TW202413441A - Use of tnf-alpha binding proteins and il-7 binding proteins in medical treatment - Google Patents

Abstract

The present disclosure relates to the use of TNF-α binding proteins and interleukin 7 (IL-7) binding proteins in the treatment of autoimmune and/or inflammatory conditions. The disclosure also relates to methods of treating diseases with TNF-α binding proteins and IL-7 binding proteins. The disclosure further relates to bispecific antibodies binding to TNF-α and IL-7, uses of such bispecific antibodies, pharmaceutical compositions comprising such bispecific antibodies and methods of their manufacture.

Description

Use of TNF-α binding protein and IL-7 binding protein in medical treatment

The present invention relates to the use of TNF-α (tumor necrosis factor-α) binding protein and interleukin 7 (IL-7) binding protein in the treatment of autoimmune and/or inflammatory conditions. The present invention also relates to methods of treating diseases with TNF-α binding protein and IL-7 binding protein. The present invention further relates to bispecific antibodies that bind to TNF-α and IL-7, uses of such bispecific antibodies, pharmaceutical compositions comprising such bispecific antibodies, and methods of making the same. Other aspects of the present invention will be apparent from the following description.

Dysregulation of innate and adaptive immune pathways plays a key role in human autoimmune diseases such as inflammatory bowel disease (IBD) and rheumatoid arthritis (RA).

IBD is a multifactorial, chronic, relapsing, immune-mediated disease consisting of two major subtypes: Crohn's Disease (CD) and ulcerative colitis (UC). Although the etiology and pathogenesis of IBD are not fully understood, it is thought to be caused by abnormal innate and adaptive immune responses to intestinal microorganisms in genetically susceptible individuals. In the absence of an adequately regulated response to intestinal bacterial flora, intestinal resident macrophages produce elevated levels of pro-inflammatory interleukins and cytokines. This serves to recruit innate immune cells from the blood to the tissues. The most important of these recruited cells are immature monocytes, which, after entering the lamina propria, are triggered to differentiate into pro-inflammatory macrophages in response to elevated levels of interleukins, such as TNF-α.

TNF-α is a pleiotropic interleukin expressed by activated macrophages, NK cells, and T cells, as well as non-immune cells such as endothelial cells and fibroblasts. Anti-TNF-α therapy is clinically validated for the treatment of IBD and can induce rapid disease remission, mucosal healing, and improved quality of life in approximately 60% of patients with CD or UC. However, clinical studies have shown that a large proportion of IBD patients are unable to achieve sustained remission (Gisbert et al. (2020) J Crohn's and Colitis). The exact reasons for the inadequate response to anti-TNF-α therapy are unclear. Additional effective treatments for IBD are still needed.

In one aspect, a method of treating an autoimmune and/or inflammatory condition in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of an IL-7 inhibitor and a therapeutically effective amount of a TNF-α inhibitor.

In another aspect, an IL-7 inhibitor and a TNF-α inhibitor are provided for use in treating autoimmune and/or inflammatory conditions.

In another aspect, the use of an IL-7 inhibitor and a TNF-α inhibitor for the manufacture of a medicament for the treatment of autoimmune and/or inflammatory conditions is provided.

In another aspect, an IL-7 and TNF-α binding protein is provided, comprising: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In another embodiment, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable excipient and the following IL-7 and TNF-α binding proteins: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In another embodiment, a method for treating autoimmune and/or inflammatory conditions in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of an IL-7 and TNF-α binding protein, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an IL-7 and TNF-α binding protein, wherein the IL-7 and TNF-α binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53 of CDRL3.

In another embodiment, an IL-7 and TNF-α binding protein or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an IL-7 and TNF-α binding protein is provided for treating autoimmune and/or inflammatory conditions, wherein the IL-7 and TNF-α binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In another embodiment, an IL-7 and TNF-α binding protein or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an IL-7 and TNF-α binding protein is provided, which is used to manufacture a medicament for treating autoimmune and/or inflammatory conditions, wherein the IL-7 and TNF-α binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

Other aspects and embodiments of the present invention will be apparent from the following detailed description.

Inflammatory bowel disease is a chronic, disabling autoimmune disease that places a heavy burden on patients and society. Current standard of care treatments have limited ability to induce lasting clinical and endoscopic remission. Without wishing to be bound by theory, it is expected that targeting the TNF-α and IL-7 pathways will increase mucosal healing, increase disease remission, and minimize or no increase in treatment-induced toxicity. In the examples provided herein, the combination of an IL-7 inhibitor and a TNF-α inhibitor showed enhanced activity in blocking IFNg secretion compared to a single TNF-α or IL-7 control, indicating that the combined use of an IL-7 inhibitor and a TNF-α inhibitor provides a promising therapeutic intervention for the treatment of autoimmune and/or inflammatory diseases.

As used herein, the term "IL-7 inhibitor" means a molecule that partially or completely destroys the natural function of IL-7. The IL-7 inhibitor can be a small organic molecule or an IL-7 binding domain. As used herein, the term "TNF-α inhibitor" means a molecule that partially or completely destroys the natural function of TNF-α. The TNF-α inhibitor can be a small organic molecule or a TNF-α binding domain. Such IL-7 binding domains and TNF-α binding domains can be provided as binding domains in separate proteins or as binding domains in a single protein. As used herein, "IL-7 binding domain and TNF-α binding domain" can exist in the form of separate proteins. As used herein, "IL-7 and TNF-α binding protein" is a single protein containing both an IL-7 binding domain and a TNF-α binding domain. IL - 7 binding domain

As used herein, the term "IL-7-mediated signaling" means the biological effects induced by the IL-7 receptor complex when it binds to its ligand IL-7. IL-7-mediated signaling thus includes (but is not necessarily limited to) one or more or all of the following: IL-7-induced phosphorylation of signal transducer and activator of transcription 5 (STAT-5), IL-7-induced TH17 cell expansion and IL-7-induced TH17 cell survival, IFNg production, Bcl2 expression, α4β7 integrin expression.

As used herein, the term "IL-7 binding domain" refers to antibodies and other protein constructs that are capable of binding to IL-7. This term does not include naturally occurring cognate receptors.

In one embodiment, the IL-7 binding domain used according to the uses and methods disclosed herein is an antibody. TNF - α Binding Domain

As used herein, the term "TNF-α-mediated signaling" means the biological effects induced by the TNF-α receptor complex when it binds to its ligand TNF-α. TNF-α-mediated signaling thus includes (but is not necessarily limited to) one or more or all of the following: NFkB activation, apoptosis, necroptosis, interleukin secretion, MIP-1α production, MIP-1β production, IL-8 production, cell proliferation, macrophage differentiation.

As used herein, the term "TNF-α binding domain" refers to antibodies and other protein constructs that are capable of binding to TNF-α. The terms "TNF-α binding domain" and "anti-TNF-α antigen binding domain" are used interchangeably herein. This term does not include naturally occurring homologous receptors.

In one embodiment, the TNF-α binding domain used according to the uses and methods disclosed herein is an antibody. IL - 7 and TNF - α Binding Protein

As used herein, the term "IL-7 and TNF-α binding protein" refers to antibodies and other protein constructs that contain an IL-7 binding domain and a TNF-α binding domain and are capable of binding to IL-7 and TNF-α.

In one embodiment, the IL-7 and TNF-α binding protein used according to the uses and methods disclosed herein is an antibody. Treatment of inflammatory or autoimmune diseases

In one aspect, an IL-7 inhibitor and TNF-α inhibitor or IL-7 and TNF-α binding protein as described herein is provided for use in treating inflammatory or autoimmune diseases.

In one embodiment, the IL-7 inhibitor is an IL-7 binding domain and the TNF-α inhibitor is a TNF-α binding domain. The IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding proteins described herein can be used to treat autoimmune and/or inflammatory diseases for which an IL-7 inhibitor is indicated or a TNF-α inhibitor is indicated or both an IL-7 and TNF-α inhibitor are indicated.

In some embodiments, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease (Crohn's disease); ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease Disease); dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; anti- Diseases mediated by antigen-antibody complexes, including glomerulonephritis; sepsis; sarcoidosis; immunopathological responses to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonitis, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD and type 1 diabetes; and any combination thereof. Inhibition of IL-7-induced IL-7R-mediated signaling may be useful in the treatment of inflammatory (non-autoimmune) diseases, such as asthma, in which elevated IL-17 or IL-2 has been implicated. In another embodiment, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; and ulcerative colitis, and any combination thereof. In another embodiment, the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). In another embodiment, the IL-7 binding domain and TNF-α binding domain described herein, or the IL-7 and TNF-α binding proteins, can be used to treat autoimmune and/or inflammatory diseases, such as IBD, in individuals who have been inadequately responsive to treatment with anti-TNF-α therapy, such as initial therapy or first line therapy.

In some embodiments, the inhibition of IL-7 by the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding proteins described herein affects the survival, expansion and function of autoreactive effector T cells while preserving regulatory T lymphocytes. In some embodiments, the inhibition of IL-7 by the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding proteins described herein inhibits the formation of allogeneic lymphoid tissue. In some embodiments, the inhibition of IL-7 by the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding proteins described herein can help restore homeostasis by inhibiting the survival of innate lymphoid cells (ILCs).

In some embodiments, the IL-7 binding domains in the uses and/or methods described herein or the IL-7 and TNF-α binding proteins disclosed herein are capable of antagonizing the biological effects of IL-7 and are capable of antagonizing at least one of IL-7R-mediated TH17 expansion and IL-7R-mediated TH17 survival. The terms inhibition, antagonism and neutralization are used synonymously herein. The terms are not intended to indicate a requirement for total neutralization; partial neutralization is also encompassed - corresponding to a reduction but not complete elimination of a biological effect. In some embodiments, the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding proteins disclosed herein are capable of antagonizing the biological effects of IL-7 and are capable of antagonizing at least one of IL-7R-mediated TH17 expansion and IL-7R-mediated TH17 survival, wherein partial neutralization corresponds to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% reduction in the biological effect.

At the molecular level, TH17 expansion and/or survival can be observed by an increase in IL-17 production by a CD4+ T cell population (or a TH17 cell population). Therefore, in one embodiment, the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding protein disclosed herein reduces IL-17 production by a CD4+ T cell population. IL-7 receptor-mediated TH17 expansion and survival can also be observed by an increase in IFN-γ production by a CD4+ T cell population (or a TH17 cell population). Therefore, in one embodiment, the IL-7 binding domain in the uses and/or methods described herein or the IL-7 and TNF-α binding protein disclosed herein antagonizes (reduces) IFN-γ production by a CD4+ T cell population. At the molecular level, the IL-7 binding domain or IL-7 and TNF-α binding protein disclosed herein can inhibit IL-7 receptor-mediated STAT-5 phosphorylation.

In some embodiments, at the molecular level, we can observe and measure the blocking effect of the IL-7 binding proteins described herein by analysis of, for example, IL-7-induced p-STAT5 or Bcl-2. In some embodiments, at the cellular level, we can observe and measure the blocking effect by analysis of TH17 secretion, such as IL-17 or IFNγ. Exemplary assays are described in PCT Application No. PCT/US2009/053136 (WO2010/017468). In an exemplary pSTAT-5 assay, PBMCs can be stimulated with IL-7 in the presence and absence of a test agent. The cells can then be quantitatively assessed for the level of pSTAT-5, for example, by staining for pSTAT-5 (e.g., using a labeled anti-pSTAT-5 antibody, such as ALEXA FLUOR 647 mouse anti-Stat5 (pY694, BD [#612599])), followed by fluorescence activated cell sorting. The level of phosphorylated STAT-5 can also be measured by ELISA. Those agents that reduce the level of phosphorylated STAT-5 can be potential therapeutic candidates for autoimmune diseases.

In some embodiments, the present invention provides a method of treating an autoimmune disease in a human subject, comprising administering to the subject an IL-7 binding domain and a TNF-α binding domain or an IL-7 and TNF-α binding protein in an amount sufficient to reduce IL-7R-mediated STAT-5 phosphorylation. In some embodiments, the IL-7 binding protein disclosed herein blocks or inhibits IL-7-mediated STAT5 phosphorylation directly downstream of IL-7R. In some embodiments, the IL-7 binding domain or an IL-7 and TNF-α binding protein disclosed herein downregulates the surface expression of activation markers and interleukin receptors responsible for lymphocyte migration to the CNS on active TH1 cells.

Antagonists of the invention, such as IL-7 binding domains, may be capable of reducing phosphorylated STAT-5 levels by at least: 20%, 50%, 75%, 80%, 85%, 90%, 95%, or 100% when compared to STAT-5 levels in the absence of the antagonist, or when compared to negative controls or untreated cells. The antagonist may have an IC _{50 of 50 μg/ml, 25 μg/ml or less, 10 μg/ml or less, 5 μg/ml or less, or 2 μg/ml or less. In one embodiment, the antagonist has an IC 50 of} less than or equal to 1 μg/ml, less than or equal to 0.75 μg/ml, less than or equal to 0.5 μg/ml, less than or equal to 0.25 μg/ml, or less than or equal to 0.1 μg/ml. In one embodiment, the antagonist has an _IC50 of less than or equal to 50 ng/ml, less than or equal to 40 ng/ml, less than or equal to 30 ng/ml, less than or equal to 20 ng/ml, or less than or equal to 10 ng/ml. In one embodiment, the antagonist has an _IC50 of 5 ng/ml.

The antagonists disclosed herein may be particularly effective in inhibiting the expansion of TH17 cells. The expansion of TH17 cells may be determined in a TH17 expansion assay, which may include stimulating a naive T cell population to expand in the presence and absence of a test agent, subsequently stimulating the cells to produce IL-17, and assessing the level of IL-17 produced by the cells in the presence and absence of the test agent. In an exemplary assay, human CD4+ T cells may be differentiated into TH17 by stimulating with T cell receptor activation in the presence of IL-1, IL-6, and IL-23. After 5 days of differentiation, CCR6+ cells may be sorted to produce an enriched TH17 population. This population can then be stimulated with human IL-7 and the supernatant can be assayed for increases in IL-17 and IFN-γ. Since the presence of an antagonist of this interaction during the incubation period should prevent TH17 cell expansion, resulting in a decrease in IL-17 and IFN-γ production, the ability of test agents such as the antigen-binding fragments of the invention to inhibit IL-7R induction by IL-7 can be assayed.

The IL-7 binding domain or IL-7 and TNF-α binding protein may be capable of inhibiting IL-17 secretion by 20% or more in such an assay compared to a negative control. More typically, the IL-7 binding protein is capable of inhibiting IL-17 secretion by 50%, 75%, 85%, or 90% or more relative to the control. In some embodiments, the IL-7 binding domain or IL-7 and TNF-α binding protein may exhibit an IC50 of less than or equal to 50 μg/ml in the assay. In other embodiments, the IC50 may be less than or equal to 20 μg/ml, 10 μg/ml, or 5 μg/ml. Thus, in another aspect, the invention provides a method for treating an autoimmune disease or inflammatory disorder comprising administering to a patient an IL-7 binding domain or IL-7 and TNF-α binding protein disclosed herein in an amount sufficient to reduce TH17 cell counts in the patient.

In one aspect, a method for treating an autoimmune and/or inflammatory condition in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of an IL-7 inhibitor and a therapeutically effective amount of a TNF-α inhibitor or a therapeutically effective amount of an IL-7 and TNF-α binding protein. In one embodiment, the IL-7 inhibitor is an IL-7 binding domain and the TNF-α inhibitor is a TNF-α binding domain. In one embodiment, a method for treating a disease or condition for which an IL-7 inhibitor, or a TNF-α inhibitor, or both an IL-7 and TNF-α inhibitor are prescribed in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of an IL-7 binding domain and a TNF-α binding domain as described herein or an IL-7 and TNF-α binding protein.

As used herein, the term "therapeutically effective" refers to the amount of IL-7 inhibitor, TNF-α inhibitor or IL-7 and TNF-α binding protein that will induce the desired biological response in a subject, such as a human subject. It may vary depending on the IL-7 inhibitor, TNF-α inhibitor or IL-7 and TNF-α binding protein, the disease and its severity, and the age and weight of the subject to be treated.

In some embodiments, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, cardiac arrest, heart failure, Reperfusion after visceral surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis ; autoimmune diseases, such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; group Immunopathological reactions to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; and any combination thereof. Inhibition of IL-7-induced IL-7R-mediated signaling may be useful in the treatment of inflammatory (non-autoimmune) diseases, such as asthma, that have been implicated in elevated IL-17 or IL-2. In another embodiment, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis and any combination thereof. In another embodiment, the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). In another embodiment, a method of treating an autoimmune and/or inflammatory condition in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of an IL-7 inhibitor and a therapeutically effective amount of a TNF-α inhibitor or a therapeutically effective amount of an IL-7 and TNF-α binding protein in an individual who has not responded sufficiently to treatment with an anti-TNF-α therapy, e.g., as an initial therapy or first line therapy.

In some embodiments, the subject is a human. In addition to subjects who may develop a disease in the future, those in need of treatment may also include subjects who already have a medical disease.

Therefore, in one embodiment, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein can be used for preventive or prophylactic treatment. In this case, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein are administered to an individual to prevent or delay the onset of one or more aspects or symptoms of the disease. The individual may be asymptomatic. The individual may have a genetic predisposition to the disease. In some embodiments, an effective amount of IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein is administered to such individuals. In some embodiments, the effective amount for prevention or treatment is an amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

The IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein can also be used in a treatment method. The term "treatment" covers the alleviation, reduction or prevention of at least one aspect or symptom of a disease. For example, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein can be used to improve or reduce one or more aspects or symptoms of the diseases described herein.

In some embodiments, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein are used in an amount effective for therapeutic, prophylactic or preventive treatment. In some embodiments, the therapeutically effective amount of the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein is an amount that effectively improves or alleviates one or more aspects or symptoms of the disease. In some embodiments, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein can also be used to treat, prevent or cure the diseases described herein. In some embodiments, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein have an overall beneficial effect on individual health, for example, it can increase the expected life span of an individual.

The use of the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding proteins described herein need not affect complete cure or eradication of every symptom or manifestation of a disease to constitute a viable therapeutic treatment. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease condition, but does not necessarily eliminate every manifestation of the disease to be considered a suitable therapeutic agent. Similarly, a treatment administered prophylactically need not be completely effective in preventing the onset of the disease in order to constitute a viable prophylactic agent. Simply reducing the impact of the disease (e.g. by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reducing the likelihood that an individual will develop the disease (e.g. by delaying disease onset) or get worse is sufficient.

In some embodiments, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein can be used to treat a subject suffering from or suspected of having a disease or condition described herein. In some embodiments, the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein described herein are administered to a subject suffering from or suspected of having a disease or condition described herein.

In one aspect, the present invention provides the use of an IL-7 inhibitor and a TNF-α inhibitor or a IL-7 and TNF-α binding protein for the manufacture of a medicament for the treatment of an autoimmune and/or inflammatory disease. In one embodiment, the IL-7 inhibitor is an IL-7 binding domain and the TNF-α inhibitor is a TNF-α binding domain. In some embodiments, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, cardiac arrest, heart failure, etc. Reperfusion after visceral surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis ; autoimmune diseases, such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; group Immunopathological reactions to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; and any combination thereof. Inhibition of IL-7-induced IL-7R-mediated signaling may be useful in the treatment of inflammatory (non-autoimmune) diseases, such as asthma, that have been implicated in elevated IL-17 or IL-2. In another embodiment, the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis, and any combination thereof. In another embodiment, the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD).

In some embodiments, treatment may include further monitoring the individual for a disease or condition. Treatment may include a single treatment. Treatment may include repeated treatments. Treatment may include repeated treatments for the remaining life of the individual. Treatment may include daily treatment. Treatment may include treatment every two weeks. In some embodiments, treatment may be selected based on an assessment of the patient or a sample obtained from the patient.

In some embodiments, the IL-7 binding domain used in the methods of therapy or treatment disclosed herein reduces B cell activation, reduces autoantibody production, and/or reduces B cell antigen presentation. In some embodiments, the IL-7 binding domain used in the methods of therapy or treatment disclosed herein, wherein the IL-7 binding domain binds IL-7, inhibits IL-7 receptor-mediated signaling, and does not restrict the development or function of regulatory T cells (T _reg ). In some embodiments, the IL-7 binding domain used in the methods of therapy or treatment disclosed herein inhibits signaling, activation, interleukin production, and/or proliferation of CD4 ⁺ and CD8 ⁺ T cells. In some embodiments are IL-7 binding domains for use in the methods of therapy or treatment disclosed herein, wherein blocking of IL-7 binding domains of IL-7 mediated signaling reduces inflammatory responses.

In some embodiments, the TNF-α binding domain is used in the methods of treatment or therapy disclosed herein, wherein the TNF-α binding domain reduces macrophage activation, reduces interleukin and cytokine production, inhibits intestinal epithelial cell death, and maintains intestinal barrier function. In some embodiments, the TNF binding domain is used in the methods of treatment or therapy disclosed herein, wherein the TNF binding domain reduces inflammatory response by blocking TNF-mediated signaling. Definitions related to IL - 7 binding domain, TNF - α binding domain, and IL - 7 and TNF - α binding protein

IL-7 and TNF-α binding domains used according to the uses and methods described herein are further described below. Such IL-7 and TNF-α binding domains may also form part of IL-7 and TNF-α binding proteins.

The term "antibody" as used herein refers in the broadest sense to molecules having immunoglobulin-like domains (e.g., IgG, IgM, IgA, IgD, or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies (including bispecific antibodies) and heterojunction antibodies; single variable domains (e.g., single domain antibodies (DABs)), antigen-binding antibody fragments, Fab, F(ab') ₂ , Fv, disulfide-linked Fv, single chain Fv, disulfide-linked scFv, bifunctional antibodies, TANDABS, etc. and modified forms of any of the foregoing (for a summary of alternative "antibody" forms, see Holliger and Hudson, Nature Biotechnology , 2005, Vol. 23, No. 9, 1126-1136).

In one embodiment, the IL-7 and TNF-α binding protein is a bispecific antibody. In another embodiment, the IL-7 and TNF-α binding protein is a bispecific antibody having an immunoglobulin format.

In some embodiments, the IL-7 binding domain or TNF-α binding domain disclosed herein may be derived from rats, mice, primates (e.g., cynomolgus macaques, old mainland monkeys, or great apes) or humans. The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein may be human, humanized, or chimeric antibodies. The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein may include a constant region, which may be any isotype or subclass. The constant region may have an IgG isotype, such as IgG1, IgG2, IgG3, IgG4, or a variant thereof. The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein constant region may be IgG1. In some embodiments, the IL-7 binding protein is an IgG1k antibody.

As used herein, "about" means plus or minus 10%.

The terms "complete,""whole," or "intact" antibodies are used interchangeably herein to refer to heterotetrameric proteins. An intact antibody is composed of two identical heavy chains (HC) and two identical light chains (LC) linked by covalent disulfide bonds. This _H2L2 structure folds to form three functional domains including two antigen binding fragments, called "Fab" fragments, and an "Fc" crystallizable fragment. The _Fab fragment is composed of the variable domains variable heavy chain ( _VH ) or variable light chain ( _VL ) at the amino terminus and the constant domains CH1 (heavy) and CL (light) at the carboxyl terminus. The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. Fc can induce effector function by binding to receptors on immune cells or by binding to C1q (the first component of the classical complement pathway). The five classes of antibodies, IgM, IgA, IgG, IgE, and IgD, are defined by different heavy chain amino acid sequences called µ, α, γ, ε, and δ, respectively, each of which can pair with a kappa or lambda light chain. Most antibodies in serum belong to the IgG class, and there are four isotypes of human IgG (IgG1, IgG2, IgG3, and IgG4), whose sequences differ primarily in the hinge region. In some embodiments, the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein disclosed herein is human IgG1. In some embodiments, the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein disclosed herein is a disulfide-linked α2β2 tetramer. In some embodiments, the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein disclosed herein comprises two light (κ) chains and two heavy (IgG1) chains.

Fully human antibodies can be obtained using a variety of methods, such as using yeast-based libraries or transgenic animals (e.g., mice) that can generate a human antibody repertoire. Yeast that present human antibodies bound to the relevant antigen on their surface can be selected using FACS (fluorescence activated cell sorting) based methods or by capture on beads using labeled antigens. Transgenic animals that have been modified to express human immunoglobulin genes can be immunized with the relevant antigen, and antigen-specific human antibodies isolated using B cell sorting techniques. Human antibodies generated using these techniques can then be characterized for desired properties such as affinity, developability, and selectivity.

In some embodiments, alternative antibody formats may be used. Alternative antibody formats include alternative scaffolds, wherein one or more CDRs of an IL-7 antibody and/or a TNF-α antibody may be arranged on a suitable non-immunoglobulin protein scaffold or framework, such as an affinity antibody, SpA scaffold, LDL receptor class A domain, avidin multimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301), or an EGF domain.

The term "domain" refers to a folded polypeptide structure that retains its tertiary structure independently of the rest of the polypeptide. In general, domains are responsible for discrete functional properties of a polypeptide and in many cases can be added, removed, or transferred to other polypeptides without loss of function of the rest of the protein and/or the domain.

The term "single variable domain" refers to a folded polypeptide domain comprising a sequence that characterizes an antibody variable domain. Thus, a single variable domain includes complete antibody variable domains, such as _VH , VHH and _VL, as well as modified antibody variable domains, for example, antibody variable domains in which one or more loops have been replaced by sequences that do not characterize antibody variable domains, or antibody variable domains that have been truncated or include N-terminal or C-terminal extensions, and folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain. A single variable domain can bind an antigen or antigenic determinant independently of different variable regions or domains. "Domain antibodies" or "DABs" may be considered to be the same as "single variable domains". The single variable domain may be a human single variable domain, and also includes single variable domains from other species such as rodents (e.g., as disclosed in WO 00/29004 A1), nurse sharks, and camel family VHH DABs. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, dromedaries, and chestnut llamas that produce heavy chain antibodies that naturally do not contain light chains. Such VHH domains can be humanized according to standard techniques available in the art, and such domains are considered "single variable domains". As used herein, _VH includes Camelid VHH domains.

Antigen binding fragments, IL-7 binding domain fragments, TNF-α binding domain fragments, functional fragments, biologically active fragments or immunologically effective fragments may contain part of the heavy chain or light chain variable sequence. The length of the fragment is at least 5, 6, 8 or 10 amino acids. Alternatively, the length of the fragment is at least 15, at least 20, at least 50, at least 75 or at least 100 amino acids.

The antigen-binding fragment can be provided by arranging one or more CDRs on a non-antibody protein framework. As used herein, "protein framework" includes but is not limited to immunoglobulin (Ig) frameworks, such as IgG frameworks, which can be four-chain or two-chain antibodies, or it can only include the Fc region of an antibody, or it can include one or more constant regions from an antibody, which constant regions can be of human or primate origin, or it can be an artificial chimera of human and primate constant regions.

The protein backbone may be an Ig backbone, such as an IgG or IgA backbone. An IgG backbone may comprise some or all domains of an antibody (i.e., CH1, CH2, CH3, _VH , _VL ). An IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein may comprise an IgG backbone selected from IgG1, IgG2, IgG3, IgG4, or IgG4PE. For example, the backbone may be IgG1. The backbone may consist of, comprise, or be part of the Fc region of an antibody.

