functionally equivalent analog of an Fc region may contain one or more amino acid residues added or deleted from the N-terminus or C-terminus of the protein, e.g., additions and / or deletions of 30 or fewer or 10 or fewer. 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 contain most of all Ig domains that make up the Fc region, as defined above; for example, IgG and IgA Fc regions, as defined herein, must contain most of the CH2-encoding sequence and most of the CH3-encoding sequence. Thus, the CH2 domain alone or the CH3 domain alone is not considered an Fc region. Fc region can refer to this region alone or in the context of an Fc fusion polypeptide.

[0080] When referring to an antigen-binding protein of the present invention, the binding of the protein to an 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 biologics. Thus, under specified conditions, the antigen-binding protein will preferentially bind to a particular target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of a tumor, such as ROR1) and will not bind in significant amounts to other proteins or polysaccharides present in a sample or subject. Specific binding can be determined by methods known in the art. For antibody-antigen complexes, specific binding of an antigen to an antibody is typically determined by a method known in the art. -5 Less than 10 moles -6 Less than 10 moles -7 Less than 10 moles, e.g., about 10 -7 Less than 10 moles -8 Less than 10 moles -9 Less than 10 moles, or even about 10 -10 Submolar K d It has.

[0081] 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) between the antigen-binding protein and its target. Similarly, the binding specificity of an antigen-binding protein to a target can be defined by the relative dissociation constant (Kd) of an antibody to a target compared to the dissociation constants for the antigen-binding protein and another non-target molecule.

[0082] Typically, the Kd of an antibody for a target is 2-fold, preferably 5-fold, and more preferably 10-fold less than the Kd for other non-target molecules, such as unrelated or accompanying substances in the environment. More preferably, the Kd is less than 50-fold, even more preferably less than 100-fold, and even more preferably less than 200-fold.

[0083] The value of the dissociation constant can be determined directly by well-known methods, and even for complex mixtures, it can be calculated by methods such as those described by Caceci MS and Cacheris WP (1984, Byte, 9, 340-362). For example, Kd can be established using a double-filter nitrocellulose filter binding assay such as that described by Wong I and Lohman™ (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or by using, for example, Octet surface plasmon resonance.

[0084] One method for assessing binding affinity is ELISA. Other standard assays for assessing the binding ability of a ligand, such as an antibody, to a target are known in the art, including, for example, Western blot, RIA, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of an antibody can also be assessed by standard assays known in the art, such as surface plasmon resonance, for example, using a Biacore™ system analysis.

[0085] 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 particular epitope. The monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, 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).

[0086] In a preferred embodiment of the invention, the therapeutic agent is an antigen-binding protein comprising a first antigen-binding domain and a second antigen-binding domain, and the autoregulatory element is located between the first antigen-binding domain and the second antigen-binding domain. The term "between" may be interpreted to mean that the autoregulatory element is located at the C-terminus of the first antigen-binding domain and the N-terminus of the second antigen-binding domain. In one embodiment of the invention, the antigen-binding protein comprises one or more autoregulatory elements. In a preferred embodiment of the invention, the antigen-binding protein is a bispecific antibody comprising a first autoregulatory element at the C-terminus of the first antigen-binding domain and a second autoregulatory element at the N-terminus of the second antigen-binding domain.

[0087] In one embodiment of the invention in which the antigen binding protein comprises an scFv, an autoregulatory element may be located within the scFv in a peptide linker sequence between the heavy and light chain variable regions such that cleavage of the autoregulatory element renders the scFv non-functional.

[0088] In a preferred embodiment of the invention, the first and second antigen-binding domains of the antigen-binding protein are scFv proteins covalently linked by a peptide linker. In a preferred embodiment, the autoregulatory element is present within the peptide linker.

[0089] As noted below, the sequences of each of the light and heavy chain variable domains referred to above may differ from the sequences provided. For example, the light / heavy chain variable domains may comprise sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth in the Sequence Listing. Alternatively, the light / heavy chain variable domain sequences may differ at up to 10 amino acid positions, although preferably there are fewer than 10 amino acid substitutions, such as up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions.

[0090] As mentioned above, in some embodiments, the light chain variable domain and the heavy chain variable domain comprise amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences set forth above. For example, the light chain framework regions, heavy chain framework regions, light chain variable domain, and heavy chain variable domain may comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitution in the amino acid sequences set forth above. If there are variations in the sequences of the light chain variable domain and the heavy chain variable domain, the amino acid substitutions are preferably not in the CDRs. In particular, the light chain framework region and / or the heavy chain framework region of the antibody as described above may comprise an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described above. Furthermore, the light chain framework region and / or the heavy chain framework region may comprise at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid substitution in the amino acid sequence as described above. Preferably, the amino acid substitutions are conservative substitutions as described above. For example, the framework region may comprise such substitutions to humanize the sequence. Preferably, the framework region is humanized.

[0091] The sequences of each light chain variable domain and heavy chain variable domain of the second antigen-binding domain mentioned above may differ from the predetermined sequence. For example, the light chain / 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 set forth in the Sequence Listing. Alternatively, the light chain / heavy chain variable domain sequences may differ at up to 10 amino acid positions, but preferably there are fewer than 10 amino acid substitutions, such as up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions. Preferably, there are no substitutions in the heavy chain / light chain CDRs.

[0092] Antigen-binding proteins containing antigen-binding domains can be prepared by proteolytic hydrolysis of antibodies or by expression in mammalian cells of DNA encoding the fragments. Antibody fragments can be obtained by conventional methods such as pepsin or papain digestion of whole antibodies. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to produce a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent and, optionally, a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see U.S. Pat. Nos. 4,036,945 and 4,331,647 and references 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. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

[0093] Other methods of cleaving antigen-binding proteins and antibodies, such as separating heavy chains to form monovalent light and heavy chain fragments, further cleaving the fragments, or other enzymatic, chemical, or genetic techniques, may also be used so long as the fragment binds to an intact antibody or the antigen recognized by the antigen-binding protein.

[0094] In one embodiment of the invention, the antigen binding protein is exogenously expressed in CAR-T cells, CAR-M cells, or engineered NK cells.

[0095] Location of the cleavage site within the antigen-binding protein In one embodiment of the present invention, the autoregulatory element is positioned in an exposed position within the antigen binding protein such that the autoregulatory element is accessible to endogenous factors.

[0096] In one embodiment of the invention, the antigen binding protein comprises an autoregulatory element, wherein the autoregulatory element is located within a peptide linker sequence.

[0097] In one embodiment of the invention, the antigen binding protein comprises an autoregulatory element, wherein the autoregulatory element is located within a peptide linker sequence within the first and / or second antigen binding domain.

[0098] In one embodiment, the autoregulatory element may be adjacent to and C-terminal to the first antigen-binding domain. The term "adjacent" means that the autoregulatory element is positioned so as not to affect the function of the first antigen-binding domain, but is located within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the first antigen-binding domain. The autoregulatory element may be located within a peptide linker sequence. The linker sequence may 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). In one embodiment of the present invention, the autoregulatory element is located directly C-terminal to the peptide linker having the sequence GGGGS (SEQ ID NO. 59).

[0099] In one embodiment of the present invention, the autoregulatory element may be located between the first and second antigen-binding domains of the antigen-binding protein, hi one embodiment, the autoregulatory element may be located in the hinge region located between the first and second antigen-binding domains.

[0100] In one embodiment of the invention, the antigen-binding protein comprises a constant region, and the autoregulatory element is located within the hinge region of the constant region, between CH1 and CH2. In a preferred embodiment, there are two autoregulatory elements, each located within the hinge region of the constant region, between CH1 and CH2.

[0101] ROR1-binding antigen-binding protein In a preferred embodiment, the antigen-binding protein of the present invention comprises an autoregulatory element comprising a granzyme B cleavage site. 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), the first antigen-binding domain binding to an epitope of ROR1 comprising amino acid Gln-261, and an autoregulatory element comprising a granzyme B cleavage site.

[0102] In a preferred embodiment of the 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 an epitope of ROR1 that includes 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 set forth in SEQ ID NO 7.

[0103] The antigen-binding proteins disclosed in this document that specifically bind to ROR1 and CD3 are disclosed in WO2019 / 008379, which is incorporated herein by reference.

[0104] 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 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; the heavy chain variable domain comprises 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; and the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0105] The antigen-binding protein specifically binds to a ROR1 polypeptide and specifically binds to CD3. The first and / or second antigen-binding domains may be monoclonal antibodies or antigen-binding fragments thereof. In certain embodiments, both the first and second antigen-binding domains are monoclonal antibodies or antigen-binding fragments thereof.

[0106] As shown above, the sequence of each CDR may differ from the given sequence at up to two amino acid positions. That is, a CDR may contain one or two amino acid substitutions compared to the given sequence. However, even if one or more CDRs contain amino acid substitutions, the antibody can still selectively bind to ROR1. Preferably, the amino acid substitutions are conservative substitutions.

[0107] 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.

[0108] In some embodiments, the heavy chain complementarity determining region 3 (HCDR3) comprises an amino acid sequence selected from any of the sequences set forth in SEQ ID NOs: 25, 26, 27, and 28. Preferably, the HCDR3 comprises an amino acid sequence selected from any of the sequences set forth in SEQ ID NOs: 26, 27, and 28.

[0109] Preferably, the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 29, 30, 31, 32, 33 and 34. More preferably, the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 30, 31, 32, 33 and 34.

[0110] Preferably, the heavy chain variable domain comprises the amino acid sequence set forth as 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 as one of SEQ ID NOs: 36, 37, 38, 39 and 40.

[0111] 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 combinations of these light and heavy chain regions, resulting in 25 different constructs.

[0112] Thus, in some embodiments, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 36, 37, 38, 39, and 40. In particular embodiments, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 30, and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 36, 38, and 39.

[0113] In other embodiments, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO:31 and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs:36, 37, 38, 39, and 40.

[0114] In a further embodiment, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO:32 and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs:36, 37, 38, 39 and 40.

[0115] In an alternative embodiment, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 33, and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0116] In various embodiments, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 34, and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 36, 37, 38, 39, and 40.

[0117] Similarly, in some embodiments, the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO:36, and the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs:30, 31, 32, 33, and 34.

[0118] In other embodiments, the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO:37 and the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs:30, 31, 32, 33, and 34.

[0119] In a further embodiment, the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 38, and the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0120] In an alternative embodiment, the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 39, and the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0121] In various embodiments, the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 40, and the light chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0122] In certain embodiments, (a) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 29 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 35; (b) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 30 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 36; (c) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 31 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 37; (d) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 32 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 38; (e) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 33 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 39; or (f) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 34, and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 40.

[0123] 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. Furthermore, bispecific antibodies that bind to tumor antigens and CD3 are also known in the art, such as those described in 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).

[0124] The second antigen-binding domain that selectively binds to the CD3 subunit of the T cell receptor (TCR) may 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 as 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 as 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 as one of SEQ ID NOs: 42, 43, 44, 45, and 46, and the heavy chain variable domain comprises the amino acid sequence set forth as one of SEQ ID NOs: 48, 49, 50, 51, and 52.

[0125] In certain embodiments of the second antigen-binding domain, (a) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 41 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 47; (b) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 42 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 48; (c) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 43 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 49; (d) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 44 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 50; (e) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 45 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 51; or (f) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 46, and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 52.

[0126] 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 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 55; the heavy chain variable domain comprises 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; and the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0127] The first antigen-binding domain may have the structure of an antibody fragment, such as Fab, F(ab')2, or Fv, which contains the variable regions of the heavy and light chains and is capable of binding to an epitope determinant on ROR1. Similarly, the second antigen-binding domain may have the structure of an antibody fragment, such as Fab, F(ab')2, or Fv, which contains the variable regions of the heavy and light chains and is capable of binding to an epitope determinant on CD3. These antibody fragments retain the ability to selectively bind to antigens, as described above. Methods for producing these fragments are known in the art (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988).

[0128] In a further group of embodiments, the antigen-binding domain may have the structure of an Fv antibody, which is usually about 25 kDa and contains a complete antigen-binding site with three CDRs for each heavy and light chain. H and V L can be expressed in a host cell from two separate nucleic acid constructs. H and V LWhen expressed discontinuously, the chains of Fv antibodies are typically held together by noncovalent interactions. However, because these chains tend to dissociate upon dilution, methods have been developed to crosslink the chains via glutaraldehyde, intermolecular disulfides, or peptide linkers. Thus, in one example, an Fv can be a disulfide-stabilized Fv (dsFv), in which the heavy and light chain variable regions are chemically linked by a disulfide bond.

[0129] In a further example, the Fv fragment comprises V fragments connected by a peptide linker. H Chain and V L These single-chain antigen-binding proteins (scFv) contain V chains linked by oligonucleotides. H and V L scFvs are prepared by constructing a structural gene containing DNA sequences encoding the V domains. This structural gene is inserted into an expression vector, which is then introduced into host cells, such as mammalian cells or E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Exemplary linkers include the amino acid sequence 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). Single-chain antibody dimers (scFV2s) are also contemplated.

[0130] The antigen-binding proteins disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). Generally, the antigen-binding protein is derivatized so that binding to the ROR1 polypeptide and the CD3 subunit is not adversely affected by the derivatization or labeling. For example, the antigen-binding protein can be operably linked by chemical coupling, genetic fusion, non-covalent association, or other methods to one or more other molecular entities, such as other antibodies, detection agents, pharmaceutical agents, and / or proteins or peptides that mediate association of antibodies with other molecules (such as a streptavidin core region or a polyhistidine tag).

[0131] Bispecific antibodies can be produced by crosslinking two or more antibodies (of the same or different types). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), which have two distinct reactive groups separated by a suitable spacer. Such linkers are available, for example, from Pierce Chemical Company (Rockford, IL).

[0132] In certain embodiments, the ROR1 antigen-binding domain may be an scFv antibody. In some embodiments, the CD3 antigen-binding domain is an scFv antibody. In various embodiments, both the ROR1 antigen-binding domain and the CD3 antigen-binding domain are scFv antibodies. These two scFv antibodies may be covalently linked using a short peptide linker of 5 to 20 amino acids.

[0133] In a preferred embodiment, the autoregulatory element is located within this short peptide linker.

[0134] 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.

[0135] The antibody may be labeled with a detectable moiety or marker as described above. The antibody may also be labeled with a radioactively labeled amino acid. Examples of radioactive labels 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 may be used for both diagnostic and therapeutic purposes.

[0136] Means of detecting such labels are well known to those of skill in the art. Thus, for example, radioactive labels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light, enzymatic labels are usually detected by providing the enzyme with a substrate and detecting the reaction product produced by the enzyme acting on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0137] An antibody can be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups, which may be useful to improve the biological properties of the antibody, such as increasing serum half-life or increasing tissue binding.

