Bifunctional antibody for promoting wound healing reaction and pharmaceutical composition thereof

Abstract

The invention relates to the technical field of biology, in particular to a bifunctional antibody for promoting wound healing reaction and a pharmaceutical composition thereof, at least comprising: a first protein functional region targeting CD4, the first protein functional region being an anti-CD4 antibody or an antigen binding fragment thereof; a second protein functional region, the second protein functional region targeting TGF [beta] 1 / [beta] 2 (especially not targeting TGF [beta] 3), the second protein functional region being an anti-TGF [beta] 1 / [beta] 2 (especially not anti-TGF [beta] 3) antibody or an antigen binding fragment thereof; the double antibody can be safely combined with CD4, but the function of the CD4 is not influenced; by combining with CD4, the double antibody can specifically inhibit a TGF-beta1 signal of CD4 + T cells, so that the inhibition effect of TGF-beta1 on the CD4 + T cells is relieved, and the wound healing reaction mediated by the TGF-beta1 is released; meanwhile, the double antibodies can inhibit TGF-beta2 so as to inhibit the fibrosis promotion capability of the TGF-beta2, and have no inhibition capability on TGF-beta3, so that the wound healing promotion capability of the double antibodies is reserved, and the double antibodies can be used for reducing the scar area after wound healing.

Description

Technical Field

[0001] This invention patent belongs to the field of biotechnology, specifically relating to a bifunctional antibody and its pharmaceutical composition that promote wound healing response. Background Technology

[0002] Hypertrophic scars from burns are very common. These scars are particularly prevalent after burns and are typically raised, red, hardened, and may cause abnormal sensations. This pathological scarring can lead to severe functional impairment, psychological distress, and costly long-term healthcare.

[0003] Wound healing is the intrinsic process by which skin is restored to its integrity after injury. While scarring is a common byproduct, the observation of scarless wound healing in early human fetuses suggests that it is not a significant component of this response. This has led to a large body of research attempting to understand the mechanisms behind scar formation and, in turn, prevent it.

[0004] TGFβ was first identified in 1981 (Robert et al., PNAS, 78: 5339-5343. (1981)). Transforming growth factor β is a multifunctional cytokine named for its ability to convert normal fibroblasts into cells that do not depend on anchorage growth.

[0005] The TGFβ family generally includes TGFβ1, TGFβ2, and TGFβ3. TGFβ1 was first isolated from platelets, while TGFβ2 was isolated from osteocytes. Human TGFβ and mouse TGFβ are highly conserved: human TGFβ1 differs from mouse TGFβ1 by only one amino acid, human TGFβ2 differs from mouse TGFβ2 by only three amino acids, and human TGFβ3 is identical to mouse TGFβ3.

[0006] Biologically active TGFβ generally exists in dimer form. When it binds to the TGFβ Type II receptor (TGFβRII) on the cell surface, it binds to the adjacent TGFβ Type I receptor (TGFβRI), forming a hexamer. Subsequently, due to TGFβ binding, the serine / threonine kinase activity of TGFβRII is activated. At this point, TGFβRII phosphorylates some key serine residues on TGFβRI, activating its serine / threonine kinase activity. This phosphorylation process continues downstream, phosphorylating adjacent regulatory SMAD (R-SMAD), mainly including SMAD2, 3, 9, etc. (R-SMAD is usually fixed inside the cell membrane by SARA proteins). Phosphorylated R-SMAD has a very high affinity for free Co-SMAD in the cytoplasm (such as SMAD4), thus forming R-SMAD-Co-SMAD polymers. It will enter the cell nucleus, bind to DNA together with transcription factors, and initiate the expression of downstream genes (Sporn et al., Science, 233:532 (1986)).

[0007] However, TGFβ cannot directly act on receptors on the cell surface. Under normal circumstances, only a small amount of free TGFβ can bind to receptors, while most TGFβ is bound to latency-associated peptide (LAP) and is inactive. There are four forms of TGFβ, and TGFβ can only be activated when the TGFβ-LAP polymer is cleaved by proteases in the extracellular microenvironment to form a free state. For a general review of TGFβ and its functions, please refer to the relevant literature (Joan Massague, TGFβ in Cancer. Cell, 134(2):215-230 (2008)).