The protein scaffold may be a derivative of a scaffold selected from the group consisting of CTLA-4, lipocalin, protein A derived molecules such as the Z domain (affimer, SpA), A domain (affinity multimer/maximer) of protein A; heat shock proteins such as GroE1 and GroES; ferrotransferrin (trans-host); DARPin; peptide aptamers; C-type lectin domains (tetranectin); human gamma-crystallin and human ubiquitin (affilins); PDZ domains; kunitz-type domains of human protease inhibitors; and fibronectin/adnectin; which have been protein engineered to obtain binding to antigens other than the natural ligand.

As used herein, the term "antagonist antibody" refers to an antibody or fragment thereof that is capable of completely or partially inhibiting the biological activity of an antigen (e.g., IL-7 or TNF-α), such as by binding to the antigen by completely or partially blocking binding to a ligand or reducing the biological activity of the antigen.

"Antigen binding site" refers to a site on an antigen binding protein that is capable of specifically binding to an antigen, which may be a single variable domain, or it may be a pair of _VH / _VL domains such as may be present on a standard antibody. A single chain Fv (ScFv) domain may also provide an antigen binding site.

As used herein, the term "chimeric antigen receptor"("CAR") refers to an engineered receptor consisting of an extracellular antigen binding domain (which is usually derived from a monoclonal antibody or fragment thereof, such as a _VH domain and a _VL domain in the form of an scFv), optionally a spacer, a transmembrane region, and one or more intracellular effector domains. CARs have also been referred to as chimeric T cell receptors or chimeric immune receptors (CIRs). CARs are genetically introduced into hematopoietic cells (such as T cells) to redirect the T cell specificity of a desired cell surface antigen, thereby generating a CAR-T therapeutic.

"Humanized antibody" refers to a type of engineered antibody whose CDRs are derived from non-human donor immunoglobulins and the remaining immunoglobulin-derived portions of the molecule are derived from one or more human immunoglobulins. In addition, the framework support residues may be altered to maintain binding affinity (see, e.g., Queen et al. Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al. Bio / Technology , 9:421 (1991)). Suitable human acceptor antibodies may be antibodies homologous to the nucleotide and amino acid sequences of the donor antibody selected from known databases, such as the KABAT database, the Los Alamos database, and the Swiss Protein database. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody may be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies capable of supplying light chain constant regions or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains do not need to originate from the same acceptor antibody. Prior art describes several methods for producing such humanized antibodies - see, for example, EP-A-0239400 and EP-A-054951.

As used herein, the term "spacer" refers to an oligopeptide or polypeptide used to connect the transmembrane domain to the target binding domain. This region may also be referred to as a "hinge" or "handle." The size of the spacer can vary depending on the location of the target epitope in order to maintain a set distance (e.g., 14 nm) after CAR: target binding.

As used herein, the term "transmembrane domain" refers to the portion of the CAR molecule that crosses the cell membrane.

As used herein, the term "intracellular effector domain" (also referred to as "signaling domain") refers to the domain in the CAR that is responsible for intracellular signaling after the antigen binding domain binds to the target. The intracellular effector domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including the secretion of cytokines.

Those skilled in the art will appreciate that the _VH and/or _VL domains disclosed herein can be incorporated into CAR-T therapeutics, for example in the form of scFv.

In some embodiments, the IL-7 binding domain of the present invention exhibits cross-reactivity between human IL-7 and IL-7 from another species, such as cynomolgus macaque IL-7. In one embodiment, the IL-7 binding domain of the present invention specifically binds to human and macaque IL-7. In some embodiments, the TNF-α binding domain of the present invention exhibits cross-reactivity between human TNF-α and TNF-α from another species, such as macaque TNF-α. In one embodiment, the TNF-α binding domain of the present invention specifically binds to human and macaque TNF-α. This is particularly applicable because drug development often requires testing lead candidate drugs in mouse systems before testing the drugs in humans. Providing a drug that can bind to both human and macaque species allows us to test results in these systems and perform side-by-side comparisons of data using the same drug. This avoids the complexity of needing to find a drug that works on macaque IL-7 and isolate a drug that works on human IL-7 and/or on macaque TNF-α, and also avoids the need to use non-identical drugs to compare results in humans and macaques. Cross-reactivity between other species used in disease models (such as dogs or mice) is also envisioned.

As appropriate, the binding affinity of the IL-7 binding domain to at least cynomolgus macaque IL-7 and to human IL-7 and/or the binding affinity of the TNF-α binding domain to at least cynomolgus macaque TNF-α and to human TNF-α differs by no more than 2 or 5 fold. In some embodiments, the IL-7 binding proteins disclosed herein are species specific.

Affinity, also known as "binding affinity", is the strength of binding at a single interaction site, i.e., the strength of binding between a molecule (e.g., an IL-7 binding domain or TNF-α binding domain of the present invention) and another molecule (e.g., its target antigen) at a single binding site. The binding affinity of an IL-7 binding domain or TNF-α binding domain to its target can be determined by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., surface plasmon resonance (SPR) analysis using a BIACORE instrument). For example, the SPR method described in Example 2 can be used to measure binding affinity.

Avidity (also called functional affinity) is the cumulative binding strength at multiple interaction sites, e.g., the sum of the binding strengths of two molecules (or more, e.g., in the case of bispecific or multispecific molecules) to each other at multiple sites, e.g., taking into account the valency of the interaction.

In one embodiment, the equilibrium dissociation constant (KD) of the IL-7 binding domain-IL-7 interaction or the TNF-α binding domain-TNF-α interaction is about 100 nm or less, about 10 nM or less, about 2 nM or less, or about 1 nM or less. Alternatively, the KD may be between about 5 and about 10 nM; or between about 1 and about 2 nM. The KD may be between about 1 pM and about 500 pM; or between about 500 pM and about 1 nM. In one embodiment, the equilibrium dissociation constant (KD) of the IL-7 binding protein-IL-7 interaction is 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the KD may be between 5 nM and 10 nM; or between 1 nM and 2 nM. The KD may be between 1 pM and 500 pM; or between 500 pM and 1 nM. One skilled in the art will appreciate that the smaller the KD value, the stronger the binding. The reciprocal of the KD (i.e., 1/KD) is the equilibrium association constant (KA) with units of M ^{- 1} . One skilled in the art will appreciate that the larger the KA value, the stronger the binding. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 30 to 90 pM. In some embodiments, the KD of the IL-7 binding protein disclosed herein is about 30 to about 80 pM, about 30 to about 70 pM, about 30 to about 60 pM, about 30 to about 50 pM, about 30 to about 55 pM, or about 30 to about 40 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 40 to about 80 pM, about 40 to about 70 pM, about 40 to about 60 pM, about 40 to about 50 pM, about 40 to about 55 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 30 to about 55 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 31, about 34, about 46, about 53, about 69, about 73, about 75, or about 87 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 31, about 34, about 46, about 53, about 69, about 73, about 75, or about 87 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 34 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 67 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 30 to about 55 pM at 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 36 pM at about 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 45 to about 90 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 45 to about 90 pM at 37°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 67 pM at 37°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 30 to 90 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 30 to 80 pM, 30 to 70 pM, 30 to 60 pM, 30 to 50 pM, 30 to 55 pM, or 30 to 40 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 40 to 80 pM, 40 to 70 pM, 40 to 60 pM, 40 to 50 pM, 40 to 55 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 30 to 55 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 31, 34, 46, 53, 69, 73, 75, or 87 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 31, 34, 46, 69, 75, or 87 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 34 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 67 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 30 to 55 pM at 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 36 pM at 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 45 to 90 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 45 to 90 pM at 37°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 67 pM at 37°C.

In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 31, 34, 46, 53, 69, 73, 75, or 87 pM ± 15%. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 31, 34, 46, 69, 75, or 87 pM ± 15%. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 34 pM ± 15%. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 67 pM ± 15%. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 30 to 55 pM at 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 36 pM ± 15% at 25°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 45 to 90 pM. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 45 to 90 pM at 37°C. In some embodiments, the KD of the IL-7 binding domain or TNF-α binding domain disclosed herein is 67 pM ± 15% at 37°C.

Those skilled in the art will appreciate that surface plasmon resonance (SPR) is a suitable method for measuring binding affinity and determining binding kinetics, see, e.g., Day et al., Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods, Protein Science (2002), 11:1017-1025; and Hearty et al., "Measuring antibody-antigen binding kinetics using surface plasmon resonance" Methods Mol Biol (2012) 907:411-4.

The dissociation rate constant (kd) or "dissociation rate" describes the stability of the IL-7 binding domain-IL-7 complex, or the stability of the TNF-α binding domain-TNF-α complex, i.e., the fraction of the complex that decays per second. For example, a kd of 0.01 s ^{- 1} is equivalent to a complex that decays by 1 ^% per second. In one embodiment, the dissociation rate constant (kd) is about 1× ^10-3 s ^{- 1} or less, about 1× ^10-4 s ^{- 1} or less, ^about 1× ^10-5 s ^{- 1} or less, or about ¹ × ^10-6 s ^{- 1} or ^less . The kd may be between about 1× ^{10-5 s - 1} and ^about 1× ^{10-4 s - 1} ; or between about 1× ^10-4 s ^{- 1} and about 1× ^10-3 s ^{- 1} . In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein ^is about ^2.06 × ^10-4 s ^{- 1} or less ^{, about 1.58×10-4 s-1 or less, about 1.7×10-4 s - 1 or less , or about 5.68×10-4 s-1 or less, about 6.78×10-4 s - 1 or less , about} 8.26× ^10-4 s ^{- 1} or less, ^{about 5.15} × ^10-4 s ^{- 1} or less. In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is about ^1.58 × ^10-4 s ^{- 1} or less. In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 5.68× ^10-4 s ^{- 1} or less. In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is about 2.06× ^10-4 s ^{- 1} or less, about 1.58× ^10-4 s ^{- 1} or less, or about ^1.7 × ^10-4 s ^{- 1} or less ^{at 25} ° ^C . In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein at 37° C. is about 5.68×10 ^{− 4} s ^{− 1} or less, about 6.78×10 ^{− 4} s ^{− 1} or less, about 8.26×10 ^{− 4} s ^{− 1} or less, or about 5.15×10 ^{− 4} s ^{− 1} or less.

In one embodiment, the ^dissociation rate constant (kd) is 1× ^10-3 s ^{- 1} or less, 1× ^10-4 s ^{- 1} or less, 1× ^10-5 s ^{- 1} or less, or 1× ^10-6 s ^{- 1} or less. The ^kd may be between 1× ^10-5 s ^{- 1 and 1 × 10-4 s- 1 ;} or between 1 ^{× 10-4} s ^{- 1} and ¹ × ^10-3 s ^{- 1} . In some embodiments, the kd of the IL- ⁷ binding domain or TNF-α binding domain disclosed herein is 2.06× ^10-4 s ^{- 1} or less, 1.58× ^10-4 s ^- 1 or less ^, 1.7 ^{× 10-4} s ^{- 1} or less, or ^{5.68×10-4 s-1 or less, 6.78×10-4 s - 1 or less , 8.26} × ^10-4 s ^{- 1} or less, or 5.15 ^{×10-4 s - 1 or} less. In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is ^1.58 × ^10-4 s ^{- 1} or less. In some embodiments, the kd of the IL-7 binding domain or ^TNF -α binding domain disclosed herein is 5.68× ^10-4 s ^{- 1} or ^less . In some embodiments, ^the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is 2.06× ^10-4 s ^{- 1} or less, 1.58× ^10-4 s ^{- 1} or less, or 1.7× ^10-4 s ^{- 1} or less at 25°C. In some embodiments, the kd of the IL-7 binding domain or TNF-α binding domain disclosed herein is 5.68× ^{10-4 s - 1} or less, ^6.78 × ^10-4 s ^{- 1} or less, 8.26× ^10-4 s- ¹ or less, or 5.15× ^{10-4 s- 1 or} less ^{at 37 ° C} .

The association rate constant (ka) or "association rate" describes the rate at which the IL-7 binding domain-IL-7 or TNF-α binding domain-TNF-α complex is formed. In one embodiment, the association rate constant (ka) is about 6.49×10 ⁶ M ^{- 1} s ^{- 1} , about 4.65×10 ⁶ M ^{- 1} s ^{- 1} , about 3.17×10 ⁶ M ^{- 1} s ^{- 1} , about 8.28×10 ⁶ M ^{- 1} s ^{- 1} , about 1.47×10 ⁷ M ^{- 1} s ^{- 1} , about 1.10×10 ⁷ M ^{- 1} s ^{- 1} , or about 5.90×10 ⁶ M ^{- 1} s ^{- 1} . In one embodiment, the association rate constant (ka) is about 6.49×10 ⁶ M ^{- 1} s ^{- 1} , 4.65×10 ⁶ M ^{- 1} s ^{- 1} , or about 3.17×10 ⁶ M ^{-1 s-1 at 25° C. In one embodiment, the association rate constant (ka) is about 8.28×10 6 M- 1 s - 1 , about 1.47 ×} 10 ⁷ M ^{- 1} s ^{- 1} , about 1.10×10 ⁷ M ^{- 1} s ^{- 1} , or about 5.90×10 ⁶ M ^{- 1} s ^{- 1} at 37° C. In one embodiment, the association rate constant (ka) is 6.49×10 ⁶ M ^{- 1} s ^{- 1} , 4.65×10 ⁶ M ^{- 1} s ^{- 1} , 3.17×10 ⁶ M ^{- 1} s ^{- 1} , 8.28×10 ⁶ M ^{- 1} s ^{- 1} , 1.47×10 ⁷ M ^{- 1} s ^{- 1} , 1.10×10 ⁷ M ^{- 1} s ^{- 1} , or 5.90×10 ⁶ M ^{- 1} s ^{- 1} . In one embodiment, the association rate constant (ka) is 6.49×10 ⁶ M ^{- 1} s ^{- 1} , 4.65×10 ⁶ M ^{- 1} s ^{- 1} , or 3.17×10 ⁶ M ^{- 1} s ^{- 1} at 25°C. In one embodiment, the association rate constant (ka) at 37° ^C. is 8.28× ^{10 6} M ⁻¹ s ^{−1 ,} 1.47×10 ⁷ M ⁻¹ s ⁻¹ , 1.10×10 ⁷ M ^{−1 s −1 ,} or 5.90× ^{10 6 M −1 s −1 .}

As used throughout this specification, the term "neutralize" means that the in vitro or in vivo biological activity of IL-7 is reduced in the presence of an IL-7 binding domain as described herein, compared to the activity of IL-7 in the absence of the IL-7 binding domain; or the in vitro or in vivo (as appropriate) biological activity of TNF-α is reduced in the presence of a TNF-α binding domain as described herein, compared to the activity of TNF-α in the absence of the TNF-α binding domain. In the case of IL-7, neutralization can be due to blocking the binding of IL-7 to its receptor, preventing IL-7 from activating its receptor, downregulating IL-7 or its receptor, or affecting one or more of the effector functions. In the case of TNF-α, neutralization can be due to one or more of blocking the binding of TNF-α to its receptor, preventing TNF-α from activating its receptor, downregulating TNF-α or its receptor, or affecting effector function.

The reduction or inhibition of biological activity can be partial or complete. Neutralizing IL-7 binding proteins can neutralize the activity of IL-7 by reducing the threshold of B cell activation by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to the activity of IL-7 in the absence of the IL-7 binding domain. Neutralization can be determined or measured using one or more assays known to those skilled in the art or described herein. For example, FIG. 2B, FIG. 2C and FIG. 2D. Neutralizing the TNF-α binding domain can neutralize the activity of TNF-α by reducing the threshold of B cell activation by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to the activity of TNF-α in the absence of the TNF-α binding domain. Neutralization can be determined or measured using one or more assays known to those skilled in the art or described herein.

It will be apparent to those skilled in the art that the term "derived" is intended to define a source not only in the sense that it is the physical source of a material, but also to define materials that are structurally identical to that material but that are not derived from the referenced source.

"Isolated" means that a molecule, such as an IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein, is removed from an environment in which it may be found in nature. For example, a molecule can be purified away from the substance in which it would normally exist in nature. For example, the IL-7 binding domain, the TNF-α binding domain, or the IL-7 and TNF-α binding protein can be purified to at least 95%, 96%, 97%, 98%, or 99% or greater relative to a medium containing the IL-7 binding domain, the TNF-α binding domain, or the IL-7 and TNF-α binding protein. The IL-7 binding domain, the TNF-α binding domain, or the IL-7 and TNF-α binding protein disclosed herein can be an isolated IL-7 binding domain, a TNF-α binding domain, or a TNF-α/IL-7 bispecific antibody.

"CDR" is defined as the complementary determining region amino acid sequence of an antigen binding protein. These complementary determining region amino acid sequences are the hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Therefore, "CDR" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy chain and light chain CDRs, or at least two CDRs.

Throughout this specification, the amino acid residues in the variable domain regions within the variable domain sequences and the full-length antigen binding sequences (e.g., within the antibody heavy chain sequence or the antibody light chain sequence) are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the examples follow the Kabat numbering convention. For further information, see Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987). Variants

It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those set forth in Chothia et al. (1989) Nature 342: 877-883. The structure and protein fold of the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein may mean that other residues are considered part of the CDR sequence and those skilled in the art will understand this.

Other numbering conventions for CDR sequences that are available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide a "minimum binding unit". The minimum binding unit can be a sub-portion of a CDR.

Table 1 below shows one definition using the numbering conventions for each CDR or binding unit. The Kabat numbering scheme is used in Table 1 to number the variable domain amino acid sequences. It should be noted that some of the CDR definitions may vary depending on the individual publication used. Table 1 Kabat CDR Chothia CDR AbM CDR Contact CDR Minimum binding unit H1 31-35/35A/ 35B 26-32/33/34 26-35/35A/35B 30-35/35A/35B 31-32 H2 50-65 52-56 50-58 47-58 52-56 H3 95-102 95-102 95-102 93-101 95-101 L1 24-34 24-34 24-34 30-36 30-34 L2 50-56 50-56 50-56 46-55 50-55 L3 89-97 89-97 89-97 89-96 89-96

Thus, an IL-7 and TNF-α binding protein is provided that comprises the following CDRs: CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, CDRL3 of SEQ ID NO: 11, or CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. In another embodiment, an IL-7 and TNF-α binding protein is provided, comprising any one or combination of the following CDRs: CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, CDRL3 of SEQ ID NO: 11, and CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. The CDRs may be modified by at least one amino acid substitution, deletion, or addition, wherein the variant IL-7 and TNF-α binding protein substantially retains the biological properties of the unmodified protein, such as binding to IL-7 and/or TNF-α.

It should be understood that each of the CDRs H1, H2, H3, L1, L2, L3 of TNF-α or IL-7 can be modified alone or in combination with any other CDR in any arrangement or combination. In one embodiment, the CDR is modified by substitution, deletion or addition of up to 3 amino acids (e.g., 1 or 2 amino acids, such as 1 amino acid). Typically, the modification is a substitution, particularly a conservative substitution, for example, as shown in Table 2 below. Table 2: Side chain Member Hydrophobicity Met, Ala, Val, Leu, Ile Neutral hydrophilicity Cys, Ser, Thr Acidic Asp、Glu Alkalinity Asn, Gln, His, Lys, Arg Residues that affect chain orientation Gly, Pro Aromatic Trp, Tyr, Phe

For example, in a variant CDR, the flanking residues comprising the CDR as part of an alternative definition (e.g., Kabat or Chothia) may be substituted with conservative amino acid residues. In some embodiments, an IL-7 binding protein comprising a variant CDR as described above may be referred to herein as a "functional CDR variant."

Thus, in another embodiment, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to IL-7 and comprises a CDRH3 or a variant thereof CDRH3 of SEQ ID NO: 8. In one embodiment, the IL-7 binding domain comprises CDRH1 or a variant thereof CDRH1 of SEQ ID NO: 6, CDRH2 or a variant thereof CDRH2 of SEQ ID NO: 7, CDRH3 or a variant thereof CDRH3 of SEQ ID NO: 8, CDRL1 or a variant thereof CDRL1 of SEQ ID NO: 9, CDRL2 or a variant thereof CDRL2 of SEQ ID NO: 10, and CDRL3 or a variant thereof CDRL3 of SEQ ID NO: 11. In some embodiments, IL-7 binding domains provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein include those having one or more variant CDRs that can bind to IL-7 and also neutralize IL-7 activity.

Disclosed herein is an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein that binds to IL-7 and comprises a heavy chain variable region of SEQ ID NO: 4. An IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein may comprise a light chain variable region of SEQ ID NO: 5. In some embodiments, an IL-7 binding protein provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein binds to and neutralizes IL-7. In one embodiment, an IL-7 binding protein provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain variable region of SEQ ID NO: 4 and a light chain variable region of SEQ ID NO: 5.

CDRs L1, L2, L3, H1, H2, and H3 tend to structurally exhibit one of a finite number of main-chain configurations (canonical). A particular canonical structural class of a CDR is defined by both the length of the CDR and the loop packing, which is determined by residues located at key positions in both the CDR and the framework regions (structurally determined residues or SDRs). Martin and Thornton (1996; J Mol Biol 263:800-815) have developed an automated method for defining "key residue" canonical templates. Cluster analysis is used to define canonical classes of CDR sets, and canonical templates are subsequently identified by analyzing buried hydrophobicity, hydrogen-bonded residues, and conserved glycine and proline residues. The CDRs of antibody sequences can be assigned to canonical classes by comparing the sequences to key residue templates and scoring each template using an identity or similarity matrix.

Based on the canonical class of DRSPAI-L7B antibodies, it can be predicted that functional antibody binding is maintained in the presence of the following CDR substitutions, where the amino acids before the Kabat number are the original amino acid sequence and the amino acid sequence at the end of the Kabat number are the substituted amino acids: CDRL1 Canonical : K24R S27aN, S27aD, S27aT, S27aE, S31,N, S31T, S31K, S31G L27bV D27cL, D27cY, D27cV, D27cI, D27cS, D27cN, D27sF, D27cH, D27cG, D27cT Y32F, Y32N, Y32A, Y32H, Y32S, Y32R I33M, I33L, I33V, I33R N34H CDRL2 Canonical : G51A CDRL3 typical : Q89S, Q89G, Q89F, Q89L, Q90N, Q90H S91N, S91F, S91G, S91R, S91D, S91H, S91T, S91Y, S91V N92Y, N92W, N92T, N92S, N92R, N92Q, N92Q, N92H, N92A, N92D V93E, V93N, V93G, V93H, V93T, V93S, V93R, V93A D94Y, D94T, D94V, D94L, D94H, D94N, D94I, D94W, D94P, D94S L96P, L96Y, L96R, L96I, L96W, L96F CDRH1 Typical : Y32I, Y32H, Y32F, Y32T, Y32N, Y32C, Y32E, Y32D G33Y, G33A, G33W, G33T, G33L, G33V V34M, V34I, V34L, V34T, V34W H35E, H35N, H35Q, H35S, H35Y, H35T CDRH2 Typical : I51L, I51V, I51T, I51S, I51N, I51M G55D Y59L CDRH3 Typical : Y102H, Y102V, Y102I, Y102S, Y102D, Y102G

As discussed above, the specific canonical structural classes of CDRs are defined by both the length of the CDRs and the loop packing, which is determined by residues located at key positions in both the CDRs and the framework regions. Therefore, substitutions can also be made based on canonical classes in the framework residues of the IL-7 binding proteins of the invention while retaining functional antibodies. Such substitutions may include (using Kabat numbering): Light chain: I, L or V at position 2; Q, L or E at position 3; M or L at position 4; I or V at position 48; and/or Heavy chain: V, I or L at position 2; L or V at position 4; L, I, M or V at position 20; T, A, V, G or S at position 24; F, Y, T or G at position 27; F, L, I, V or S at position 29; W or Y at position 47; I, M, L or V at position 48; I, L, F, M or V at position 69; R, K, V or I at position 71; A, L, V, Y or F at position 78; L or M at position 80; Y or F at position 90; R, K, G, S, H, N, T, A and/or L at position 94.

Thus, the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein provided for use according to the uses and methods described herein may have any of the above substitutions within the described positions. Multiple substitutions may exist for each variant CDR, each heavy chain or light chain variable region, each heavy chain or light chain, and each IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein provided for use according to the uses and methods described herein, and thus, any combination of substitutions may be present in the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein, provided that the canonical structure of the CDR is maintained. For the avoidance of doubt, the above substitutions should not be construed as limiting the possible CDR substitutions that may be performed while still maintaining a functional anti-IL-7, TNF-α, or TNF-α/IL-7 antibody.

The _VH or _VL or heavy chain (HC) or light chain (LC) sequences disclosed herein may be variant sequences with up to 10 amino acid substitutions, additions or deletions. For example, the variant sequence may have up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions. The sequence variation may exclude one or more or all of the CDRs, for example, the CDRs are identical to the _VH or _VL (or HC or LC) sequence, and the variation is in the remainder of the _VH or _VL (or HC or LC) sequence, such that the CDR sequence is fixed and complete.

Alternatively, the heavy chain variable region may have 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence of SEQ ID NO: 4; and the light chain variable region may have 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, 99% or greater, or 100% identity to the amino acid sequence of SEQ ID NO: 5.

The heavy chain variable region may be a variant of the amino acid sequence of SEQ ID NO: 4, which may contain 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution, insertion or deletion. The light chain variable region may be a variant of the amino acid sequence of SEQ ID NO: 5, which may contain 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitution, insertion or deletion. IL - 7 binding domain

In a specific embodiment, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to IL-7 and comprises a heavy chain and light chain variable domain combination of SEQ ID NO: 4 and SEQ ID NO: 5, or an IL-7 binding protein having heavy chain and light chain variable domains that are at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 4 and SEQ ID NO: 5, respectively. In one embodiment, an IL-7 binding protein for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to and neutralizes IL-7.

Any of the heavy chain variable regions of the IL-7 binding domain provided for use according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein can be combined with a suitable constant region. Any of the light chain variable regions of the IL-7 binding domain provided for use according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein can be combined with a suitable constant region. The constant regions disclosed herein can be human constant regions.

The present invention also provides nucleic acid molecules encoding a polypeptide sequence of any of the IL-7 binding domains for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein. The nucleic acid molecule may comprise sequences encoding: (i) one or more CDRHs, heavy chain variable sequences, or full-length heavy chain sequences; and (ii) one or more CDRLs, light chain variable sequences, or full-length light chain sequences, wherein (i) and (ii) are on the same nucleic acid molecule. Alternatively, a nucleic acid molecule encoding an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein may comprise a sequence encoding each of: (a) one or more CDRHs, heavy chain variable sequences, or full-length heavy chain sequences; or (b) one or more CDRLs, light chain variable sequences, or full-length light chain sequences, wherein (a) and (b) are on separate nucleic acid molecules. In some embodiments, the nucleic acid comprises a sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the nucleic acid sequence of SEQ ID NO: 13 encoding the light chain. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 13 encoding the light chain. In some embodiments, the nucleic acid comprises a sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleic acid sequence of SEQ ID NO: 14 encoding heavy chain. In some embodiments, the nucleic acid comprises a nucleic acid sequence of SEQ ID NO: 14 encoding heavy chain. In some embodiments, the nucleic acid further comprises a sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleic acid sequence of SEQ ID NO: 15 encoding a signal peptide.