[0138] In certain embodiments, the ROR1 antigen-binding domain may be part of a chimeric antigen receptor (CAR). The CAR may include a spacer sequence or hinge region that links the ROR1-binding domain and the transmembrane domain and spatially separates the ROR1-binding domain from the endodomain of the CAR. The flexible spacer / hinge region allows the ROR1-binding domain to orient in different directions, enabling ROR1 binding. In this document, the terms "flexible spacer," "spacer sequence," and "hinge region" are used interchangeably as equivalent terms. Those skilled in the art will recognize that any suitable spacer sequence can be used. In a preferred embodiment, an autoregulatory element is located within this flexible spacer / hinge region. In a preferred embodiment, the hinge region is set forth as SEQ ID NO: 82. In a preferred embodiment, the autoregulatory element is located directly N-terminal to the hinge region. In a preferred embodiment, the autoregulatory element replaces the first 1, 2, 3, 4, or 5 amino acids of the hinge region. In a preferred embodiment, the autoregulatory element is a granzyme B cleavage site. In a preferred embodiment, the combination of the autoregulatory element and hinge region is set forth as SEQ ID NO: 83 or 84.

[0139] Factor IXa and X antigen-binding proteins 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 FIX / FIXa and a second antigen-binding domain that selectively binds 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 FIXa and a second antigen-binding domain that selectively binds 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.

[0140] In a preferred embodiment, the first antigen-binding domain and the second binding domain comprise a light chain variable domain and a heavy chain variable domain, respectively, wherein the light chain variable domain comprises light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3, and the heavy chain variable domain comprises heavy chain complementarity 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; the heavy chain variable domain comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3, wherein 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; the heavy chain variable domain comprises heavy chain complementarity determining regions (HCDRs) 1, HCDR2 and HCDR3, wherein 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; The sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

[0141] In a specific embodiment, for the first antigen-binding domain, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 72 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 73, and for the second antigen-binding domain, the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 72 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 74.

[0142] 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 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, and the antigen binding protein also comprises an autoregulatory element comprising a thrombin cleavage site.

[0143] Antigen-binding proteins can be produced by crosslinking two or more antibodies (of the same or different types). Suitable crosslinkers include heterobifunctional (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate), which have two distinct reactive groups separated by a suitable spacer. Such linkers are available, for example, from Pierce Chemical Company (Rockford, IL).

[0144] Polynucleotides, Vectors and Host Cells The present invention also encompasses polynucleotides, vectors, and expression vectors encoding the antigen binding proteins, antibodies, or binding fragments thereof described herein.

[0145] The present invention also relates to polynucleotides encoding any of the antigen-binding proteins or fragments described herein. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymer of any length of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, genomic DNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may be provided in isolated or purified form.

[0146] A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated into a polypeptide (in the case of mRNA) in vivo. 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' (carboxy) terminus. For the purposes of this disclosure, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence. In one embodiment, the polynucleotide comprises a sequence encoding a VH or VL amino acid sequence as described above. The polynucleotide may encode the VH or VL sequence of a specific antigen-binding protein, antibody, or binding fragment thereof, as disclosed herein.

[0147] An antigen-binding protein, antibody, or binding fragment thereof may then be produced from or delivered in the form of an expressible polynucleotide encoding it. If the antigen-binding protein or antibody contains more than one chain, the polynucleotide may encode one or more antibody chains. For example, the polynucleotide may encode an antibody light chain, an antibody heavy chain, or both. Two polynucleotides may be provided, one encoding an antibody light chain and the other encoding the corresponding antibody heavy chain. Such a polynucleotide or pair of polynucleotides may be co-expressed to generate an antibody.

[0148] Polynucleotides can be synthesized by methods well known in the art, for example, by the method described in Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0149] The nucleic acid molecules of the present invention may be provided in the form of expression cassettes comprising regulatory sequences operably linked to the insert sequence, thus allowing for the expression of the antibodies of the present invention in vivo. These expression cassettes are in turn typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes may be administered directly to the host subject. Alternatively, a vector comprising the polynucleotide may be administered to the host subject. Preferably, the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector may be any vector carrying a sufficient amount of genetic information to allow for the expression of a polypeptide, such as an antibody or binding fragment thereof, as defined above.

[0150] Expression vectors containing such polynucleotide sequences are also disclosed. Such expression vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, that may be required and correctly oriented to allow expression of the peptides of the invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook J et al. (1989, Molecular cloning: a laboratory manual; Cold Spring Harbor: New York: Cold Spring Harbor Laboratory Press).

[0151] One skilled in the art may use the sequences described herein to clone or generate cDNA or genomic sequences, for example, as described in the examples below. Cloning of these sequences into a suitable eukaryotic expression vector, such as pcDNA3 (Invitrogen) or its derivatives, followed by transfection of mammalian cells (such as CHO cells) with the appropriate combination of light and heavy chain-containing vectors results in the expression and secretion of the antibodies described herein.

[0152] One skilled in the art can create analogs of the antigen binding proteins, antibodies, or binding fragments thereof described herein by using specific binding domains of the antigen binding protein or antibody sequences and expressing them in a different context, such as a polypeptide, such as a fusion protein, as is well known in the art.

[0153] Also disclosed are cells that have been modified to express the 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 that may be modified by insertion of a vector or expression cassette encoding an antigen binding protein or antibody of the invention include mammalian HEK293, CHO, HeLa, NS0, and COS cells. Preferably, the cell line selected is not only stable, but also capable of mature glycosylation.

[0154] Such cell lines may be routinely cultured to produce the antigen binding protein, antibody, or binding fragment thereof, or may be used therapeutically or prophylactically to deliver the antigen binding protein, antibody, or binding fragment thereof to a subject, or the polynucleotide, expression cassette, or vector of the invention may be administered to the subject's cells ex vivo and the cells returned to the subject.

[0155] Pharmaceutical Composition When used in the methods of the invention, the therapeutic agent, preferably an antigen binding protein as defined above, may be provided as a pharmaceutical composition comprising the therapeutic agent or antigen binding protein. The present invention therefore encompasses pharmaceutical compositions comprising a therapeutic agent or antigen binding protein and a pharmaceutically acceptable carrier for use in the methods of the invention.

[0156] As used herein, a "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity or absorption delaying agents, or the like. Preferably, the carrier is suitable for parenteral administration, e.g., 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, liposome, micelle, liquid crystal, nanocrystal, nanoparticle, emulsion, microparticle, microsphere, nanosphere, nanocapsule, polymer or polymeric carrier, surfactant, suspending agent, complexing agent such as cyclodextrin or adsorbent molecule such as albumin, surface-active particle, and chelating agent. In further embodiments, polysaccharides include hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives (e.g., methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate butyrate, hydroxypropylmethylcellulose acetate), chitosan and its derivatives, [beta]-glucan, arabinoxylan, carrageenan, pectin, glycogen, fucoidan, chondrotin, dermatan, heparan, heparin, pentosan, keratan, alginic acid, cyclodextrin, and salts and derivatives thereof (including esters and sulfates).

[0157] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, buffered water, and physiological saline. Other examples of carriers include ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by using coating materials such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0158] Pharmaceutical compositions may contain pharmaceutically acceptable antioxidants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, as described above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Furthermore, the absorption of injectable pharmaceutical forms may be prolonged by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0159] Therapeutic compositions must usually be sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0160] Sterile injectable solutions can be prepared by incorporating the required amount of an active agent (e.g., an antibody) in an appropriate solvent containing one or a combination of the ingredients listed above, as needed, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying (lyophilization), which yield a powder of the active agent and any additional desired ingredients from a previously sterile-filtered solution.

[0161] Pharmaceutical compositions may contain additional active ingredients, as well as the therapeutic agents defined above. As noted above, compositions of the present invention may contain one or more antigen-binding proteins. They may also contain additional therapeutically or prophylactically active agents.

[0162] Depending on the route of administration, the antigen binding protein, antibody, or binding fragment thereof may be coated with a material to protect the antigen binding protein or antibody from the action of acids and other natural conditions that may inactivate or denature the antigen binding protein or antibody.

[0163] In a preferred embodiment, the pharmaceutical composition according to the present invention is in a form selected from the group consisting of an aqueous solution, a gel, a hydrogel, a film, a paste, a cream, a spray, an ointment, or a wrap.

[0164] In further embodiments, the pharmaceutical compositions described herein may be administered intravenously, subcutaneously, intraocularly, intramuscularly, intraarticularly, intradermally, intraperitoneally, intraspinally, or by other parenteral routes of administration, such as by injection or infusion. Administration may be rectal, oral, ophthalmic, topical, epidermal, or mucosal. Administration may be topical, including by inhalation. In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously. In one embodiment, the pharmaceutical composition may be administered by inhalation. In one embodiment, a metered dose delivery device containing the pharmaceutical composition is used.

[0165] Also disclosed herein are kits comprising the therapeutic or other compositions of the invention and instructions for use. The kits may further comprise one or more additional reagents, such as additional therapeutic or prophylactic agents as discussed herein.

[0166] Methods for the prevention and treatment of diseases The methods of the invention can be for the prevention or treatment of a disease or condition in a subject, in which case the method comprises administering to the subject a prophylactically or therapeutically effective amount of said therapeutic agent, such as an antigen binding protein.

[0167] In therapeutic applications, a therapeutic agent such as an antigen-binding protein is administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially suppress the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms, or an increase in the frequency or length of symptom-free periods. An amount sufficient to accomplish this is defined as a "therapeutically effective amount." The effective amount for a given purpose will depend on the subject's weight and general condition, as well as the severity of the disease or injury. In prophylactic applications, a polypeptide or composition is administered to a subject not yet exhibiting symptoms of a disorder or condition in an amount sufficient to prevent or delay the onset of symptoms. Such an amount is defined as a "prophylactically 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.

[0168] In certain embodiments, a therapeutic agent, such as an antigen-binding protein, may be linked (directly or indirectly) to another moiety. The other moiety may be an additional therapeutic agent, such as a drug. The other moiety may be a detectable label. The other moiety may be a binding moiety, such as an antibody or polypeptide binding domain, specific for a therapeutic target. The antigen-binding protein of the present invention may be a bispecific antibody.

[0169] Additional therapeutic agents or detectable labels may be directly attached to the antigen-binding proteins of the present invention, for example, by chemical conjugation. Methods for conjugating drugs or labels to antigen-binding proteins are known in the art. For example, carbodiimide conjugation (Bauminger S and Wilchek M, 1980, Methods Enzymol., 70, 151-159) can be used to conjugate various drugs, including doxorubicin, to antibodies or peptides. The water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularly useful for conjugating functional moieties to binding moieties.

[0170] Other methods of conjugating moieties to therapeutic agents may also be used, such as oxidation of appropriate reactants with sodium periodate followed by reductive alkylation, or glutaraldehyde cross-linking. However, regardless of the method chosen to generate the conjugates of the present invention, it is recognized that it must be determined that the antigen-binding protein or antibody maintains its targeting ability and that the functional moiety maintains its relevant function.

[0171] Additional therapeutic agents linked to the antigen binding protein may include therapeutically beneficial polypeptides or polynucleotides encoding polypeptides. Examples of such polypeptides include anti-proliferative or anti-inflammatory cytokines.

[0172] The antigen-binding protein may be linked to a detectable label. By "detectable label" is meant that the antigen-binding protein is linked to a moiety that can be detected outside the body and non-invasively from the target site when it is located at the target site after administration of the antigen-binding protein to a patient.

[0173] Typically, the label is or contains a radioactive atom useful for imaging. Suitable radioactive atoms include 99mTc and 123I for scintigraphy studies. Other labels include, for example, spin labels for magnetic resonance imaging (MRI) (again 123I, 131I, 111In, 19F, 13C, 15N, 17O, gadolinium, manganese, iron, etc.). Clearly, a sufficient amount of the appropriate atomic isotope must be attached to the antibody to render the molecule readily detectable.

[0174] Radiolabels or other labels can be incorporated using known methods. For example, antigen-binding proteins, antibodies, or fragments thereof can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors, e.g., containing fluorine-19 instead of hydrogen. Labels such as 99mTc, 123I, 186Rh, 188Rh, and 111In can be attached, for example, through a cysteine ​​residue in a polypeptide. Yttrium-90 can be attached through a lysine residue. Preferably, the detectable label comprises a radioactive atom, e.g., technetium-99m or iodine-123. Alternatively, the detectable label can be selected from the group including iodine-123; iodine-131; indium-111; fluorine-19; carbon-13; nitrogen-15; oxygen-17; gadolinium; manganese; and iron.

[0175] Therapeutic agents of the present invention, preferably antigen-binding proteins of the present invention, can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those of skill in the art, the route and / or method of administration will vary depending on the desired results. Preferred routes of administration of antigen-binding proteins, antibodies, or compositions of the present invention include intravenous, subcutaneous, intraocular, intramuscular, intradermal, intraperitoneal, spinal, or other parenteral routes of administration, such as administration by injection or infusion. As used herein, "parenteral administration" refers to a method of administration other than enteral and topical administration, typically by injection. Administration may be by rectal, oral, ocular, topical, epidermal, or mucosal routes. Administration may be local, including peritumoral, juxtatumoral, intratumoral, at the tumor margin, intralesional, perilesional, by intracavitary injection, intravesicular, or by inhalation. In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously.

[0176] The appropriate dosage of a therapeutic agent can be determined by a skilled physician. The actual dosage of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration and is not toxic to the patient. The selected dosage will depend on various pharmacokinetic factors, including the activity of the particular therapeutic agent employed, the route of administration, the time of administration, the excretion rate of the therapeutic agent, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0177] An appropriate dosage of an antigen-binding protein may be, for example, within the range of about 0.1 μg to about 100 mg per kg of body weight of the patient being treated. For example, an appropriate dosage may be about 1 μg / kg to about 50 mg / kg of body weight per week, about 100 μg / kg to about 25 mg / kg of body weight per week, or about 10 μg / kg to about 12.5 mg / kg per week.

[0178] Suitable dosages are 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.

[0179] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0180] The antigen-binding protein may be administered in a single dose or multiple doses. Multiple doses may be administered via the same or different routes and to the same or different sites. Alternatively, the antigen-binding protein may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency of administration may vary depending on the half-life of the antigen-binding protein in the patient and the desired duration of treatment. The dosage and frequency of administration may also vary depending on whether the treatment is preventative or therapeutic. In preventative applications, a relatively low dosage may be administered at relatively infrequent intervals over an extended period of time. In therapeutic applications, a relatively high dosage may be administered, for example, until the patient shows partial or complete improvement in disease symptoms.

[0181] Co-administration of two or more agents may be achieved in many different ways. In one embodiment, the therapeutic agent of the present invention and the other agent may be administered together in a single composition. In another embodiment, the therapeutic agent and the other agent may be administered in separate compositions as part of a combination therapy. For example, the therapeutic agent may be administered before, after, or simultaneously with the other agent.

[0182] Disease to be diagnosed, treated or prevented The therapeutically effective amount of an antigen-binding protein (or nucleic acid encoding an antigen-binding protein) depends on the severity of the disease and the patient's general condition. A therapeutically effective amount of an antigen-binding protein may provide either subjective relief of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer. As noted above, these compositions may be administered in combination with other therapeutic agents, either simultaneously or sequentially.

[0183] If the therapeutic activity of a therapeutic agent, such as an antigen binding protein, remains within a defined range, the therapeutic agent retains its full therapeutic potential; whereas, if the therapeutic activity is such that it may cause an adverse event, alteration of the activity of the endogenous factor leads to cleavage of the autoregulatory element, ultimately inactivating the therapeutic agent and reducing the risk of adverse event development.