[0008] One of the main focuses of recent research is the role of growth factor TGF-β in wound healing and scar formation. The three isoforms (TGF-β1, TGF-β2, and TGF-β3) appear to have overlapping functions, primarily mediating their effects through the intracellular SMAD pathway. Initial studies suggested that TGF-β1 is responsible for the fibrotic scar formation response, while scarless healing observed in fetal wounds is attributed to increased TGF-β3 levels. However, the reality appears to be much more complex, and simply altering the ratio of TGF-β isoforms is unlikely to result in scarless wound healing. Other aspects of the TGF-β system look promising, including downstream mediators CTGF, the proteoglycan core proteoglycan, and the binding protein p311. Other proposed mechanisms that may contribute to the pathogenesis of hypertrophic scars include excessive inflammation, excessive angiogenesis, matrix metalloproteinases, altered growth factor levels, and delayed apoptosis of fibrotic myofibroblasts due to p53 gene alterations or wound tension. Further work is necessary to understand the fundamental mechanisms of pathological scar formation if effective treatments for hypertrophic scars following burns are to be developed.

[0009] Scars are an inevitable product of the wound healing process. Wound healing occurs in two forms: complete repair, where the wound is repaired by cells with the same structure as the original damaged tissue, such as the scarless healing of early fetal wounds or the healing of superficial wounds; and almost all other wounds heal with scarring during epithelialization. This type of scar is a product of the normal tissue repair process and is therefore called a "normal scar." When the wound repair process is abnormal, a large amount of extracellular matrix components, mainly collagen, are deposited, leading to excessive proliferation of dermal tissue and the appearance of an "abnormal scar," also known as a "hypertrophic scar (HS)" or "keloid (K)."

[0010] Hypertrophic scars and keloids are a type of dermal fibrosis, similar to conditions like pleural adhesions, cirrhosis, and pulmonary fibrosis. They are pathological conditions characterized by excessive deposition of extracellular matrix (ECM) components, including type I and III collagen, in tissues, which are difficult for the body to absorb or remodel. Therefore, the term "bnormal scar" is used to distinguish them from "normal scar." Although keloids share many similarities with hypertrophic scars and are often described together as HS and Keloids, Keloids are also classified as benign tumors due to their tendency to grow in a tumor-like manner. Based on the numerous damages caused to the body by HS and Keloids, they are also referred to as "healing skin trauma."

[0011] Bioactive factors are associated with hypertrophic scarring. Following trauma, a series of complex biological processes occur in the wound area: cell infiltration, neutrophil aggregation, increased macrophage numbers, increased collagen and matrix synthesis by fibroblasts, and granulation tissue formation. The diversity and synthesis of cells in the wound area are regulated by multiple growth factors. Among them, platelet-derived growth factor (PDGF), transforming growth factor (TGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), and insulin-like growth factor (IGF-1) are highly active and associated with wound healing responses.

[0012] Among these, TGF-β is most closely related to scar hyperplasia. The biological effects of TGF-β are diverse; it can inhibit epidermal cells, endothelial cells, and hematopoietic cells, while stimulating the growth and promoting the function of fibroblasts. In fact, TGF-β can be synthesized by various cells, such as platelets, macrophages, lymphocytes, fibroblasts, and keratinocytes, and almost all cells have TGF-β receptors. Therefore, TGF-β may be the family of growth factors with the most extensive target cells. Cromack measured the TGF-β content in mouse wound fluid and found that TGF-β levels increased in the early post-injury period and decreased as the wound closed. Adding TGF-β to fibroblast cultures of fetal skin can induce the expression of type I collagen genes, which were previously not expressed, leading to scar hyperplasia. Gallivan, Bullard, and others found that the collagenase content in 6-month-old fetal skin was higher than that in adult skin, while the TGF-β content was relatively low, which is the basis for early scarless wound healing in fetuses. Exogenous TGF-β can cause scarring of early fetal wounds, partly because it reduces the synthesis of collagenase and collagen breakdown, leading to scar hyperplasia.