Variants whose sequences are partially altered, such as by chemical modification and/or insertion, deletion or substitution of one or more amino acid residues, or those having 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 98% or greater, or 99% or greater identity to any of the sequences described above, may exhibit IL-7 binding potency within 10-fold or within 5-fold of that exhibited by DRSPAI-L7B (as demonstrated by EC50 or surface plasmon resonance analysis). DRSPAI-L7B is a human IgG1 that potently (Kd 67 pM) inhibits IL-7-mediated signaling by binding to interleukin 7 (IL-7). DRSPAI-L7B is a disulfide-bonded α2β2 tetramer composed of two light (κ) chains and two heavy (IgG1) chains. The heavy chain constant region contains two point mutations, L235A and G237A (LAGA), which reduce the binding of mAb to Fcγ receptors and prevent Fc-mediated effector functions, including CDC and ADCC. DRSPAI-L7B comprises a heavy chain with amino acids of SEQ ID NO: 2 and a light chain with amino acids of SEQ ID NO: 3.

In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain having at least 80%, 85%, 90% or 95% sequence identity with an amino acid of SEQ ID NO: 19, 21 or 23. In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain having an amino acid of SEQ ID NO: 19, 21 or 23. In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a light chain having at least 80%, 85%, 90% or 95% sequence identity with an amino acid of SEQ ID NO: 18, 20 or 22. In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a light chain having amino acids of SEQ ID NO: 18, 20, or 22. In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain having amino acids of SEQ ID NO: 19 and a light chain having amino acids of SEQ ID NO: 18. In some embodiments, an IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain having amino acids of SEQ ID NO: 21 and a light chain having amino acids of SEQ ID NO: 20. In some embodiments, the IL-7 binding domain provided for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises a heavy chain having amino acids of SEQ ID NO: 23 and a light chain having amino acids of SEQ ID NO: 22. TNF - α Binding Domain

In one aspect, the TNF-α binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein comprises CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In one embodiment, the TNF-α binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein comprises a VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and/or a VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55.

In another embodiment, the TNF-α binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein comprises a VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and a VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55.

In another embodiment, the TNF-α binding domain for use according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein comprises the VH domain of SEQ ID NO: 54 and the VL domain of SEQ ID NO: 55.

In another embodiment, the TNF-α binding domain used according to the uses and methods described herein comprises adalimumab, golimumab, infliximab, certolizumab or entercept, or a TNF-α binding domain comprising the VH and VL regions of such antibodies. In another embodiment, the TNF-α binding domain used according to the uses and methods described herein comprises adalimumab or a TNF-α binding domain comprising the VH and VL regions of adalimumab. In another embodiment, the TNF-α binding domain used according to the uses and methods described herein comprises adalimumab with M252Y/S254T/T256E modifications.

The term "antigenic determinant" as used herein refers to the portion of the antigen that contacts a specific binding domain of the IL-7 binding domain or the TNF-α binding domain, also known as a complementary site. An antigenic determinant may be linear or conformational/non-continuous. A conformational or non-continuous antigenic determinant comprises amino acid residues that are separated by other sequences (i.e., not in a continuous sequence in a primary sequence of an antigen assembled by tertiary folding of a polypeptide chain). Although the residues may come from different regions of the polypeptide chain, they are very close in the three-dimensional structure of the antigen. In the case of a polymeric antigen, a conformational or non-continuous antigenic determinant may include residues from different peptide chains. Specific residues contained in the antigenic determinant can be determined by computer modeling programs or by three-dimensional structures obtained by methods known in the art, such as X-ray crystallography. Epitope localization can be performed using various techniques known to those skilled in the art, such as those described in publications such as Methods in Molecular Biology " Epitope Mapping Protocols " , Mike Schutkowski and Ulrich Reineke ( Volume 524 , 2009 ) and Johan Rockberg and Johan Nilvebrant ( Volume 1785 , 2018) . Exemplary methods include peptide-based methods such as peptide scanning, in which a series of overlapping peptides are screened for binding using techniques such as ELISA or by in vitro presentation of large libraries of peptide or protein mutants, such as on phage. Detailed epitope information can be determined by structural techniques including X-ray crystallography, solution nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy (cryo-EM). Mutation induction such as alanine scanning is an effective method in which binding loss analysis is used for epitope localization. Another method is hydrogen/deuterium exchange (HDX) combined with proteolysis and liquid chromatography mass spectrometry (LC-MS) analysis to characterize non-continuous or conformational epitopes.

In one aspect, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to human IL-7 at one or more amino acid residues within SEQ ID NO: 1. In one embodiment, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to human IL-7 at one or more amino acid residues within SEQ ID NO: 12. In another embodiment, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided that binds to human IL-7 at one or more amino acid residues within SEQ ID NO: 16.

In another aspect, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided, which protects residues of SEQ ID NO: 12 of IL-7 from deuterium exchange in HDX-MS analysis. In one embodiment, an IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein is provided, which protects residues 67 to 81 (SEQ ID NO: 1) of IL-7 from deuterium exchange in HDX-MS analysis. In one embodiment, the IL-7 binding domain for use according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein binds to a sequence having at least about 50%, 60%, 70%, 80%, 90% or 95% identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein binds to a sequence having at least about 50%, 60%, 70%, 80%, 90% or 95% identity to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein binds to IL-7 at a site adjacent to the IL-7Rα and ɣ-chain interaction site on the folded IL-7 domain. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein exhibits binding specificity to IL-7 at an antigenic determinant comprising at least 5 consecutive amino acids of the sequence of SEQ ID NO: 12. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein exhibits binding specificity to IL-7 at an antigenic determinant comprising at least 5 consecutive amino acids of the sequence of SEQ ID NO: 16. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein exhibits binding specificity to IL-7 at an antigenic determinant comprising at least 10 consecutive amino acids of the sequence of SEQ ID NO: 12. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein exhibits binding specificity to IL-7 at an antigenic determinant comprising at least 10 consecutive amino acids of the sequence of SEQ ID NO: 16.

In some embodiments, the affinity of an IL-7 binding domain (used according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein) for an antigen can be determined by a competitive binding assay. In some embodiments, the competitive binding assay is an immunoassay. In some embodiments, the competitive binding assay is, for example, an ELISA or a radioimmunoassay.

In some embodiments, the reduction or inhibition of biological activity can be partial or complete. Relative to the activity of IL-7 in the absence of the IL-7 binding domain, the neutralizing IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein can neutralize the activity of IL-7 by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Neutralization can be determined or measured using one or more assays known to those skilled in the art or described herein.

In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein targets the membrane-bound IL-7 receptor α (CD127), which forms a heterodimeric receptor (CD132) with a common γ chain when IL-7 binds. In some embodiments, the IL-7 binding domain used according to the uses and methods described herein or as part of the IL-7 and TNF-α binding protein targets the soluble IL-7 receptor α (sCD127).

Competition between an IL-7 binding domain of the invention and a reference IL-7 binding domain (e.g., a reference antibody or IL-7 receptor) can be determined by one or more techniques known to those skilled in the art, such as ELISA, FMAT, surface plasmon resonance (SPR), or ForteBio Octet Bio-Layer Interferometry (BLI). Such techniques may also be referred to as antigenic determinant grouping. There are several possible reasons for this competition: the two domains may bind to the same or overlapping antigenic determinants, there may be steric inhibition of binding, or binding of the first domain may induce a conformational change in the antigen that prevents or reduces binding of the second domain.

The reduction or inhibition of biological activity can be partial or complete. A neutralizing antigen binding domain used according to the uses and methods described herein or as part of an IL-7 and TNF-α binding protein can neutralize the activity of IL-7 by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to the activity of IL-7 in the absence of the antigen binding domain.

Neutralization can be determined or measured using one or more assays known to those skilled in the art or described herein. IL - 7 and TNF - α Binding Protein

In one embodiment, an IL-7 and TNF-α binding protein is provided, comprising: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and/or CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In addition, an IL-7 and TNF-α binding protein is provided, wherein the binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10 and/or CDRL3 of SEQ ID NO: 11; and b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52 and CDRL3 of SEQ ID NO: 53.

In one embodiment, an IL-7 and TNF-α binding protein is provided, wherein the binding protein comprises: a) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 4 and/or a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 5; and/or b) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 54 and/or a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 55.

In another embodiment, an IL-7 and TNF-α binding protein is provided, wherein the binding protein comprises: a) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 4 and/or a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 5; and b) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 54 and/or a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 55.

In another embodiment, an IL-7 binding and TNF-α protein is provided, wherein the binding protein comprises: a) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 5; and b) a VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 54 and a VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 55.

In another embodiment, an IL-7 binding and TNF-α protein is provided, wherein the binding protein comprises: a) a VH domain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 5; and b) a VH domain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 54 and a VL domain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 55.

In another embodiment, an IL-7 binding and TNF-α protein is provided, wherein the binding protein comprises: a) a VH domain having an amino acid sequence of SEQ ID NO: 4 and a VL domain having an amino acid sequence of SEQ ID NO: 5; and b) a VH domain having an amino acid sequence of SEQ ID NO: 54 and a VL domain having an amino acid sequence of SEQ ID NO: 55.

In another aspect, the IL-7 binding and TNF-α protein comprises a bispecific antibody. In one embodiment, the IL-7 and TNF-α binding protein is an IgG-like bispecific antibody format. In another embodiment, the IL-7 and TNF-α binding protein is an IgG-like bispecific antibody format selected from the group consisting of: biantibodies, L-antibodies, common light chain antibodies, antibodies with modified cysteine bridges between the heavy chain and the light chain, chimeric heavy chain/light chain antibodies, Cross-Mab, mAb pairs, and Het-mAb. In one embodiment, the IL-7 and INF-α binding bispecific antibody is a Het-mAb.

As used herein, "Het-mAb" is an IgG-like molecule that can target two different antigenic determinants on the same or different targets, with 4 different chains; 2 heavy chains and 2 light chains. These chains contain a set of mutations in the Fc portion of the molecule to drive heavy chain dimerization, and a set of mutations on the fAb portion to drive correct heavy chain/light chain pairing, thereby forming a bispecific mAb of the κ/κ or λ/κ subtype. Suitable mutations for driving heavy chain dimerization are disclosed in WO2012/058768 and WO2013/063702. In some embodiments, the first heavy chain contains mutations T350V L351Y F405A Y407V and the second heavy chain contains mutations T350V T366L K392L T394W. Suitable mutations for driving HC/LC pairing are disclosed in WO 2014/082179, WO 2015/181805, and WO 2017/059551. In some embodiments, the IL-7 and TNF-α binding protein contains TNF-α light chain, TNF-α heavy chain, and IL-7 light chain and IL-7 heavy chain. In some embodiments, TNF-α and IL-7 heavy chains contain mutations that direct the correct pairing of heavy chains. In some embodiments, the fAb portion of the TNF-α heavy and light chains and the fAb portion of the IL-7 heavy and light chains contain mutations that direct the correct pairing of the heavy and light chains. In other embodiments, the TNF-α and IL-7 heavy chains contain mutations that direct the correct pairing of the heavy chains, and the fAb portion of the TNF-α heavy and light chains and the fAb portion of the IL-7 heavy and light chains contain mutations that direct the correct pairing of the heavy and light chains.

In another embodiment, the IL-7 and TNF-α bispecific antibody further comprises an Fc mutation that disables effector function. In one embodiment, the IL-7 and TNF-α bispecific antibody further comprises a substitution of alanine residues at positions 235 and 237 (EU index number) of the heavy chain constant region, i.e., L235A and G237A.

In one embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and/or a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 or 63; and/or b) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and/or a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 58 or 62.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 or 63; and/or b) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 58 or 62.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 58 or 63; and b) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 58 or 62.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having an amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having an amino acid sequence of SEQ ID NO: 59 or 63; and b) a heavy chain having an amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having an amino acid sequence of SEQ ID NO: 58 or 62.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59; and b) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 58.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 57 and a light chain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 59; and b) a heavy chain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 56 and a light chain having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence of SEQ ID NO: 58.

In another embodiment, the IL-7 and TNF-α binding protein comprises: a) a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59; and b) a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58.

In another embodiment, a bispecific antibody of IL-7 and TNF-α is provided, comprising: an IL-7 binding domain comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and/or CDRL3 of SEQ ID NO: 11; and a TNF-α binding domain comprising CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In another embodiment, the IL-7 and TNF-α bispecific antibody is Het-mAb.

In another embodiment, the IL-7 and TNF-α bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, a bispecific antibody for IL-7 and TNF-α is provided, comprising: an IL-7 binding domain comprising a VH domain having an amino acid sequence of SEQ ID NO: 4 and a VL domain having an amino acid sequence of SEQ ID NO: 5; and a TNF-α binding domain comprising a VH domain having an amino acid sequence of SEQ ID NO: 54 and a VL domain having an amino acid sequence of SEQ ID NO: 55.

In another embodiment, the bispecific antibody is Het-mAb.

In another embodiment, the bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, a bispecific antibody for IL-7 and TNF-α is provided, comprising: an IL-7 binding domain comprising a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59; and a TNF-α binding domain comprising a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58.

In another embodiment, the bispecific antibody is Het-mAb.

In another embodiment, the bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, the bispecific antibody comprises canonical substitutions in the framework region of the IL-7 binding domain.

The "percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity value" expressed as a percentage, which is calculated over the entire length of the query sequence using an appropriate algorithm or software such as BLASTN, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene after a pairwise global sequence alignment has been performed using an appropriate algorithm or software such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign. Importantly, the query nucleic acid sequence may be described by a nucleic acid sequence identified within one or more of the claims herein.

The "percent identity" between a query amino acid sequence and a subject amino acid sequence is an "identity" value expressed as a percentage, which is calculated over the entire length of the query sequence using a suitable algorithm or software such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle or EMBOSS infoalign, after a pairwise global sequence alignment has been performed using a suitable algorithm/software such as BLASTP, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. Importantly, the query amino acid sequence may be described by an amino acid sequence identified within one or more of the claims herein.

The query sequence may be 100% identical to the subject sequence, or it may include up to an integer number of amino acid or nucleotide changes compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the target sequence. Such changes include at least one amino acid deletion, substitution (including conservative and non-conservative substitutions), or insertion, and wherein such changes may occur at the amino-terminal or carboxyl-terminal position of the query sequence or anywhere between those terminal positions, interspersed individually between amino acids or nucleotides in the query sequence, or within one or more contiguous groups in the query sequence.

The % identity can be determined across the entire query sequence length (including CDRs). Alternatively, the % identity can exclude one or more or all of the CDRs, such that all CDRs are 100% identical to the subject sequence, and the % identity variation is in the remainder of the query sequence (e.g., framework sequence) such that the CDR sequences are fixed and intact. In some embodiments, the variant sequence substantially retains the biological properties of the unmodified protein, such as DRSPAI-L7B. Modification

Those skilled in the art will appreciate that post-translational modifications may occur in the production of IL-7 binding domains, TNF-a binding domains, or IL-7 and TNF-a binding proteins (such as antibodies in host cells) used in accordance with the uses and methods described herein. For example, this may include cleavage of certain leader sequences; addition of various sugar moieties in various glycosylation patterns; non-enzymatic glycosylation; deamidation; oxidation; disulfide perturbations and other cysteine variants, such as free sulfhydryls, racemic disulfide bonds, thioethers, and trisulfide bonds; isomerization; C-terminal lysine cleavage; and N-terminal glutamine cyclization. The present invention encompasses the use of IL-7 binding proteins that have undergone or have undergone one or more post-translational modifications. Therefore, the "IL-7 binding domain", "TNF-α binding domain" or "IL-7 and TNF-α binding protein" or "antibody" of the present invention respectively include the "IL-7 binding domain", "TNF-α binding domain" or "IL-7 and TNF-α binding protein" or "antibody" as defined above that have undergone post-translational modifications as described herein.

Glycosylation is a non-enzymatic chemical reaction between reducing sugars (such as glucose) and free amine groups in proteins and is usually observed at the epsilon amine of the lysine side chain or at the N-terminus of the protein. Glycosylation can occur during production and storage only in the presence of reducing sugars.

Deamination can occur during production and storage, and is an enzymatic reaction that converts asparagine (N) to iso-aspartic acid (iso-aspartic acid/iso-aspartate) and aspartic acid (aspartic acid/aspartate) (D) primarily in a ratio of approximately 3:1. Therefore, this deamination reaction is associated with the isomerization of aspartate (D) to iso-aspartate. Both the deamination of asparagine and the isomerization of aspartate involve the intermediate succinimide. To a lesser extent, glutamine residues can be deamidated in a similar manner. Deamination can occur in the CDR, Fab (non-CDR region), or Fc region.

Oxidation can occur during production and storage (i.e., in the presence of oxidizing conditions) and results in covalent modifications of proteins, either directly induced by reactive oxygen species or indirectly by reaction with secondary byproducts of oxidative stress. Oxidation occurs primarily with methionine residues, but can occur at tryptophan and free cysteine residues. Oxidation can occur within the CDR, Fab (non-CDR regions), or Fc regions.

Disulfide bond perturbations can occur during production and under alkaline storage conditions. Under certain circumstances, disulfide bonds can be cleaved or incorrectly formed, resulting in unpaired cysteine residues (-SH). These free (unpaired) sulfhydryl groups (-SH) can promote reorganization.

Thioether formation and disulfide racemization can occur under alkaline conditions during production or storage via beta elimination of the disulfide bridge via dehydroalanine and a persulfide intermediate back to the cysteine residue. Subsequent cross-linking of dehydroalanine and cysteine results in the formation of the thioether bond, or the free cysteine residue can reform the disulfide bond with a mixture of D-cysteine and L-cysteine.

Trisulfides result from the insertion of a sulfur atom into a disulfide bond (Cys-S-S-S-Cys) and are formed due to the presence of hydrogen sulfide in cell cultures.

N-terminal glutamic acid (Q) and glutamine (E) in the heavy and/or light chains may undergo cyclization to form pyroglutamic acid (pGlu). Most pGlu formation occurs in the production bioreactor, but it can be formed non-enzymatically depending on the pH and temperature of the process and storage conditions. Cyclization of N-terminal Q or E is commonly observed in native human antibodies.

C-terminal lysine cleavage is an enzymatic reaction catalyzed by carboxypeptidases and is commonly observed in recombinant and natural human antibodies. A variation of this process includes the removal of lysine from one or both recombinant chains by cellular enzymes from the recombinant host cells. Removal of any remaining C-terminal lysine may result upon administration to human subjects/patients.

The terms "peptide", "polypeptide" and "protein" each refer to a molecule comprising two or more amino acid residues. A peptide can be a monomer or a polymer.

In some embodiments, it may be desirable to modify the effector function of the IL-7 binding domain and/or TNF-α binding domain or IL-7 and TNF-α binding protein used according to the uses and methods described herein, for example, to enhance ADCC or CDC half-life, etc. In some embodiments, it may be desirable to modify the effector function of the IL-7 binding domain and/or TNF-α binding domain or IL-7 and TNF-α binding protein used according to the uses and methods described herein, so that it has a modification that extends the half-life. The in vivo half-life of the IL-7 binding protein in humans or in a murine animal model may be at least 6 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days, or at least 9 days.

Mutational changes in the Fc effector portion of an antibody can be used to alter the affinity of the interaction between FcRn and the antibody to modulate antibody switching. The half-life of the antibody can be extended in vivo. This can benefit the patient population because the maximum dose and maximum dosing frequency can be achieved by maintaining the _IC50 for a longer period of time in vivo. Since killing of those cells expressing CD127 may not be required, the Fc effector function of the antibody can be removed in whole or in part. This removal can increase the safety profile. For example, with respect to IgG1, M252Y/S254T/T256E (commonly referred to as "YTE" mutations) and M428L/N434S (commonly referred to as "LS" mutations) increase FcRn binding at pH 6.0 (Wang et al. 2018).

In some embodiments, the IL-7 binding domain and/or TNF-α binding domain used according to the purposes and methods described herein or the IL-7 and TNF-α binding proteins comprising the constant region may have reduced ADCC and/or complement activation or effector function. The constant region may comprise a naturally disabled constant region of the IgG2 or IgG4 isotype or a mutant IgG1 constant region. In some embodiments, the IL-7 binding domain and/or TNF-α binding domain used according to the purposes and methods described herein or the IL-7 and TNF-α binding proteins of the present invention may disable Fc. Examples of suitable modifications are described in EP0307434. One way to achieve Fc disablement includes replacing alanine residues at positions 235 and 237 (EU index number) of the heavy chain constant region, i.e., L235A and G237A (collectively referred to as "LAGA" mutations). Another example includes alanine substitutions at positions 234 and 235 (EU index number), i.e., L234A and L235A (commonly referred to as "LALA" mutations). In some embodiments, the Fc effector function of the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein disclosed herein used according to the uses and methods described herein has been disabled using LAGA mutations. Alternatively, the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein used according to the uses and methods described herein can be Fc-enabled and does not include alanine substitutions at positions 235 and 237.

Additional changes and mutations that reduce effector function include: (unless otherwise indicated, reference is made to IgG1): glycosylation N297A or N297Q or N297G; L235E; IgG4: F234A/L235A; and chimeric IgG2/IgG4. IgG2: H268Q/V309L/A330S/P331S and IgG2: V234A/G237A/P238S/H268A/V309L/A330S/P331S can reduce FcγR binding to C1q (Wang et al. 2018 and US 8,961,967).

Other mutations that reduce effector function include L234F/L235E/P331S; a chimeric antibody formed using the CH1 and hinge regions from human IgG2 and the CH2 and CH3 regions from human IgG4; IgG2m4, which is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S, and P331S); IgG2σ containing the V234A/G237A/P238S/H268A/V309L/A330S/P331S substitutions to eliminate affinity for Fcγ receptors and C1q complement proteins; IgG2m4 (H268Q/V309L/A330S/P331S, becoming IgG4); IgG4 (S228P/L234A/L235A); huIgG1 L234A/L235A (AA); huIgG4 S228P/L234A/L235A; IgG1σ (L234A/L235A/G237A/P238S/H268A/A330S/P331S); IgG4σ1 (S228P/F234A/L235A/G237A/P238S); and IgG4σ2 (S228P/F234A/L235A/ΔG236/G237A/P238S, where Δ indicates deletion) (Tam et al., Antibodies 2017, 6(3)).

In some embodiments, the IL-7 binding domain and/or TNF-α binding domain used according to the purposes and methods described herein or the IL-7 and TNF-α binding proteins disclosed herein may comprise one or more modifications selected from the mutant constant domains so that the antibody has enhanced effector function/ADCC and/or complement activation. Examples of suitable modifications are described in Shields et al. J. Biol. Chem (2001) 276: 6591-6604, Lazar et al. PNAS (2006) 103: 4005-4010, and US6737056, WO2004063351, and WO2004029207. The IL-7 binding domain and/or TNF-α binding domain or IL-7 and TNF-α binding protein used according to the uses and methods described herein may comprise a constant domain with an altered glycosylation profile, such that the IL-7 binding domain, TNF-α binding domain or IL-7 and TNF-α binding protein has enhanced effector function/ADCC and/or complement activation. Examples of methods suitable for producing IL-7 binding domains, TNF-α binding domains or IL-7 and TNF-α binding proteins used according to the uses and methods described herein with altered glycosylation profiles are described in WO2003/011878, WO2006/014679 and EP1229125. Hosts and vectors

The IL-7 binding domain or TNF-α binding domain or IL-7 and TNF-α binding protein used according to the uses and methods described herein can be prepared by any of a variety of known techniques. For example, the IL-7 binding domain, TNF-α binding domain or IL-7 and TNF-α binding protein used according to the uses and methods described herein can be purified from cells that naturally express it (e.g., antibodies can be purified from fusion tumors that produce it) or produced in a recombinant expression system.

A variety of different expression systems and purification protocols can be used to produce IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins of the invention for use according to the uses and methods described herein. Generally, host cells are transformed with a recombinant expression vector encoding the desired antigen binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein for use according to the uses and methods described herein. Depending on the expression system, the expression vector can be maintained by the host as a separate genetic element or integrated into the host chromosome.

In some embodiments, expression vectors comprising nucleic acid molecules are described herein. A recombinant host cell is also provided, comprising an expression vector as described herein. The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein described herein can be produced in a suitable host cell. A wide range of host cells can be used, including prokaryotes (including Gram-negative or Gram-positive bacteria, such as E. coli, Bacillus, Pseudomonas, Corynebacterium), eukaryotes including yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), fungi (e.g., Aspergillus), or higher eukaryotes, including insect cells and cell strains of mammalian origin. Examples of cell lines include Chinese hamster ovary (CHO) cells, PER.C6, HEK293, HeLa, or NSO. In some embodiments, the host cells described herein are CHO cells, NSO myeloma cells, COS cells, or SP2 cells. The host cell may be a non-human host cell. The host cell may be a non-embryonic host cell. Human cells may be used, thus achieving a modified human glycosylation pattern. Alternatively, other eukaryotic cell lines may be employed. In some embodiments, the selection of suitable mammalian host cells and methods for transformation, culture, expansion, screening, and product production and purification are known in the art. In some embodiments, the host cell is a yeast strain. Host cells can be cultured in a culture medium (e.g., a serum-free medium). The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein can be secreted from the host cells into the culture medium. The IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein can be purified to at least 95% or greater (e.g., 98% or greater) relative to a culture medium containing the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein.

The host cell may be an isolated host cell. The host cell is not usually part of a multicellular organism such as a plant or an animal. The host cell may be a non-human host cell.

Appropriate selection and expression vectors for use with bacterial, fungal, yeast, and mammalian host cells are known in the art.

Methods for producing IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins as described herein may include culturing host cells and recovering the IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins. In one aspect, a method for preparing IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins is provided, the method comprising maintaining host cells in culture medium to produce IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins, and isolating or purifying the IL-7 binding domains, TNF-α binding domains, or IL-7 and TNF-α binding proteins produced by host cells.

The recombinant transformed, transfected or transduced host cell may comprise at least one expression cassette, wherein the expression cassette comprises a polynucleotide encoding a heavy chain of an IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein as described herein and further comprises a polynucleotide encoding a light chain of an IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein as described herein. Alternatively, the recombinant transformed, transfected or transduced host cell may comprise at least one expression cassette, wherein a first expression cassette comprises a polynucleotide encoding a heavy chain of an IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein as described herein, and further comprises a second cassette, the second cassette comprising a polynucleotide encoding a light chain of an IL-7 binding domain, a TNF-α binding domain, or an IL-7 and TNF-α binding protein as described herein. Stably transformed host cells may contain vectors comprising one or more expression cassettes encoding the heavy and/or light chains of the IL-7 binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein or fragments thereof described herein. For example, such host cells may contain a first vector encoding a light chain and a second vector encoding a heavy chain.

The cells can be cultured under conditions that promote expression of the antigen binding domain, TNF-α binding domain, or IL-7 and TNF-α binding protein using a variety of equipment (e.g., shake flasks, roller bottles, and bioreactors). The polypeptide is recovered by known protein purification procedures. Protein purification procedures generally consist of a series of unit operations consisting of various filtration and chromatographic processes developed to selectively concentrate and isolate antigen binding proteins. The purified antigen binding protein can be formulated in a pharmaceutically acceptable composition. PHARMACEUTICAL COMPOSITIONS / ADMINISTRATION ROUTE / DOSAGE

The IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein as described herein can be incorporated into a pharmaceutical composition for the treatment of human diseases described herein. In one embodiment, the pharmaceutical composition comprises a combination of IL-7 binding and TNF-α binding domains and one or more pharmaceutically acceptable carriers and/or excipients. In one embodiment, the first pharmaceutical composition comprises a combination of IL-7 binding domains and one or more pharmaceutically acceptable carriers and/or excipients and the second pharmaceutical composition comprises a combination of TNF-α binding domains and one or more pharmaceutically acceptable carriers and/or excipients. In one embodiment, the pharmaceutical composition comprises a combination of IL-7 and TNF-α binding protein and one or more pharmaceutically acceptable carriers and/or excipients.