[0184] Single or multiple administrations of a composition comprising an antigen-binding protein disclosed herein may be performed, depending on the dosage and frequency required and tolerated by the patient. In any event, the composition should provide a sufficient amount of at least one of the antigen-binding proteins disclosed herein to effectively treat the patient. Doses may be administered once, but may be applied periodically until a therapeutic result is achieved or until side effects warrant discontinuing treatment. In one example, a dose of the antigen-binding protein is infused over 30 minutes every other day. In this example, from about one to about ten doses may be administered, e.g., three or six doses every other day. In yet another example, a continuous infusion is administered for about five to about ten days. The subject may be treated at regular intervals, such as monthly, until the desired therapeutic result is achieved. Generally, the dose is sufficient to treat or ameliorate the symptoms or signs of the disease without causing unacceptable toxicity to the patient.

[0185] Further disclosed are compositions comprising an antigen-binding protein or a nucleic acid encoding an antigen-binding protein in a carrier. The compositions may be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are left to the discretion of the treating physician to achieve the desired goal. The antigen-binding protein and / or nucleic acid may be formulated for systemic or local administration. In one example, the antigen-binding protein or nucleic acid encoding the antigen-binding protein is formulated for parenteral administration, such as intravenous administration. In some embodiments, administration is intramuscular.

[0186] The active ingredient can be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Specifically, antibody-containing liposomes can be prepared by methods such as those 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. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. The reverse phase evaporation method can be used with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with defined pore sizes to obtain liposomes with the desired diameter. The polypeptides of the present invention can be conjugated to liposomes via a disulfide exchange reaction, for example, as described in Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0187] Compositions for administration may comprise a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, including, for example, buffered saline. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the antigen-binding protein in these formulations may vary widely and is selected primarily based on fluid volume, viscosity, body weight, and the like, depending on the particular method of administration selected and the needs of the subject. In some embodiments, administration is intravenous.

[0188] Controlled-release parenteral formulations can be prepared as implants, oily injections, or particulate systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a central core of a therapeutic protein, such as a cytotoxin or drug. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries have a diameter of about 5 μm, 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).

[0189] Polymers can be used for ion-controlled release of the antigen-binding proteins disclosed herein. Various degradable and non-degradable polymer matrices for use in 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 mobile liquid at low temperatures, but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle 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). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, 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)).

[0190] Typical pharmaceutical compositions for intravenous administration include about 0.1 to 10 mg / kg of antigen-binding protein per day, or about 0.5 to 15 mg / kg of antigen-binding protein per day. Dosages of 0.1 to about 100 mg / kg per subject per day may be used, particularly when the agent is administered to a remote site, such as within a body cavity or lumen of an organ, and not to the circulatory or lymphatic system. Exemplary doses include 1 to 10 mg / kg, e.g., 2 to 8 mg / kg, e.g., 3 to 6 mg / kg. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in further detail in publications such as Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA (1995).

[0191] Antigen-binding proteins are provided in lyophilized form and can be rehydrated with sterile water before administration, but they are also provided in sterile solutions of known concentrations. Antigen-binding protein solutions are added to infusion bags containing 0.9% sodium chloride, USP, and are typically administered at a dose of 0.1-10 mg / kg or 0.5-15 mg / kg body weight. Exemplary doses include 1-10 mg / kg, e.g., 2-8 mg / kg, e.g., 3-6 mg / kg. Since the approval of Rituxan® in 1997, considerable knowledge has become available in the art regarding the administration of antibody drugs marketed in the United States. Administration of antigen-binding proteins can be by slow infusion rather than intravenous push or bolus administration. In one example, a high dose is administered, followed by lower maintenance doses. For example, a loading dose of 4 mg / kg may be infused over a period of approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg infused over a period of 30 minutes for 4 to 8 weeks if the previous dose is well tolerated.

[0192] A therapeutically effective amount of nucleic acid encoding an antigen-binding protein can be administered to a subject in need thereof. One approach to nucleic acid administration is direct immunization with plasmid DNA, such as a mammalian expression plasmid. The nucleotide sequence encoding the antigen-binding protein can be placed under the control of a promoter to increase expression of the molecule. Immunization with nucleic acid constructs is well known in the art, and several methods for delivering nucleic acids to organisms are described in, for example, U.S. Patent Nos. 5,643,578, 5,593,972, 5,817,637, and 5,880,103. These methods include liposomal delivery of nucleic acids.

[0193] In another approach using nucleic acids, antigen-binding proteins can be expressed by attenuated viral hosts or vectors, or bacterial vectors, and then administered to a subject. Recombinant vaccinia virus, adeno-associated virus (AAV), herpesvirus, retrovirus, cytomegalovirus, poxvirus, or other viral vectors can be used to express antibodies. For example, vaccinia vectors are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the disclosed antigen-binding proteins (see Stover, Nature 351:456-460, 1991).

[0194] In one embodiment, nucleic acids encoding antigen-binding proteins are directly introduced into cells. For example, nucleic acids can be loaded into gold microspheres using standard methods and then introduced into the skin using a device such as Bio-Rad's Heliosae Gene Gun. The nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter.

[0195] Typically, DNA is injected into muscle, but it can also be injected directly into other sites. The dosage for injection is typically about 0.5 mg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0196] In some examples, DNA encoding an antigen-binding protein is administered to a subject to provide in vivo production of the antigen-binding protein, for example, using the subject's cellular machinery. Immunization with nucleic acid constructs is well known in the art and is taught, for example, in U.S. Patent Nos. 5,643,578, 5,593,972, and 5,817,637. U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding an organism, including liposomal delivery of nucleic acids. Those skilled in the art can apply such methods to the production of antigen-binding proteins.

[0197] One approach to nucleic acid administration is direct administration via 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.

[0198] Treatment of Hemophilia A Embodiments of the invention in which the autoregulatory element comprises a thrombin, factor IXa, factor Xa, factor XIa, factor XIIIa, or APC cleavage site are useful for the treatment of hemophilia A. Embodiments of the invention in which the autoregulatory element comprises a thrombin cleavage site are useful for use in the treatment of hemophilia A. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for the treatment of hemophilia A comprises a thrombin cleavage site. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for the treatment of hemophilia A comprises an autoregulatory element set forth in SEQ ID NO: 1, 2, 3, or 4. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for the treatment of hemophilia A comprises an autoregulatory element set forth in SEQ ID NO: 1.

[0199] 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 FIXa / FIX as defined herein, and a second antigen-binding domain that specifically binds FX / FXa as defined herein, and a thrombin cleavage site as defined herein.

[0200] Accordingly, also disclosed is a method of treating hemophilia A in a subject, the method comprising administering to the subject a therapeutically effective amount of the disclosed antigen binding proteins and / or nucleic acids encoding the antigen binding proteins, thereby treating the hemophilia.

[0201] The present invention also relates to the disclosed antigen binding proteins for use in the treatment of hemophilia A. Furthermore, the present invention also relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for the treatment of hemophilia A.

[0202] Embodiments of the invention in which the autoregulatory element comprises a thrombin, factor IXa, factor Xa, factor XIa, factor XIIIa, or APC cleavage site are useful for preventing prothrombotic risk. Embodiments of the invention in which the autoregulatory element comprises a thrombin cleavage site are useful for use in preventing prothrombotic risk. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for preventing prothrombotic risk comprises a thrombin cleavage site. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for preventing prothrombotic risk comprises an autoregulatory element set forth in SEQ ID NO: 1, 2, 3, or 4. In a preferred embodiment of the invention, an antigen binding protein of the invention useful for preventing prothrombotic risk comprises an autoregulatory element set forth in SEQ ID NO: 1.

[0203] Accordingly, in another embodiment of the present invention, a method of preventing a prothrombotic risk 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 a nucleic acid encoding the antigen binding protein, thereby preventing the prothrombotic risk.

[0204] The present invention also relates to the disclosed antigen binding proteins for use in the prevention of prothrombotic risk. Furthermore, the present invention also relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for the prevention of prothrombotic risk.

[0205] Cancer Treatment Embodiments of the invention in which the autoregulatory element comprises a cleavage site for granzyme A, granzyme B, granzyme K, granzyme H, granzyme M, cathepsin B, cathepsin L, cathepsin W, or a matrix metalloprotease such as MMP-2, MMP-9, and MMP-28 are suitable for use in the treatment of cancer. Embodiments of the invention in which the autoregulatory element comprises a granzyme B cleavage site are suitable for use in the treatment of cancer. In preferred embodiments of the invention, the antigen binding protein comprises an autoregulatory element set forth in any one of SEQ ID NOs:5-18. In preferred embodiments of the invention, the antigen binding protein comprises an autoregulatory element set forth in SEQ ID NO:7. In preferred embodiments of the invention, the antigen binding protein comprising a granzyme B cleavage site comprises a first antigen-binding domain that specifically binds to ROR1, as defined herein. In a preferred embodiment of the invention, the antigen binding protein comprising a granzyme B cleavage site comprises a first antigen binding domain that specifically binds to ROR1, as defined herein, and a second antigen binding domain that specifically binds to CD3, as defined herein.

[0206] Accordingly, also disclosed are methods of treating cancer in a subject, the methods comprising administering to the subject a therapeutically effective amount of the disclosed antigen binding proteins and / or nucleic acids encoding the antigen binding proteins, thereby treating the cancer.

[0207] The disclosed antigen binding proteins can be cytotoxic to cancer cells.

[0208] Preferably, the cancer is leukemia (such as chronic lymphocytic leukemia (CLL), acute lymphoblastic 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, etc. Furthermore, ROR1 is expressed in a subset of cancer stem cells.

[0209] The present invention also relates to the disclosed antigen binding proteins for use in the treatment of cancer. Additionally, the present invention relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for the treatment of cancer.

[0210] Preferably, the cancer is leukemia (such as chronic lymphocytic leukemia (CLL), acute lymphoblastic 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, etc. Furthermore, ROR1 is expressed in a subset of cancer stem cells.

[0211] The cancer or tumor need not be completely eliminated for the composition to be effective. For example, the antibody can reduce the tumor by a desired amount, e.g., by 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.

[0212] Administration of an antigen binding protein of the invention may result in 5%, 10%, 20%, 50%, 75%, 90%, 95% or 99% depletion of malignant cells, i.e., reduction of malignant cells.

[0213] In another example, the subject may be administered an effective amount of an additional agent, such as a chemotherapeutic agent. The method may include administering one or more additional agents known in the art.

[0214] Severe toxicities associated with immunotherapy mechanisms of action (MoA), including but not limited to cytokine release syndrome (CRS) and neurotoxicity, result in acute systemic inflammatory syndrome (ASS), characterized by fever and multiple organ dysfunction. Acute systemic inflammatory syndrome can be associated with (i) chimeric antigen receptor (CAR) T-cell therapy, (ii) therapeutic antibodies, and (iii) haploidentical allogeneic stem cell transplantation (HAC). Granzyme B levels are elevated in subjects progressing toward severe MOA-associated toxicities. Embodiments of the invention in which the autoregulatory element comprises a cleavage site for granzyme A, granzyme B, granzyme K, granzyme H, granzyme M, cathepsin B, cathepsin L, cathepsin W, or a matrix metalloproteinase such as MMP-2, MMP-9, and MMP-28 are suitable for use in preventing MoA-associated toxicity associated with immunotherapy, such as cytokine release syndrome. Embodiments of the invention in which the autoregulatory element comprises a granzyme B cleavage site are suitable for use in preventing MoA-associated toxicity associated with immunotherapy. In preferred embodiments of the invention, the antigen binding protein comprises an autoregulatory element set forth in any one of SEQ ID NOs:5-18. In preferred embodiments of the invention, the antigen binding protein comprises an autoregulatory element set forth in SEQ ID NO:7.

[0215] In another embodiment of the present invention, a method for preventing MoA-associated 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 proteins and / or nucleic acids encoding the antigen binding proteins, thereby preventing MoA-associated toxicity.

[0216] The present invention also relates to the disclosed antigen binding proteins for use in the prevention of MoA-associated toxicity associated with immunotherapy. Furthermore, the present invention also relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for the prevention of MoA-associated toxicity.

[0217] Other antigen binding proteins that may benefit from the introduction of granzyme B autoregulatory elements to prevent MoA-associated toxicity include muromonab, basiliximab, daclizumab, blinatumumab, and alemtuzumab.

[0218] In another embodiment of the present invention, a method of preventing cytokine release syndrome 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 a nucleic acid encoding the antigen binding protein, thereby preventing cytokine release syndrome.

[0219] The present invention also relates to the disclosed antigen binding proteins for use in the prevention of cytokine release syndrome. Further, the present invention relates to the use of the disclosed antigen binding proteins in the manufacture of a medicament for the prevention of cytokine release syndrome.

[0220] Other antigen-binding proteins that may benefit from the introduction of a granzyme B autoregulatory element to prevent cytokine release syndrome include muromonab, basiliximab, daclizumab, blinatumumab, and alemtuzumab. [Example]

[0221] Example 1 - Rationale FVIII mimetic antibodies in hemophilia A Coagulation factor VIII (FVIII) is a key cofactor in the coagulation cascade, facilitating the catalytic conversion of activated factor IX (FIXa) and factor X (FX) to activated FXa through complex formation. In hemophilia A (an X-linked monogenic disorder affecting 1 in 5,000 male births), FVIII deficiency inhibits coagulation, resulting in a life-threatening idiopathic bleeding disorder. Standard treatment, FVIII replacement therapy, requires frequent intravenous infusions of FVIII. However, approximately 30% of treated patients develop neutralizing anti-FVIII antibodies, rendering FVIII replacement therapy ineffective and significantly reducing survival.

[0222] Emicizumab, a humanized IgG4 bispecific antibody approved in late 2017, is a FVIII mimetic molecule that acts by simultaneously binding to FIXa and FX, promoting the FIXa-mediated catalytic conversion of FX to activated FXa, leading to thrombin generation and restoration of clot formation. Emicizumab has redefined the treatment of hemophilia A by reducing bleeding episodes by 80%, with prophylaxis achievable 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 were observed in emicizumab-treated patients, prompting the FDA to issue a black box warning against the use of this therapy.

[0223] The mechanisms by which FVIII and emicizumab maintain the coagulation cascade are fundamentally different (Lenting, Denis et al. 2017). FVIII circulates in an inactive form and requires activation to FVIIIa to promote thrombin generation. Thrombin further activates protein C (PC), inhibiting FVIIIa through a negative feedback loop that prevents excessive procoagulant activity. In contrast, emicizumab remains constitutively active and is not "switched off" by activated PC (aPC) after clot formation, thereby promoting a prothrombotic state as long as it remains in the circulation. This dangerous state could be overcome by the development of an autoregulatory FVIII-mimetic antibody. By introducing a thrombin-cleavable peptide that acts as a built-in "switch" mechanism, it is possible to cleave the autoregulatory antibody 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.