[0013] TGF-β promotes wound healing but is also a major cytokine stimulating pathological scar hyperplasia. Low concentrations of TGF-β1 act as potent chemotactic agents for macrophages and neutrophils. Through autocrine and paracrine processes, local TGF-β concentrations increase, thereby activating macrophages to increase TGF-β mRNA expression and promoting fibroblast proliferation. High concentrations of TGF-β can induce the production of other growth factors, such as IL-1, FGF, TNF-α, PDGF, and TGF-α. TGF-β strongly promotes collagen I synthesis and stimulates fibronectin synthesis, providing a network for collagen accumulation and facilitating the migration of inflammatory cells to the wound site. TGF-β can also reduce the synthesis of intercellular matrix proteins, inhibit the activity of metalloproteinases, and promote scar hyperplasia.

[0014] The TGF-β superfamily members (TGF-β1, TGF-β2, and TGF-β3) play crucial roles in different stages of wound healing, but their functions differ significantly and are closely related to scar formation. TGF-β1 promotes fibroblast proliferation, collagen I, and extracellular matrix (ECM) deposition, leading to an imbalance in ECM degradation and acting as a major driver of hypertrophic scars and keloids. It may also exacerbate fibrosis through compensatory upregulation. Unlike TGF-β1 and TGF-β2, which promote fibrosis, TGF-β3 has the opposite effect. It inhibits excessive collagen deposition, promotes ECM remodeling, regulates epithelial-mesenchymal transition (EMT) in normal skin, and reduces scar contraction. In clinical practice, increased TGF-β3 receptor sensitivity is observed in keloids, potentially limiting fibrosis through a negative feedback mechanism.

[0015] Different TGF-β isoforms can exhibit synergistic and antagonistic interactions. For example, TGF-β1 / 2 activates the SMAD pathway via the ALK5 receptor, while TGF-β3 may inhibit excessive fibrosis via the ALK1 receptor. In scar formation, the pro-fibrotic effect of TGF-β1 / 2 is often partially offset by the repair function of TGF-β3. Intralesional injection of recombinant TGF-β3 peptides can significantly improve appearance.

[0016] Bispecific antibodies, also known as bifunctional antibodies, are specific antibodies that simultaneously target two different antigens. They can be produced through immunosorting and purification, or through genetic engineering. Genetic engineering offers flexibility and advantages in terms of binding site optimization, synthetic formulation, and yield. Currently, more than 45 bispecific antibodies have been identified. Many developed bispecific antibodies are in the IgG-ScFv form, also known as the Morrison form. Due to its similarity to naturally occurring IgG, this form offers advantages in antibody engineering, expression, and purification, making it an ideal form for bispecific antibodies.

[0017] Based on the different roles of different TGF-β subtypes in wound healing response, the technical challenge in this field is to design bispecific antibodies using genetic engineering techniques that can specifically and selectively inhibit different TGF-β subtypes, thereby achieving optimal wound healing effects. Summary of the Invention

[0018] In view of this, and addressing the shortcomings of existing technologies, the purpose of this application is to provide a bifunctional antibody and its pharmaceutical composition that promote wound healing. Through extensive preliminary basic research, the inventors discovered that TGF-β1-dominated fibrosis and scar hyperplasia are primarily due to TGF-β1's inhibition of the CD4+ T cell subtype Th2 cells, thereby suppressing collagen III production in the early wound healing response. This leads to an increased collagen I / III ratio, resulting in excessive collagen I deposition, affecting wound healing, and causing scar and keloid formation. TGF-β2 directly promotes collagen fiber production, while TGF-β3 can inhibit excessive collagen deposition and reduce scar contraction. In summary, TGF-β1 dominates fibrosis, TGF-β2 participates in fibrosis, and TGF-β3 has anti-fibrotic potential. A balance among these three is crucial for normal healing; imbalance leads to abnormal scarring.