Such compositions contain pharmaceutically acceptable carriers, as known and required by accepted medical practice.

The pharmaceutical composition of the present invention can be used for therapeutic or prophylactic applications. In one embodiment, a pharmaceutical composition comprising 1-500 mg of the IL-7 binding domain disclosed herein and 1-500 mg of the TNF-α binding domain disclosed herein is provided. In another embodiment, a pharmaceutical composition comprising 1-500 mg of the IL-7 and TNF-α binding protein disclosed herein is provided. In another embodiment, a pharmaceutical composition is provided herein, which comprises 1-500 mg of IL-7 and TNF-α binding protein, which is an antibody comprising the following: a) a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59 and b) a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58.

The IL-7 binding domain and TNF-α binding domain used according to the uses and methods of the present invention can be presented as independent pharmaceutical compositions or as a single pharmaceutical composition. In one embodiment, the IL-7 binding domain and TNF-α binding domain used according to the uses and methods of the present invention can be presented as independent pharmaceutical compositions. In another embodiment, the IL-7 binding domain and TNF-α binding domain used according to the uses and methods of the present invention can be presented as a single pharmaceutical composition.

In some embodiments, when presenting a pharmaceutical formulation, the therapeutic agent of the present invention (the IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein used according to the purposes and methods of the present invention) is presented in a unit dosage form. In some embodiments, the dosing regimen will be determined by medical professionals and/or clinical factors. As is well known in the medical art, the dosage for any one patient depends on many factors, including the patient's body size, body surface area, age, compound to be administered, sex, administration time and route, general health, and other drugs to be administered simultaneously. Exemplary dosages may vary according to the size and health of the individual being treated and the condition being treated. In some embodiments, when the IL-7 binding domain and the TN-α binding domain are in separate proteins, the dosage is determined separately and independently. For example, in some embodiments, the disclosed antibodies or functional fragments can be administered at a dosage of 1 to 100 mg/kg. In some embodiments, the pharmaceutical compositions disclosed herein are administered multiple times at such dosages. In some embodiments, the dosage is administered once or multiple times, for example, daily, weekly, biweekly or monthly, hourly, or at recurrence, relapse or progression of the disease or condition being treated. In some embodiments, when the IL-7 binding domain and the TN-α binding domain are in separate proteins, they are administered at the same treatment time interval. In some embodiments, when the IL-7 binding domain and the TN-α binding domain are in separate proteins, they are administered at different treatment time intervals.

In another embodiment, a pharmaceutical composition comprising a bispecific antibody for IL-7 and TNF-α is provided, wherein the bispecific antibody comprises: an IL-7 binding domain comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and/or CDRL3 of SEQ ID NO: 11; and a TNF-α binding domain comprising CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53.

In another embodiment, the IL-7 and TNF-α bispecific antibody is Het-mAb.

In another embodiment, the IL-7 and TNF-α bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, a pharmaceutical composition comprising a bispecific antibody for IL-7 and TNF-α is provided, wherein the bispecific antibody comprises: an IL-7 binding domain comprising a VH domain having an amino acid sequence of SEQ ID NO: 4 and a VL domain having an amino acid sequence of SEQ ID NO: 5; and a TNF-α binding domain comprising a VH domain having an amino acid sequence of SEQ ID NO: 54 and a VL domain having an amino acid sequence of SEQ ID NO: 55.

In another embodiment, the IL-7 and TNF-α bispecific antibody is Het-mAb.

In another embodiment, the IL-7 and TNF-α bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, a pharmaceutical composition comprising a bispecific antibody for IL-7 and TNF-α is provided, wherein the bispecific antibody comprises an IL-7 binding domain comprising a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59; and a TNF-α binding domain comprising a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58.

In another embodiment, the IL-7 and TNF-α bispecific antibody is Het-mAb.

In another embodiment, the IL-7 and TNF-α bispecific antibody comprises canonical substitutions in the CDRs of the IL-7 binding domain.

In another embodiment, the IL-7 and TNF-α bispecific antibody comprises canonical substitutions in the framework region of the IL-7 binding domain.

In some embodiments, the pharmaceutical composition comprises a composition for parenteral, percutaneous, intraluminal, intraarterial, intrathecal and/or intranasal administration or by direct injection into a tissue. In some embodiments, when the IL-7 binding domain and the TN-α binding domain are in separate proteins, they are administered in the same pharmaceutical composition. In some embodiments, when the IL-7 binding domain and the TN-α binding domain are in separate proteins, they are administered in different pharmaceutical compositions. When administered separately, this can be performed simultaneously or sequentially in any order (by the same or by different administration routes). Such sequential administration can be close in time or remote in time. The dosages and relative times of administration of the IL-7 binding domain and TNF-α binding domain or pharmaceutical compositions thereof and other therapeutically active agents will be selected to achieve the desired combined therapeutic effect.

In some embodiments, the pharmaceutical composition is administered to a patient via infusion or injection. In one embodiment, a pharmaceutical composition for intravenous administration is provided, comprising an IL-7 binding domain and a TNF-α binding domain or an IL-7 and TNF-α binding protein. In one embodiment, a pharmaceutical composition for subcutaneous administration is provided, comprising an IL-7 binding domain and a TNF-α binding domain or an IL-7 and TNF-α binding protein. In some embodiments, the pharmaceutical composition described herein is administered to an individual intra-arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, by intravenous (i.v.) infusion, or intraperitoneally. In some embodiments, the IL-7 binding domain and the TNF-α binding domain, or the IL-7 and TNF-α binding proteins or pharmaceutical compositions thereof are administered to a subject by intradermal or subcutaneous injection.

In some embodiments, the pharmaceutical composition is prepared by a method known per se for preparing a pharmaceutically acceptable composition for administration to an individual, so that an effective amount of IL-7 binding domain and TNF-α binding domain or IL-7 and TNF-α binding protein is combined with a pharmaceutically acceptable carrier in the form of a mixture. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, Easton, Pa., USA, 2000). On this basis, the composition may include (but not exclusively) a solution of a substance, which is combined with one or more pharmaceutically acceptable carriers or diluents and contained in a buffer solution having a suitable pH and isotonic with a physiological fluid.

The pharmaceutical compositions disclosed herein can be formulated into a variety of forms and administered by a variety of different means. The pharmaceutical formulations can be administered orally, rectally or parenterally as needed in the form of formulations containing conventionally acceptable carriers, adjuvants and vehicles. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular or intrasternal injection and infusion techniques. Administration includes injection or infusion, including intraarterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalation, transdermal, transmucosal, sublingual, intrabuccal and topical (including epidermal, dermal, enema, eye drops, ear drops, intranasal, vaginal) administration. In some exemplary embodiments, the route of administration is via injection, such as intramuscular, intravenous, subcutaneous or intraperitoneal injection.

Liquid formulations may include oral formulations, intravenous formulations, intranasal formulations, ophthalmic formulations, ocular formulations, aerosols, and the like. In certain embodiments, a combination of formulations is administered. In certain embodiments, the composition is formulated for an extended release profile.

The pharmaceutical composition of the present invention can be administered with other therapeutic agents or therapeutic combinations. In some embodiments, the treatment for an individual can be surgery, a nutritional regimen, physical activity, immunotherapy, a pharmaceutical composition, cell transplantation, blood fusion, or any combination thereof.

In some embodiments, an IL-7 binding domain and a TNF-α binding domain, or an IL-7 and TNF-α binding protein, "retains its biological activity" in a pharmaceutical formulation if the biological activity at a given time is within about 10% (within the assay error) of the biological activity exhibited when the pharmaceutical formulation is prepared, as determined, for example, in an antigen binding assay.

In some embodiments, the compositions disclosed herein may further comprise a chemotherapeutic agent, a cytotoxic agent, an interleukin, a growth inhibitor, an anti-hormone agent and/or a cardioprotectant. Such molecules are suitably present in the composition in an amount effective for the intended purpose.

A kit is further provided, which comprises a pharmaceutical composition and instructions for use. For convenience, the kit may comprise a reagent in a predetermined amount and the instructions for use.

In some embodiments, the present invention discloses a kit comprising an IL-7 binding domain and a TNF-α binding domain or an IL-7 and TNF-α binding protein disclosed herein. In some embodiments, the kit may be a diagnostic kit. In some embodiments, the kit comprises an IL-7 binding domain and a TNF-α binding domain or an IL-7 and TNF-α binding protein disclosed herein, and instructions for use. In some embodiments, the kit comprises components and instructions for use for measuring the IL-7 content in a sample. In some embodiments, the kit comprises components and instructions for use for measuring the TNF-α content in a sample. The kit may provide a unit or device (e.g., a device having a needle coupled to an aspirator) for obtaining a sample from an individual. The kit may include a plurality of syringes, ampoules, foil packets, or blister packages each containing a single unit dose of the kit components described herein. The container of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation) and/or opaque. The kit may include a device suitable for administering the components, such as a syringe, an inhaler, a dropper, a tweezer, a measuring spoon, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden swab), or any such delivery device. In some embodiments, the device may be, for example, a medical implant device packaged for surgical insertion. The kit disclosed herein may include one or more reagents or tools that enable the method to be performed. In some embodiments, the reagent or instrument includes one or more of the following: a suitable buffer (aqueous solution) a support having holes on which a quantitative reaction can be performed. The kit may be a specific kit for a specific tissue sample. In addition, the kit disclosed herein may include a control.

In addition to the above components, instructions for use may also be provided in the kit. Such instructions may be present in the kit in various forms, such as printed information on a suitable medium or substrate (e.g., one or more pieces of paper with information printed thereon), in the form of a package of the kit, in the form of a package insert, etc. In some embodiments, the instructions for use may be provided on a computer-readable medium (e.g., a jump/thumb drive, a CD, etc.) on which information has been recorded, or provided at a URL that can be used via the Internet for accessing information at a website. Device

Another aspect of the invention provides a prefilled syringe or autoinjector device comprising an IL-7 binding domain and a TNF-α binding domain as described herein, or an IL-7 and TNF-α binding protein or composition. In some embodiments, a composition stored in a container, a prefilled syringe, a syringe, or an autoinjector device contains an IL-7 binding domain disclosed herein. In some embodiments, a composition stored in a container, a prefilled syringe, a syringe, or an autoinjector device contains a TNF-α binding protein disclosed herein. In some embodiments, a composition stored in a container, a prefilled syringe, a syringe, or an autoinjector device contains an IL-7 and TNF-α binding protein disclosed herein. Examples Example 1 : DRSPAI-L7B in cynomolgus macaques

A study was described to evaluate DRSPAI-L7B in cynomolgus macaques. In Part 1, three single dose IV injections at 0.1, 1, and 10 mg/kg were evaluated. In Part 2, 30 mg/kg was repeat dosed (four SC injections) in vehicle-controlled animals from Part 1. Blood samples were collected for evaluation of drug PK as well as free and total IL-7 levels and pharmacodynamic activity of DRSPAI-L7B via measurements of STAT5 phosphorylation. Total antibody levels

The total antibody content was determined on GYROLAB using a universal antigen capture and detection method. The maximum detectable peak serum concentration (C _max ) and its observation time (T _max ) were determined using the data obtained from the assay. In addition, a non-compartmental PK model was used to calculate AUC, total serum elimination rate (CL); steady-state distribution volume (Vss ) and terminal half-life (t½). (Table 3) Table 3 : Average PK parameters of DRSPAI-L7B (IV administration ) for single- ascending dose part 1 and repeated dose part 2 Part 1 Dosage (mg/kg) C _max (ug/mL) AUC _inf (hr*ug/mL) Half-life (hr) CL (mL/hr/Kg) V _ss (mL/Kg) 0.1 3.12 (2.80 -3.55) 721 ( 710 - 733 ) 350 (270 - 410) 0.14 (0.14 - 0.14) 82 (71 - 90) 1.0 37.2 (27.8 - 48.4) 8250 (7520 - 9060) 470 (350 - 560) 0.12 (0.11 - 0.13) 71 (58 - 80) 10.0 307 (262 -337) 93100 (76800 - 112000) 420 (340 - 490) 0.11 (0.09 - 0.13) 66 (58 - 73) Portion 2 (30 mg/kg) Period C _max (ug/mL) AUC _i0-168 (hr*ug/mL) First dose 320 (258-398) 41600 (32700-47300) Second dose (168h after administration) 463 (407-516) N/A The third dose (168 hours after administration) 556 (490-602) N/A Fourth dose 911 (688-1270) 119000 (94100-169000)

No target-mediated drug disposition (TMDD) was observed, and the increase in serum Cmax and AUC was dose-proportional with increasing doses from 0.1 mg/kg to 10 mg/kg. DRSPAI-L7B was slowly cleared with a mean half-life of approximately 17 ± 3.9 days, and drug accumulation was 2.8-fold with repeated dosing. The data are presented in Figures 1A, 1B, and 1C.

The presence of anti-drug antibodies (ADA) was assessed using an acid dissociation bridging assay with capture using DRSPAI-L7B. No ADA was detected during Part 1 or Part 2 of the study.

A further study was conducted to determine the PK and PD of DRSPAI-L7B in stone crab macaques following subcutaneous (SC) administration. Four single-dose IV injections at 0.1, 1, 3, and 10 mg/kg were evaluated. These doses were administered by SC injection on day 1.

Systemic exposure of DRSPAI-L7B was determined by calculating the area under the serum concentration-time curve (AUC) from the start of dosing to the last quantifiable time point (AUC _0-t ) using the linear upper/log lower trapezoidal method. The maximum observed peak serum concentration (Cmax) and its observation time (Tmax) were determined by PK SUBMIT . Table 4 : Serum PK parameters of DRSPAI-L7B (SC administration ) Parameters Dosage of DRSPAI-L7B (mg/kg) 0.1 1 3 10 AUC _0-t (µg.h/ml) average value 943 6960 18900 60200 Min 687 3870 17100 52700 Max 1120 9130 21300 69700 C _max (µg/ml) average value 2.01 18.1 34.2 119 Min 1.76 16.9 29.5 108 Max 2.21 19.0 39.1 128 _Tmax (h) average value 168 96 96 96 Min 96 48 96 96 Max 168 96 96 168

At the highest dose of 10 mg/kg, the sex-averaged mean Cmax was 119 µg/mL (range 108 to 128 µg/mL) and the mean AUC _{0 - t} was 60 200 µg.h/mL (range 52 700 to 69 700 µg.h/mL).

At all doses, there was a dose-dependent increase in total IL-7 levels, indicating target engagement of DRSPAI-L7B. There was also a dose-dependent inhibition of IL-7-induced STAT5 phosphorylation in total Th and Tc lymphocytes at ≥1 mg/kg and a dose-dependent reduction in Bcl-2 expression in Th lymphocytes at ≥3 mg/kg.

Low levels of anti-DRSPAI-L7B antibodies were detected in two of three monkeys given 1.0 mg/kg. In one male, this resulted in reduced target binding and a lower AUC. Example 2 : Binding affinity of DRSPAI - L7B to IL - 7

The kinetics and affinity of DRSPAI-L7B binding to human and cynomolgus macaque IL-7 were assessed by surface plasmon resonance (SPR) at 25°C and 37°C using a Biacore 8K instrument. The affinity of DRSPAI-L7B for human IL-7 was approximately 34 pM at 25°C and approximately 67 pM at 37°C (Table 5). The affinity of DRSPAI-L7B for cynomolgus macaque IL-7 was approximately 53 pM at 25°C and approximately 75 pM at 37°C (Table 5). Table 5 : Binding kinetics and affinity of DRSPAI-L7B for human and cynomolgus macaque IL -7 DRSPAI -L7B at 25 °C Analyte Average ka (1/Ms) Average kd (1/s) Average KD (pM) SD (KD, pM) hIL-7 6.49E+06 2.06E-04 31 5 hIL-7 4.65E+06 1.58E-04 34 7 cynoIL-7 3.17E+06 1.70E-04 53 6 DRSPAI -L7B at 37 °C Analyte Average ka (1/Ms) Average kd (1/s) Average KD (pM) SD (KD, pM) hIL-7 8.28E+06 5.68E-04 69 5 hIL-7 1.47E+07 6.78E-04 46 8 hIL-7 1.10E+07 8.26E-04 75 12 hIL-7 5.90E+06 5.15E-04 87 3 hIL-7 : Geometric mean KD (pM)* 67.4 (58.4 , 76.3) cynoIL-7 6.17E+06 4.62E-04 75 5 *Geometric mean KD (pM, confidence interval 85%) from all experiments Example 3 : Inhibition of IL - 7 signaling - functional analysis

All human samples were obtained with informed consent from patients in accordance with ICH GCP based on protocols approved by national, regional or site ethics committees or institutional review boards (IRBs). Disease PBMCs were supplied by approved external human tissue suppliers. PBMCs were stored frozen at -80°C until use.

Blood from healthy volunteers was provided by a blood donor: blood was drawn by venous puncture and transferred to a can or blood bag containing sodium heparin (1 U/mL). For whole blood analysis or PBMC isolation, blood was collected and used within 1 hour. Different donors were used for each experiment. Cells were thawed by removing from -80°C storage and immediately placed in a water bath at 37°C. After transferring the cell suspension to a 15 mL centrifuge tube, warm medium (RPMI + 10% heat-inactivated FCS, 1% penicillin/streptomycin and 1% GlutaMax) was added very slowly to gradually reduce the DMSO concentration. Once the volume has increased to 10 mL, cells are centrifuged and washed again before being counted and resuspended in an appropriate volume of assay medium to give 5 × 10 ⁶ cells per 1 mL.

All antibodies arrived aliquoted for long-term storage at -80°C. For experiments, antibody aliquots were thawed and stored at 4°C for no longer than 8 weeks. Reagent Suppliers AIM V Medium Gibco Recombinant human IL-7 R&D Systems BD Phosflow™ Lysis/Fixation Buffer 5× BD Bioscience BD Phosflow™ Osmotic Buffer III BD Biosciences Flow Cytometry Staining Buffer eBioscience FcR blocking reagent Miltenyi Biotec BD Phosflow™ PE Mouse Anti-STAT5 (pY694) BD Bioscience Reagent Suppliers Mouse anti-human CD8 FITC (Clone: SK1) Biolegend Mouse anti-human CD4 PerCP/Cy5.5 (Clone: RPA-T4) Biolegend Mouse anti-human CD3 BV510 (strain: SK7) Biolegend Anti-mouse Ig, κ/Negative control compensatory particles set BD Bioscience BD FACSDiva CS&T Research Beads BD Bioscience

All antibody treatments and rhIL-7 stimulation were made up in culture medium at 4× final assay concentration (F.A.C) before mixing 1:1 and incubating for 5 minutes at room temperature. Culture medium was added in the absence of antibody or IL-7 stimulation.

For whole blood: aliquot 100 μL of the antibody:IL-7 mixture into FACS tubes, followed by the addition of 100 μL of whole blood. Mix tubes gently by vortexing and incubate at 37°C in a humidified incubator for 20 minutes. At the end of the stimulation period, add 2.5 mL of pre-warmed PHOSFLOW lysis buffer (1×) and incubate samples at 37°C for an additional 10 minutes. Add 2 mL of PBS to the suspension and centrifuge the tubes to pellet the cells (300×g, 5 minutes at room temperature). Discard the supernatant and wash the cells twice more in PBS. After the final wash, resuspend the cells in 500 μL of Permeabilization Buffer III (pre-chilled to -20°C) and vortex to mix. Incubate the tubes on ice for 30 minutes, then wash once in 3 mL of PBS. Resuspend the cells in 100 μL of PBS and transfer to a 96-well round-bottom plate for staining.

For PBMC treatment: 50 μL of the antibody:IL-7 mixture was added to each well of a 96-well round-bottom tissue culture plate. 50 μL of PBMC suspension was added to each treatment well (2.5×10 ⁵ cells/well). The plate was gently mixed on a rotating plate shaker and then incubated at 37°C in a humidified incubator for 20 minutes. At the end of the stimulation period, 250 μL of pre-warmed PHOSFLOW lysis buffer (1×) was added and the samples were incubated for an additional 10 minutes at 37°C. After fixation, cells were pelleted by centrifugation (300×g, 5 minutes at room temperature) and washed twice in 200 μL PBS. The cell pellet was resuspended in 100 μL of Permeabilization Buffer III (pre-chilled to -20°C) and pipetted up and down to mix. The cells were incubated on ice for 30 minutes, then washed once in 200 μL of PBS and resuspended in PBS.

For all samples: After permeabilization, the plates were centrifuged (300 × g, 5 min at room temperature) and the cell pellets were resuspended in 25 μL FcR blocking reagent diluted 1:5 in PBS containing 3% BSA. The cells were incubated for 10 min at room temperature and then 2.5 μL anti-CD3 (BV510), 2.5 μL anti-CD4 (PerCP/Cy5.5), 2.5 anti-CD8 (FITC), 7.5 μL anti-pSTAT5 (PE) and 35 μL flow cytometry staining buffer were added (total 50 μL/sample). In the case of exclusion staining for the control group, an equivalent volume of staining buffer was added instead. The plate was mixed briefly on a rotating plate shaker and incubated on ice for 30 minutes in the dark. Cells were washed in 200 μL staining buffer and resuspended in 200 μL staining buffer for analysis on a FACS Canto II the same day.

Check the performance of the instrument using the Cell Counter Set and Tracking (CST) Beads. This is a QC check of the instrument, setting the baseline and optimizing the voltages of each laser before use. The results of the calibration are stored in the CST software on the instrument.

Compensation of the instrument was performed according to the manufacturer's instructions using anti-mouse IgG, kappa/negative control compensation beads. The relevant antibodies used to stain cells during the experiment were used to label the appropriate compensation bead type. Compensation of the experiments was performed using the auto-compensation facility available within the FACS Diva software using the appropriately labeled beads. After analysis of the compensated samples, the appropriate compensation settings were calculated and applied to each experimental staining panel. pSTAT5 FACS analysis: Cells were collected using FSC-A and FSC-H curves to exclude doublets. Single cells were collected using FSC-A and SSC-A curves and gated around live lymphocytes. These single cells were harvested into AmCyan and FSC-A curves and gated around the CD3+ population. PerCP-Cy5.5 and FITC curves were generated on CD3 ⁺ to identify CD4 ⁺ and CD8 ⁺ populations. For pSTAT5, histograms of PE fluorescence were generated for each subset. Positive gates were set based on unstimulated samples and the PE positivity statistical percentage was used for data analysis.

Data were analyzed using FlowJo software (version 10) and results were generated in Microsoft Excel (2010) spreadsheet format using the batch analysis facility within FlowJo software. Cell populations were tabulated in Excel spreadsheets as % Parental. This % Parental was converted to percent response by normalizing to unstimulated controls or to percent inhibition by normalizing to unstimulated controls and subtracting this value from 100 (theoretical maximal percent response).

Where concentration response curves were generated, individual donor data were fit with nonlinear logistic curve fitting regression and the average (mean and median), range, SD and SEM of _IC50 from all donors were calculated in GraphPad Prism (version 6). Where statistical testing was performed, two-way analysis of variance with Sidak's correction for multiple comparisons was applied. p values < 0.05 were considered statistically significant. * indicates p <0.05; ** p ≤ 0.01; *** p ≤ 0.001; and **** p ≤ 0.0001.

In whole blood analysis, STAT5 phosphorylation was assessed by flow cytometry in CD4 ⁺ T cells after 20 minutes of stimulation with 1 ng/ml recombinant human IL-7 (rhIL-7; 58 pM). As shown in Figure 2A and Table 6, DRSPAI-L7B prevented IL-7 signaling through STAT5 in a concentration-dependent manner, with a median IC ₅₀ of 34 pM (5.1 ng/mL). A1290 prevented IL-7 signaling through STAT5 in a concentration-dependent manner, with a median IC ₅₀ of 18 pM (0.00275 µg/ml). A1291 prevented IL-7 signaling through STAT5 in a concentration-dependent manner, with a median IC ₅₀ of 16 pM (0.00246 µg/ml). A1294 prevents IL-7 signaling through STAT5 in a concentration-dependent manner with a median IC ₅₀ of 76 pM (0.0114 µg/ml). Immortalized T lymphoblastoid CCRF-CEM cells were stimulated with 34 pg/mL (2 pM) recombinant human IL-7 (rhIL-7) in the presence or absence of DRSPAI-L7B. STAT5 phosphorylation was assessed by MSD on cell lysates. DRSPAI-L7B potently blocked IL-7-induced pSTAT5 (Table 6; IC ₅₀ <1 pM). Table 6. Summary of the efficacy of DRSPAI-L7B in functional assays analyze IL-7 DRSPAI-L7B IC ₅₀ ng/ml pM ng/ml pM pSTAT5 in CCRF-CEM cells 0.034 2 <0.15 ＜1 pSTAT5 in whole blood 1 58.8 5.1 34 T _eff and T _mem proliferation (CD4 ⁺ ) ^§ 20 1,176 78 520 T _eff and T _mem proliferation (CD4 ⁺ ) ^§ 10 588 79.5 530 IFN-γ production by PBMC 10 588 29.4 195.8 IL-17 production in T _mem 20 1,176 40.5 270 ^§Analyses were performed using EC80 stimulation, where IL-7 -induced STAT5 phosphorylation in healthy and diseased T cells was measured for each cell batch

PBMCs from healthy donors or IBD patients (two Crohn's disease and one ulcerative colitis) were stimulated with rhIL-7 in the presence of DRSPAI-L7B or anti-RSV antibody (isotype control). Stimulated cells were fixed and STAT5 phosphorylation in CD8 ⁺ (Figure 2B), CD4 ⁺ (Figure 2C) and CD3 ⁺ (Figure 2D) T cells was assessed by flow cytometry. Data are shown as pSTAT5 increase relative to unstimulated conditions, mean±SD, n=3. * p<0.05, ** p≤0.01, *** p≤0.001, two-way ANOVA was matched with Sidak's multiple comparison correction. Figure 2B, Figure 2C and Figure 2D. Example 4 : Dynamic light scattering analysis

Dynamic light scattering (DLS) analysis was performed on DRSPAI-L7B, A1290, A1291, and A1294 to characterize higher-order aggregated species and sample heterogeneity.

Each of the individual antibodies was concentrated to ≥10 mg/ml and the buffer was changed by dialysis into either 50 mM sodium phosphate pH 7.5 or 50 mM sodium acetate pH 5.0 buffer followed by standardization to 10 mg/ml using a 0.22 µm syringe filter. After filtration, all samples were stressed at 40°C for 2 weeks at a target concentration of 10 mg/ml. Samples were evaluated after incubation in both buffers under stressed and unstressed conditions. 100 µl samples were run in triplicate at 25°C on a Wyatt DynaPro DLS plate reader using 96-well Corning Costar 3635 plates sealed with Corning 6575 seals.

Data were analyzed using DYNAMICS v7.1.9 software. The data were distributed into the following peaks: Peak 1 = 0.1-1 nm, Peak 2 = 1-10 nm, Peak 3 = 10-100 nm, Peak 4 = 100-1000 nm, Peak 5 = 1000-10000 nm. Data were filtered using the following criteria: Amplitude must be between 0 and 1, Baseline limit 1±0.01, and Sum of all squares (SOS) must be less than 100. Rh >8 nm and mean mass % at Peak 2 <98% are associated with severe aggregation risk.