[0224] Bispecific T cell engagers for adoptive immunotherapy Bispecific T cell engagers (TCEs) are bispecific antibodies that target the cytotoxic activity of T cells against malignant tumor cells. While this is a highly effective mechanism of action, it is also associated with the occurrence of severe adverse events, including cytokine release syndrome (CRS) and neurotoxicity, resulting in life-threatening immune hyperactivation in a significant number of patients. Indeed, a 100% adverse event rate has been observed with the therapeutic use of TCEs, and grade 3 and 4 life-threatening toxicities have been observed affecting more than 50% of patients treated with currently approved TCEs (blinatumomab, mocenutuzumab, and teclistamab). While mitigation measures such as tumor burden reduction, corticosteroid administration, and dose escalation are effective, they do not eliminate these toxicities. These limitations constrain optimal dosing, shorten treatment duration, and increase monitoring burden, particularly for complex or vulnerable patients. Therefore, the development of autoregulatory TCEs (AR_TCEs) that are inactivated in a threshold-limited manner and in response to biological cues before the onset of grade 3 / 4 life-threatening toxicity could efficiently reduce the incidence of adverse events, expand the therapeutic index of TCEs, and ultimately improve outcomes for cancer patients.

[0225] Upon engagement with both targets, TCE clusters CD3 on the T cell surface, leading to activation and the formation of an immune synapse. This stimulates the local release of cytotoxic enzymes, including granzyme B, resulting in target cell apoptosis and elimination. This T cell-mediated release of cytokines and proteolytic enzymes is required for efficient cancer cell killing, and at low levels is associated with TCE's mechanism of action. However, findings from TCE and CAR-T cell studies have shown that life-threatening MoA-associated toxicity is a hallmark of overdrive T cell activation, during which granzyme B levels can increase by up to 40-fold (Kochenderfer, Somerville et al. 2017). Therefore, inserting a granzyme B-sensitive peptide between the targeting and effector arms of TCE could facilitate drug degradation and inactivation upon exposure to elevated granzyme B levels above the threshold associated with the development of MoA-associated toxicity, thereby reducing the risk of serious adverse events.

[0226] Chimeric antigen receptor (CAR)-T cell therapy Chimeric antigen receptor (CAR) T cells are cell therapies genetically engineered to express chimeric cell surface receptors that target tumor-associated antigens (TAAs) of interest and have proven clinically effective by redirecting the cytotoxic activity of T cells to eliminate cancer cells. The mechanism of action of CAR T cells is comparable to that of bispecific TCEs, but they are associated with a high risk of severe MoA-related toxicities characteristic of T cell redirecting technologies, including cytokine release syndrome and neurotoxicity.

[0227] The cytotoxicity of CAR-T is similar to that of TCE and involves the release of granzyme B as a result of overactivation of CAR-T cells. Therefore, the development of autoregulatory AR_CAR-T cells by inserting granzyme B-sensitive peptides into the structure of the CAR receptor would enable cell surface cleavage and removal of the receptor in response to cues associated with the development of MoA-associated toxicity, thereby significantly improving the safety profile of this class of therapeutic agents without affecting their efficacy.

[0228] Example 2 - Materials and Methods Bispecific antibody production and purification Emicizumab was obtained from surplus vials of commercially available Hemlibra™, provided by the Katharine Dormandy Centre for Hemophilia and Thrombosis (Royal Free Hospital, London). The production and purification of the autoregulatory 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, with the final product demonstrating >99% purity by size-exclusion chromatography.

[0229] NVG-111 and autoregulatory AR_TCE variants were produced in-house by transient transfection in the mammalian Expi293 expression system (Gibco). High-density Expi293™ cells were routinely subcultured in Expi293 expression medium at 37°C, 8% CO2, and 130 rpm. For transfection, cells were cultured the day before in Expi293 expression medium at a concentration of 1 x 10 per ml. 6The cells were seeded at a viable cell count of 1000 kJ / ml. The next day, the desired volume of cell suspension was transfected with plasmids containing the coding sequences of the different antibodies cloned into the pcDNA3.1hygro(+) expression vector using ExpiFectamin™ according to the manufacturer's instructions. 16–18 h after transfection, cells were fed with a production enhancer as recommended to increase protein yield, and the medium was incubated for an additional 5 days. The product supernatant was harvested and clarified by both centrifugation (4000 g, 20 min) to remove cells and filtration through a 0.22 μm filter. The different variants were purified by affinity chromatography using a 5 mL HiTrap™ column with MabSelect™ Prism A resin (Cytiva) on an AEKTA avant 25 system (Cytiva). The purified protein was eluted with 50 mM glycine, 150 mM NaCl, pH 3 and immediately neutralized with 2% v / v of 2 M Tris-base (pH 8.3). After purification, the antibody was buffer-exchanged into a formulation solution containing 10 mM histidine, 150 mM NaCl, 0.02% Tween 80, pH 6.0, and stored at -80°C.

[0230] Chromogenic assay The FVIII mimetic activity of emicizumab and AR_Ab8 was assessed by measuring FIX-mediated activation of FX to FXa using the FXa-specific S-2765 chromogenic substrate. In a polystyrene 96-flat-bottom half-well plate (Greiner Bio-one), 50 μL of a coagulation factor solution containing 40 μM phospholipid-TGT (Rossix AB), 280 nM plasma-derived human FX (Coagadex®, Bio Product Laboratory), 6 nM plasma-derived human FIXa (Haematologic Technologies), and 10 mM CaCl2 in running buffer (50 mM Tris-base, 150 nM NaCl, 0.1% BSA-protease-free, pH 7.8) was mixed with 25 μL of 2 nM purified antibody and incubated at 37°C for 15 min. Next, 25 μL of S-2765 chromogenic substrate (Chromogenix) diluted to 1.2 mM in running buffer was added to each well, and the kinetics of color conversion was immediately recorded at 405 nm on a plate reader (SpectraMax m3, Molecular Devices) for 20 min at 37°C (one measurement per min, vortexing for 2 s before each measurement).

[0231] Activated partial thromboplastin time (aPTT) Citrated pools of human plasma from at least 20 healthy volunteers were spiked with 200 Bethesda units of a neutralizing polyclonal antibody (PAHFVIII-S, Haematologica Technologies) to remove FVIII activity. Induced hemophilia A plasma was spiked with 350 nM emicizumab or AR_Ab8 and incubated for 15 minutes at room temperature. Clotting times were measured using aPTT synthaSil reagent (Werfen Limited) in an ACL Top 700 coagulometer (Werfen Limited).

[0232] Thrombin cleavage assay To assess bispecific antibody inactivation by thrombin, a 2 nM solution of emicizumab or AR_Ab8 was mixed with 2 U / mL of 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 stopped at different time points, from 15 min to 3 h, by adding 500 nM of PPACK inhibitor (Merck - Millipore) solution v / v, and samples were frozen for further analysis.

[0233] Antibody cleavage was detected by SDS-Page and Western blotting. Samples were loaded undiluted onto NuPAGE 4-12% bis-Tris precast gels (Invitrogen) and run at 120-180V in MOPS-SDS running buffer (Invitrogen). For molecular weight comparison, 5 μL of prestained PageRuler Protein Ladder (Thermofisher) was added to separate wells. Proteins were further transferred to a nitrocellulose blotting membrane by wet transfer in NuPAGE transfer buffer (Invitrogen). The membrane was saturated in TBS.T buffer (50 mM Tris-base, 150 mM NaCl, 0.1% Tween 20, pH 7.8) with 5% nonfat milk for 1 hour on an orbital shaker at room temperature, and then blotted overnight at 4°C with horseradish peroxidase (HRP)-conjugated polyclonal goat anti-human Fc antibody (#31413, Invitrogen) diluted 1:5000 in TBS.T + 5% milk. The membrane was washed three times in TBS.T and incubated with SuperSignal West Pico PLUS chemiluminescent substrate (Thermofisher) for 5 minutes at room temperature. Images were captured using a GelDoc system (Bio-Rad).

[0234] Residual FVIII mimetic activity of the samples was measured using a chromogenic assay as previously described. Samples were diluted 1:2 to achieve a bispecific antibody concentration of 1 nM.

[0235] Prothrombotic thrombin generation assay (TGA) Abnormal thrombin generation was measured using a calibrated automated thrombogram (CAT) method with a thrombinoscope (Diagnostica stago). FVIII activity was neutralized in pooled human plasma using 200 BU of anti-FVIII antibody as previously described. The neutralized plasma was added with 600 nM emicizumab or AR_Ab8 in combination with 0.5 U / mL aPCC (FEIBA, Takeda Pharmaceuticals) and incubated for 15 min at room temperature. For each condition, 80 μL of plasma was mixed with 20 μL of Tissue Factor PPP low trigger (Diagnostica stago) in an Immulon 2HB U-bottom plate (Diagnostica stago) and incubated for 10 min at 37°C. The thrombin generation reaction was initiated by automatic injection of 20 μL of prewarmed FluCa reagent, and data were recorded for 60 min. Endogenous thrombin potential (ETP) is calculated as the area under the thrombogram curve and represents the total amount of thrombin produced.

[0236] Tail clip assay The tail clip assay was used to evaluate the procoagulant potential of FVIII-mimetic bispecific antibodies in FVIII-deficient mice, and the treatment was performed as previously described (Ferriere et al., Blood 2020). Briefly, FVIII-deficient male mice (8–12 weeks old) were retroorbitally injected with 3 mg / kg emicizumab or AR_Ab8 24 h before treatment. A second retroorbital injection with a mixture of both human FIX and FX at 100 U / kg was administered 5 min before amputation of the distal tip of the tail 3 mm under ketamine (100 mg / kg) / xylazine (10 mg / kg) anesthesia. Control animals received a single retroorbital injection of 2 U / mouse recombinant human FVIII. The amputated tail was then immersed in prewarmed saline (37°C) and blood was collected for 30 min. The amount of hemoglobin in the blood collection tube was measured using a spectrophotometer at 416 nm, and the bleeding volume was quantified by calculation using a standard curve.

[0237] In vivo thrombosis model The thrombogenic potential of a FVIII-mimetic bispecific antibody was evaluated in vivo using an experimental protocol aimed at reproducing the combined effects of emicizumab and aPCC in mice. Eight- to ten-week-old C57Bl / 6 male mice (Charles River Laboratory) received an initial intravenous (IV) injection of 2.5 U of aPCC (FEIBA, Takeda Pharmaceuticals) combined with 250 μg of the bispecific antibody, followed by three booster injections of aPCC (IV, 2.5 U per injection) at 24, 48, and 72 hours. Control animals received only aPCC without the bispecific antibody. At 96 hours, all animals were bled from the caudal vena cava by cardiac puncture under non-reversible gas anesthesia (isoflurane) on EDTA anticoagulant (final concentration 0.05 M). All animals were humanely euthanized by cervical dislocation. Immediately after bleeding, platelet counts were performed using an automated cell counter (Scil Vet ABC Plus, Horiba Medical).

[0238] For thrombosis analysis, lungs were removed and fixed in 10% neutral-buffered formalin (CellPath limited) for 24 hours at room temperature. Fixed organs were briefly washed with PBS, embedded in OCT matrix (CellPath limited), and frozen in liquid nitrogen vapor. Tissue sections were prepared from frozen lung blocks and processed for immunofluorescence (IF) staining of platelets and blood vessels. Lung sections were washed with PBS for 10 minutes to remove the OCT matrix, followed by a detergent-based antigen retrieval step with a 10-minute incubation in PBS + 0.05% Tween 20 (PBS.T) and 0.5% Triton X-100. Sections were washed 3 times for 5 minutes with PBS.T, and nonspecific binding sites were saturated with 3% bovine serum albumin (BSA) in PBS.T (w / v) for 1 hour at room temperature (RT). Platelets and endothelial cells were detected by incubating sections 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. Slides were washed 3 times for 5 minutes in PBS.T and then incubated for 1 hour at room temperature on an orbital shaker with Alexa 488 donkey anti-rat (A48269) and Alexa-555 donkey anti-goat (A32816) polyclonal antibodies diluted in PBS.T + 1% BSA. After a final 3 x 5 minute wash step, slides were mounted under coverslips with a drop of ProLong Diamond antifade reagent (ThermoFisher Scientific) containing DAPI and allowed to dry in the dark for 24 hours.

[0239] Images were acquired using a 20x objective on a Z1 inverted microscope (Zeiss). To analyze thrombus formation, a single lung section with a section surface area of ​​>10 4 μm 2All individual blood vessels (identified by CD31 staining) were imaged. CD31 staining was used to manually delineate the vascular borders using Fiji software (open source). Using the αIIb (platelet) staining channel, a signal intensity threshold was empirically defined based on the signal-to-noise ratio, converted into a binary occlusion mask to define positive and negative areas of occlusion, and applied to all images from the same experiment. The occlusion rate for each individual blood vessel was calculated as the percentage of the surface occupied by the occlusion mask within the vascular border using Fiji software (open source software, GNU general public license).

[0240] Protease-mediated cleavage assay using TCE Granzyme B-specific cleavage of autoregulatory AR_TCE variants was evaluated in vitro. Recombinant human granzyme B protein (Bio-Techne) was first activated with mouse cathepsin C (Bio-Techne) as recommended in the manufacturer's instructions. 1 μg / mL of NVG-111 or AR_TCE was mixed with various 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 hours. The catalytic reaction was stopped by adding a broad-spectrum protease inhibitor cocktail (Tebu-Bio) to a final concentration of 1× (1:10 dilution), and samples were frozen and stored for further analysis. Antibody cleavage was visualized by SDS-PAGE electrophoresis and Western blotting using undiluted samples according to the protocol described above. The membrane was blotted overnight at 4°C with HRP-labeled protein L (Genscript) diluted 1:2000 in TBS.T + 5% milk.

[0241] To assess the susceptibility of AR_TCE to various proteases, cleavage assays were performed as described using a panel of representative proteases at a single concentration of 50 nM: activated FIIa, FVIIa, and FXa (Prolytix), caspase-3, caspase-8 (Bio-Techne), and caspase-9 (Abcam), and matrix metalloproteinases MMP-3 and MMP-9 (Abcam).

[0242] The remaining amount of intact antibody was quantified using a dedicated ELISA specific for the intact molecule. Polystyrene 96-flat-bottom half-well plates (Greiner Bio-one) were coated overnight at 4°C with 2 μg / mL of ROR1 protein (Abcam) diluted in carbonate buffer (12.2 mM NaCO3, 35 mM NaHCO3, pH 9.6). The plates were washed three times with TBS.T (50 mM Tris-base, 150 mM NaCl, 0.1% Tween 20, pH 7.8), and nonspecific binding sites were saturated with TBS.T + 3% BSA for 1 h at 37°C. After three washing steps, samples were diluted in TBS.T + 1% BSA and incubated for 2 h at 37°C. Unbound samples were washed three times with TBS. Bound, intact antibody was detected via the His-tag motif (released upon cleavage) present in the CD3-binding arm using an HRP-conjugated monoclonal anti-His tag antibody (clone J099B12, Biolegend) diluted 1:2500 in TBS + 1% BSA and incubated for 2 h at 37 °C. After the final three wash steps, the plate was incubated with slow-kinetic 3,3',5,5'-tetramethylbenzidine (TMB) substrate and developed for 5–15 min. The reaction was stopped by adding 2 M H2SO4 v / v, and the optical density was read at 450 nm within 30 min on a SpectraMax m3 (Molecular Devices).