[0019] Based on the different mechanisms of action of TGF-β family members in wound healing, to achieve optimal wound healing, it is necessary to inhibit TGF-β1 and TGF-β2 while preserving the function of TGF-β3. Inhibition of TGF-β1 requires specific targeting of TGF-β1 signaling on CD4+ T cells. Therefore, a CD4 / TGF-β1 bispecific antibody needs to be designed to relieve the inhibitory effect of TGF-β1 on CD4+ T cells and release its mediated wound healing response. Simultaneously, this bispecific antibody needs to retain its activity in inhibiting TGF-β2 and must not have cross-reactivity with TGF-β3. In summary, this invention requires the design of an antibody with three characteristics: (1) specifically targeting TGF-β1 signaling on CD4+ T cells; (2) broadly inhibiting TGF-β2 signaling; and (3) having no effect on TGF-β3 signaling.

[0020] Given the high conservation of amino acid sequences among TGF-β1, TGF-β2, and TGF-β3, screening for neutralizing antibodies against TGF-β1 often results in cross-inhibition of TGF-β2 / 3. Therefore, in this invention, it is necessary to select antibodies that inhibit only TGF-β1 / 2.

[0021] This invention utilizes genetic engineering to design a bispecific antibody targeting CD4 and TGF-β1 / 2. This bispecific antibody can safely bind to CD4 without affecting CD4 function. By binding to CD4, the bispecific antibody specifically inhibits TGF-β1 signaling in CD4+ T cells, thereby relieving the inhibitory effect of TGF-β1 on CD4+ T cells and releasing its mediated wound healing response. Simultaneously, this bispecific antibody inhibits TGF-β2, thereby suppressing its pro-fibrotic ability, while having no inhibitory ability on TGF-β3, thus retaining its wound-healing-promoting ability.

[0022] Through in-depth experimental research and creative work, the inventors have screened out a bispecific CD4 / TGF-β antibody that can promote Th2 cell activation. Among several candidate CD4 antibodies, only the bispecific CD4 / TGF-β combination constructed from ipalizumab and anti-human TGFβ1 / 2 monoclonal antibodies can effectively promote Th2 cell activation, and the activated Th2 cells can effectively promote wound healing. In this invention, the bispecific CD4 / TGF-β combination constructed from ipalizumab and anti-human TGFβ1 / 2 monoclonal antibodies is named WH405.

[0023] The inventors have discovered that WH405 is capable of:

[0024] (1) It effectively binds to CD4 molecules on the surface of human helper T cells, relieves the immunosuppression of helper T cells by TGFβ1, promotes the activation of Th2 cells, and thus effectively promotes the wound healing response mediated by Th2 cells.

[0025] (2) Effectively inhibits TGF-β2, inhibits fibrosis, and promotes wound healing.

[0026] (3) It has no inhibitory effect on TGF-β3 and retains the wound healing ability of TGF-β3.

[0027] This leads to the following invention:

[0028] A first aspect of the present invention provides a bispecific antibody that binds to human CD4 and TGF-β1 / 2, the antibody comprising:

[0029] The first protein functional region targets CD4, and the first protein functional region is an anti-CD4 antibody or its antigen-binding fragment;

[0030] The second protein functional region targets TGF-β1 / 2, and the second protein functional region is an antibody against TGF-β1 / 2 or its antigen-binding fragment;

[0031] The heavy chain variable region of the first protein functional region contains amino acid sequences HCDR1-HCDR3 as shown in SEQ ID:1-SED IDNO:3, and its light chain variable region contains amino acid sequences LCDR1-LCDR3 as shown in SEQ ID:4-SED ID NO:6.

[0032] and,

[0033] The heavy chain variable region of the second protein functional region contains amino acid sequences HCDR1-HCDR3 as shown in SEQ ID:7-SED IDNO:9, and its light chain variable region contains amino acid sequences LCDR1-LCDR3 as shown in SEQ ID:10-SED ID NO:12.