A1290, A1291 and A1294 exhibited a severe aggregation risk, all of which exhibited hydrodynamic radius (Rh) values of >8 nm. DRSPAI -L7B had an Rh value of <8 nm and thus did not present this aggregation risk. γc Shared Gamma Chain BV510 Brilliant Violet 510 CD(X) Cluster of differentiation (X) CHO Chinese Hamster Ovarian Cells CST Cell Counter Equipment and Tracking DMSO Dimethyl sulfoxide FAC Final analysis concentration FACS Fluorescence Activated Cell Sorting FqV Fc receptor FCS Fetal bovine serum FITC Fluorescence isothiocyanate FSC-(A/H/W) Forward scatter - (area/height/width) HEK Human embryonic kidney cells IBD Inflammatory bowel disease IC ₅₀ Inhibit concentration 50% IL-7 Interleukin 7 IL-7Rα Interleukin 7 receptor alpha JAK Janus kinase (e)LNB (Electronic) Laboratory laptop mAbs Monoclonal antibody PBMCs Peripheral blood mononuclear cells PBS Phosphate Buffered Saline PE Phycoerythrin PerCP/Cy5.5 Peridin Chlorophyll Protein Complex/Cyanine 5.5 SD Standard Deviation SEM Standard error of the mean SSC-(A/H/W) Side scattering - (area/height/width) pSTAT5 Phosphorylation of STAT5 rhIL-7 Recombinant human IL-7 RPMI Roswell Park Memorial Institute medium STAT5 Signal transducer and activator of transcription 5 Example 5 : Effect of DRSPAI-L7B on interleukin production in human PBMCs

DRSPAI-L7B was used to interrogate the role of IL-7 in Th1 and Th17 function and differentiation. The effect of DRSPAI-L7B on interleukin secretion from healthy PBMCs stimulated with IL-7 in the presence of CD3 agonist antibodies was assessed.

Blood from healthy volunteers was provided by a blood donor: blood was drawn by venous puncture and transferred to a can or blood bag containing sodium heparin (1U/mL). Blood was collected and used for PBMC isolation. The blood was diluted 2× with PBS, layered onto an Accuspin catheter containing 15 mL of polysucrose, and centrifuged at 800 rcf for 20 minutes without interruption. The plasma was carefully removed with a dropper, and the layer containing PBMCs was carefully transferred to a 50 mL test tube. PBMCs were washed twice in 50 mL PBS (250 rcf, 10 minutes), and then resuspended in 50 mL RPMI+10% FCS+L-glutamine and counted using Vi-cell XR.

Resuspend PBMCs in medium at 5×10 ⁶ cells/mL. Prepare antibody dilutions of anti-RSV antibody (isotype control) and DRSPAI-L7B at 2.5 times the desired final concentration in medium. Mix equal volumes of cell suspension and antibody dilution (150 μL) and incubate at room temperature for 30 min. Prepare IL-7 at 5 times the final concentration in medium and add 20 μL to the desired wells of a 96-well U-bottom polystyrene plate that has been pre-coated with 10 μg/mL anti-CD3 overnight at 4°C. Add medium to all remaining wells. 80 μL of mixed cell suspension/antibody dilution (2×106 cells/well) was added to each well of a 96-well plate and incubated at 37°C, 5% CO2 for 48 hours.

After 48 hours, the plate was centrifuged at 300 rcf for 5 minutes and the supernatant was removed without disturbing the pellet and transferred to a new 96-well U-bottom plate. The supernatant was used for ELISA/MSD immediately after capture or stored at -80°C until further use.

Anti-RSV IgG1 is available at a stock concentration of 2.8 mg/mL. On the day of use, thaw and dilute to an appropriate concentration. DRSPAI-L7B is available at a stock concentration of 11.33 mg/mL. On the day of use, thaw and dilute to an appropriate concentration. Recombinant human IL-7 was purchased from R&D systems. Resuspend the freeze-dried protein to 25µg/mL in sterile PBS + 0.1% BSA and store 50μL aliquots at -20°C. On the day of use, thaw and dilute to an appropriate concentration. Anti-CD3 clone HIT3a. The stock solution is 1 mg/mL. Dilute to an appropriate concentration on the day of use. Other Materials Reagent company RPMI Gibco FCS Interior L-Glutamine Interior 96-well flat bottom TC treated polystyrene plate Costar 96-well U-bottom TC-treated polystyrene plate Greiner DPBS Gibco Aluminum Foil Cover Beckman Coulter Disc seals Greiner bio-one U-plex Biomarker Panel 1 (Human) Mesoscale Discovery

ECL signals were derived from the MSD instrument and converted to concentrations using standard curves for each analyte. For data normalized curves, the mean of anti-CD3 ⁺ IL-7 samples was set to 100% and all other values were normalized to that mean. Antibody concentrations were logarithmically transformed and plotted against interleukin concentrations. IL-7 (anti-CD3 only) samples are not shown in each curve for comparison. For curve fitting, the following nonlinear fit from Graphpad Prism was used: log (inhibitor) vs. response (three parameters), and IC ₅₀ values were calculated by Graphpad Prism.

DRSPAI-L7B inhibited IFN-γ (Figure 3A) and IL-10 (Figure 3B) secretion in a concentration-dependent manner (IFN-γ average _IC50 = 195.8 ± 101 pM; IL-10 average _IC50 = 207.2 ± 86 pM). Data represent the mean of n = 6 independent donors ± SEM evaluated in 3 independent experiments. Figure 3C, Figure 3D, Figure 3E, Figure 3F, Figure 3G show the inhibition of IL-2 by DRSPAI-L7B in the presence of rhIL-7 and anti-CD3. After IL-7 stimulation in 5 donors, IL-2 production increased. This increase was completely inhibited by DRSPAI-L7B. Example 6 : DRSPAI-L7B inhibits interleukin production by memory T cells

To accurately determine the role of IL-7 in Th17 cell function and differentiation, the "poised Th17" assay was used to analyze the secretion of Th17-related interleukins. T _mem cells were isolated from healthy donors and cultured with IL-7 in the presence of DRSPAI-L7B.

Collect human whole blood from donors in a blood donation unit (BDU). Typically, 200 mL is collected from each donor by venous puncture, and the anticoagulant citrate-dextrose solution (ACD, Sigma, catalog number C3821) is immediately added to each sample. Add ACD at 15% (e.g., 30 mL ACD is added to 200 mL blood). Within 2 hours of collection, and using a microbiological safety cabinet, evenly distribute the blood into pre-filled LEUCOSEP tubes (Greiner, catalog number 227288) at 30 mL (max) per tube. Centrifuge the blood at 800×g for 15 minutes at room temperature in a swing bucket rotor without a brake applied. PBMCs were washed in PBS (500×g, 10 min) and then resuspended in 10 mL PBS and counted using a NucleoCounter. Cells were centrifuged again at 500×g and the cell pellets were resuspended in FACS buffer at a concentration of 5×10^7 cells/mL.

Human CD4 ⁺ memory T cells (T _mem ) EasySep enrichment mix was added to the cell suspension at 50 µL/mL. Cells were incubated for 10 minutes at room temperature, after which EasySep magnetic particles were added at 50 µL/mL. The suspension was again incubated for 10 minutes at room temperature, after which the cell suspension was placed in the EasySep magnet for 10 minutes. The cell suspension was then transferred to a fresh tube while still in the magnet to ensure negative selection. Cells were centrifuged at 500 × g for 10 minutes and resuspended in cell culture medium. Cell counts were determined using a NucleoCounter.

The cells were allowed to stand overnight in culture medium at 37°C and 5% CO _2. The next day, the cells were centrifuged and resuspended in assay medium at a concentration of 2.5×10^5 cells/mL. Recombinant hIL-7 was added to the cells at a final concentration of 20 ng/mL. The cells were then cultured in the presence of DRSPAI-L7B at 37°C and 5% CO ₂ for 4 days, after which they were stimulated with 10 nM PMA and 1 µM ionomycin for 16 hours at 37°C and 5% CO _2. IL-17 from the supernatant was detected using MSD

After 16 hours of PMA and ionomycin stimulation, cells were centrifuged and 40 µL of supernatant was transferred to MSD plates pre-blocked with 0.5% Blocker B. The MSD plates were coated with anti-IL-17A capture antibody. The samples were incubated for 2 hours at room temperature with shaking, after which the plates were washed with PBS and 0.05% Tween-20. 10 µL of IL-17 specific detection antibody labeled with MSD SULFO-TAG reagent was then added and the samples were incubated for another 2 hours at room temperature with shaking. The plates were washed again three times in PBS and 0.05% Tween-20, and 2× Read Buffer T was added to the samples. The plates were read on the Sector Imager. Detection of other interleukins

The U-Plex MSD kit is designed to enable the study of the effects of DRSPAI-L7B on the secretion of IL-6, IL-10, IFN-γ, TNF-α and CCL3. MSD U-Plex plates are prepared by coating the plates with linker-coupled capture antibodies. Each capture antibody is biotinylated and has a unique linker assigned to it. The supernatant is diluted 1/100 and transferred to the MSD plate. The samples are incubated for 1 hour at room temperature while shaking, after which the plates are washed with PBS and 0.05% Tween-20. 50 µL of specific detection antibodies labeled with MSD SULFO-TAG reagent are then added and the samples are incubated for another hour at room temperature while shaking. The plates were again washed three times in PBS and 0.05% Tween-20, and 2× Read Buffer T was added to the samples. The plates were read on a Sector Imager.

The DRSPAI-L7B antibody was raised at a stock concentration of 11.33 mg/mL in 20 mM histidine, 180 mM trehalose, 40 mM arginine, 8 mM methionine, 0.05 mM EDTA pH 6.0. The antibody was diluted to a concentration of 20 µg/mL in assay medium. A 1/3 serial dilution was then made in assay medium to generate a 10 point dose response curve. The dilutions were transferred to the assay plate, which ensured a maximum final assay concentration of 10 µg/mL for the antibody.

BRL-54319MM (Rapamycin) was used as a positive control in the assay at a final assay concentration of 1 µM in the assay medium.

Resuspend recombinant human IL-7 to 25 µg/mL in sterile PBS and store 50 µL aliquots at -20°C. Thaw aliquots and dilute to appropriate concentration on the day of use. Reagent company Pre-filled Leucosep tubes Greiner Human IL-17 Basic Kit MSD Easy50 EasySep Magnet Stemcell Phosphate buffered saline, Ca2+/Mg2+ free (PBS) Gibco IMDM Gibco 96-well Costar round-bottom polystyrene sterile plate, with lid, transparent Costar Heat-inactivated fetal bovine serum Hyclone Xvivo15 Lonza Penstrep Invitrogen Easysep Human Memory CD4+ T Cell Enrichment Kit Stern cell L-Glutamine Invitrogen MEM non-essential amino acids Invitrogen HEPES Invitrogen PMA-phorbol 12-myristate 13-acetate Sigma Ionomycin Sigma Sodium pyruvate Invitrogen U-plex Biomarker Set 1 (Human) Mesoscale Discovery CellTiter-Glo Promega

FACS buffer: sterile PBS containing 2% heated inactivated FBS. Cell culture medium: 450mL IMDM, 50mL FBS, 5mL penicillin-streptomycin, 5mL L-glutamine, 5mL non-essential amino acids, and 5mL sodium pyruvate. Assay medium: 500mL Xvivo 15, 5mL Penstrep, 5mL L-glutamine, 5mL HEPES, and 5mL sodium pyruvate.

All data were normalized to the average of 8 high control wells and 8 low control wells on each plate. A four-parameter curve fit of the following form was then applied. Where a is the minimum value, b is the Hill slope, c is the XC50, and d is the maximum value. Data are presented as the mean _IC50 and the standard deviation of the mean of n experiments.

CD4 ⁺ T _mem cells isolated from healthy donor blood were cultured with IL-7 in the presence of DRSPAI-L7B for 4 days, after which PMA/ionomycin was added for 16 hours and subsequently captured. Interleukin secretion in the supernatant was assessed by MSD. DRSPAI-L7B treatment caused concentration-dependent inhibition of IL-17 (Fig. 4A, IC ₅₀ =270±31.59 pM), TNF-α (Fig. 4B, IC ₅₀ =127.1±89.83 pM), IL-6 (Fig. 4C, IC ₅₀ =202.07±99.64 pM), IL-10 (Fig. 4D, IC ₅₀ =197.48±148.91), INFγ (Fig. 4E, IC ₅₀ =157.98±89.55), and CCL3 (Fig. 4F, IC ₅₀ =163.83±85.96) secretion. Dose-dependent inhibition was also observed for IL-6, IL-10, IFNγ, TNF-α, and CCL3. Inhibition of these interleukin production is expressed as a percentage of that achieved with rapamycin (1 µM) positive control. Example 7 : T cell population analysis

Given the central role of T cells in MS, T cell populations were analyzed in PBMCs from RRMS, PPMS, and SPMS patients.

All human samples were obtained with informed consent from patients according to ICH GCP based on protocols approved by national, regional or site ethics committees or institutional review boards (IRBs).

Healthy PBMCs were isolated from BDU blood and stored frozen in liquid nitrogen until use. Disease PBMCs were supplied by an approved external human tissue supplier. Healthy control blood was drawn by venous puncture and transferred to a container with sodium heparin anticoagulant (1 U/mL). Blood was collected and used within 1 hour for PBMC isolation.

Use pre-prepared and frozen healthy control human PBMCs and prepare PBMCs by layering blood on 15 mL of Ficoll. Centrifuge tubes at 800 g for 20 minutes with the brake off. Transfer the mononuclear cell layer at the interface to a 50 mL Falcon tube, wash by filling to 45 mL with PBS, and centrifuge at 300 g for 10 minutes. Resuspend the pellet in freezing medium A (60:40 FCS: medium), add 5% of the original blood volume dropwise, and then add an equal volume of freezing medium B (80:20 FCS: DMSO) to reduce osmotic shock. Transfer cells to cryovials (1 mL (approximately 1×10 ⁷ cells) per vial) and freeze at -80°C in a Mr Frosty freezer for up to 1 week, then transfer to liquid nitrogen for long-term storage. PBMC recovery

Thaw cells by removing from liquid nitrogen storage and immediately placing in a water bath at 37°C until thawed. After transferring the cell suspension to a 50 mL centrifuge tube, very slowly add medium (RPMI + 10% heated inactivated FCS, 1% penicillin/streptomycin, and 1% glutamine) to gradually decrease the DMSO concentration. Once the volume has increased to 30 mL, the cells are centrifuged at 300 g for 10 min and resuspended in 5 mL of medium, then counted, made up to 15 mL with medium, centrifuged as above, and the cells resuspended in an appropriate volume of medium to give 5 × 10 ⁶ cells/1 mL.

In each of 5 separate experiments, two healthy control (HC), two RRMS, one PPMS, and one SPMS donor PBMC samples were thawed as above, and the cells were processed as follows. Different donors were used for each experiment, and the HC were broadly matched to the disease patient donors used in age and sex. T cell phenotype flow cytometric analysis

After resuspending PBMCs in medium at 5×10 ⁶ /mL, transfer 100 μL of cells to FACS tubes (for complete staining) and an additional 50 μL of HC samples (for FMO control tubes). Wash cells by adding 2 mL of FACS buffer, centrifuge at 300 g for 5 minutes, and resuspend pellets in the residual volume. Add 5 μL of Human FcX Trustain Blocking Solution over 10 minutes, followed by 100 μL of Antibody Staining Mix and incubate at room temperature for 30 minutes. Wash cells by adding 2 mL of FACS buffer, centrifuge at 300 g for 5 minutes, and resuspend pellets in the residual volume. Add 500 μL of diluted Live/Dead Fixable Aqua Dead Cell Stain and incubate at room temperature for 25 minutes. Wash cells by adding 2 mL FACS buffer, centrifuge at 300 g for 5 minutes, and resuspend the pellet in the residual volume. Add 200 μL FACS buffer and analyze samples on the same day using a BD FACS Canto II.

Check the performance of the instrument using the Cell Counter Set and Tracking (CST) Beads. This is a QC check of the instrument, setting the baseline and optimizing the voltages of each laser before use. The results of the calibration are stored in the CST software on the instrument.

Compensation of the instrument was performed using UltraComp compensation beads according to the manufacturer's instructions. The relevant antibodies used to stain cells during the experiment were used to label the compensation beads. Compensation of the experiment was performed using the automatic compensation facility available within the FACS Diva software using the appropriately labeled beads. After analysis of the compensated samples, the compensation settings were calculated and applied to each experimental staining panel. Reagent PBS (without Ca ^{2 +} and Mg ^{2 +} ) RPMI 1640 L-Glutamine 200 mM Penicillin/Streptomycin FCS is not activated by heating DMSO FcX Trustain Blocking Reagent BD Horizon Brilliant Staining Buffer Anti-human CD8 AF488 (Clone: RPA-T8) Anti-human CD25 PE (clone: BC96) Anti-human CD4 PerCP/Cy5.5 (Clone: RPA-T4) Anti-CD45RO PE/Cy7 (Clone: UCHL1) Anti-CCR7 AF647 (Strain: G043H7) Anti-CD20 APC/Fire750 (Clone: 2H7) Anti-CD127 BV421 (Clone: A019D5) Anti-CD14 BV510 (Clone: M5E2) Anti-CD19 BV510 (Clone: SJ25C1) Anti-CD56 BV510 (clone 5.1H11) Anti-CD16 BV510 (Clone: 3G8) Aqua can fix Live/Dead dye UltraComp eBeads Compensation Particle Set BD FACSDiva CS&T Research Beads Equipment Description Muse Cell Analyzer (Counter) BD Canto II flow cytometer

Samples were acquired on a BD FACS Canto II flow cytometer using BD Biosciences FACS Diva software (v8.0.1). The resulting compensated .fcs files were analyzed with FlowJo software (v10.0.8) and results were generated in Excel using the batch analysis facility within the software.

T cell populations were analyzed in PBMCs from RRMS, PPMS, and SPMS patients. Data were generated from patients in which all patients were given treatment (10/10 RRMS patients were given natalizumab, and all progressive MS patients were given steroids and/or symptomatic treatment) and a decrease in T _reg cells was observed. CD4 ⁺ (Figure 5A), CD8 ⁺ (Figure 5B), and regulatory T cells (Figure 5C) from healthy controls and MS patients were analyzed by flow cytometry based on the expression of CD45RO, CCR7, CD127, and CD25 on the cell surface. No differences were seen between healthy and disease T cell populations in either the CD4 ⁺ or CD8 ⁺ subsets. Compared with HC, RRMS and PPMS T _reg populations were significantly reduced, no differences were found in Treg values in SPMS patients. ***0<0.0001, *0<0.05 as tested by one-way analysis of variance using Dunnett multiple comparison test. Data presented represent mean ± SEM of n=5 to 10 donors per group analyzed in 5 independent experiments. Effect memory=CD45RO ^{+ CCR7-} , central memory=CD45RO ⁺ CCR7 ⁺ , naive=CD45RO ^- CCR7 ⁺ , effector=CD45RO ^{- CCR7-} , T _reg = ^{CD127low /} -CD25 ⁺ . Example 8 : STAT5 phosphorylation

All human samples were obtained with informed consent from patients according to ICH GCP based on protocols approved by national, regional or site ethics committees or institutional review boards (IRBs).

Healthy PBMCs were isolated from BDU blood and stored frozen in liquid nitrogen until use. Diseased PBMCs were supplied by an approved external human tissue supplier.

Blood from healthy control group was drawn by venous puncture and transferred into a container with sodium heparin anticoagulant (1 U/mL). Blood was collected and used within 1 hour for PBMC isolation.

Use pre-prepared and frozen healthy control human PBMCs and prepare PBMCs by layering blood on 15 mL of Ficoll. Centrifuge tubes at 800 g for 20 min with the brake off. Transfer the mononuclear cell layer at the interface to a 50 mL Falcon tube, wash by filling to 45 mL with PBS, and centrifuge at 300 g for 10 min. Resuspend the pellet in freezing medium A (60:40 FCS: medium), add 5% of the original blood volume dropwise, and then add an equal volume of freezing medium B (80:20 FCS: DMSO) to reduce osmotic shock. Cells were transferred to cryovials (1 mL (approximately 1×10 ⁷ cells) per vial) and frozen at -80°C in Mr Frosty freezers for up to 1 week, then transferred to liquid nitrogen for long-term storage.

Thaw cells by removing from liquid nitrogen storage and immediately placing in a water bath at 37°C until thawed. After transferring the cell suspension to a 50 mL centrifuge tube, very slowly add medium (RPMI + 10% heated inactivated FCS, 1% penicillin/streptomycin, and 1% glutamine) to gradually decrease the DMSO concentration. Once the volume has increased to 30 mL, cells are centrifuged (300 g for 10 min), resuspended in 5 mL of medium and counted, then topped up to 15 mL with medium, centrifuged as above, and resuspended in an appropriate volume of medium to give 5 × 10 ⁶ cells/mL.

In each of 5 separate experiments, two healthy control (HC), two RRMS, one PPMS, and one SPMS donor PBMC samples were thawed as above, and the cells were processed as follows. A different HC donor was used for each experiment, broadly matched in age and sex to the disease patient donors used .

After resuspending PBMCs in medium at 5×10 ⁶ /mL, 450 μL of cells were transferred to a 15 mL Falcon tube and incubated with 5 mL PBS containing 5 μL near-infrared live/dead stain. Cells were washed by adding 9 mL of full medium, centrifuged (300 g, 5 min), and cell pellets were resuspended in AIM V serum-free medium at 5×10 ⁶ cells/mL. All antibody treatments and rhIL-7 stimulation were made in AIM V serum-free medium at 4× final assay concentration before mixing 1:1 (IL-7:mAb) and incubating for 10 min at room temperature. Medium was added in the absence of antibody or IL-7 stimulation. The final concentration of IL-7 used was 1 ng/mL (57 pM). The final concentration of mAb used was 500 ng/mL (3.33 nM).

Add 100 μL of PBMC suspension to four FACS tubes per donor (5×105 cells/assay). Add 100 μL of Antibody:IL-7 mix to appropriate FACS tubes (medium only, IL-7 only, IL-7+DRSPAI-L7B, or IL-7+anti-RSV isotype control antibody). Mix tubes gently before incubating at 37°C in a humidified incubator for 20 minutes. At the end of the stimulation period, centrifuge cells at 300 g for 5 minutes, resuspend the cell pellet, and add 250 μL of pre-warmed PHOSFLOW Fixation Buffer (1×). Incubate samples at 37°C for an additional 10 minutes. After fixation, cells were pelleted by centrifugation (300 g, 5 min) and washed in 2 mL PBS. The cell pellet was resuspended in 100 μL of ice-cold Permeabilization Buffer III and gently vortexed to mix. Cells were incubated on ice for 30 min, then washed once in 1 mL PBS and centrifuged (300 g, 5 min). Cells were washed in 2 mL PBS, then centrifuged (300 g, 5 min).

After permeabilization, cell pellets were resuspended in 25 μL FcR blocking reagent diluted 1:5 in FACS buffer. Cells were incubated at room temperature for 10 minutes, followed by the addition of the detection antibody staining cocktail: 5 μL anti-CD3 (PerCP/Cy5.5), 5 μL anti-CD4 (AF488), 5 μL anti-CD8 (APC), 20 μL anti-pSTAT5 (PE), and 15 μL FACS buffer (50 μL total/assay). In the case of excluding the staining agent for the FMO control group, an equivalent volume of FACS buffer was added instead. The tubes were mixed briefly and incubated at room temperature (RT) for 30 minutes in the dark. Cells were washed in 2 mL FACS buffer, centrifuged (300 g, 5 min), and pellets were resuspended in 100 μL FACS buffer for analysis on the same day using a FACS Canto II flow cytometer.

The performance of the FACS Canto II instrument is checked using the Cell Counter Set and Tracking (CST) Beads. This is a QC check of the instrument, setting the baseline and optimizing the voltages of each laser before use. The results of the calibration are stored in the CST software on the instrument.

Compensation of the instrument was performed using UltraComp compensation beads according to the manufacturer's instructions. The relevant antibodies used to stain cells during the experiment were used to label the appropriate compensation bead type. For compensation of live/dead cell dyes, 1 μL of undiluted dye was added to 1 drop of ArcAmine reaction beads, incubated for 30 minutes, washed, and ArcAmine negative beads were added just prior to running samples. Compensation of experiments was performed using the automated compensation facility available within FACS Diva software using appropriately labeled beads. After analysis of the compensated samples, compensation settings were calculated and applied to each experimental staining panel. Antibodies and Reagents

For experiments, antibody aliquots were thawed and stored at 4°C for no longer than 8 weeks. DRSPAI-L7B (anti-IL-7) and anti-RSV isotype control. Reagent Suppliers PBS (without Ca ²⁺ and Mg ²⁺ ) Life Technologies RPM1 1640 Life Technologies L-Glutamine 200mM Life Technologies Penicillin/Streptomycin Life Technologies FCS is not activated by heating Life Technologies AIM V Medium Gibco Recombinant human IL-7 R&D Systems BD Phosflow™ Lysis/Fixation Buffer 5× BD Bioscience BD Phosflow™ Osmotic Buffer III BD Biosciences FcX Trustain Blocking Reagent BioLcgend BD Phosflow™ PE Mouse Anti-STAT5 (pY694) BD Bioscience Mouse anti-human CD8 APC (clone: SK1) BioLegend Mouse anti-human CD4 BioLegend Reagent Suppliers AF488 (Strain: RPA-T4) Mouse anti-human CD3 PerCP/Cy5.5 (Strain: SK7) BioLegend Near-infrared fixable Live/Dead stain Life Technologies UltraComp eBeads Compensation Particle Set Life Technologies ArcAmine Reaction Beads Life Technologies BD FACSDiva CS&T Research Beads BD Biosciences Equipment Description Muse Cell Analyzer (Counter) BD Canto II flow cytometer

Samples were acquired on a BD FACS Canto II flow cytometer using BD BioSciences FACS Diva software (v8.0.1). The resulting compensated .fcs files were analyzed with FlowJo software (v10.0.8) and results were generated in Excel using the batch analysis facility within the software.

To determine the ability of disease cells to respond to IL-7 and confirm the efficacy of DRSPAI-L7B in samples from MS patients, PBMCs isolated from healthy controls or MS patient donors were stimulated with 1 ng/mL (58 pM) IL-7 in the presence of non-saturating (IC90 = 500 ng/mL (3.3 nM)) concentrations of DRSPAI-L7B or anti-RSV IgG1κ isotype control antibody.

PBMCs from healthy donors or MS patients (10 healthy, 10 RRMS, 5 PPMS, and 5 SPMS) were stimulated with rhIL-7 in the presence of DRSPAI-L7B or anti-RSV antibodies (isotype control). STAT5 phosphorylation in CD4 ⁺ (Figure 6A) and CD8 ⁺ (Figure 6B) T cells was assessed by flow cytometry. IL-7 stimulation induced STAT5 phosphorylation in CD4 ⁺ and CD8 ⁺ T cells derived from healthy donors and MS patients at 1 ng/mL (58 pM, approximately 200-fold higher than that reported in the disease). IL-7 stimulation was almost completely abolished by 500 ng/mL or 3.3 nM (non-saturating concentration) of DRSPAI-L7B. The data shown are normalized to the IL-7 treatment condition, mean ± SEM. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p < 0.0001. One-way ANOVA with Tukey's multiple comparison correction. Similar results were observed upon IL-7 stimulation of PBMCs derived from IBD patients. Example 9 : IL-7 levels in disease

All human samples were obtained with informed consent from patients in accordance with ICH GCP based on protocols approved by national, regional or site ethics committees or institutional review boards (IRBs). Samples were stored at -80°C until required.