[0243] Preparation and culture of T cells Human peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis chambers of healthy individuals and purified by Ficoll-Plaque density gradient. PBMCs were cultured at 37°C, 8% CO2, and 5 × 10 cells / mL in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U / mL recombinant human interleukin-2 (IL-2, Miltenyi Biotec). 5 The cells were cultured at a density of 1000 cells / ml for 5 days, which allowed for the gradual elimination of non-T cell related lineages.

[0244] Short-term co-culture assay NVG-111 and AR_TCE variants were characterized in a short-term coculture assay to assess T cell engagement and cytotoxicity against the ROR1+ mantle cell lymphoma Jeko-1 cell line. To track target cells during coculture, Jeko-1 cells were pre-stained with CellTrace™ Violet (CTV; C34557; Life Technologies) according to the manufacturer's instructions. Jeko-1 cells were then mixed with excess purified human T cells (prepared as described above) at a target-to-effector cell ratio of 1:5 and co-cultured for 48 hours with increasing concentrations of NVG-111 or AR_TCE.

[0245] At the end of the assay, cells were spun down at 300 x g for 5 minutes and washed once with PBS. The pellet was resuspended in a PBS solution containing Live / Dead fixable green dye (dilution 1:800, L23101, Life Technology) and APC-conjugated anti-human CD69 antibody (dilution 1:50, 310910, Biolegend). Cells were then incubated at 4°C for 60 minutes, washed once with PBS, and analyzed by flow cytometry on a CytoFlex (Beckman Coulter Life Sciences). Gating on CTV staining was used to distinguish T cells (CTV-) from Jeko-1 cells (CTV+). Cytotoxicity was measured as the percentage of dead cells (Live / Dead marker-positive staining) within the Jeko-1 population. T cell activation was measured by the mean fluorescence intensity (MFI) of the T cell population. All flow cytometry data were analyzed using FlowJo software (BD Biosciences).

[0246] Long-term co-culture assay The effect of autoregulation on cytokine release in vitro was assessed in a long-term coculture assay using excess target cells to simulate continuous T cell engagement. Purified human T cells were prepared as described above and mixed with excess Jeko-1 cells at a target-to-effector ratio of 10:1. These cells were cocultured for up to 120 hours in the presence or absence of 1 μg / mL NVG-111 or AR_TCE. Coculture samples were collected every 24 hours and centrifuged at 300g for 5 minutes to separate cells from the supernatant. Cells were then collected and stored at -80°C for further analysis. The cell pellets were washed once with PBS and incubated in Live / Dead fixable green dye solution as described above. Cells were washed again with PBS and fixed for 20 minutes at 4°C using CytoFix / CytoPerm reagent (BD Biosciences).

[0247] To assess cytotoxicity and T cell activation, fixed cells were probed with Pacific Blue-conjugated anti-human CD3 antibody (dilution 1:50, 300417, Biolegend) and PE-conjugated anti-human CD25 antibody (dilution 1:50, 356134, Biolegend) using the protocol described above and analyzed by flow cytometry. T cells and target cells were distinguished using a CD3 gating strategy. Cytotoxicity was measured as the percentage of dead cells in the CD3- population (Jeko-1 cells), and T cell activation was measured as the mean MFI of CD25 expression in the T cell population (CD3+). Human INFγ release from activated T cells was measured in coculture supernatants using an ELISA MAX Deluxe set (Biolegend) according to the manufacturer's recommendations. AR_TCE cleavage was detected using undiluted supernatant samples by SDS-Page electrophoresis and protein L immunoblotting as described above.

[0248] In vivo models of severe toxicity related to the mechanism of action of TCE, such as cytokine release syndrome The mechanism of action of AR_TCE and its potential to reduce the risk of severe toxicity associated with excessive T cell engagement were tested in an in-house model of adoptive immunotherapy in mice xenografted with triple-negative breast cancer (TBNC) solid tumors or disseminated pancreatic cancer cells (both ROR1+).

[0249] Eight- to ten-week-old NOD.SCID gamma (NSG) male mice (Charles River Laboratory) were first induced to express stable circulating levels of either NVG-111 or the autoregulatory AR_TCE-3 variant using a proprietary method. For the TNBC model, 2 × 10 MDA-MB-231 cell line were transfected. 6 were injected subcutaneously into the mammary fat pad of mice, and the resulting solid tumors were allowed to grow for 25 days. 6PANC-1 cells were injected directly into the peritoneal cavity and the mice were allowed to recover for 8 days. Once tumors formed, all animals were treated with six cycles (TNBC) or four cycles (pancreatic) of purified human T cell injections. T cells were prepared as described above, resuspended in 100 μL of PBS, and injected intravenously into the tail vein of mice. The first injection consisted of 10 cells per mouse. 7 T cells, subsequent injections were given at half this dose, and injections were spaced on a 4 day / 3 day schedule.

[0250] During the T cell administration phase, animal weights were recorded daily and normalized to the weight measured on the day of the first injection (day 25 for TNBC and day 8 for pancreatic cancer). The study protocol imposed an ethical limit of 20% weight loss from baseline, and animals that fell more than 1 g below this value were humanely culled. Furthermore, the general health of the mice and the appearance of the solid tumors (TNBC model) were monitored daily by staff from an independent animal facility. Signs of tumor distress, inflammation, or ulceration were grounds for ethical culling.

[0251] In the TNBC model, solid tumor progression was monitored every 2–3 days. The mean tumor diameter (d) was measured using micrometer calipers on two different axes, and the volume (V) was calculated using the sphere formula (Equation 1).

[0252]

number

[0253] The tumor volumes calculated on days 15 and 18 were averaged and used as a reference 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 intraperitoneally injected with 200 μg / mouse of D-luciferin (Melford Laboratories) diluted in 200 μL of PBS. 15 minutes after injection, BLI images were acquired using an IVIS Lumina II in vivo imaging system (Perkin-Elmer). Images were processed using Fiji software.

[0254] At the completion of the study or at early cull, mice were bled via cardiac puncture from the caudal vena cava under non-reversible gas anesthesia (isoflurane) on EDTA anticoagulant (final concentration 0.05 M). All animals were humanely euthanized 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 by ELISA using an ELISA MAX Deluxe set (Biolegend) for INFγ and a DuoSet system (DY-2906-05, Bio-Techne) for granzyme B, according to the manufacturer's recommendations. In the TNBC model, tumors were also excised and photographed with a millimetric ruler, and their volumes were calculated as described above.

[0255] Quantification of metastatic progression At the end of the study, livers from mice implanted with TNBC solid tumor models were removed and fixed in 10% neutral-buffered formalin (CellPath Limited) for 24 hours at room temperature before processing into paraffin blocks. Paraffin sections were prepared for immunofluorescence by deparaffinization in xylene for 3 × 5 minutes, rehydration with 100% ethanol for 3 × 5 minutes, and equilibration in HO. Sections were washed in PBS for 10 minutes and then subjected to a detergent-based antigen retrieval step involving a 10-minute incubation in PBS + 0.05% Tween 20 (PBS.T) and 0.5% Triton X-100. Sections were then washed in PBS.T for 3 × 5 minutes, and nonspecific binding sites were saturated with 3% bovine serum albumin (BSA) in PBS.T (w / v) for 1 hour at room temperature (RT). 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 invaded MDA-MB-231 cancer cells. Slides were washed 3 times for 5 minutes with PBS.T and then incubated for 1 hour at RT on an orbital shaker with Alexa-555 donkey anti-goat (A32816, Thermofisher) polyclonal antibody diluted 1:2000 in PBS.T + 1% BSA. After a final 3 x 5-minute wash step, slides were mounted under coverslips with a drop of ProLong Diamond antifade reagent with DAPI (ThermoFisher Scientific) and allowed to dry in the dark for 24 hours. Images were acquired with a PhenoImager HT slide scanner (Akoya Bioscience), and metastatic nodules in each section were manually quantified with diameters measured using Fiji software (open source software, GNU general public license).

[0256] Generation of CAR-T cells Lentiviruses encoding ROR1-targeted CAR receptors or AR_CAR variants were generated by triple transfection into the human embryonic kidney (HEK)-293T cell line using a second-generation lentiviral packaging system consisting of pMD2.G for viral envelope, pCMV delta R8.2 for packaging, and a transgene plasmid containing a CAR expression cassette. Cells were plated at 1.65 × 10 cells per 10 cm dish. 6 Cells / dish were seeded in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) supplemented with 10% FBS and incubated overnight. The seeded cells were transfected using GeneJuice (Millipore) according to the manufacturer's instructions. Supernatants containing functional lentivirus were harvested 48 hours later and either used immediately or stored at -80°C.

[0257] Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and purified by density gradient using Ficoll-Paque Plus (GE Healthcare) as previously described. Isolated PBMCs were activated overnight with 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 cultured in 0.5 mL of RPMI medium supplemented with 10% FBS. 5 Activated PBMCs were dispensed into each well and completed with 1.5 mL of lentivirus-containing supernatant and 100 U / mL of human IL-2. Transduced cells were incubated on retronectin-coated plates for 5 days before evaluating transduction efficacy by flow cytometry. Transduced cells were centrifuged at 300 g for 5 minutes and washed once with PBS. The cells were then incubated with PE-labeled Protein L (ACROBiosystems) diluted 1:50 in PBS for 60 minutes at 4°C to detect CAR receptor expression. Stained cells were washed once with PBS and analyzed by flow cytometry using a CytoFlex (Beckman Coulter Life Sciences).

[0258] Granzyme B cleavage in CAR-T cells Granzyme B cleavage of the AR_CAR receptor was evaluated in vitro. 5 CAR-T cells were resuspended in 200 μL of RPMI medium and incubated with 100 nM activated human granzyme B for 2 hours at 37°C. Cells were centrifuged at 300 g for 5 minutes and washed once with PBS. The presence of remaining CAR receptors on the cell surface was detected by flow cytometry using the protein L detection method described above.

[0259] Short-term co-culture assay with CAR-T cells AR_CAR-T cells were tested in a short-term coculture assay similar to the method described previously for TCE. A fixed concentration of ROR1+ Jeko-1 cells was mixed with different amounts of CAR-T cells to achieve a range of effector-to-target ratios between 0.04:1 (target cell excess) and 10:1 (CAR-T cell excess) and cocultured for 48 hours. Cytotoxicity was measured as the percentage of target cell death identified by positive staining for live / dead markers, while CAR-T cell activation was measured by the mean fluorescence intensity (MFI) of cell surface CD69 staining by flow cytometry.

[0260] Long-term co-culture assay with CAR-T cells The continuous engagement and killing of CAR-T cells was tested in a long-term coculture assay with excess target cells. CAR-T cells were mixed with excess Jeko-1 cells at an effector-to-target ratio of 1:10 and cocultured for up to 5 days. Coculture samples were harvested every 24 hours, and target cell death was measured by flow cytometry as previously described.

[0261] Example 3 - Design of a prototype FVIII mimetic bispecific antibody with an autoregulatory peptide Prothrombin (coagulation factor II) is a central component of the coagulation cascade, and its activation to thrombin (FIIa) is one of the major outputs of the coagulation process. Abnormally elevated levels of this enzyme are also a hallmark of thrombosis and prothrombotic events. Therefore, autoregulation of bispecific antibodies with FVIII-mimetic activity (such as emicizumab) using elevated thrombin enzyme activity as a trigger is likely to prevent the occurrence of thrombotic events.

[0262] To design AR_Ab8, a prototype FVIII-mimetic bispecific antibody with autoregulatory activity controlled by thrombin levels, we inserted an 8-amino acid P4 to P4' peptide (LTPRGVRL) into the sequence of emicizumab, which was identified by Gallwitz and coworkers (Gallwitz, Enoksson et al. 2012) as the consensus recognition sequence for thrombin-mediated cleavage. The peptide binds to the K receptors of both heavy chains to allow for degradation and inactivation of the autoregulatory bispecific antibody. 228 and Y 229 It was inserted within the hinge region between residues (Figure 2). Thrombin-mediated cleavage yields three non-functional fragments containing the Fc region and two separate Fab domains.

[0263] After purification and purification, the migration pattern of AR_Ab8 was compared to that of emicizumab by electrophoresis under non-reducing conditions. Both constructs showed similar profiles, with a strong predominance of intact antibody (Figure 3A). Notably, no degradation products were observed with AR_Ab8, indicating the absence of premature nonspecific cleavage of the thrombin-sensitive peptide during the purification and purification process.

[0264] Example 4 - In vitro procoagulant activity of a prototype FVIII mimetic antibody with thrombin-mediated autoregulation To assess whether the structural changes in the hinge region due to the insertion of the thrombin-sensitive peptide have a disruptive effect on therapeutic function, the FVIII-mimetic ability of AR_Ab8 and emicizumab was evaluated in vitro.

[0265] Both antibodies successfully catalyzed the FIXa-mediated conversion of FX to FXa in a chromogenic assay, and the overlapping kinetic curves (Fig. 3B) suggest that the insertion of a cleavable peptide into AR_Ab8 does not affect its ability to bridge FIXa and FX and restore FVIII-like activity. Additionally, in human FVIII-deficient plasma, which exhibits significantly increased clotting times in the aPTT assay compared with pooled healthy volunteers (mean ± SD; 95.6 ± 12.6 vs. 29.6 ± 0.7 s, respectively), emicizumab and AR_Ab8 added at 350 nM corrected the clotting times (22.0 ± 0.3 and 28.1 ± 2.0 s, respectively) to values ​​similar to those of the normal control, with no significant difference between the two antibodies (Fig. 3C).

[0266] Example 5 - Therapeutic potential of autoregulatory FVIII mimetic antibodies in vivo without loss Next, the procoagulant potential of AR_Ab8 and its parent antibody, emicizumab, was tested in vivo using a hemophilia A mouse model (FVIIIKO) in a bleeding assay optimized specifically for FVIII-mimetic bispecific antibodies as described by Ferriere and colleagues (Ferriere, Peyron et al. 2020). In this model, a 3 mm distal tail was amputated and bleeding monitored over a 30-minute period. Untreated FVIIIKO animals exhibited a robust bleeding phenotype, with a mean blood loss of 900 ± 92 μL (mean ± SD). Control animals administered 2 U / mouse of recombinant human FVIII showed complete correction of the bleeding tendency (Figure 4A). As expected, partial correction of bleeding was observed with a single intravenous injection of 3 mg / kg emicizumab 24 hours before the test, reducing the mean blood loss to 677 ± 198 μL. Interestingly, treatment with the same dose of AR_Ab8 resulted in a mean blood loss of 609 ± 182 μL, correcting the bleeding phenotype at the same rate as the parent antibody, emicizumab, thus demonstrating that insertion of a thrombin-sensitive peptide into the core structure of the bispecific antibody does not impair its procoagulant potential and therapeutic efficacy (Figure 4B).

[0267] Example 6 - Thrombin-mediated inactivation of autoregulatory FVIII mimetic antibodies By inserting a thrombin-cleavable peptide into the structure of AR_Ab8, this prototypical autoregulatory antibody could be inactivated and degraded upon exposure to high concentrations of thrombin.We therefore assessed the susceptibility of AR_Ab8 to thrombin-mediated cleavage in vitro.