[0034] The first protein functional region is selected from Fab, Fab', Fd, Fv, dAb, ScFv, complementarity-determining region fragment, humanized antibody or chimeric antibody;

[0035] And / or,

[0036] The second protein functional region is selected from Fab, Fab', Fd, Fv, dAb, ScFv, complementarity-determining region fragments, humanized antibodies, or chimeric antibodies;

[0037] In some embodiments, the first antigen binding site is capable of binding an amino acid sequence as shown in positions 77, 79, 96, 121-124, 127-134, and 163 of SEQ ID NO: 19.

[0038] In some embodiments, the amino acid sequence of the heavy chain variable region of the first protein functional region is shown in SEQ ID NO:21, and the amino acid sequence of the light chain variable region of the first protein functional region is shown in SEQ ID NO:23.

[0039] In some embodiments, the amino acid sequence of the heavy chain variable region of the second protein functional region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region of the second protein functional region is shown in SEQ ID NO:27.

[0040] In some embodiments, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin, that is, the bispecific antibody exists in the form of IgG-ScFv.

[0041] In some embodiments, the Th2 cells and CD4+ T cells are located in the paracortical region of the lymph node.

[0042] In some embodiments, the paracortical lymph node region is the paracortical lymph node region of a mouse.

[0043] In some embodiments, the bispecific antibody is any of the following:

[0044] (1) The first protein functional region is a single-chain antibody, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-ScFv;

[0045] (2) The first protein functional region is a variable region fragment, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-Fv;

[0046] (3) The first protein functional region is a variable region fragment, the second protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of Fab-Fv-Fc;

[0047] (4) The second protein functional region is a variable region fragment, the first protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of Fab-Fv-Fc;

[0048] (5) The first protein functional region is an antigen-binding fragment, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-Fab;

[0049] (6) The first protein functional region is an antigen-binding fragment, the second protein functional region is a single-chain antibody, and the bispecific antibody exists in the form of Fab-ScFv-Fc;

[0050] (7) The first protein functional region is a single-chain antibody, the second protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of Fab-ScFv-Fc.

[0051] (8) The first protein functional region is a single-chain antibody, the second protein functional region is a single-chain antibody, and the bispecific antibody exists in the form of ScFv-Fc-ScFv or ScFv-ScFv-Fc;

[0052] In some embodiments, the bispecific antibody is any of the following:

[0053] (1) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the second protein functional region through a linker;

[0054] (2) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the second protein functional region through a linker;

[0055] (3) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker;

[0056] (4) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker;

[0057] (5) The first protein functional region is Fv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker;

[0058] (6) The first protein functional region is Fv, the second protein functional region is Fab, the C end of the first protein functional region is hinged to the Fc region, and the second protein functional region is connected to the N end of the first protein functional region through a connector;

[0059] (7) The first protein functional region is Fab, the second protein functional region is Fv, the C end of the second protein functional region is hinged to the Fc region, and the first protein functional region is connected to the N end of the second protein functional region through a connector;

[0060] (8) The first protein functional region is Fab, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the heavy chain of the second protein functional region through a linker;

[0061] (9) The first protein functional region is Fab, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the heavy chain of the second protein functional region through a linker;

[0062] (10) The first protein functional region is Fab, the second protein functional region is ScFv, the first protein functional region is connected to the C end of the Fc region through a linker; the second protein functional region is hinged to the Fc region;

[0063] (11) The first protein functional region is ScFv, the second protein functional region is ScFv, the first protein functional region is connected to the C end of the Fc region through a connector, and the second protein functional region is hinged to the Fc region.

[0064] (12) The first protein functional region is ScFv, the second protein functional region is ScFv, the second protein functional region is hinged to the Fc region, and the second protein functional region is connected to the C end of the first protein functional region through a connector.

[0065] In some embodiments, the hinge is an immunoglobulin hinge.

[0066] In some embodiments, the hinge is a hinge of the immunoglobulin Fc region.

[0067] In some embodiments, the first protein functional region is ScFv, the second protein functional region is IgG, and the C-terminus of the first protein functional region VH is connected to the C-terminus of the second protein functional region CH3 via a linker.