Measurement of IL-7 was performed following the method SOP. Briefly, the method followed the MSD kit protocol with a 1:2 sample dilution using diluent 43. The diluted samples were incubated on pre-coated IL-7 MSD plates and subsequently detected with a ruthenium-tagged anti-IL-7 antibody. The amount of IL-7 in the plasma sample was proportional to the ECL signal read using an MSD Sector Imager 6000 reader. The IL-7 method used the MSD V-PLEX Human IL-7 Kit (Catalog No. K151RCD).

ECL signals for standards, controls, and unknown samples were derived from the MSD instrument and exported to Softmax Pro GxP for analysis. The back-calculated concentrations for each method were interpolated using the standard curve and a 4-parameter curve fitting model with 1/y2 weights. A summary of IL-7 concentrations was transferred to Microsoft Excel.

All data were transferred from Microsoft Excel 2016 to GraphPad Prism v.6 for plotting and statistical analysis. Data were copied to Prism's "Column Tables" based on sample matrix (serum) and analyte (IL-7). Statistical tests including the Shapiro-Wilk normality test were performed on each data set to determine whether each data set was normally distributed. If the data were not normally distributed, the data were transformed using the formula y=log(y) to normalize the distribution. Statistical tests were performed again on the transformed data to confirm normal distribution.

For serum samples, groups were compared using one-way analysis of variance (ANOVA). Data were tested for distribution using the Shapiro-Wilk test for normality in the row statistics function. Group differences in non-normally distributed data were assessed using the Kruskal-Wallis unpaired nonparametric analysis with Dunn's multiple comparison test to compare mean ranks of rows to each other. Data were also transformed using y=log(y) to normalize the distribution of the data. After transformation, the Shapiro-Wilk test was performed using the row statistics function to confirm that the transformation produced a normal distribution of the data. The transformed normally distributed data were then compared with each other using ordinary one-way unpaired ANOVA with Tukey's multiple comparison test for statistically significant differences.

IL-7 levels were quantified in serum samples from patients with healthy controls (HC, n=10), Crohn's disease (n=15), ulcerative colitis (UC, n=15), systemic lupus erythematosus (SLE, n=15), and primary Sjögren's syndrome (pSS, n=15). IL-7 was significantly increased in Crohn's disease, UC, and SLE (7.3-, 9.44-, and 5.53-fold, respectively) compared with healthy controls. A smaller increase in IL-7 was also present in the pSS group (3.55-fold vs. HC). Data are mean ± SD. ** p≤0.01, *** p≤0.001, **** p≤0.0001; serum analyses using one-way ANOVA with Dunn's multiple comparison correction. Figures 7A and 7B. Example 10 : DRSPAI-L7B generated

Transfect cell lines with plasmids. This plasmid contains codon-optimized DRSPAI-L7B heavy and light chain genes, each under the transcriptional control of a separate human EF1α promoter. The light chain constant region is human κ, and the heavy chain constant region is human IgG1 containing a LAGA substitution. The LAGA substitution corresponds to L235A/G237A. Expand and select transfected cell lines based on the expression of DRSPAI-L7B. Example 11 : Antigenic Determinant Binding Instruments Waters Synapt G2-Si Mass Spectrometer Acquity M-Class UPLC LEAP H/DX PAL Liquid-Handling Robot Solutions Quenching Solution: 400 mM potassium phosphate, 6 M guanidine hydrochloride, 0.5 M TCEP pH 2.5 (after 1:1 mixing with sample - quench buffer pH - adjusted with NaOH to obtain this pH during mixing). Dilution Buffer: H2O pH 7.0 containing 50 mM sodium phosphate, 100 mM NaCl. Proteins: IL-7: Concentration: 0.68 mg/ml (33 μM) in PBS. Sequence before treatment (SEQ ID NO: 1): MFHVSFRYIFGLPPLILVLLPVASS DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHHHHHH Secretory leader (underlined above)

DRSPAI-L7B was produced in HEK cells and used at 15 mg/ml (100µM) in PBS. HDX

Dilution buffer is: Non-deuterated: H2O containing 50mM sodium phosphate, 100mM NaCl, pH7.0 Deuterated: D2O containing 50mM sodium phosphate, 100mM NaCl, pD 6.6

For initial testing, the DRSPAI-L7B was tested in a single operation to check the resolution quality and signal strength.

For HDX experiments, IL-7:mAb mixtures were prepared as follows: "Apo" samples contained 20 µl concentrated IL-7, 15 µl PBS, and 25 µl dilution buffer. "mAb" samples contained 20 µl concentrated IL-7, 15 µl DRSPAI-L7B, and 25 µl dilution buffer. This gave a final nominal concentration of 50 µM for IL-7 and 25 µM for DRSPAI-L7B. Samples were prepared on ice and kept at 0°C until analysis.

For mAb binding experiments, samples were subjected to a standard deuteration method using a 10-fold dilution into deuterated buffer, which was performed using a LEAP H/DX PAL robot. Protein samples were placed in a rack at 0°C. 6 µl of protein sample was transferred to a vial maintained at 20°C, followed by the addition of 54 µl of dilution buffer (non-deuterated for time point 0, deuterated for other time points) to initiate hydrogen exchange. After the appropriate incubation time, 50 µl of this sample was withdrawn and transferred to a pre-chilled vial (0°C) containing 50 µl of quench solution (400 mM sodium phosphate, 6 M guanidine hydrochloride, 0.5 M tris-carboxyethylphosphine [pH 2.5 after mixing with sample]). After mixing and incubation for 1 min, another 90 µl of this sample was withdrawn and transferred to the HDX manager where it was injected via a 100 µl loop at 50 µl/min onto an immobilized pepsin column (Enzymate BEH, 2.1×30 mm, Waters #186007233, maintained at 15°C). The resulting peptides were eluted in 0.2% formic acid and captured on a Vanguard BEH C18 pre-column (2.1×5 mm; Waters #186003975). The capture column was then switched in unison with an analytical reverse phase column (BEH C18, 1×100 mM, Waters #186002346) and the peptides were eluted using the following gradient, where A=0.2% formic acid, 0.03% trifluoroacetic acid/water, B=0.2% formic acid in acetonitrile: Steps Time(min) Flow rate (uL-/min) %A %B Curve 1. initial 40.000 88.0 12.0 2. 8.00 40.000 64.0 36.0 6 3. 9.00 40.000 5.0 95.0 6 4. 10.00 40.000 5.0 95.0 6 5. 10.50 40.000 88.0 12.0 6 6. 11.50 40.000 5.0 95.0 6 7. 13.00 40.000 5.0 95.0 6 8. 13.50 40.000 88.0 12.0 6 9. 15.00 40.000 88.0 12.0 6

During the gradient phase, the pepsin column was washed with 2 × 80 µl of pepsin wash buffer (2 M guanidine-HCl, 0.8% formic acid, 5% acetonitrile 5% propan-2-ol pH 2.5) at 25 µl/min and returned to 0.2% formic acid at 25 µl/min in preparation for the next sample. The eluate from the analytical column was analyzed using ESI-MS using a Waters Synapt G2-Si mass spectrometer operated in positive resolution mode, where continuous data were collected. Lockspray data containing leucine-enkephalin and Glu-Fib ions were also collected. For peptide identification samples, MSe data were acquired (alternating between low and high energy conditions in the collision cell) to provide segmented data to assist in stable peptide identification. For HDX samples, a single low energy acquisition (with dual electrospray ion source) was performed.

Initial samples were run in MS ^e mode to generate peptide search lists using ProteinLynx Global Server v3.0.2 (Waters). The HDX sample run was repeated twice with deuteration periods of 0, 0.5, and 5 min. The peptide search list was imported into DynamX v3.0 (Waters) and filtered to obtain high-quality peptides for searching in HDX samples (minimum intensity >10,000; peptide score >7.0). HDX sample data were then imported and processed to determine the deuteration of each identified peptide in each sample. Peptide and ion assignments were manually checked and optimized when necessary.

To interpolate the peptide-level data to the residue-level data (to enable heatmaps and structure views), the algorithm used to generate heatmaps by DyanamX was used, where for each residue the data from the shortest overlapping peptide was used (if two overlapping peptides were of the same length, the peptide closest to the N-terminus was used).

Two peptides in the region of residues 67 to 81 (numbered from the full-length unprocessed construct) showed stronger protection in the presence of mAb. (SEQ ID NO: 12 - FKRHICDANKEGMFL) (SEQ ID NO: 16 - FKRHICDANKEGMF)

Likewise, the region covering residues 67 to 81 shows a clear conservation signal. In addition, the heat map data were mapped onto the available 3D structure of IL-7 (in complex with IL-7Rα) and the protected region covers residues 67-81 (FKRHICDANKEGMFL). This region is far from the region where IL-7 interacts with the receptor IL-7Rα. The antigenic determinant is located adjacent to the interaction site of IL-7Rα and the ɣ-chain on the folded protein. Example of a bispecific protein associated with IL - 7 and TNF - α

In the examples herein, the following antibodies are described: Identifier SEQ ID NO: T7 HET mAb 1 56, 57, 58, 59 T7 HET mAb 2 60, 61, 62, 63 Control Antibody

In the examples herein, the following control antibodies were used: Identifier describe TNF control 1 Adalimumab plus T7 Het mAb 1 Fc mutation TNF control 2 Adalimumab plus T7 Het mAb 2 Fc mutation TNF control 3 Adalimumab plus YTE Fc mutation IL-7 control 1 DRSPAI-L7B plus Fc mutation of T7 Het mAb 2 IL-7 control 2 DRSPAI-L7B Negative control 1 RSV mAb plus Fc mutation of T7 Het mAb 2 Example 12 : Performance, Purification, and Quality Control

Antibodies were transiently expressed in HEK293 6E cells using BalanCD HEK293 medium (Irvine Scientific) and purified from filtered supernatants by protein A affinity chromatography (MabSelect SuRE resin or MagSepharose PrismA beads, Cytiva) followed by size exclusion chromatography (Superdex S200, Cytiva). Standard precautions to minimize endotoxin contamination were maintained at all stages of purification, and all preparations were filter sterilized by vacuum filtration (0.2 μM).

In addition, some antibody batches have been produced in CHO cells.

The proportions of high molecular weight (HMW), low molecular weight (LMW) and monomeric species within each bispecific sample were characterized by standard analytical size exclusion chromatography (aSEC) or mixed-mode size exclusion chromatography (mmSEC). Samples were analyzed on an Agilent 1260 Infinity (Agilent, UK) with a DaD detector running Chemstation software (Agilent, UK).

Bispecific sample composition was characterized by reversed phase high performance liquid chromatography (RP-HPLC) or reversed phase liquid chromatography mass spectrometry (RP-LCMS). For off-line RP-HPLC analysis, samples were separated using an acetonitrile gradient (supplemented with trifluoroacetic acid and acetic acid) on a Waters Acquity HPLC system (Waters, UK) and UV data were processed using Empower software (Waters, UK). For RP-LCMS, samples were deglycosylated with PNGase F and separated using a similar gradient on a Waters Acquity HPLC system (Waters, UK). The column eluent was analyzed by UV detection and introduced into an on-line Waters Synapt G2-Si mass spectrometer (Waters, UK). UV data and ESI mass spectra were processed together using Protein Metrics Intact Mass software (Protein Metrics Inc, USA) to provide semi-quantitative species ratios. Analytical SEC RP-LCMS molecular HMW% Monomer (%) LMW (%) Corrected bispecificity T7 HET mAb 2 batches 1.2 98.8 0.0 98.9 1.1 98.9 0.0 98.2 T7 HET mAb 1 batch 0.0 100.0 0.0 94.8 0.0 100.0 0.0 98.6 0.0 100.0 0.0 94.1 T7 HET mAb 1 0.0 100.0 0.0 90.6 Mixed Mode SEC RP-HPLC molecular HMW% Monomer (%) LMW (%) Bispecific T7 HET mAb 1 1.2 97.1 1.7 82.4 Example 13 : Binding affinity of T7 HET MAB 1 to TNF and IL - 7

Binding of T7 HET MAB 1, TNF controls 1 and 3, and IL-7 control 1 to recombinant human TNFα and IL-7 was assessed using a Biacore T200 (Cytiva) surface plasmon resonance instrument.

The antibodies were captured on protein AG and immobilized on flow cells 2, 3 and 4 of a CM5 series S sensor chip by primary amine coupling. TNFα or IL-7 analytes were passed over the captured antibodies at different concentrations including 25 nM, 6.25 nM, 1.56 nM, 0.39 nM and 0.098 nM. The association phase lasted for 240 sec at 30 µl/min, followed by a 300 sec dissociation step. 0 nM (i.e., buffer only) injections were used for double reference binding curves. 50 mM NaOH was used for chip surface regeneration between cycles. Analysis was performed in HBS-EP ⁺ buffer at 25°C and 37°C. Biacore T200 control software version 1.0 was used to perform the experiments. Data were analyzed using Biacore T200 Evaluation Software version 1.0, and affinity and kinetics were determined where possible using the software's inherent 1:1 kinetic fit.

Binding of T7 HET MAB 1, TNF controls 1 and 3, and IL-7 control 1 molecules to recombinant human TNFα and IL-7 was also assessed using a Biacore 8K (Cytiva) surface plasmon resonance instrument. Antibodies were captured on protein AG and immobilized to flow cell 2 of all 8 channels of a CM5 Series S sensor chip by primary amine coupling. TNFα or IL-7 analytes were passed over the captured antibodies at different concentrations including 25nM, 6.25nM, 1.56nM, 0.39nM, and 0.098nM. The association phase lasted 240 seconds at 30µl/min, followed by a 1800 second dissociation step. A 0nM (i.e., buffer only) injection was used for the double reference binding curves. Surface regeneration between cycles was performed with 50 mM NaOH. Analyses were performed in HBS-EP ⁺ buffer at 25°C and 37°C. Biacore 8K Control software version 3.0 was used to perform the experiments. Data were analyzed using Biacore 8K Insight evaluation software version 3.0, and affinities and kinetics were determined where possible using the software's inherent 1:1 kinetic fit.

All affinity values generated using Biacore T200 and Biacore 8K were combined to determine the geometric mean. Table 7 shows the affinity of all antibodies to human TNFα and IL-7. The results show that the binding affinity of T7 HET MAB 1 to human TNFα and IL-7 is comparable (within 2-fold) to the relevant TNF Control 1 and 3 and IL-7 Control 1 molecules. No data were generated for binding to TNFα protein at 37°C. Table 7 Binding of T7 HET MAB 1, Control 1 and 3, and IL-7 Control 1 molecules to recombinant human TNF-α and IL-7 using SPR ND = Not determined antibody antigen Temperature ( ℃ ) Average _KD (pM) T7 HET MAB 1 Human TNFα 25 82.6 37 ND TNF control 1 Human TNFα 25 63.9 37 ND T7 HET MAB 1 Human IL-7 25 33.6 37 30.9* IL-7 control 1 Human IL-7 25 14.4 37 37.5 Affinity values are the geometric mean of n=3-4 replicates, except for *, where n=2

In addition, binding of T7 HET MAB 2 and additional batches of T7 HET MAB 1 to recombinant human TNFα and IL-7 was evaluated using the reagents, methods, and data analysis described. molecular Target KD (M) Compound level review Temperature ℃ TNF control 1 TNF-α Human 2.95E-10 25 TNF control 3 TNF-α Human 1.32E-10 25 IL-7 control 2 IL-7 Human 1.29E-11 25 IL-7 control 2 IL-7 Human 3.09E-11 37 T7 HET MAB 1 TNF-α Human 1.32E-10 25 T7 HET MAB 1 IL-7 Human 2.82E-11 25 T7 HET MAB 1 IL-7 Human 3.80E-11 37 IL-7 control 1 IL-7 Human 1.91E-11 37 IL-7 control 1 IL-7 Human 1.00E-11 25 T7 HET MAB 2 IL-7 Human 5.37E-11 25 T7 HET MAB 2 IL-7 Human 2.09E-12 Instrument Limit Dynamics 25 T7 HET MAB 2 TNF-α Human 2.14E-10 Remove top density to improve fit 25 TNF control 2 TNF-α Human 2.04E-10 Remove top density to improve fit 25 IL-7 control 1 IL-7 Human 5.13E-11 25 IL-7 control 1 IL-7 Human 2.00E-12 Instrument Limit Dynamics 25 T7 HET MAB 2 TNF-α Human 4.07E-10 25 T7 HET MAB 2 IL-7 Human No binding due to no catch 25 T7 HET MAB 1 IL-7 Human 7.59E-11 25 T7 HET MAB 1 TNF-α Human 9.33E-11 25 T7 HET MAB 1 IL-7 Human 7.59E-11 25 T7 HET MAB 1 IL-7 Human 6.17E-11 25 T7 HET MAB 1 TNF-α Human 4.17E-10 25 T7 HET MAB 1 TNF-α Human 4.57E-10 25 Negative control 1 IL-7 Human Negative control 25 Negative control 1 TNF-α Human Negative control 25 TNF control 2 TNF-α Human 3.98E-10 25 TNF control 2 TNF-α Human 3.72E-10 25 IL-7 control 1 IL-7 Human 1.02E-10 25 IL-7 control 1 IL-7 Human 1.02E-10 25 Example 14 : IL - 7 MSD SET at 25 °C

Binding of T7 HET MAB 1 to biotinylated recombinant human IL-7 was measured at room temperature (25°C). IL-7 control 1 and human IgG1 isotype antibody were included as positive and negative controls, respectively.

Binding was measured in 384-well plates using MSD-SET (MesoScale Discovery Solution Equilibrium Titration) analysis in an 11-point curve format. MSD-SET determines the equilibrium affinity of the antibody in the solution phase. The method relies on the detection of free antigen at equilibrium over a range of antibody concentrations being titrated. All test antibodies were measured in triplicate on the same plate.

A fixed concentration of biotinylated human IL-7 (60 pM) was incubated with the test antibody, starting with 1 nM (row 1) and 0.5 nM (row 13), and diluted (2-fold) in the plate from rows 1-12 and rows 13-24, respectively. All incubation samples were diluted in PBSF (PBS + 0.1% BSA without IgG). Rows 6 and 18 were excluded from the titration and served as high and low control rows, respectively. Row 6 contained only 1× antigen, which produced a high signal due to high free antigen. Row 18 contained only PBSF to simulate 100% binding of antigen to antibody and should therefore produce a low signal. The incubation plate was incubated for 24 hours at room temperature.

After 24 hours, the same antibody (5 nM in PBS) was coated onto the standard binding MSD plate for 30 minutes at room temperature. The antibody position in the MSD plate was the same as that in the incubation plate. After 30 minutes of incubation, the coating antibody solution was discarded, and 80μl of starting block (Thermo Scientific, #37538) was added to the plate and repeated three times. The plate was then washed three times with MSD wash buffer (PBSF + 0.05% Tween). Then 20µl of incubation solution was added to the MSD plate and kept at room temperature for 2.5 minutes while shaking at 700rpm. After incubation, the plate was washed once with 80µl of MSD wash buffer. After 3 minutes of incubation, the captured antigen on the plate was detected with sulfo-tagged streptavidin (250 ng/ml). The plate was washed three times with MSD wash buffer, 35 µl of read buffer T 1× was added, and the plate was read on an MSD sector S 600 imager.

The raw data obtained from the MSD Sector S 600 reader were imported into Activitybase and analyzed using the protocol TIGHT_BINDING_FC_2C_BIOP. Activity base outputs are pKD, antigen binding site concentration, Z prime, 95% confidence interval and R ² . See Table 8 for the results.

The positive control (IL-7 control 1) binds to human IL-7, while the negative isotype control antibody shows no binding to IL-7. All analytical results met the acceptance criteria of R ² >0.96. The antigen active binding concentration calculated in the analysis of the T7 HET MAB 1 results ([ABC] Ag, values not shown) was within 10 times of the calculated affinity and therefore met the acceptance criteria. The reported results are the average of n=3. The results show that the affinity of T7 HET MAB 1 binding to human IL-7 is comparable to that of IL-7 control 1 (10.2pM and 4.17pM, respectively). Table 8 Affinity of binding of T7 HET MAB 1 and IL - 7 control 1 to human IL - 7 analyzed by MSD - SET at 25 °C . antigen Antibody strain name Average KD (pM) 95 % confidence limit (pM) 95 % confidence limit (pM) Human IL-7 T7 HET MAB 1 10.20 1.78 89.3 Human IL-7 IL-7 control 1 4.17 0.15 31.2 Affinity values are the arithmetic mean of n=3 replicates. Example 15 : Dual conjugation of T7 HET MAB 1 with recombinant human TNF - α and IL-7 using BLI

Dual binding of T7 HET MAB 1 to human TNF-α and human IL-7 was determined using a binding assay on a Sartorius Octet RED384 biolayer interferometry (BLI) instrument.

Human IL-7 at 200 nM was captured onto anti-Penta His buffer and the biosensor was read for 240 seconds. Loaded sensors were immersed in T7 HET MAB 1 (diluted to 20 µg/ml in PBSF) or PBSF for 240 seconds. Thereafter, the sensors were immersed in PBSF or human TNF-α (diluted to 200 nM in PBSF) or PBSF for 240 seconds. The sensors were then immersed back in buffer for the dissociation phase for 240 seconds. A blank sensor was included to check for non-specific binding of proteins to the sensor. Regeneration of the biosensor tip was performed using 10 mM glycine pH 1.5. The analysis was performed at 25°C with a plate shaker speed of 1000 rpm. The data were compared to baseline, but no kinetic model was applied to the data. Although no kinetic model was applied, the dual binding data generated on the Sartorius Octet RED384 instrument were analyzed using the instrument-native data analysis software (v11.1). There was no non-specific binding of the protein to the anti-Penta His sensor or to human TNF-α and human IL-7. Simultaneous binding of T7 HET MAB 1 to human TNF-α and IL-7 was successfully demonstrated. Table 9 Dual binding of T7 HET MAB 1 to recombinant human TNF - α and IL-7 using BLI Sensor load Combination 1 Combination 2 Combine Human IL-7 T7 HET MAB 1 Human TNF-α Combine Human IL-7 Buffer Human TNF-α No binding Buffer T7 HET MAB 1 Human TNF-α No binding Buffer Buffer Human TNF-α No binding Example 16 : Potency against TNF : L929 analysis ( for T7 HET MAB 2 and T7 HET MAB 1 )

L929 cells are murine aneuploid fibrosarcoma cells. In this assay, they are treated with actinomycin B (to sensitize the cells), followed by the addition of recombinant human TNF-α to initiate apoptosis and subsequent cell death (TNF enhances the cytotoxicity of actinomycin B, which targets DNA topoisomerase II). The resulting amount of cell death is then quantified using the Promega Cell Titre Glo (a homogenous method for determining the number of viable cells in culture based on the quantification of the ATP present (an indicator of metabolically active cells)). The luminescence values measured by the Cell Titer luminescence readout can then be plotted to fit the curve, enabling the determination of the potency of the molecule (IC50). This assay is used to test the ability of antibodies to neutralize TNF-α and block cell death.

L929 cells were seeded at 10,000 cells/well in 100 µl RPMI 1640 (without phenol red) in a 96-well flat-bottom plate and incubated overnight at 37°C, 5% CO _2. The medium was removed and the cells were sensitized in 80 µl medium containing 1.25 mg/ml actinomycin D for 1 hour. For neutralization studies, 0.7ng/ml - 15ug/ml (= 5pM -100nM T7 HET MAB 1 (or control molecule) was pre-incubated with 1ng/ml (19pM) human TNF-α at a 1:1 ratio for 1 hour at room temperature. After 1 hour pre-incubation with actinomycin D, 20ml of antibody-antigen complex was added to each well. Only 20ml of medium was added to the wells as a negative control. The plates were incubated at 37°C, 5% _CO2 for 18 hours. After this treatment period, cell viability was determined by the cell titer-Glo Luminescent Assay Kit according to the manufacturer's instructions (Promega, Manison USA).

Raw data were generated on a Pherastar FS multimode plate reader. Raw luminescence counts were analyzed in ActivityBase, derived pIC50 values were obtained from each independent experiment, and the geometric mean and range between these values were calculated. The calculated geometric mean and pIC50 value range were also converted to picomoles. Neutralization of human TNFα by T7 HET MAB 1 and control

The geometric means were calculated from TNF control 3 and T7 HET MAB 1, N=12.

For negative control 1, the geometric mean was calculated from N = 6. This molecule was inactive in this assay. PIC50 ( geometric mean ) PIC50 ( Range ) IC50pM ( geometric mean ) IC50pM ( Range ) T7 HET MAB 1 9.62 9.56-9.72 240 190 - 275 TNF control 3 10.07 9.99-10.18 85 66 - 102 Negative control 1 ＜8 N/A N/A N/A

In addition, the recombinant human TNF - α potency of T7 HET MAB 2 and additional batches of T7 HET MAB 1 were also evaluated using the reagents and methods described above. molecular IC50 (pM) T7 HET MAB 2 269.1535 T7 HET MAB 2 269.1535 T7 HET MAB 2 223.8721 T7 HET MAB 2 239.8833 T7 HET MAB 2 263.0268 T7 HET MAB 2 281.8383 TNF control 3 100 TNF control 3 104.7129 TNF control 3 89.12509 TNF control 3 85.1138 TNF control 3 93.32543 TNF control 3 97.72372 Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive TNF control 2 97.72372 TNF control 2 77.62471 TNF control 2 102.3293 IL-7 control 1 Inactive IL-7 control 1 Inactive IL-7 control 1 Inactive molecular IC50 (pM) T7 HET MAB 2 114.8154 T7 HET MAB 2 112.2018 T7 HET MAB 2 102.3293 TNF control 3 42.65795 TNF control 3 34.67369 TNF control 3 38.90451 TNF control 3 28.84032 TNF control 3 38.90451 TNF control 3 44.66836 T7 HET MAB 1 125.8925 T7 HET MAB 1 120.2264 T7 HET MAB 1 107.1519 T7 HET MAB 1 85.1138 T7 HET MAB 1 109.6478 T7 HET MAB 1 85.1138 T7 HET MAB 1 112.2018 T7 HET MAB 1 123.0269 T7 HET MAB 1 114.8154 Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive TNF control 2 33.11311 TNF control 2 33.88442 TNF control 2 38.90451 TNF control 2 27.54229 TNF control 2 37.15352 TNF control 2 30.90295 IL-7 control 1 Inactive IL-7 control 1 Inactive IL-7 control 1 Inactive IL-7 control 1 Inactive IL-7 control 1 Inactive IL-7 control 1 Inactive Example 17 : Efficacy against IL - 7 : CCRF - CEM analysis ( T7 HET MAB 2 and T7 HET MAB 1T7 HET MAB 1 ) ​

CCRF-CEM cells are human T lymphoblastoid cells and express IL-7 receptors on the cell surface. Binding of IL-7 to the IL-7 receptor causes receptor dimerization and activation, recruitment of JAK proteins, and phosphorylation of STAT5. Phosphorylated STAT5 (pSTAT5) can be detected in cell lysates using a sandwich immunoassay specific for pSTAT5. After stimulation with a fixed concentration of IL-7. Binding of the antibody to IL-7 in this system disrupts the interaction between the cytokine and its receptor, which in turn prevents IL-7-mediated STAT5 phosphorylation. The pSTAT5 present in these cells can be normalized to wells containing only IL-7 and wells without IL-7 to determine the percentage of inhibition. These values can then be used to plot curve fits, enabling the determination of molecular potency (IC50).