[0268] When exposed to 2 U / mL human α-thrombin for up to 180 min, the parent antibody emicizumab (2 nM) showed no signs of cleavage; only the band corresponding to the intact antibody was detectable on Western blot (Figure 5A). In contrast, AR_Ab8 was gradually cleaved, with degradation fragments detectable as soon as 15 min of exposure. Furthermore, the presence of two sets of fragments, approximately 90–100 kDa and 50–55 kDa, indicates that both autoregulatory peptides present in AR_Ab8 were efficiently cleaved, resulting in nearly complete degradation of the autoregulatory antibody after 180 min of exposure (Figure 5A). These results were confirmed using a chromogenic assay to assess the residual FVIII mimetic activity of each sample after exposure to α-thrombin. As expected, the activity of emicizumab remained unchanged, whereas AR_Ab8 showed a significant decrease in FVIII mimetic activity as soon as 30 min of exposure to α-thrombin, with the residual activity gradually decreasing. After 180 min of exposure, nearly complete inactivation was achieved, with the mean residual activity dropping to only 1.5 ± 1.3% of that of emicizumab (Figure 5B). Next, we compared the sensitivity of several cleavable peptides to thrombin exposure. FVIII mimetic antibody constructs were designed as bispecific tandem-scFvs, each containing (or not containing) different thrombin-sensitive peptides. The peptides tested included the previously described consensus LTPRGVRL, as well as peptides with single amino acid modifications at position P1 (LTPRDVRL and LTPRLVRL), and the naturally occurring human coagulation factor V (FV) cleavage site at position R1753 (WYLRSNNG). After 20 min of exposure to α-thrombin, the peptides exhibited different levels of sensitivity, as indicated 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, whereas the LVRL site showed much lower sensitivity to thrombin exposure with 56.7% residual FVIII-mimetic activity, and the DVRL site showed no cleavage at all. Interestingly, the naturally occurring FV_R1573 cleavage site showed only minimal inactivation when exposed to thrombin, retaining 88.7% of its FVIII-mimetic activity after 20 min of exposure (Figure 5C).

[0269] To determine whether thrombin-mediated inactivation could effectively reduce the thrombotic risk of FVIII-mimetic antibodies, we tested the thrombin generation profiles of both AR_Ab8 and emicizumab in combination with activated prothrombin complex concentrate (aPCC). This assay, previously reported by Hartmann and colleagues (Hartmann, Feenstra et al. 2018), mimics the prothrombotic potential of emicizumab observed clinically when administered in combination with this bypassing agent. Indeed, the combination of 600 nM emicizumab and 0.5 U / mL aPCC in hemophilia A plasma resulted in an abnormally high amount of thrombin generation, with a 1.79-fold increase in the maximum peak compared to pooled normal plasma (Figure 6A, B). However, thrombin generation was significantly lower in the presence of AR_Ab8, and the peak value was not significantly different from that of normal plasma (1.15-fold). This suggests that autoregulation was efficiently induced to prevent excessive thrombin generation (Fig. 6B).

[0270] Example 7 - Thrombin-mediated autoregulation reduces the prothrombotic phenotype associated with FVIII mimetic antibodies in vivo Next, we evaluated the in vivo thrombin-mediated autoregulation potential of FVIII-mimetic bispecific antibodies to control therapeutic activity and reduce thrombotic risk using a mouse model that recapitulates the prothrombotic potential of emicizumab in the context of aPCC. C57Bl / 6 mice were initially injected with 2.5 U of aPCC alone or in combination with 250 μg of emicizumab or AR_Ab8. All animals then received three booster doses of aPCC every 24 hours, sustaining elevated levels of bypassing agents and reversing the prothrombotic phenotype (Figure 7A).

[0271] Total platelet counts were measured in blood samples taken from mice 30 minutes after the last aPCC injection. As expected, the combination of emicizumab and aPCC induced a significant platelet-consuming coagulopathy, with a mean platelet count of 908 × 10 in the aPCC-only control group. 3 Platelets / μL was 621 × 10 3However, in mice treated with aPCC and AR_Ab8, the platelet count in peripheral blood was completely corrected, with an average of 958 × 10 3 The platelets / µL were not significantly different from the control group, indicating that the presence of thrombin-mediated autoregulation effectively manages the risk of coagulopathy associated with FVIII mimetic antibodies (Figure 7B).

[0272] Next, thrombus formation in the lungs was evaluated using immunofluorescence microscopy (Figure 8). Immunofluorescent staining for PECAM (vessels) and integrin aIIb (platelets) was then performed, further confirming that these structures were platelet-rich thrombi partially occluding pulmonary vessels (Figure 8A). Images of these immunofluorescent stains were subsequently used to define a quantification mask to measure the percentage of vascular occlusion, defined as the percentage of platelet-positive staining within a delimited area of ​​the vessel (Figure 8A). Blinded lung sections showed a visible increase in the mean occlusion rate in mice treated with emicizumab compared to the control group treated with aPCC alone, with several vessels exhibiting a luminal occlusion rate exceeding 20% ​​(Figure 8B). In contrast, mice treated with the autoregulatory antibody AR_Ab8 showed a decrease in the occurrence of highly occluded vessels, and the distribution of occlusion rates approached that of the control group (Figure 8B). This again demonstrates the ability of thrombin-mediated autoregulation to reduce the risk of adverse events associated with FVIII mimetic antibodies.

[0273] Example 8 - Design of ROR1 x CD3 bispecific engagers using granzyme B-sensitive autoregulatory peptides To demonstrate that autoregulation can be achieved across different indications using a non-canonical antibody format, a bispecific T cell engager (TCE) incorporating autoregulation was designed to prevent mechanism-of-action (MoA)-related adverse events, one of the major risks associated with this class of therapeutic agents. Granzyme B is an immune cell-specific enzyme released by activated T cells in the context of T cell redirection immunotherapies such as TCE. Interestingly, circulating levels of granzyme B are strongly increased in patients progressing to severe MoA-related adverse events, making this enzyme an attractive target for inducing TCE autoregulation and reducing the risk of MoA-related toxicity associated with T cell redirection immunotherapies.

[0274] The autoregulatory bispecific T cell engager (AR_TCE) was designed based on the ROR1 × CD3 bispecific tandem scFv (td_scFv) NVG-111. The central GSGGGGS linker was replaced with a granzyme B-cleavable peptide (8 amino acids long) flanked on both sides by GGGGS extenders (Figure 9A). Several granzyme B-sensitive peptides were initially tested, and AR_TCE-1 and AR_TCE-2 were designed using peptides Gzmb-1 and Gzmb-2, respectively, derived from the mouse or human P4-P4' granzyme B cleavage sequence of the BH3-interacting-domain death agonist (BID), a well-described target of granzyme B. AR_TCE-3 used the peptide GZMB-3, derived from the granzyme B cleavage consensus sequence described by Wee and coworkers (Wee, Er et al. 2011) (Figure 9B). 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 of approximately 55 kDa, the expected size of a td_scFv antibody, indicating premature cleavage of the granzyme B-sensitive peptide and the absence of degradation of the autoregulated constructs (Figure 9C).

[0275] Example 9 - Cytotoxicity and T cell engagement capacity of autoregulatory AR_TCE The cytotoxicity and T cell engagement capabilities of AR_TCE were evaluated alongside those of the parent NVG-111 in a short-term killing assay. ROR1+ Jeko-1 target cells were incubated with an excess of purified human T cells for 48 hours, using a target-to-effector ratio of 5:1, along with increasing concentrations of either antibody. Both AR_TCE-1 and AR_TCE-3 exhibited similar T cell activation potential compared to the parent NVG-111, as measured by cell surface expression of CD69 (Figure 10A). Furthermore, all three constructs induced comparable T cell-mediated target cell killing (Figure 10B), thus demonstrating that the insertion of a granzyme B-cleavable peptide into the linker of autoregulatory AR_TCE does not impair its mechanism of action in vitro.

[0276] Example 10 - Autoregulation allows threshold-dependent inactivation of TCE by exposure to granzyme B. To determine whether the presence of a granzyme B-cleavable peptide inserted into AR_TCE confers efficient sensitivity to granzyme B exposure, several AR_TCE variants were tested in an in vitro cleavage assay. After 2 h of incubation with 100 nM recombinant activated granzyme B, both AR_TCE-1 and AR_TCE-2 showed partial cleavage, with a faint band between 25 kDa and 30 kDa corresponding to the cleaved single scFv detectable by electrophoresis and protein L immunoblotting. Residual intact material was also present around 55 kDa (Figure 11A). In contrast, the AR_TCE-3 variant exhibited much higher sensitivity to granzyme B, achieving complete cleavage within 2 h with no detectable intact material (Figure 11A).

[0277] Subsequent detection of remaining intact antibody by ELISA showed, as expected, that exposure of the parental antibody NVG-111 to increasing concentrations of granzyme B had no effect (Figure 11B). In contrast, AR_TCE-1 showed a slight decrease in intact antibody levels at the highest concentration of 100 nM, consistent with the partial degradation observed by Western blot, whereas AR_TCE-3 was gradually cleaved with increasing concentrations of granzyme B until it was completely inactivated (Figure 11B). This observation highlights a dose-dependent, threshold-mediated autoregulatory inactivation mechanism for TCE, where AR_TCE-3 remains intact at low concentrations of granzyme B but shows complete inactivation at higher concentrations associated with a higher risk of severe MoA-related adverse events.

[0278] Example 11 - Specificity and sensitivity of granzyme B cleavable peptides used in autoregulation To further explore the relationship between autoregulatory peptide sequence and susceptibility to granzyme B exposure, we generated several additional peptides from the consensus peptide GZMB-3 by modifying the P4-P4' sequence. These new peptides (GZMB-4 to -14) were further used to generate AR_TCE variants as previously described (Figure 12A). Interestingly, upon exposure to a single dose of granzyme B (50 nM), the diversity of autoregulatory peptides exhibited a wide range of cleavage susceptibility, ranging from constructs (AR_TCE-2, -13) that showed almost no signs of inactivation, similar to the parent antibody NVG-111, to highly sensitive variants (AR_TCE-3, -8) at the other end of the spectrum, where only approximately 25% of the intact antibody remained after 2 hours of exposure (Figure 12A). This diversity in granzyme B susceptibility supports the ability to fine-tune the kinetics of autoregulation and select specific sequences that provide AR_TCE inactivation only at granzyme B levels associated with MoA-related adverse events.

[0279] Additionally, the specificity of the GZMB-3 peptide as a substrate for the proteolytic activity of granzyme B was assessed in vitro. AR_TCE-3, chosen for its reported presence in the circulatory system and tumor microenvironment, was exposed to equimolar concentrations (50 nM) of a panel of proteases representing different protease families for 2 hours. In this assay, AR_TCE-3 exhibited unique proteolytic sensitivity to granzyme B, which significantly inactivated the molecule while leaving it intact when exposed to the other proteases tested in this panel (Figure 12B). This selectivity for the proteases used to induce autoregulation significantly reduces the risk of nonspecific premature inactivation of AR_TCE.

[0280] Example 12 - Granzyme B-mediated autoregulation reduces TCE-induced cytokine release in vitro Two AR_TCEs, one with low sensitivity to granzyme B exposure (AR_TCE-1) and the other with high sensitivity (AR_TCE-3), were characterized in combination with NVG-111 in a long-term killing assay to assess continuous target cell killing and progressive T cell engagement. Purified human T cells were co-cultured with excess ROR+ Jeko-1 target cells (effector-to-target cell ratio 1:10) in the presence of the bispecific antibody. Similar to previous observations in the short-term killing assay (Figure 10), the cytotoxic potential of all three antibodies over 120 hours of co-culture was similar, with maximum target cell death of 60.5%, 58.7%, and 55.7% for NVG-111, AR_TCE-1, and AR_TCE-3, respectively (Figure 13A). However, long-term T cell activation, as measured by increased cell surface expression of CD25 (IL-2 receptor α subunit), began to gradually plateau after 96 h with AR_TCE compared with NVG-111, with the strongest effect observed with the highly sensitive AR_TCE-3 molecule. Indeed, mean CD25 expression at 120 h was reduced by 14.2% and 21.5% with AR_TCE-1 and AR_TCE-3, respectively (Figure 13B).

[0281] Additionally, the apparent reduction in T cell activation correlated with a strong and significant decrease in human interferon-γ (IFNγ) release. At 120 h, T cells cocultured in the presence of AR_TCE-1 and AR_TCE-3 released levels of IFNγ comparable to 77.3% and 52.3%, respectively, of cells cocultured with the parental NVG-111 (Figure 13C). This decrease in cytokine release also correlated with visible autoregulation and inactivation of AR_TCE. Indeed, while NVG-111 remained intact in the coculture supernatant throughout the assay, AR_TCE-1 and AR_TCE-3 showed evidence of cleavage, along with the presence of fragmentation by-products (Figure 13D). As expected, antibody fragments were only detected after 120 h for the less sensitive AR_TCE-1 construct, whereas inactivation was observed as early as 72 h for the more sensitive AR_TCE-3 molecule (Figure 13D). Overall, these results demonstrate that antibody engineering incorporating a granzyme B-mediated autoregulatory mechanism into TCEs can significantly reduce cytokine release induced by T cell engagement and activation, while preserving unaffected cytotoxicity. Additionally, the kinetics of antibody inactivation and the resulting effect on cytokine release can be modified by careful selection of the autoregulatory peptide sequence.

[0282] Example 13 - Autoregulation reduces the occurrence of severe in vivo toxicities associated with TCE mechanisms of action such as cytokine release syndrome in a triple-negative breast cancer model The mechanism of granzyme B-mediated autoregulation was further validated in an in vivo model recapitulating features of severe TCE-associated MoA-related toxicity. In this model, immunocompromised NSG mice were stably induced with either NVG-111 or the AR_TCE-3 variant at concentrations (average ranging from 0.66 to 2.01 μg / mL) known to produce rapid toxicity with the non-regulatory parent antibody (Figure 14A). Mice were then subcutaneously implanted with the ROR1+ MDA-MB-231 triple-negative breast cancer (TNBC) cell line, and the resulting solid tumors were treated with six cycles of purified human T cells over a 21-day period (from day 25 to day 46).

[0283] Weight loss and animal survival are the primary outcomes of TCE-induced toxicity in this model. As expected, animals treated with NVG-111 showed rapid signs of toxicity 24 h after the second T cell injection, with sudden and significant weight loss occurring as early as day 30 (Figure 14B). Due to subsequent worsening weight loss and overall deterioration in the animals' well-being, four of six animals were ethically culled by day 36 (Figure 14C), and the remaining animals were removed from the protocol. In contrast, animals treated with the autoregulatory AR_TCE-3 molecule showed no signs of toxicity, with the same stable weight fluctuations as the vehicle group (Figure 14B). Consequently, all animals in this group were maintained in the protocol until the end of the study, day 46 (Figure 14C). Interestingly, these results suggest that the autoregulatory response to granzyme B release is extremely rapid and may control the development of toxicity within 24 h after T cell injection. Human cytokines released by T cells were also measured in plasma samples collected on the day of cull for both groups of mice. Animals treated with AR_TCE-3 had significantly reduced circulating levels of both human IFNγ and granzyme B compared to NVG-111, indicating reduced T cell activation that correlated with improved survival (Figure 15).