[0068] In some embodiments, the first protein functional region is ScFv, the second protein functional region is IgG, and the C-terminus of the first protein functional region VH is connected to the C-terminus of the second protein functional region CL via a linker.

[0069] In some embodiments, the first protein functional region is ScFv, the second protein functional region is IgG, and the C-terminus of the first protein functional region VL is connected to the N-terminus of the second protein functional region VH via a connector.

[0070] In some embodiments, the first protein functional region is ScFv, the second protein functional region is IgG, and the C-terminus of the first protein functional region VL is connected to the N-terminus of the second protein functional region VL via a connector.

[0071] In some embodiments, the first protein functional region is Fv, the second protein functional region is IgG, the C-terminus of the first protein functional region VL is connected to the N-terminus of the second protein functional region VL via a connector, and the VH of the first protein functional region is connected to the N-terminus of the VH of IgG via a connector.

[0072] In some embodiments, the first protein functional region is Fv, the second protein functional region is Fab, the C-terminus of the VL of the first protein functional region is hinged to the Fc region, the C-terminus of the CH1 domain of the heavy chain of the second protein functional region is connected to the N-terminus of the VL of the first protein functional region through a connector, and the C-terminus of the CL domain of the light chain of the second protein functional region is connected to the N-terminus of the VH domain of the first protein functional region through a connector.

[0073] In some embodiments, the first protein functional region is Fab, the second protein functional region is Fv, the C-terminus of the VL of the second protein functional region is hinged to the Fc region, the CH1 domain of the heavy chain of the first protein functional region is connected to the N-terminus of the VL of the second protein functional region through a connector, and the C-terminus of the CL domain of the light chain of the first protein functional region is connected to the N-terminus of the VH domain of the second protein functional region through a connector.

[0074] In some embodiments, the first protein functional region is Fab, the second protein functional region is IgG, and the N-terminus of the first protein functional region VH is connected to the C-terminus of the heavy chain of the second protein functional region via a linker.

[0075] In some embodiments, the first protein functional region is Fab, the second protein functional region is IgG, and the C-terminus of the first protein functional region CH1 is connected to the N-terminus of the heavy chain of the second protein functional region via a linker.

[0076] In some embodiments, the first protein functional region is Fab, the second protein functional region is ScFv, the N-terminus of the VH of the first protein functional region is connected to the C-terminus of the Fc region via a connector, and the C-terminus of the VL of the second protein functional region is hinged to the Fc region.

[0077] In some embodiments, the first protein functional region is ScFv, the second protein functional region is ScFv, the N-terminus of VH of the first protein functional region is connected to the C-terminus of the Fc region via a connector, and the C-terminus of VL of the second protein functional region is hinged to the Fc region.

[0078] In some embodiments, the first protein functional region is ScFv, the second protein functional region is ScFv, the C-terminus of the VL of the second protein functional region is hinged to the Fc region, and the N-terminus of the VH of the second protein functional region is connected to the C-terminus of the VL of the first protein functional region through a connector.

[0079] In some embodiments, the hinge is an immunoglobulin hinge.

[0080] In some embodiments, the hinge is a hinge of the immunoglobulin Fc region.

[0081] In some embodiments, the first protein chain is the amino acid sequence shown in SEQ ID NO:33; and the second protein chain is the amino acid sequence shown in SEQ ID NO:35.

[0082] In some embodiments, the ScFv is linked to the C-terminus of the IgG heavy chain via a linker fragment, wherein the linker fragment is (GGGGS)m, where m is an integer, and GGGGS (SEQ ID NO:80) is a constituent unit of the linker.

[0083] In some embodiments, the immunoglobulin includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0084] In some embodiments, the immunoglobulin has a constant region derived from human antibodies;

[0085] In some embodiments, the constant region of the immunoglobulin is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

[0086] In some embodiments, the bispecific antibody, wherein,

[0087] In some implementations, its heavy chain constant region is the human IgG1 chain C region, the human IgG2 chain C region, or the human IgG4 chain C region, and its light chain constant region is the human Ig kappa chain C region or the human Ig lambda chain C region.