CCRF-CEM cells were stimulated with dexamethasone [Sigma-Aldrich D4902] and incubated overnight at 37°C, 5% CO2. Human IL-7 was prepared at a 15 pM concentration in assay medium (PR-FREE RPMI [Gibco 32404-014] + 10% FBS [Gibco 10100-147]). 10 uL of this IL-7 preparation was dispensed into all rows of a 384-well V-bottom plate (Greiner 781280) using MultiDrop, except rows 11 and 23. 10 μL of assay medium was dispensed into rows 11 and 23.

Test antibodies were prepared at 3 nM concentration and serially diluted 1:4 over 10 points. Rows 11 and 12 contained assay matrix only. 10 μL from this plate was transferred to the plate containing human IL-7. The plate was incubated on an orbital rocker at room temperature for 30 minutes to allow pre-complexation of antibodies with IL-7.

Collect dexamethasone-stimulated CCRF-CEM cells from the incubator and count 250 μL on a Beckman Coulter ViCell. Centrifuge the culture at 1500 rpm for 5 minutes and discard the spent medium. Resuspend the cell pellet to a concentration of 5×10 ⁶ cells/mL. Dispense 10 μL of this cell suspension onto the plate containing the antibody and IL-7. Incubate the plate at 37°C, 5% CO2 for 20 minutes.

Dispense 10 μL of 4× lysis buffer (2.4 mL 10× lysis buffer [Cell Signaling 9803], 2 mL dH2O, 200 μL protease inhibitor [Sigma P8340], 200 μL phosphatase inhibitor cocktail II [Sigma P5726], 200 μL phosphatase inhibitor cocktail III [Sigma P0044]) into each well and shake the plate vigorously for 10 seconds to lyse the cells. Store these cell lysates on ice for pSTAT5 immunoassay.

One plate from the MSD pSTAT5 Immunoassay Kit (MSD K15163D) was used for every 96 wells used on a 384-well plate. 1× Tris wash buffer was prepared from the 10× stock provided in the kit by diluting 10-fold with dH2O. 150 μL of MSD Blocker A (3% w/v MSD Blocker A powder in 1× Tris Wash Buffer) was dispensed into each well of the plate. The plate was covered with an aluminum foil seal and incubated on an orbital rocker at room temperature for 1 hour. Following this incubation, the plate contents were discarded by flicking into a sink and 250 μL of 1× Tris Wash Buffer was dispensed into each well. Flick the plate contents and dispense buffer two more times to wash the plate. Transfer 25 μL of cell lysate from the plate on ice to the MSD plate. Seal the plate as before and incubate on an orbital rocker for 1 hour at room temperature. Prepare antibody diluent solution by adding 150 μL 2% Blocker D-M, 30 μL 10% Blocker D-R, 1 mL Blocker A solution, and 1.82 mL 1× Tris Wash Buffer. Dilute pSTAT5 detection antibody from 50× stock to 1× by adding 60 μL to 2.94 mL of prepared diluent. The plates were washed 3 times as before and 25 μL of the detection antibody solution was dispensed into each well. The plates were sealed as before and incubated on an orbital rocker for 1 hour at room temperature. After this incubation, the plates were washed as before. 1× Read Buffer T was prepared from 4× Read Buffer T stock (MSD R92TC) by diluting 4-fold in dH20 and 150 μL was dispensed per well and the plates were read on an MSD Sector S 600 imager.

Raw data were generated using MSD Workbench (MSD) on an MSD Sector S 600 imager. Raw MSD counts were exported as delimited text files and further analysis was performed in ActivityBase. Mean pIC50 values were obtained from each independent experiment, and the geometric mean and range between these values were calculated. The calculated geometric mean and pIC50 value range were also converted to picomoles.

The potency (IC50) of T7 HET MAB 1 against human IL-7 was in the single digit picomolar range and was slightly decreased (less than 2-fold) relative to IL-7 control 1, which is expected since T7 HET MAB 1 has only half the amount of anti-IL-7 fAb arm compared to IL-7 control 1. Table 10 : Human IL - 7 neutralization potency of T7 HET MAB 1 and control . pIC50 (geometric mean) pIC50 (range) IC50 (pM, geometric mean) IC50 (pM, range) T7 HET MAB 1 11.46 11.25 - 11.58 3.47 2.63 - 5.62 TNF control 2 Inactive Inactive Inactive Inactive IL-7 control 1 11.59 11.49 - 11.68 2.57 2.09 - 3.24 Negative control 1 Inactive Inactive Inactive Inactive

In addition, the potency of T7 HET MAB 2 against recombinant human IL-7 was evaluated using similar reagents, methods, and data analysis as described above. Neutralization potency of T7 HET MAB 1 and control against human IL - 7 and human TNF - α molecular XC50 (M) T7 HET MAB 2 2.45E-12 T7 HET MAB 2 2.82E-12 T7 HET MAB 2 2.04E-12 T7 HET MAB 2 2.24E-12 T7 HET MAB 2 2.63E-12 T7 HET MAB 2 2.09E-12 TNF control 3 Inactive TNF control 3 Inactive TNF control 3 Inactive TNF control 3 Inactive TNF control 3 Inactive TNF control 3 Inactive IL-7 control 2 6.17E-13 IL-7 control 2 1.02E-12 IL-7 control 2 7.41E-13 IL-7 control 2 6.76E-13 IL-7 control 2 7.08E-13 IL-7 control 2 1.10E-12 Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive Negative control 1 Inactive TNF control 2 Inactive TNF control 2 Inactive TNF control 2 Inactive TNF control 2 Inactive TNF control 2 Inactive TNF control 2 Inactive IL-7 control 1 9.12E-13 IL-7 control 1 7.94E-13 IL-7 control 1 1.12E-12 IL-7 control 1 7.24E-13 IL-7 control 1 9.33E-13 IL-7 control 1 8.71E-13 Example 18 : Efficacy of T7 HET mAb 1 in a mixed lymphocyte reaction ( MLR ) assay.

The MLR assay is an ex vivo cellular immunoassay in which peripheral blood mononuclear cells from two different donors are combined in culture. In these co-cultures, T cells are activated and produce proinflammatory interleukins in response to allogeneic allo-tissue compatible molecules expressed on antigen presenting cells from the opposing donor. The MLR assay was developed to compare the potency of T7 HET mAb 1 with that of TNF control mAb 1 and IL-7 control mAb 1. Since IL-7 is not expressed in PBMCs, human recombinant IL-7 was added to the culture to stimulate the IL-7 receptor pathway. IFNg inhibition in the MLR assay using TNF control 6 and IL - 7 control 1 alone or in combination .

Cryopreserved PBMCs (STEMCELL Technologies) from individual donors were thawed and resuspended at 1-2×10 ⁶ cells/ml. PBMCs from two donors were combined 1:1 and 100 μl was dispensed into duplicate wells of a 96-well round bottom plate (n=3 different MLR co-culture combinations). Recombinant human IL-7 was added to the cultures at a final concentration of 10 ng/ml. Co-cultures were treated with the following antibodies: (A) 2 μg/ml TNF Control 6-Negative Control + 5 μg/ml Negative Control; (B) 2 μg/ml TNF Control 6; (C) 5 μg/ml IL-7 Control 1; (D) 2 μg/ml TNF Control 6 + 5 μg/ml IL-7 Control 1. Co-cultures were incubated for 48 hours at 37°C and 5% _CO2 . IFNg secretion was quantified using the Meso Scale Discovery Immunoassay Kit (Figure 8). The amount of IFNg secreted by samples treated with 2 μg/ml TNF Control 6-Negative Control + 5 μg/ml Negative Control was normalized to 100% or maximum signal. The amount of IFNg secreted by samples treated with the test antibody was calculated as a percentage of the maximum signal.

Treatment of MLR co-cultures with 2 μg/ml TNF control 6 antibody reduced IFNg secretion by approximately 82%. Treatment of MLR co-cultures with 5 μg/ml IL-7 control 1 antibody reduced IFNg secretion by approximately 81%. Treatment of MLR co-cultures with the combination of 2 μg/ml TNF control 6 + 5 μg/ml IL-7 control 1 antibodies reduced IFNg secretion by approximately 97%. Therefore, the combination of TNF control 6 + IL-7 control 1 exhibited enhanced activity in blocking IFNg secretion compared to either TNF control 6 or IL-7 control 1 antibody alone (Figure 8). IFNg inhibition of T7 HET mAb 1 compared to TNF control 1 or IL - 7 control 1 in MLR analysis.

Cryopreserved PBMCs (STEMCELL Technologies) from separate donors were thawed and resuspended at 1-2×10 ⁶ cells/ml. PBMCs from two donors were combined 1:1 and 100 μl was dispensed into replicate wells of a 96-well round bottom plate (n=9 different MLR co-culture combinations). Recombinant human IL-7 was added to the cultures at a final concentration of 10 ng/ml. Co-cultures were treated with increasing concentrations of the following antibodies: negative control, T7 HET mAb 1, TNF control 1, IL-7 control 1. Interleukin secretion was quantified using the Meso Scale Discovery Immunoassay Kit. Data were recorded and normalized (by subtraction) to the mean response of all concentrations relative to the negative control and the average of the replicates was taken. For IFNg, IL-6, and IL-8, a four-parameter logistic curve was fitted with the maximum response shared between multiple antibodies (Figure 9). IC50 estimates and minimum response for IFNg are reported (Table 11). Changes in IC50 and minimum response between T7 HETmAb and TNF control 1 and IL-7 control 1 are reported as fold changes, with no change tests performed with 95% CI and p values. All analyses were performed in R, version 4.0.3.

As shown in Figure 9 and summarized in Table 11, T7 HETmAb inhibited IFNg production in a concentration-dependent manner with an estimated IC50 = 60.5 pM and a 95% CI of (25.9 pM-140.9 pM). Similarly, treatment with TNF Control 1 resulted in an estimated IC50 = 50.0 pM and a 95% CI of (14.0 pM-178.6 pM). Therefore, despite the fact that T7 HETmAb has an arm that targets TNFa, there was no significant difference between the IC50s generated by the two molecules in the MLR analysis compared to TNF Control 1 (p = 0.6077) (Table 11). IL7 Control 1 inhibited IFNg production in a dose-dependent manner with an estimated IC50 = 10.3 pM and a 95% CI of (2.2-47.7) (Table 11). However, despite an IC50 approximately 6-fold lower than that of T7 HETmAb, IL7 control 1 achieved significantly lower IFNg inhibition (minimal response) at the highest dose tested (p = < 0.0001) (Table 11).

For IL-6, T7 HETmAb, TNF Control 1, and IL7 Control 1 all inhibited IL-6 production in a dose-dependent manner, and all three achieved similar levels of inhibition (minimal response) (Figure 9). As shown in Table 11, T7 HETmAb inhibited IL-6 with an IC50 of 8.8 pM and a 95% CI of (1.0-73.9). TNF Control 1 produced an IC50 of 1.7 pM and a 95% CI of (0.1-33.1). IL7 Control 1 produced an IC50 of 0.8 pM and a 95% CI of (0.01-56.1). As shown in Table 11, for IL-6, there was no statistical difference between the IC50s produced by T7 HETmAb and TNF Control 1 (p=0.3277) or IL7 Control 1 (p=0.2739).

For IL-8, T7 HETmAb inhibited IL-8 production with an estimated IC50 = 53.7 pM and 95% CI of (18.5 pM-155.5 pM). TNF Control 1 inhibited IL-8 with an estimated IC50 = 11.0 pM and 95% CI of (2.9 pM-41.7 pM) (Table 11). Therefore, for IL-8, T7 HETmAb was approximately 5-fold less potent than TNF Control 1 (p = 0.0192) (Table 11). IL7 Control 1 did not inhibit IL-8 production within the dose range tested, indicating that IL-8 is independent of IL-7 signaling (Figure 9 and Table 11). Table 11. Summary of IC50 and minimum response of T7 HETmAb , TNF Control 1 , and IL7 Control 1 for inhibition of interleukin production in MLR analysis Interleukin antibody IC50 (pM) IC50 lower limit CL IC50 Upper limit CL p-value Minimal response (% change relative to mean RSV response) p-value T7 HETmAb 1 60.5 25.9 140.9 -96.5 IFNγ TNF control 1 50.0 14.0 178.6 0.6077 -88.8 0.0041 IL7 comparison 1 10.3 2.2 47.7 0.0002 -76.0 ＜ 0.0001 T7 HETmAb 1 8.8 1.0 73.9 -97.9 IL-6 TNF control 1 1.7 0.1 33.1 0.3277 -97.2 0.7001 IL7 comparison 1 0.8 0.01 56.1 0.2739 -94.6 0.2335 T7 HETmAb 1 53.7 18.5 155.5 -78.1 IL-8 TNF control 1 11.0 2.9 41.7 0.0192 -74.8 0.4071 IL7 comparison 1 7.9* n/a n/a n/a -36.9 0.0006

IC50 Values are scaled to original interleukin concentrations ( that is , Give the concentration of the reaction between the minimum and maximum reaction ) Report , Trust limit is 95 % . P The value corresponds to log10 Interleukin concentration scale T7 HETmAb Of IC50 No changes in the test. * Please do not explain ; The curve is actually flat , Therefore, it is estimated IC50 Contains high uncertainty. Confidence interval is very large and not reported ( n / a ) The minimum response is reported as the change from the mean negative control response. % ; p The value corresponds to T7 HETmAb The test of minimal response without change.

The maximum IFNg inhibition observed by T7 HET mAb 1 in the dose-response MLR analysis was further analyzed by comparing each compound at the highest dose used, 5 μg/ml (Figure 9). Data were recorded and a mixed model was fitted in which the donor combination was a random effect for IFNg and the treatment was fixed. A Tukey test was performed to assess differences between compounds. If there were multiple observations for each donor combination, treatment, and experiment, the average was taken to avoid pseudoreplication. The designated inhibition percentage for each compound was calculated as the percentage reduction in interleukin secretion compared to the negative control (Figure 10 and Table 12). T7 HET mAb 1 inhibited IFNg secretion by 96.3% compared to the negative control. Compared to negative controls, TNF control 1 and IL-7 control 1 inhibited IFNg secretion by 87.45 and 69.4%, respectively. The maximum degree of inhibition achieved by T7 HET mAb was significantly greater than the inhibition observed with TNF control 1 (p = 0.016 ) or IL-7 control 1 (p < 0.001) (Table 12). Table 12. Statistical analysis of the maximum inhibition observed for IL - 7 control 1 , TNF control 1 , and T7 HET mAb compared to negative controls at a concentration of 5 μg / ml . Contrast P-value Fold change 95% fold change CI, lower limit 95% fold change CI, upper limit Significance level Inhibition percentage 95% inhibition percentage CI, lower limit 95% inhibition percentage CI, upper limit Negative control - IL7 control 1 0.021 3.265 1.155 9.228 * 69.4% 13.4% 89.2% Negative control - TNF control 1 0.000 7.918 2.802 22.378 **** 87.4% 64.3% 95.5% Negative control - T7 HET mAb 1 0.000 27.063 9.576 76.486 **** 96.3% 89.6% 98.7% In the MLR assay , T7 HETmAb 1 inhibited other soluble analytes compared to TNF control 1 or IL - 7 control 1 .

To identify other soluble markers for the additivity of T7 HETmAb 1, supernatants from MLR samples treated at the highest tested concentration (5 μg/ml) were analyzed using Luminex 65-plex compared to TNF control 1 or IL7 control 1. Analyte concentrations from Luminex analysis were log10 or sqrt transformed depending on the presence of zero (log10 if no zero, sqrt otherwise) and analyzed by a linear mixed effects model (fixed effect: treatment, random effect: MLR, MLR-treatment interaction) followed by Tukey post hoc comparisons. The fold changes in concentrations of HETmAb relative to isotype control or parental antibody shown in the heat map (Figure 11) were calculated by taking the ratio of the geometric means. An offset of 0.1 was added to all values to prevent individual values from being zero and setting the geometric mean to zero. Software used: R version 4.0.3. The heat map shown in Figure 11 shows (1) the fold change of T7 HETmAb analyte concentration relative to negative control, IL7 control and TNF control 1; (2) p-values for the no change test (* = p < 0.05 and ** = p < 0.01).

Compared with IL7 control 1 and TNF control 1, T7 HETmAb significantly inhibited the following analytes (Figure 11): • Interleukins : IL-17A, IFNγ, GM-CSF, IL-5, M-CSF, IL-13, LIF, IL-8, IL-1ɑ, IL-6, IL-10, IL-21 • Trending factors : MIP-1ɑ, MIP-1β, MCP1, MDC, CX3CL1, CXCL11, MIG, CCL24, CCL26 • Soluble receptors : CD40L, IL2R, TRAIL, TWEAK, TNFRII • Growth factors : MMP-1, VEGF-A In the MLR analysis , T7 HETmAb 1 inhibited IL - 7- dependent markers compared with TNF control 1 or IL - 7 control 1 .

To assess IL-7-dependent cell surface and cytoplasmic proteins, the above MLR assay was extended to a five-day co-culture assay. In this five-day MLR assay, IL-7-dependent CD4+ T cell expression of the gut-homing integrin α4β7, the anti-apoptotic factor Bcl-2, and the proliferation marker Ki-67 was detected using flow cytometry. After 5 days of incubation, cells were processed for flow cytometry. CD4+ cells were gated and the geometric mean fluorescence intensity of cell surface α4β7 and intracellular Bcl-2 and Ki-67 was measured. Data represent n=16 independent MLRs with technical replicates. For each MFI type, a separate linear mixed effects model was fitted, each model including antibody, stimulation and their interaction as fixed effects, and donor as a random effect. Contrasts were calculated by comparing the negative control with IL7 control 1, TNF control 1, and T7 HETmAb 1 (***p < 0.001, ****p < 0.0001, adjusted using Tukey's HSD). Analysis was performed using R 4.0.3. As shown in Figure 12, both T7 HETmAb 1 and IL7 control 1 reduced the expression of IL-7-induced α4β7, Bcl-2, and Ki-67 in CD4+ T cells compared to the negative control. There was no statistical difference between TNF control 1 and the negative control (Figure 12). Fc -mediated induction of M2 macrophages by T7 HETmAb in the MLR assay .

Anti-TNF antibodies have been shown to induce alternative activation of macrophages (termed M2 macrophages), which have anti-inflammatory activity through Fc receptor recognition. MLR analysis was used to compare the induction of M2 macrophages by T7 HETmAb and TNF control 6. Peripheral blood mononuclear cells (PBMCs) were enriched from whole blood by Ficol gradient centrifugation. PBMCs from donors 1-5 were stained with CellTrace Violet following the manufacturer's recommendations, and cells from donors 6-10 were stained with CellTrace CFSE. MLRs were established by mixing cells from donors 1-5 with cells from donors 6-10 in a 1:1 ratio. Samples were incubated at 37°C for 48 hours to establish an inflammatory environment. The cells were then supplemented with 100 μL of medium containing the test antibody (with or without FcR blockers) and the plates were incubated for an additional 5 days at 37°C. After the incubation period, the cells were washed as previously described and incubated with a cocktail of fluorescently labeled antibodies against surface antigens. Sample data were collected using a Cytek Aurora Spectral Flow Cytometer with SpectroFlo™ Software (v2.0). Prior to sample collection, QC beads were used to confirm optimal instrument settings. Data were analyzed using FlowJo software (v 10). Percent positive values (CD206+CD14+ cells) were exported to a Microsoft Excel spreadsheet using the TABLE Editor in FlowJo software. Cell populations were tabulated as % of parental in an Excel spreadsheet and then copied and pasted into GraphPad Prism (V9) software for plotting and statistical analysis. For statistical analysis, pairwise one-way ANOVA with LSD test was used (***p<0.005, ****p<0.0001).

TNF control 6 significantly increased the frequency of M2 macrophages in the MLR assay compared to PBS and negative control 1 treated co-cultures. T7 HETmAb had a similar effect and also significantly increased M2 macrophages. Blocking the Fc receptor by adding FcRX abolished the induction of M2 macrophages, demonstrating that this effect is dependent on Fc receptor signaling (Figure 13).