[0284] Example 14 - Autoregulatory AR_TCE exhibited antitumor efficacy without loss in vivo To assess whether the decreased toxicity and cytokine release observed in mice treated with autoregulatory TCE were associated with a reduced therapeutic effect, tumor progression was measured in each group. AR_TCE-3 demonstrated a clear, non-detrimental effect, with significantly reduced tumor progression detectable as early as day 34 compared with the vehicle group (Figure 16A). Indeed, by the end of the study, tumor volume had progressed 18.5 ± 0.94-fold (mean ± SD) from day 15 in the vehicle group, whereas it had progressed only 8.0 ± 3.4-fold in the AR_TCE-3-treated animals. Additionally, tumors in three of six mice in the vehicle group progressed to the ulcerated stage, leading to early ethical culling at day 40 (Figure 16A). These data were supported by postmortem tumor excision volume measurements (Figure 16B), which averaged 910 mm in the vehicle group. 3and only 578 mm in AR_TCE-3 treated animals. 3 Interestingly, although data are incomplete due to early culling of NVG-111-treated mice, tumor progression in this group was not different by day 36 compared with mice receiving AR_TCE-3 (4.6±2.3-fold and 5.2±1.7-fold progression, respectively), supporting the non-destructive effect of autoregulatory AR_TCE (FIG. 16A).

[0285] Example 15 - Autoregulation allows sustained treatment with AR_TCE to reduce TNBC metastatic growth in vivo AR_TCE-3, with its improved safety profile, enabled longer treatment periods in mice compared with its parent drug, NVG-111 (Figure 14). Therefore, the long-term therapeutic effects of sustained exposure to AR_TCE-3 were evaluated in the metastatic progression of a TNBC model. Animals in the vehicle group exhibited obvious metastatic nodules on the surface of the liver (Figure 17A), confirmed by the presence of cancer cell clusters in liver sections (Figure 17B). While no such nodules were observed in mice treated with AR_TCE-3, quantification of metastatic nodules detected by immunofluorescence staining further demonstrated a significant reduction in the number of large cancer cell clusters (d > 100 μm) infiltrating liver tissue in AR_TCE-3-treated animals compared with mice in the vehicle group (Figure 17C). Therefore, it was confirmed that AR technology does not affect therapeutic efficacy, while reduced toxicity allows for an extended treatment period compared with NVG-111.

[0286] Overall, the results obtained in this model, which exhibits severe toxicity related to the mechanism of action of TCE, demonstrated that the incorporation of autoregulation via a granzyme B-cleavable peptide efficiently controls the risk of severe adverse events inherent in T cell engager therapy, thus enabling long-term treatment with sustained therapeutic efficacy against tumor progression and metastatic dissemination.

[0287] Example 16 - Autoregulation improves survival in vivo while preserving therapeutic efficacy in a pancreatic cancer model The results obtained in the TNBC model were further confirmed in a different cancer type using a shorter xenograft model established by intraperitoneal injection of the ROR+ PANC-1 pancreatic cancer cell line and a 14-day treatment period with four cycles of human T cell administration (Figure 18A). Similar to the results in the TNBC xenograft model, NVG-111-treated mice showed rapid signs of toxicity after T cell administration, resulting in increased mortality, with four of six mice requiring ethical culling between days 16 and 20. In contrast, only one of six animals treated with the autoregulatory AR_TCE-3 required ethical culling at day 21 (Figure 18B).

[0288] To evaluate cytokine release in response to T cell engagement, a single blood sample was collected from all groups of mice 24 hours after the second injection (e.g., on day 13). Mice treated with AR_TCE-3 showed significantly lower levels of granzyme B and significantly less IFNγ release compared to NVG-111, suggesting that the autoregulatory mechanism of AR_TCE-3 efficiently controlled T cell activation over a 24-hour period (Figure 18C). The therapeutic effect of AR_TCE was further verified by bioluminescence analysis of tumor progression. Multiple foci of tumor growth were observed in three of five mice in the vehicle group at the end of the study (day 22), whereas none of the mice treated with AR_TCE-3 showed tumor progression (Figure 19).

[0289] Example 17 - Application of Granzyme B-mediated autoregulation to a ROR1-targeted CAR-T cell model 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 engineered from ROR1-targeted CAR-T cell therapy. Eight residues (sequence P4 to P4') of the granzyme B-sensitive peptide GZMB-3, previously used in the autoregulatory AR_TCE-3, were inserted into the upper region of the chimeric receptor (Figure 20A). The autoregulatory peptide was inserted either directly between the ROR1-targeting scFv and the hinge region of the chimeric receptor (AR_CAR-T27) or by substituting the first five residues of the hinge region (AR_CAR-T22).

[0290] Both the autoregulatory AR_CAR-T variants and the parental CAR-T cells were generated by lentiviral infection of PBMCs obtained from healthy donors. CAR-T cells were subsequently exposed to 100 nM of activated human granzyme B in vitro for 2 hours to assess the susceptibility of the AR_CAR-T variants to the proteolytic activity of granzyme B. As assessed by flow cytometry, both AR_CAR-T22 and AR_CAR-T27 showed significant cleavage and removal of the chimeric receptor from the cell surface, with 39.5% and 38.0% of the remaining CAR receptor detected by protein L (Figure 20B). In contrast, the parental CAR-T cells were unaffected by exposure to granzyme B.

[0291] Example 18 - Autoregulation maintains the cytotoxic potential and continuous engagement capacity of AR_CAR-T cells The cytotoxicity of AR_CAR-T cells was compared with that of parental ROR1-targeted CAR-T cells in a short-term killing assay using various cell concentrations. The ROR1+ Jeko-1 target cell line was cocultured with CAR-T cells for 48 hours using effector-to-target ratios ranging from 0.04 (target cell excess) to 10 (CAR-T cell excess). Interestingly, both autoregulatory AR_CAR-T cell variants exhibited activation levels similar to those of the parental CAR-T cells at all cell ratios tested (Figure 21A). In addition, activation levels correlated with comparable target cell killing potency (Figure 21B). Next, the function and ability of autoregulatory AR_CAR-T cells to promote sequential killing was evaluated in a long-term killing assay, which reflects progressive CAR-T cell engagement. CAR-T cells were cocultured with excess ROR+ Jeko-1 target cells (effector-to-target ratio 1:10) for up to 5 days. Consistent with previous observations, CAR-T cell engagement and cytotoxicity were not compromised in the presence of autoregulatory agents. Progressive cell death was observed, with maximal target cell death observed at 120 hours, at 72.4%, 74.8%, and 79.1% for AR_CAR-T22, AR_CAR-T27, and parental CAR-T cells, respectively (Figure 22).

[0292] These results show that modifying the CAR receptor to introduce a granzyme B-sensitive peptide does not impair CAR T cell function, thus demonstrating that autoregulation can be successfully applied to genetically modified CAR T cells to enable granzyme B-mediated inactivation and reduce the risk of serious adverse events associated with the CAR T cell mechanism of action.

[0293] Array List SEQ ID NO: 1 - Thrombin cleavage recognition sequence consensus sequence TIFF2025538963000003.tif4159SEQ ID NO: 2- Thrombin cleavage recognition sequence P1' G~L TIFF2025538963000004.tif4159SEQ ID NO: 3- Thrombin cleavage recognition sequence P1' G~D TIFF2025538963000005.tif4159SEQ ID NO: 4- Thrombin cleavage recognition sequence of human coagulation factor V (FV) TIFF2025538963000006.tif4159SEQ ID NO: 5: Granzyme B cleavage recognition sequence of mouse BID protein GZMB-1 TIFF2025538963000007.tif4159SEQ ID NO: 6: Granzyme B cleavage recognition sequence GZMB-2 of mouse BID protein TIFF2025538963000008.tif4159SEQ ID NO: 7: Granzyme B cleavage recognition sequence GZMB-3, consensus TIFF2025538963000009.tif4159SEQ ID NO: 8: Granzyme B cleavage recognition sequence P10-P10' sequence of GZMB-4 and GZMB-2 TIFF2025538963000010.tif4159SEQ ID NO: 9: Granzyme B cleavage recognition sequence GZMB-5, P4 I-V TIFF2025538963000011.tif4159SEQ ID NO: 10: Granzyme B cleavage recognition sequence GZMB-6, P4 I to L TIFF2025538963000012.tif4159SEQ ID NO: 11: Granzyme B cleavage recognition sequence GZMB-7, P2 P~G TIFF2025538963000013.tif4159SEQ ID NO: 12: Granzyme B cleavage recognition sequence GZMB-8, P2 P~A TIFF2025538963000014.tif4159SEQ ID NO: 13: Granzyme B cleavage recognition sequence GZMB-9, P1' S~A TIFF2025538963000015.tif4159SEQ ID NO: 14: Granzyme B cleavage recognition sequence GZMB-10, P2' L to E and P4' E to Q TIFF2025538963000016.tif4159SEQ ID NO: 15: Granzyme B cleavage recognition sequence GZMB-11, P2' L to E and P4' E to V TIFF2025538963000017.tif4159SEQ ID NO: 16: Granzyme B cleavage recognition sequence GZMB-12, P1 D-N TIFF2025538963000018.tif4159SEQ ID NO: 17: Granzyme B cleavage recognition sequence GZMB-13, P1 D~E TIFF2025538963000019.tif4159SEQ ID NO: 18: Granzyme B cleavage recognition sequence GZMB-14, P1 D~Q TIFF2025538963000020.tif4159SEQ ID NO: 19: ROR1-binding antigen-binding domain LCDR 1 TIFF2025538963000021.tif4159SEQ ID NO: 20: ROR1-binding antigen-binding domain LCDR 2 TIFF2025538963000022.tif4159

[0294] SEQ ID NO: 21: ROR1-binding antigen-binding domain LCDR 3 TIFF2025538963000023.tif4159SEQ ID NO: 22: ROR1-binding antigen-binding domain HCDR 1 TIFF2025538963000024.tif4159SEQ ID NO: 23: ROR1-binding antigen-binding domain HCDR 2 TIFF2025538963000025.tif4159SEQ ID NO: 24: ROR1-binding antigen-binding domain consensus HCDR 3 TIFF2025538963000026.tif4159 (wherein X1 is A or T, and X2 is S, K, or R) SEQ ID NO: 25: ROR1-binding antigen-binding domain HCDR 3, clone F heavy chain CDR3 TIFF2025538963000027.tif4159SEQ ID NO: 26: ROR1-binding antigen-binding domain HCDR 3, humanized 1 heavy chain CDR3 TIFF2025538963000028.tif4159SEQ ID NO: 27: ROR1-binding antigen binding domain HCDR 3, humanized 2 and 5 heavy chain CDR3 TIFF2025538963000029.tif4159SEQ ID NO: 28: ROR1-binding antigen-binding domain HCDR 3, humanized 3 and 4 heavy chain CDR3 TIFF2025538963000030.tif4159SEQ ID NO: 29: ROR1-binding antigen-binding domain, clone F light chain variable region (underlined; CDRs) TIFF2025538963000031.tif9168SEQ ID NO: 30: ROR1-binding antigen-binding domain, humanized 1 light chain variable region (underlined; CDRs) TIFF2025538963000032.tif9168SEQ ID NO: 31: ROR1-binding antigen-binding domain, humanized 2 light chain variable regions (underlined; CDRs) TIFF2025538963000033.tif9168SEQ ID NO: 32: ROR1-binding antigen-binding domain, humanized 3 light chain variable region (underlined; CDR) TIFF2025538963000034.tif9168SEQ ID NO: 33: ROR1-binding antigen-binding domain, humanized 4 light chain variable regions (underlined; CDRs) TIFF2025538963000035.tif9168SEQ ID NO: 34: ROR1-binding antigen-binding domain, humanized 5 light chain variable region (underlined; CDRs) TIFF2025538963000036.tif9168SEQ ID NO: 35: ROR1-binding antigen-binding domain, clone F heavy chain variable region (underlined; CDRs) TIFF2025538963000037.tif9168SEQ ID NO: 36: ROR1-binding antigen-binding domain, humanized heavy chain variable region 1 (underlined; CDRs) TIFF2025538963000038.tif9168SEQ ID NO: 37: ROR1-binding antigen-binding domain, humanized dual heavy chain variable region (underlined; CDR) TIFF2025538963000039.tif9168SEQ ID NO: 38: ROR1-binding antigen-binding domain, humanized 3 heavy chain variable region (underlined; CDRs) TIFF2025538963000040.tif9168SEQ ID NO: 39: ROR1-binding antigen-binding domain, humanized 4 heavy chain variable region (underlined; CDR) TIFF2025538963000041.tif9168SEQ ID NO: 40: ROR1-binding antigen-binding domain, humanized 5 heavy chain variable region (underlined; CDRs) TIFF2025538963000042.tif9168

[0295] SEQ ID NO: 41: CD3-binding antigen-binding domain, murine light chain variable region (underlined; CDRs) TIFF2025538963000043.tif9168SEQ ID NO: 42: CD3-binding antigen-binding domain, humanized 1 light chain variable region (underlined; CDRs) TIFF2025538963000044.tif9168SEQ ID NO: 43: CD3-binding antigen-binding domain, humanized 2 light chain variable regions (underlined; CDRs) TIFF2025538963000045.tif9168SEQ ID NO: 44: CD3-binding antigen-binding domain, humanized 3 light chain variable region (underlined; CDR) TIFF2025538963000046.tif9168SEQ ID NO: 45: CD3-binding antigen-binding domain, humanized 4 light chain variable regions (underlined; CDRs) TIFF2025538963000047.tif9168SEQ ID NO: 46: CD3-binding antigen-binding domain, humanized 5 light chain variable region (underlined; CDRs) TIFF2025538963000048.tif9168SEQ ID NO: 47: CD3-binding antigen-binding domain, mouse heavy chain variable region (underlined; CDRs) TIFF2025538963000049.tif9168SEQ ID NO: 48: CD3-binding antigen-binding domain, humanized heavy chain variable region (underlined; CDRs) TIFF2025538963000050.tif9168SEQ ID NO: 49: CD3-binding antigen-binding domain, humanized dual heavy chain variable region (underlined; CDRs) TIFF2025538963000051.tif9168SEQ ID NO: 50: CD3-binding antigen-binding domain, humanized 3 heavy chain variable regions (underlined; CDRs) TIFF2025538963000052.tif9168SEQ ID NO: 51: CD3-binding antigen-binding domain, humanized 4 heavy chain variable region (underlined; CDRs) TIFF2025538963000053.tif9168SEQ ID NO: 52: CD3-binding antigen-binding domain, humanized 5 heavy chain variable region (underlined; CDRs) TIFF2025538963000054.tif9168SEQ ID NO: 53: CD3-binding antigen-binding domain LCDR 1 TIFF2025538963000055.tif4161SEQ ID NO: 54: CD3-binding antigen-binding domain LCDR 2 TIFF2025538963000056.tif4161SEQ ID NO: 55: CD3-binding antigen-binding domain LCDR 3 TIFF2025538963000057.tif4161SEQ ID NO: 56: CD3-binding antigen-binding domain HCDR 1 TIFF2025538963000058.tif4161SEQ ID NO: 57: CD3-binding antigen-binding domain HCDR 2 TIFF2025538963000059.tif4161SEQ ID NO: 58: CD3-binding antigen-binding domain HCDR 3 TIFF2025538963000060.tif4161SEQ ID NO: 59: Linker sequence TIFF2025538963000061.tif4161SEQ ID NO: 60: Linker sequence TIFF2025538963000062.tif4161