[0088] In some embodiments, the constant regions of the immunoglobulins are humanized. For example, the heavy chain constant regions are all IgG4 chain C region, ACCESSION: P01861 or IgG2 chain C region, ACCESSION: P01859; the light chain constant regions are all Ig kappa chain C region, ACCESSION: P01834 or Ig lambda chain C region, ACCESSION: P0DOX8.

[0089] In some implementations, the first protein functional region and the second protein functional region are independently one, two, or more.

[0090] In some embodiments, the immunoglobulin is one; the single-chain antibody is two, and preferably two identical single-chain antibodies.

[0091] In some embodiments, the immunoglobulin is IgG, IgA, IgD, IgE or IgM; preferably IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0092] In some embodiments, the single-chain antibody is attached to the C-terminus of the heavy chain of an immunoglobulin. Since immunoglobulins have two heavy chains, one immunoglobulin molecule is linked to two single-chain antibody molecules. Preferably, the two single-chain monomer molecules are identical.

[0093] In some embodiments, the single-chain antibody consists of two chains, with one end of each single-chain antibody attached to the C-terminus or N-terminus of the two heavy chains of the immunoglobulin, respectively.

[0094] In some embodiments, a disulfide bond exists between the VH and VL of the single-chain antibody. The method of introducing disulfide bonds between the VH and VL of an antibody is well known in the art, for example, see US patent application US5747654; Rajagopal et al., Prot. Engin. 10 (1997) 1453-1459; Reiter et al., Nature Biotechnology 14 (1996) 1239-1245; Webber et al., Molecular Immunology 32 (1995) 249-258; Reiter et al., Immunity 2 (1995) 281-287; Reiter et al., JBC 269 (1994) 18327-18331; Reiter et al., Inter. J. of Cancer 58 (1994) 142-149; or, Reiter et al., Cancer Res. 54 (1994) 2714-2718; which are incorporated herein by reference.

[0095] In some embodiments, the antibody can selectively bind to human CD4 molecules, human TGF-β1 molecules, and human TGF-β2 molecules.

[0096] In some embodiments, the antibody is in a concentration of less than 10 -9 M's EC 50 The EC binds to human CD4 protein. 50 Detected by ELISA.

[0097] In some embodiments, the antibody has an IC50 of less than 100 pM against human TGFβ1 in a TGFβ reporter luciferase activity assay. 50 .

[0098] In some embodiments, the antibody has an IC50 of less than 5 nM against human TGFβ2 in a TGFβ reporter luciferase activity assay. 50 .

[0099] On the other hand, the present invention provides an isolated nucleic acid molecule that encodes a bispecific antibody as described above.

[0100] On the other hand, the present invention provides a carrier comprising the above-described isolated nucleic acid molecules.

[0101] On the other hand, the present invention provides a host cell comprising the isolated nucleic acid molecules described above, or comprising the vector described above.

[0102] On the other hand, the present invention provides a method for a bispecific antibody, which includes culturing the host cells described above under suitable conditions, and recovering the bispecific antibody from the cell culture.

[0103] On the other hand, the present invention provides a conjugate comprising a bispecific antibody and a conjugation portion, wherein the bispecific antibody is any one of the bispecific antibodies described in the present invention, and the conjugation portion is a detectable label.

[0104] In some embodiments, the coupling portion is a small molecule compound, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

[0105] On the other hand, the present invention provides a pharmaceutical composition comprising a bispecific antibody as described in any of the preceding claims or comprising a conjugate as described above.

[0106] In some embodiments, it also includes pharmaceutically acceptable excipients.

[0107] The bispecific antibody or pharmaceutical composition of the present invention can be formulated into any dosage form known in the pharmaceutical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the necessary dose of the bispecific antibody of the present invention into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use. Furthermore, the bispecific antibody of the present invention can be present in the pharmaceutical composition in unit dose form for ease of administration. In some embodiments, the unit dose is at least 1 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg. When the pharmaceutical composition is in a liquid (e.g., injectable) dosage form, it may contain the bispecific antibody of the present invention at a concentration of at least 0.1 mg / ml, such as at least 0.25 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml.