The invention is also described in the following numbered clauses. 1. A method for treating an autoimmune and/or inflammatory condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL-7 inhibitor and a therapeutically effective amount of a TNF-α inhibitor. 2. The method of clause 1, wherein the therapeutically effective amount of the IL-7 inhibitor is a therapeutically effective amount of an IL-7 binding domain and the therapeutically effective amount of the TNF-α inhibitor is a therapeutically effective amount of a TNF-α binding domain. 3. The method of clause 1 or 2, wherein the subject is a human subject. 4. The method of any one of clauses 1-3, wherein the autoimmune and/or inflammatory condition is an inflammatory skin disease (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, cardiac arrest, heart failure, Postoperative reperfusion and post-percutaneous transluminal coronary angioplasty for stenosis, stroke, and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; tissue /immunopathological reaction to organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; or any combination thereof. 5. The method of clause 4, wherein the autoimmune and/or inflammatory condition is inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; or any combination thereof. 6. The method of clause 5, wherein the autoimmune or inflammatory condition is inflammatory bowel disease (IBD). 7. The method of any of clauses 2-6, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are administered sequentially or simultaneously. 8. The method of clause 7, wherein the administration is sequential. 9. The method of clause 7, wherein the administration is simultaneous. 10. The method of clause 9, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are contained in the same pharmaceutical composition. 11. The method of any of clauses 2-9, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are contained in different pharmaceutical compositions. 12. The method of any one of clauses 2-11, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are independently administered intra-arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, or intraperitoneally. 13. The method of clause 12, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are both administered subcutaneously. 14. An IL-7 inhibitor and a TNF-α inhibitor for use in treating autoimmune and/or inflammatory conditions. 15. An IL-7 inhibitor and a TNF-α inhibitor as used in clause 14, wherein the IL-7 inhibitor is an IL-7 binding domain and the TNF-α inhibitor is a TNF-α binding domain. 16. The use of clause 14 or 15, wherein the autoimmune and/or inflammatory condition is inflammatory skin disease (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, cardiac arrest, cardiac surgery Stenosis, stroke, and abdominal aortic aneurysm after reperfusion and percutaneous transluminal coronary angioplasty; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune Immune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; tissue /immunopathological reaction of organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; or any combination thereof. 17. The use of clause 16, wherein the autoimmune and/or inflammatory condition is inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, and any combination thereof. 18. The use of clause 17, wherein the autoimmune and/or inflammatory condition is inflammatory bowel disease (IBD). 19. The use of any one of clauses 15 to 18, wherein the IL-7 binding domain and the TNF-α binding domain are administered sequentially or simultaneously. 20. The use of clause 19, wherein the administration is sequential. 21. The use of clause 19, wherein the administration is simultaneous. 22. The use of clause 21, wherein the therapeutically effective amount of the IL-7 binding domain and the therapeutically effective amount of the TNF-α binding domain are contained in the same pharmaceutical composition. 23. The use of any one of clauses 15-21, wherein the IL-7 binding domain and the TNF-α binding domain are contained in different pharmaceutical compositions. 24. The use of any one of clauses 15-23, wherein the IL-7 binding domain and the TNF-α binding domain are administered independently intra-arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously or intraperitoneally. 25. The use of clause 24, wherein the IL-7 binding domain and the TNF-α binding domain are both administered subcutaneously. 26. The method or use of any one of clauses 2-13 and 15-25, wherein the IL-7 binding domain binds to one or more amino acid residues within the amino acid sequence of human IL-7, SEQ ID NO: 12. 27. The method or use of any one of clauses 2-13 and 15-26, wherein the IL-7 binding domain protects residues 67 to 81 of IL-7 (SEQ ID NO: 12) from deuterium exchange in HDX-MS analysis. 28. The method or use of clause 27, wherein the IL-7 binding domain protects residues 67 to 80 of IL-7 (SEQ ID NO: 16) from deuterium exchange in HDX-MS analysis. 29. The method or use of any of clauses 2-13 and 15-28, wherein the IL-7 binding domain binds to human IL-7 proximal to the IL-7Rα binding site with a KD of about 100 nM or less as measured by surface plasmon resonance analysis. 30. The method or use of any of clauses 2-13 and 15-29, wherein the IL-7 binding domain inhibits the binding of IL-7 to IL-7R as measured in an in vitro competitive binding assay as determined using surface plasmon resonance analysis. 31. The method or use of any one of clauses 2-13 and 15-30, wherein the IL-7 binding domain comprises CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11. 32. The method or use of clause 31, wherein the IL-7 binding domain comprises a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5. 33. The method or use of clause 32, wherein the IL-7 binding domain comprises a _VH domain of SEQ ID NO: 4 and a _VL domain of SEQ ID NO: 5. 34. The method or use of any one of clauses 2-13 and 15-33, wherein the IL-7 binding domain comprises a constitutive region such that the IL-7 binding domain has reduced ADCC and/or complement activation or effector function. 35. The method or use of clause 34, wherein the IL-7 binding domain comprises a heavy chain Fc domain having alanine residues at positions 235 and 237 according to EU numbering. 36. The method or use of any one of clauses 2-13 and 15-35, wherein the IL-7 binding domain comprises a backbone selected from the group consisting of a human IgG1 isotype and a human IgG4 isotype. 37. The method or use of clause 36, wherein the IL-7 binding domain is a human IgG1 isotype. 38. The method or use of any one of clauses 2-13 and 15-37, wherein the IL-7 binding domain is an antibody. 39. The method or use of clause 38, wherein the IL-7 binding domain is a monoclonal antibody. 40. The method or use of clause 39, wherein the monoclonal antibody is human, humanized or chimeric. 41. The method or use of any of clauses 2-13 and 15-40, wherein the IL-7 binding domain comprises a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 2 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 3. 42. The method or use of clause 41, wherein the IL-7 binding domain comprises a heavy chain of SEQ ID NO: 2 and a light chain of SEQ ID NO: 3. 43. The method or use of any of clauses 2-13 and 15-42, wherein the IL-7 binding domain binds to IL-7 with a KD of 50 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. 44. The method or use of any of clauses 2-13 and 15-43, wherein the IL-7 binding domain binds to IL-7 and (i) inhibits IL-7-dependent IFN-γ or IL-10 secretion by peripheral blood mononuclear cells with an _IC50 of 1 nM or less, and/or (ii) inhibits IL-7-dependent STAT5 phosphorylation in CD4+ T cells with an _IC50 of 1 nM or less. 45. The method or use of any one of clauses 2-13 and 15-44, wherein the IL-7 binding domain inhibits signaling, activation, interleukin production and/or proliferation of CD4 ⁺ T cells and/or CD8 ⁺ T cells. 46. The method or use of any one of clauses 2-13 and 15-45, wherein the TNF-α binding domain comprises adalimumab, golimumab, infliximab, certolizumab or entrecept. 47. The method or use of clause 46, wherein the TNF-α binding domain comprises adalimumab or adalimumab with M252Y/S254T/T256E modifications. 48. The method or use of any one of clauses 2-13 and 15-45, wherein the TNF-α binding domain comprises CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. 49. The method or use of clause 48, wherein the TNF-α binding domain comprises a VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and a VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. 50. The method or use of clause 49, wherein the TNF-α binding domain comprises a VH domain having the amino acid sequence of SEQ ID NO: 54 and a VL domain having the amino acid sequence of SEQ ID NO: 55. 51. The method or use of clauses 2-13 and 15-50, wherein the TNF-α binding domain binds to TNF-α and reduces macrophage activation, reduces interleukin and cytokine production, inhibits intestinal epithelial cell death, or maintains intestinal barrier function. 52. The method or use of clauses 2-13 and 15-51, wherein the TNF-α binding domain reduces inflammatory responses by blocking TNF-mediated signaling. 53. The method or use of any one of clauses 2-13 and 15-52, wherein the IL-7 binding domain comprises CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and the TNF-α binding domain comprises CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. 54. The method or use of any one of clauses 2-13 and 15-53, wherein the IL-7 binding domain comprises a VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and the TNF-α binding domain comprises a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and a _VL domain having at least 90% identity to the VL amino acid sequence of SEQ ID NO: 55. 55. The method or use of any one of clauses 2 to 54, wherein the IL-7 binding domain comprises a heavy chain of SEQ ID NO: 2 and a light chain of SEQ ID NO: 3, and the TNF-α binding domain comprises adalimumab or adalimumab with M252Y/S254T/T256E modifications. 56. The method or use of any of clauses 2-13 and 15-55, wherein the IL-7 binding domain and the TNF-α binding domain are in the same protein, or wherein the IL-7 binding domain and the TNF-α binding domain are in different proteins. 57. The method or use of clause 56, wherein the IL-7 binding domain and the TNF-α binding domain are in different proteins. 58. The method or use of clause 57, wherein the IL-7 binding domain and the TNF-α binding domain are in the same protein. 59. The method or use of clause 58, wherein the IL-7 binding domain and the TNF-α binding domain are in a bispecific antibody. 60. The method or use of clause 59, wherein the bispecific antibody is selected from the group consisting of: bispecific antibodies, L-antibodies, common light chain antibodies, antibodies with a modified cysteine bridge between the heavy chain and the light chain, chimeric heavy chain/light chain antibodies, Cross-Mab, mAb pairs and Het-mAb. 61. The method or use of clause 60, wherein the bispecific antibody is a Het-mAb. 62. An IL-7 and TNF-α binding protein comprising: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. 63. The IL-7 and TNF-α binding protein of clause 62, wherein the binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10 and/or CDRL3 of SEQ ID NO: 11; and b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52 and CDRL3 of SEQ ID NO: 53. 64. The IL-7 and TNF-α binding protein of clause 62 or 63, wherein the binding protein comprises a) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and/or b) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. 65. The IL-7 and TNF-α binding protein of clause 64, wherein the binding protein comprises a) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and b) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. 66. The IL-7 and TNF-α binding protein of clause 65, wherein the binding protein comprises a) _{a VH} domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and b) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. 67. The IL-7 and TNF-α binding protein of any one of clauses 62 to 66, wherein the binding protein comprises a bispecific antibody. 68. The IL-7 and TNF-α binding protein of clause 67, wherein the bispecific antibody is a bispecific antibody, an L-antibody, a common light chain antibody, an antibody with a modified cysteine bridge between the heavy chain and the light chain, a chimeric heavy chain/light chain antibody, a Cross-Mab, a mAb pair, and a Het-mAb. 69. The IL-7 and TNF-α binding protein of clause 68, wherein the antibody is a HET mAb. 70. The IL-7 and TNF-α binding protein of any one of clauses 62 to 69, wherein the antibody further comprises an Fc mutation to extend half-life. 71. The IL-7 and TNF-α binding protein of clause 70, wherein the Fc mutation is YTE. 72. The IL-7 and TNF-α binding protein of any one of clauses 62 to 71, wherein the antibody further comprises an Fc mutation disabling effector function. 73. The IL-7 and TNF-α binding protein of clause 72, wherein the Fc mutation is LAGA. 74. The IL-7 and TNF-α binding protein of clauses 62 to 73, wherein the antibody comprises: a) a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 57 or 61 and/or a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 59 or 63; and/or b) a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 56 or 60 and/or a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 58 or 62. 75. The IL-7 and TNF-α binding protein of clause 74, wherein the antibody comprises: a) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 or 63; and/or b) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO 58 or 62. 76. The IL-7 and TNF-α binding protein of clause 75, wherein the antibody comprises: a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 59 or 63; and a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID 58 or 62. 77. The IL-7 and TNF-α binding protein of clause 76, wherein the antibody comprises: a heavy chain having an amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having an amino acid sequence of SEQ ID NO: 59 or 63; and a heavy chain having an amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having an amino acid sequence of SEQ ID NO: 58 or 62. 78. The IL-7 and TNF-α binding protein of clause 77, wherein the antibody comprises: a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 57 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 59; and a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 56 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 58. 79. The IL-7 and TNF-α binding protein of clause 78, wherein the antibody comprises: a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59; and a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58. 80. A nucleic acid sequence encoding an IL-7 and TNF-α binding protein as defined in any one of clauses 62 to 79. 81. An expression vector comprising a nucleic acid sequence of clause 80. 82. A recombinant host cell comprising a nucleic acid sequence of clause 80 or an expression vector of clause 81. 83. A method for producing an IL and TNF-α binding protein, comprising culturing a recombinant host cell as in claim 82 under conditions suitable for expressing the nucleic acid sequence(s) or vector, thereby producing a polypeptide comprising the IL-7 and TNF-α binding protein. 84. An IL-7 and TNF-α binding protein produced by the method of claim 83. 85. A cell line engineered to express the IL-7 and TNF-α binding protein of any one of claims 62 to 79 and 84. 86. A pharmaceutical composition comprising an IL-7 and TNF-α binding protein as defined in any one of claims 62 to 79 or claim 84 and a pharmaceutically acceptable excipient. 87. A pharmaceutical composition as in claim 86 comprising an IL-7 and TNF-α binding protein as defined in claim 79. 88. The IL-7 and TNF-α binding protein of any one of clauses 62-79 or clause 84 or the pharmaceutical composition of clause 86 or 87 for use in the treatment of an autoimmune and/or inflammatory disease. 89. The IL-7 and TNF-α binding protein of clause 88, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions , such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; bone Arthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; Diseases involving leukocyte infiltration; Inflammatory disorders of the central nervous system (CNS); Multiple organ injury syndrome secondary to sepsis or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex-mediated diseases, including glomerular nephritis; Sepsis; Sarcoma disease; immunopathological reaction to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; and any combination thereof. 90. The IL-7 and TNF-α binding protein for use in clause 89, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. 91. The IL-7 and TNF-α binding protein as used in clause 90, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). 92. A method for treating an autoimmune and/or inflammatory disease in a human subject in need thereof, comprising administering a therapeutically effective amount of the IL-7 and TNF-α binding protein as described in any one of clauses 62-79 or clause 84, or a pharmaceutical composition as described in clause 86 or 87. 93. A method for treating an autoimmune and/or inflammatory disease as claimed in claim 92, wherein the autoimmune or inflammatory condition is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, Myocardial infarction, cardiac arrest, postoperative reperfusion and postoperative stenosis after percutaneous transluminal coronary angioplasty, stroke and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis Arthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; Diseases involving leukocyte infiltration; Inflammatory disorders of the central nervous system (CNS); Multiple organ injury syndrome secondary to sepsis or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex-mediated diseases, including glomerular nephritis; Sepsis; Sarcoma 94. The method of clause 93, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. 95. The method of clause 94, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). 96. Use of an IL-7 and TNF-α binding protein as defined in any of clauses 62-79 or in clause 84 or a pharmaceutical composition as defined in clause 86 or 87 for the manufacture of a medicament for the treatment of an autoimmune and/or inflammatory condition. 97. Use of an IL-7 inhibitor and a TNF-α inhibitor or an IL-7 and TNF-α binding protein for the manufacture of a medicament for the treatment of an autoimmune and/or inflammatory disease. 98. Use as defined in clause 97, wherein the IL-7 inhibitor is an IL-7 binding domain and the TNF-α inhibitor is a TNF-α binding domain. 99. The use of clause 97 or 98, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, heart attack, Cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus Nephritis; autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; Immunopathological reactions to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, pleuritis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes; and any combination thereof. 100. The use of clause 99, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. 101. The use of clause 100, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). 102. The method or use of clause 59, wherein the bispecific antibody comprises: a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 59 or 63; and a heavy chain having at least 90% identity to the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity to the amino acid sequence of SEQ ID 58 or 62. 103. The method or use of clause 102, wherein the bispecific antibody comprises: a heavy chain having an amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having an amino acid sequence of SEQ ID NO: 59 or 63; and a heavy chain having an amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having an amino acid sequence of SEQ ID NO: 58 or 62. 104. The method or use of clause 103, wherein the bispecific antibody comprises: a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having an amino acid sequence of SEQ ID NO: 59; and a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58. SEQ ID NO: 1 : Human IL - 7 sequence with leader sequence MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH SEQ ID NO: 2 : DRSPAI-L7B heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGVH WVRQAPGKGLEWLA AIWTGGSTDYNSAFSS RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR NGYGESFAY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3 : DRSPAI-L7B light chain DIQMTQSPSSLSASVGDRVTITC KASESLDHDGDSYIN 5 : DRSPAI-L7B V _H QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYGVH WVRQAPGKGLEWLAAIWTGGSTDYNSAFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAY WGQGTLVTVSS SEQ ID NO: 5 : DRSPAI - L7B V _L DIQMTQSPSSLSASVGDRVTITC KASESLDHDGDSYIN : 10 : DRSPAI-L7B CDRL2 MGSNVEF SEQ ID NO: 11 : DRSPAI - L7B CDRL3 QQSNVDPLT SEQ ID NO : 12 : DRSPAI - L7B protected site 1 FKRHICDANKEGMFL SEQ ID NO : 13 : Nucleic acid sequence encoding DRSPAI - L7B light chain SEQ ID NO: 14 : Nucleic acid sequence encoding DRSPAI - L7B heavy chain SEQ ID NO: 15 : Nucleic acid sequence encoding signaling peptide ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGTGTGCACAGC SEQ ID NO: 16 : DRSPAI-L7B second protected site FKRHICDANKEGMF SEQ ID NO: 17 : Human IL - 7 sequence ( without secretory leader sequence ) DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH SEQ ID NO: 18 : A1290 light chain DIQMTQSPSSLSASVGDRVTITCKASQSVDDDGDSFINWYQQKPGKAPKLLIYVASNLESGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 19 : A1290 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWLAAIWTGGSTDYNAAFISRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20 : A1291 light chain DIQMTQSPSSLSASVGDRVTITCKASHSVDDDGDSYMNWYQQKPGKAPKLLIYMASNLESGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 21 : A1291 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWLAAIWTGGSTDYNAEFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 22 : A1294 light chain DIQMTQSPSSLSASVGDRVTITCKASQSVDDDGDSYMNWYQQKPGKAPKLLIYMASNLESGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23 : A1294 heavy chain QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGVHWVRQAPGKGLEWLAAIWSGGSTDYNVAFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 28 ： A1290 CDRH3 _NGYGESFAY SEQ ID _NO : 29 ​ ​ ​ ​ ​ ​ 33 ： A1291 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWLAAIWTGGSTDYNAEFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSS SEQ ID NO : 34 ： A1291 _VL DIQMTQSPSSLSASVGDRVTITCKASHSVDDDGDSYMNWYQQKPGKAPKLLIYMASNLESGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGGGTKVEIK SEQ ID NO : 35 ： _A1291 VH QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGLHWVRQAPGKGLEWLAAIWTGGSTDYNAEFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSS SEQ ID NO : 36 35 : A1291 CDRH2 AIWTGGSTDYNAEFSS SEQ ID NO : 36 : A1291 CDRH3 NGYGESFAY SEQ ID NO : 37 : A1291 CDRL1 KASHSVDDDGDSYMN SEQ ID NO: 38 : A1291 CDRL2 MASNLES SEQ ID NO: 39 : A1291 CDRL3 QQSNEDPLT SEQ ID NO: 40 : A1294 V _L DIQMTQSPSSLSASVGDRVTITCKASQSVDDDGDSYMNWYQQKPGKAPKLLIYMASNLESGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGGGTKVEIK SEQ ID NO: 41 : A1294 V _H 45 ： A1294 CDRL1 TYGVH SEQ ID NO: 46 ： A1294 CDRH2 AIWSGGSTDYNVAFSS SEQ ID NO: 47 ： A1294 CDRH3 NGYGESFAY SEQ ID NO: 48 ： A1294 CDRL1 KASQSVDDDGDSYMN SEQ ID NO: 49 ： A1294 CDRL2 MASNLES SEQ ID NO: 50 ： A1294 CDRL3 QQSNEDPLT SEQ ID NO: 51 ： A1294 CDRH1 TYGVH SEQ ID NO: 52 ： A1294 CDRH2 AIWSGGSTDYNVAFSS SEQ ID NO: 53 ： A1294 CDRH3 NGYGESFAY SEQ ID NO: 54 ： A1294 CDRL1 KASQSVDDDGDSYMN SEQ ID NO: 55 ： A1294 CDRL2 MASNLES SEQ ID NO: 56 ： A1294 CDRL3 QQSNEDPLT SEQ ID NO: 57 ： A1294 CDRH 54 TNF VH EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS SEQ ID NO: 55 TNF VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK SEQ ID NO:56 HC1 , HET mAb 1 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCEVTDYFPEPVTVSWNSGALTSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:57 HC2 , HET mAb 1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGVHWVRQAPGKGLEWLAAIWTGGSTDYNSAFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCRVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:58 LC1 , HET mAb 1 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDERLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:59 LC2 , HET mAb 1 DIQMTQSPSSLSASVGDRVTITCKASESLDHDGDSYINWYQQKPGKAPKLLIYMGSNVEFGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNVDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEELKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:60 HC1 , HET mAb 2 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCEVTDYFPEPVTVSWNSGALTSGVHTFPAVLESSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:61 HC2 , HET mAb 2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGVHWVRQAPGKGLEWLAAIWTGGSTDYNSAFSSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNGYGESFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCRVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELAGAPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:62 LC1 , HET mAb 2 DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDERLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSRLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:63 LC2 , HET mAb 2 DIQMTQSPSSLSASVGDRVTITCKASESLDHDGDSYINWYQQKPGKAPKLLIYMGSNVEFGVPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNVDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEELKSGTASVECLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The novel features of the present invention are described in detail in the accompanying claims. A better understanding of the features and advantages will be obtained by reference to the following embodiments describing illustrative embodiments and the accompanying drawings, in which: FIG. 1A : shows the concentration of DRSPAI-L7B in animals dosed (intravenously) with a target dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg of DRSPAI-L7B. FIG. 1B and FIG . 1C show the concentration of subcutaneous weekly dosing at repeated target doses of 30 mg/kg, FIG . 1B follows dose 1, and FIG . 1C follows dose 4. Serum samples were collected at designated time points and DRSPAI-L7B was quantified by Gyrolab immunoassay. Figure 2A : shows pSTAT5 inhibition % from whole blood obtained from healthy donors stimulated with IL-7 in the presence of DRSPAI-L7B. Figure 2B shows STAT5 phosphorylation in CD8+ T cells from peripheral blood mononuclear cells (PBMC), Figure 2C shows STAT5 phosphorylation in CD4+ T cells from peripheral blood mononuclear cells (PBMC), and Figure 2D shows STAT5 phosphorylation in CD3+ T cells from peripheral blood mononuclear cells (PBMC), obtained from healthy donors or IBD patients stimulated with rhIL-7 in the presence of DRSPAI-L7B or anti-RSV antibodies (isotype control). Figure 3A : shows inhibition of IFN-γ secretion by healthy donor PBMCs treated with increasing concentrations of DRSPAI-L7B in the presence of rhIL-7 and anti-CD3. Figure 3B shows inhibition of IL-10 secretion by healthy donor PBMC treated with increasing concentrations of DRSPAI-L7B in the presence of rhIL-7 and anti-CD3. Figures 3C , 3D , 3E , 3F , 3G show inhibition of IL-2 by DRSPAI-L7B in the presence of rhIL-7 and anti-CD3. Figure 4A : shows inhibition of IL-17 in CD4 ⁺ T _mem cells isolated from healthy donor blood and incubated with IL-7 in the presence of DRSPAI-L7B after addition of phorbol myristate acetate (PMA)/ionomycin. Figure 4B shows TNFα inhibition in CD4 ⁺ T _mem cells isolated from healthy donor blood, incubated with IL-7 in the presence of DRSPAI-L7B after addition of PMA/ionomycin. Figure 4C : shows IL-6 inhibition in CD4 ⁺ T _mem cells isolated from healthy donor blood, incubated with IL-7 in the presence of DRSPAI-L7B after addition of PMA/ionomycin. Figure 4D : shows IL-10 inhibition in CD4 ⁺ T _mem cells isolated from healthy donor blood, incubated with IL-7 in the presence of DRSPAI-L7B after addition of PMA/ionomycin. FIG4E : shows INFγ inhibition in CD4 ⁺ T _mem cells isolated from healthy donor blood, incubated with IL-7 in the presence of DRSPAI-L7B after addition of PMA/ionomycin. FIG4F : shows CCL3 inhibition in CD4 ⁺ T _mem cells isolated from healthy donor blood, incubated with IL-7 in the presence of DRSPAI-L7B after addition of PMA/ionomycin. FIG5A : shows the profile of CD4 ⁺ lymphocytes from healthy controls and MS patients analyzed by flow cytometry based on the expression of CD45RO, CCR7, CD127 and CD25 on the cell surface. Figure 5B shows the profile of CD8 ⁺ lymphocytes from healthy controls and MS patients analyzed by flow cytometry based on the expression of CD45RO, CCR7, CD127 and CD25 on the cell surface. Figure 5C shows the profile of regulatory T cells from healthy controls and MS patients analyzed by flow cytometry based on the expression of CD45RO, CCR7, CD127 and CD25 on the cell surface. Figure 6A : Shows STAT5 phosphorylation in CD4 + T cells from PBMCs obtained from healthy donors or MS patients stimulated with rhIL-7 in the presence of DRSPAI-L7B or anti-RSV antibodies (isotype control). FIG . 6B shows STAT5 phosphorylation in CD8 ⁺ T cells from PBMCs obtained from healthy donors or MS patients stimulated with rhIL-7 in the presence of DRSPAI-L7B or anti-RSV antibodies (isotype control). FIG. 7A : shows a linear curve, and FIG. 7B shows a logarithmic coordinate graph of IL-7 levels in serum samples from healthy controls (HC, n=10), Crohn's disease (n=15), ulcerative colitis (UC, n=15), systemic lupus erythematosus (SLE, n=15) and primary Sjögren's syndrome (pSS, n=15) patients. FIG. 8 : shows the inhibition of IFNg production by TNF-α control molecules and IL-7 control molecules alone or in combination. Figure 9 : Compound dose response curves in a mixed lymphocyte reaction assay are shown for T7 HetmAb 1, TNF control 1, and IL-7 control 1. Figure 10 : Maximum inhibition of IFNg secretion achieved in a mixed lymphocyte reaction assay is shown. Figure 11 : Identification of other markers of additivity of T7 HETmAb in an MLR assay is shown. Figure 12 : Inhibition of α4β7, Bcl-2, and Ki-67 expression by T7 HETmAb compared to IL7 control 1 and TNF control 1 in an MLR assay is shown. Figure 13 : Induction of M2 macrophages by T7 HETmAb and TNF control 6 in an MLR assay is shown.

TW202413441A_112119254_SEQL.xml

Claims (39)

An IL-7 and TNF-α binding protein comprising: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10, and CDRL3 of SEQ ID NO: 11; and/or b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52, and CDRL3 of SEQ ID NO: 53. The IL-7 and TNF-α binding protein of claim 1, wherein the binding protein comprises: a) CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10 and/or CDRL3 of SEQ ID NO: 11; and b) CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52 and CDRL3 of SEQ ID NO: 53. The IL-7 and TNF-α binding protein of claim 1 or 2, wherein the binding protein comprises a) _{a VH} domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and/or b) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and/or a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. The IL-7 and TNF-α binding protein of claim 3, wherein the binding protein comprises a) a _VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 4 and/or a _VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 5; and b) a _VH domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 54 and/or a _VL domain having at least 90% identity with the amino acid sequence of SEQ ID NO: 55. The IL-7 and TNF-α binding protein of claim 4, wherein the binding protein comprises a) _{a VH} domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 4 and a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 5; and b) a _VH domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 54 and a _VL domain having at least 90% identity to the amino acid sequence of SEQ ID NO: 55. The IL-7 and TNF-α binding protein of any one of claims 1 to 5, wherein the binding protein comprises a bispecific antibody. The IL-7 and TNF-α binding protein of any one of claims 1 to 6, wherein the binding protein further comprises an Fc mutation to extend half-life. The IL-7 and TNF-α binding protein of claim 7, wherein the Fc mutation is YTE. The IL-7 and TNF-α binding protein of any one of claims 1 to 8, wherein the binding protein further comprises an Fc mutation that disables effector function. The IL-7 and TNF-α binding protein of claim 9, wherein the Fc mutation is LAGA. The IL-7 and TNF-α binding protein of claim 1 to 10, wherein the antibody comprises: c) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and/or a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 or 63; and/or d) a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and/or a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO 58 or 62. The IL-7 and TNF-α binding protein of claim 11, wherein the antibody comprises: a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 57 or 61 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 59 or 63; and a heavy chain having at least 90% identity with the amino acid sequence of SEQ ID NO: 56 or 60 and a light chain having at least 90% identity with the amino acid sequence of SEQ ID 58 or 62. The IL-7 and TNF-α binding protein of claim 12, wherein the antibody comprises: a heavy chain having an amino acid sequence of SEQ ID NO: 57 and a light chain having at least an amino acid sequence of SEQ ID NO: 59; and a heavy chain having an amino acid sequence of SEQ ID NO: 56 and a light chain having an amino acid sequence of SEQ ID NO: 58. An IL-7 and TNF-α bispecific antibody comprising: an IL-7 binding domain comprising CDRH1 of SEQ ID NO: 6, CDRH2 of SEQ ID NO: 7, CDRH3 of SEQ ID NO: 8, CDRL1 of SEQ ID NO: 9, CDRL2 of SEQ ID NO: 10 and/or CDRL3 of SEQ ID NO: 11; and a TNF-α binding domain comprising CDRH1 of SEQ ID NO: 48, CDRH2 of SEQ ID NO: 49, CDRH3 of SEQ ID NO: 50, CDRL1 of SEQ ID NO: 51, CDRL2 of SEQ ID NO: 52 and CDRL3 of SEQ ID NO: 53. The bispecific antibody of claim 15, wherein the bispecific antibody is a bispecific antibody, an L-antibody, a common light chain antibody, an antibody with a modified cysteine bridge between the heavy chain and the light chain, a chimeric heavy chain/light chain antibody, a Cross-Mab, a mAb pair, or a Het-mAb. The bispecific antibody of claim 14, wherein the bispecific antibody is Het-mAb. A nucleic acid sequence encoding an IL-7 and TNF-α binding protein as defined in any one of claims 1 to 16. An expression vector comprising a nucleic acid sequence as defined in claim 17. A recombinant host cell comprising the nucleic acid sequence of claim 17 or the expression vector of claim 18. A method for producing an IL-7 and TNF-α binding protein, comprising culturing a recombinant host cell as defined in claim 19 under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising the IL-7 and TNF-α binding protein. An IL-7 and TNF-α binding protein produced by the method of claim 20. A cell line engineered to express the IL-7 and TNF-α binding protein of any one of claims 1 to 16 and 21. A pharmaceutical composition comprising an IL-7 and TNF-α binding protein as defined in any one of claims 1 to 16 or claim 21 and a pharmaceutically acceptable formulation. A pharmaceutical composition as claimed in claim 23, comprising the IL-7 and TNF-α binding protein as defined in claim 13. An IL-7 inhibitor and a TNF-α inhibitor for use in treating autoimmune and/or inflammatory conditions. The IL-7 and TNF-α binding protein of any one of claims 1 to 16 or claim 21 or the pharmaceutical composition of claim 23 or 24 for use in treating autoimmune and/or inflammatory diseases. The IL-7 and TNF-α binding protein as used in claim 25 or 26, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease (Crohn's disease); ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease Disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjogren’s syndrome (Sjogren’s syndrome), vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; immunopathological reactions to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonia, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome syndrome, GBS); COPD; type 1 diabetes; and any combination thereof. The IL-7 and TNF-α binding protein for use as claimed in claim 27, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. The IL-7 and TNF-α binding protein as used in claim 28, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). A method of treating an autoimmune and/or inflammatory condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of an IL-7 inhibitor and a therapeutically effective amount of a TNF-α inhibitor. A method for treating an autoimmune and/or inflammatory disease in a human subject in need thereof, comprising administering a therapeutically effective amount of an IL-7 and TNF-α binding protein as described in any one of claims 1 to 16 or claim 21, or a pharmaceutical composition as described in claim 23 or 24. A method for treating an autoimmune and/or inflammatory disease as claimed in claim 31 or 32, wherein the autoimmune or inflammatory condition is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemia Conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; Osteoarthritis; Lupus nephritis; Autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; Diseases involving leukocyte infiltration; Inflammatory disorders of the central nervous system (CNS); Multiple organ injury syndrome secondary to sepsis or trauma; Alcoholic hepatitis; Bacterial pneumonia; Antigen-antibody complex-mediated diseases, including glomerular nephritis; Sepsis; Myxoid neoplasia; immunopathological reaction to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes mellitus; and any combination thereof. The method of claim 32, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. The method of claim 33, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD). Use of an IL-7 and TNF-α binding protein as defined in any one of claims 1 to 16 or claim 21, or a pharmaceutical composition as defined in claim 23 or 24, for the manufacture of a medicament for the treatment of autoimmune and/or inflammatory conditions. A use of an IL-7 inhibitor and a TNF-α inhibitor or an IL-7 and TNF-α binding protein for the manufacture of a medicament for treating autoimmune and/or inflammatory diseases. The use of claim 35 or 36, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory skin diseases (including psoriasis and atopic dermatitis); systemic scleroderma and sclerosis; inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; ischemic reperfusion disorders, including surgical tissue reperfusion injury, myocardial ischemic conditions, such as myocardial infarction, Cardiac arrest, reperfusion after cardiac surgery and stenosis after percutaneous transluminal coronary angioplasty, stroke and abdominal aortic aneurysm; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; wolf Ulcerative nephritis; autoimmune diseases such as rheumatoid arthritis (RA), spondylitis, Sjögren's syndrome, vasculitis; diseases involving leukocyte infiltration; inflammatory disorders of the central nervous system (CNS); multiple organ injury syndrome secondary to sepsis or trauma; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex-mediated diseases, including glomerular nephritis; sepsis; sarcoidosis; Immunopathic reaction to tissue/organ transplantation; pulmonary inflammation (including pleurisy, alveolitis, esophageal inflammation, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchitis, allergic pneumonitis, idiopathic pulmonary fibrosis (IPF) and cystic fibrosis); psoriasis arthritis; neuromyelitis optica; Guillain-Barre syndrome (GBS); COPD; type 1 diabetes mellitus; and any combination thereof. The use of claim 37, wherein the autoimmune and/or inflammatory disease is selected from the group consisting of: inflammatory bowel disease (IBD); Crohn's disease; ulcerative colitis; and any combination thereof. The use of claim 38, wherein the autoimmune and/or inflammatory disease is inflammatory bowel disease (IBD).