[0296] SEQ ID NO: 61: ROR1xCD3 bispecific antibody amino acid sequence TIFF2025538963000063.tif28161SEQ ID NO: 62: ROR1xCD3 bispecific antibody amino acid sequence with N-terminal hexahistidine tag (NVG-111) TIFF2025538963000064.tif28161SEQ ID NO: 63: Common LCDR 1 of FIX-binding and FX-binding antigen-binding domains TIFF2025538963000065.tif4160SEQ ID NO: 64: Common LCDR 2 of FIX-binding and FX-binding antigen-binding domains TIFF2025538963000066.tif4160SEQ ID NO: 65: Common LCDR 3 of FIX-binding and FX-binding antigen-binding domains TIFF2025538963000067.tif4160SEQ ID NO: 66: FIX-binding antigen-binding domain HCDR 1 TIFF2025538963000068.tif4160SEQ ID NO: 67: FIX-binding antigen-binding domain HCDR 2 TIFF2025538963000069.tif4160SEQ ID NO: 68: FIX-binding antigen-binding domain HCDR 3 TIFF2025538963000070.tif4160SEQ ID NO: 69: FX-binding antigen-binding domain HCDR 1 TIFF2025538963000071.tif4160SEQ ID NO: 70: FX-binding antigen-binding domain HCDR 2 TIFF2025538963000072.tif4160SEQ ID NO: 71: FX-binding antigen-binding domain HCDR 3 TIFF2025538963000073.tif4160SEQ ID NO: 72: Common light chain variable region (bold; CDR) of FIX-binding and FX-binding antigen-binding domains TIFF2025538963000074.tif9160SEQ ID NO: 73: Heavy chain variable region of FIX-binding antigen-binding domain (bold; CDR) TIFF2025538963000075.tif9160SEQ ID NO: 74: Heavy chain variable region of FX-binding antigen-binding domain (bold; CDR) TIFF2025538963000076.tif9160SEQ ID NO: 75: Hinge region TIFF2025538963000077.tif4160SEQ ID NO: 76: Hinge region + SEQ ID NO: 1 (underlined portion; SEQ ID NO: 1) TIFF2025538963000078.tif5160SEQ ID NO: 77: Linker sequence TIFF2025538963000079.tif5160SEQ ID NO: 78: Linker sequence TIFF2025538963000080.tif5160SEQ ID NO: 79: Linker sequence TIFF2025538963000081.tif5160SEQ ID NO: 80: Linker sequence TIFF2025538963000082.tif5160

[0297] SEQ ID NO: 81: Linker sequence TIFF2025538963000083.tif5160SEQ ID NO: 82: Hinge region of CAR embodiment TIFF2025538963000084.tif5160SEQ ID NO: 83: Partially deleted hinge region of CAR embodiment + SEQ ID NO: 7 TIFF2025538963000085.tif5160SEQ ID NO: 84: Hinge region of CAR embodiment + SEQ ID NO: 7 TIFF2025538963000086.tif5160

[0298] Embodiment 1. A therapeutic agent comprising an autoregulatory element, wherein the autoregulatory element is a small peptide cleavable by an endogenous factor, the activity of the endogenous factor being altered as a result of the activity of the therapeutic agent, and cleavage of the autoregulatory element results in separation of the therapeutic agent into non-functional fragments.

[0299] 2. A therapeutic agent according to embodiment 1, wherein the intrinsic factor is active in the same metabolic pathway as the metabolic pathway affected by the therapeutic agent.

[0300] 3. A therapeutic agent according to embodiment 1 or 2, wherein the intrinsic factor is a protease.

[0301] 4. A therapeutic agent according to any one of the preceding embodiments, wherein the autoregulatory element comprises a proteolytic cleavage site.

[0302] 5. A therapeutic agent according to any one of the preceding embodiments, wherein the rate of cleavage of the autoregulatory element by an endogenous factor correlates with the activity level of the therapeutic agent.

[0303] 6. A therapeutic agent described in embodiment 5, wherein the rate of cleavage of the autoregulatory element by an endogenous factor correlates with the activity level of the therapeutic agent and the peptide sequence of the autoregulatory element.

[0304] 7. A therapeutic agent according to any one of embodiments 1 to 6, wherein the autoregulatory element comprises a thrombin or granzyme B cleavage recognition site.

[0305] 8. A therapeutic agent according to any one of the preceding embodiments, wherein the therapeutic agent comprises a polypeptide.

[0306] 9. The therapeutic agent of embodiment 8, wherein the therapeutic agent is an antigen-binding protein comprising a first antigen-binding domain, the first antigen-binding domain comprises a heavy chain variable domain comprising heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3, and the autoregulatory element is located at the C-terminus of the first antigen-binding domain.

[0307] 10. The antigen-binding protein of embodiment 9, wherein the first antigen-binding domain is a VHH antibody.

[0308] 11. The antigen-binding protein of embodiment 9, 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 region (LCDR) 1, LCDR2, and LCDR3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining region (HCDR) 1, HCDR2, and HCDR3, and wherein an autoregulatory element is located at the C-terminus of the first antigen-binding domain.

[0309] 12. An antigen-binding protein according to embodiment 9, 10 or 11, which forms part of a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0310] 13. The antigen-binding protein of any one of embodiments 9 to 12, wherein the antigen-binding protein comprises a first antigen-binding domain and a second antigen-binding domain, and wherein the autoregulatory element is disposed between the first and second antigen-binding domains.

[0311] 14. An antigen-binding protein according to embodiment 13, wherein the first antigen-binding domain and the second antigen-binding domain comprise a light chain variable domain and a heavy chain variable domain, respectively, the light chain variable domain comprises light chain complementarity-determining region (LCDR) 1, LCDR2 and LCDR3, and the heavy chain variable domain comprises heavy chain complementarity-determining region (HCDR) 1, HCDR2 and HCDR3.

[0312] 15. The antigen-binding protein of embodiment 13 or 14, wherein the first antigen-binding domain and the second binding domain comprise identical CDRs, comprise biparatopic CDRs, or comprise bispecific CDRs.

[0313] 16. An antigen-binding protein according to any one of embodiments 13 to 15, wherein the antigen-binding protein is an antibody or a fragment thereof.

[0314] 17. The antigen-binding protein of any one of embodiments 9 to 16, comprising 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 an scFv-Fc protein.

[0315] 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.

[0316] 19. An antigen-binding protein according to any one of embodiments 9 to 18, wherein the antigen-binding protein is an scFv protein in which the first and second antigen-binding domains are covalently linked by a peptide linker.

[0317] 20. The antigen-binding protein of any one of embodiments 9 to 19, wherein the autoregulatory element is present within the peptide linker.

[0318] 21. An antigen-binding protein according to any one of embodiments 9 to 20, wherein the autoregulatory element comprises a granzyme B cleavage site and the intrinsic factor is granzyme B.

[0319] 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).

[0320] 23. The antigen-binding protein of embodiment 22, 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 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 21; the heavy chain variable domain comprises 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; and the sequence of each complementarity-determining region may differ from the given sequence at up to two amino acid positions.

[0321] 24. The antigen-binding protein of embodiment 23, (a) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 29 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 35; (b) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 30 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 36; (c) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 31 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 37; (d) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 32 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 38; or (e) the light chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 33 and the heavy chain variable domain comprises the amino acid sequence set forth as SEQ ID NO: 39; Each of the above light chain variable domains and heavy chain variable domains may have at least 90% identity to the above amino acid sequences.

[0322] 25. An antigen-binding protein according to 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).

[0323] 26. An antigen binding protein according to any one of embodiments 22 to 25, wherein the granzyme B cleavage site comprises SEQ ID NO: 7.

[0324] 27. An antigen-binding protein according to any one of embodiments 9 to 20, wherein the autoregulatory element comprises a thrombin cleavage site and the intrinsic factor is thrombin.

[0325] 28. An antigen-binding protein according to 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.

[0326] 29. The antigen-binding protein of any one of embodiments 9 to 20 and 27 or 28, wherein the first antigen-binding domain and the second binding domain comprise a light chain variable domain and a heavy chain variable domain, respectively, wherein the light chain variable domain comprises light chain complementarity-determining region (LCDR) 1, LCDR2, and LCDR3, and the heavy chain variable domain comprises heavy chain complementarity-determining region (HCDR) 1, HCDR2, and HCDR3; 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR2 and HCDR3, wherein 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; An antigen-binding protein, wherein 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:65; the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR2 and HCDR3, wherein 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.

[0327] 30. The antigen-binding protein of embodiment 29, wherein 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.

[0328] 31. An antigen-binding protein according to any one of embodiments 9 to 20 and 27 to 30, wherein the thrombin cleavage site comprises SEQ ID NO:1.

[0329] 32. The antigen-binding protein of any one of embodiments 9 to 31, wherein the antigen-binding protein is labeled.

[0330] 33. The antigen-binding protein of embodiment 32, wherein the label is a fluorescent label, an enzyme label, or a radioactive label.

[0331] 34. A composition comprising a therapeutic agent according to any one of embodiments 1 to 33 and a pharmaceutically acceptable carrier.

[0332] 35. An isolated nucleic acid molecule encoding an antigen-binding protein of any one of embodiments 8 to 31.

[0333] 36. An isolated nucleic acid molecule according to embodiment 35, operably linked to a promoter.

[0334] 37. An expression vector comprising the isolated nucleic acid molecule of embodiment 35 or embodiment 36.

[0335] 38. An isolated host cell transformed with a nucleic acid molecule according to embodiment 35 or 36, or a vector according to embodiment 37.

[0336] 39. An antigen-binding protein according to any one of embodiments 9 to 31, wherein the antigen-binding protein is exogenously expressed in a CAR-T cell, a CAR-M cell, or an engineered NK cell.

[0337] 40. A method for treating hemophilia A, comprising administering to an individual in need thereof an antigen-binding protein described in any one of embodiments 27 to 31, or a composition described in embodiment 34.

[0338] 41. A method for preventing prothrombotic risk, comprising administering to an individual in need thereof an antigen-binding protein described in any one of embodiments 27 to 31, or a composition described in embodiment 34.

[0339] 42. A method for treating cancer, comprising administering to an individual in need thereof an antigen-binding protein of any one of embodiments 20 to 26, or the composition of embodiment 34.

[0340] 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 renal cancer.

[0341] 44. 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 described in any one of embodiments 20 to 26, or a composition described in embodiment 34.

[0342] 45. A method for preventing cytokine release syndrome, comprising administering to an individual in need thereof an antigen-binding protein described in any one of embodiments 20 to 26, or a composition described in embodiment 34.

[0343] References Ferriere, S., I. Peyron, OD Christophe, C. Kawecki, C. Casari, V. Muczynski, AC Nathwani, A. Kauskot, PJ Lenting and CV Denis (2020). "A hemophilia A mouse model for the in vivo assessment of Emicizumab function." Blood.

[0344] Gallwitz, M., M. Enoksson, M. Thorpe and L. Hellman (2012). "The extended cleavage specificity of human thrombin." PLoS One 7(2): e31756.

[0345] Harris et al. Definition and redesign of the extended substrate specificity of granzyme B, (1998) J Biol Chem: 273 pp. 27364-73.

[0346] Hartmann, R., T. Feenstra, L. Valentino, M. Dockal and F. Scheiflinger (2018). "In vitro studies show synergistic effects of a procoagulant bispecific antibody and bypassing agents." J Thromb Haemost.

[0347] 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-CD19 Chimeric Antigen Receptor T Cells Are Associated With High Serum Interleukin-15 Levels." J Clin Oncol 35(16): 1803-1813.

[0348] Lenting, P. J., C. V. Denis and O. D. Christophe (2017). "Emicizumab, a bispecific antibody recognizing coagulation factors IX and X: how does it actually compare to factor VIII?" Blood 130(23): 2463-2468.

[0349] Wee, L. J., E. P. Er, L. F. Ng and J. C. Tong (2011). "In silico prediction of the granzyme B degradome." BMC Genomics 12 Suppl 3: S11.

Claims

1. A therapeutic agent comprising an autoregulatory element, wherein the autoregulatory element is a small peptide cleavable by an endogenous factor, the activity of the endogenous factor being altered as a result of the activity of the therapeutic agent, and cleavage of the autoregulatory element results in separation of the therapeutic agent into non-functional fragments.

2. 2. The therapeutic agent of claim 1, wherein the endogenous factor is active in the same metabolic pathway as the metabolic pathway affected by the therapeutic agent, and the endogenous factor is a protease.

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

4. 4. The therapeutic agent of any one of claims 1 to 3, wherein the rate of cleavage of the autoregulatory element by the endogenous factor correlates with the activity level of the therapeutic agent, and optionally, the rate of cleavage of the autoregulatory element by the endogenous factor correlates with the activity level of the therapeutic agent and the peptide sequence of the autoregulatory element.

5. The therapeutic agent of any one of claims 1 to 4, wherein the autoregulatory element comprises a thrombin or granzyme B cleavage recognition site.

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

7. 7. The antigen-binding protein of claim 6, wherein the antigen-binding protein comprises a first antigen-binding domain, 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 region (LCDR) 1, LCDR2, and LCDR3, 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. 8. The antigen-binding protein of claim 6 or claim 7, wherein the antigen-binding protein comprises a first antigen-binding domain and a second binding domain, and the autoregulatory element is located between the first antigen-binding domain and the second binding domain.

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

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

11. 11. The antigen-binding protein of claim 6, wherein the autoregulatory element is present within the peptide linker.

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

13. 13. The antigen-binding protein of claim 12, 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 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 the heavy chain variable domain comprises 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; and the sequence of each complementarity determining region may differ from the given sequence at up to two amino acid positions.

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

15. 14. 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. 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. 12. The antigen-binding protein of any one of claims 6 to 11, wherein the autoregulatory element comprises a thrombin cleavage site, the intrinsic factor is thrombin, and optionally the first antigen-binding domain selectively binds FIXa / FIX and the second antigen-binding domain selectively binds FX / FXa.

18. the first antigen-binding domain and the second antigen-binding domain comprise a light chain variable domain and a heavy chain variable domain, respectively, the light chain variable domain comprising light chain complementarity-determining region (LCDR) 1, LCDR2, and LCDR3, and the heavy chain variable domain comprising heavy chain complementarity-determining region (HCDR) 1, HCDR2, and HCDR3; 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR2 and HCDR3, wherein 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; 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; LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 65; the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR2 and HCDR3, wherein 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.

18. The antigen-binding protein of any one of claims 6 to 11 and 17.

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

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

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

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

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

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

26. 21. 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 of any one of claims 11 to 16, or a composition of claim 20.

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

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