[0108] The bispecific antibody or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In a preferred embodiment, the bispecific antibody or pharmaceutical composition of the present invention is administered by intravenous infusion or injection.

[0109] In some implementations, the promotion of wound healing or reduction of wound scar area is achieved by increasing the ratio of Th2 cells to CD4+ T cells.

[0110] In some implementations, the application includes increasing the ratio of Th2 cells to CD4+ T cells.

[0111] In some embodiments, the wound is a full-thickness skin defect.

[0112] In some embodiments, the scar is a scar 21 days after the wound has healed. In some embodiments, the scar is a scar 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after the wound has healed.

[0113] The bispecific antibody or pharmaceutical composition provided by this invention can be used alone or in combination, or in combination with another pharmaceutically active agent. This other pharmaceutically active agent can be administered before, simultaneously with, or after the administration of the bispecific antibody or pharmaceutical composition of this invention.

[0114] In this invention, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0115] On the other hand, the present invention provides the use of the above-mentioned bispecific antibody or the above-mentioned conjugate in the preparation of a medicament for promoting wound healing.

[0116] In in vitro experiments of this invention, WH405 can selectively bind to human CD4 molecules, human TGF-β1 molecules, and human TGF-β2 molecules. WH405 can bind at concentrations of less than 10... -9 M's EC 50 The EC binds to human CD4 protein. 50 As determined by ELISA, WH405 exhibited an IC50 of less than 100 pM against human TGFβ1 in a TGFβ reporter gene luciferase activity assay. 50 WH405 exhibits an IC50 of less than 5 nM against human TGFβ2 in a TGFβ reporter gene luciferase activity assay. 50 .

[0117] The typical non-limiting range of the therapeutic or preventative effective dose of the bispecific antibody of the present invention is 0.02–50 mg / kg, for example 0.1–50 mg / kg, 0.1–25 mg / kg, or 1–10 mg / kg. It should be noted that the dosage may vary depending on the type and severity of the symptoms requiring treatment. Furthermore, those skilled in the art will understand that for any given patient, a specific dosing regimen should be adjusted over time based on the patient's needs and the physician's professional evaluation; the dosage ranges given herein are for illustrative purposes only and do not limit the use or scope of the pharmaceutical compositions of the present invention.

[0118] In this invention, the subject can be a rodent, such as a mouse, or a primate, such as a human or a cynomolgus monkey. The amino acid sequence alignment of CD4 and TGFβ1 proteins between different species is as follows: Figure 8 As shown.

[0119] Antibody therapies, particularly monoclonal antibodies (mAbs), have shown promising efficacy in treating a variety of diseases. Traditional experimental methods for obtaining these therapeutic antibodies involve immunizing animals with antigens to obtain antibodies targeting the antigens, or using affinity maturation to improve antibodies with low affinity for antigens. However, these methods require significant time and effort, and often fail to target specific epitopes on the antigen.

[0120] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Volumes 1-3, NIH Publication 91-3242, Bethesda Md).

[0121] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of the monoclonal antibody sequences in items (1)-(13) below through the VBASE2 database, the results are as follows:

[0122] (1) Ibalizumab

[0123] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:21, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:23.

[0124] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0125] HCDR1: GYTFTSYVIH (SEQ ID NO: 1);

[0126] HCDR2: YINPYNDGTDYDEKFKG(SEQ ID NO:2);

[0127] HCDR3: EKDNYATGAWFA (SEQ ID NO:3);

[0128] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0129] LCDR1: KSSQSLLYSTNQKNYLA(SEQ ID NO:4);

[0130] LCDR2: WASTRES(SEQ ID NO:5);

[0131] LCDR3: QQYYSYRT (SEQ ID NO:6);

[0132] (2) NIS793

[0133] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:27.

[0134] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0135] HCDR1: GGTFSSYA(SEQ ID NO:7);

[0136] HCDR2: IIPIFGTA (SEQ ID NO:8);

[0137] HCDR3: ARGLWEVRALPSVY(SEQ ID NO:9);

[0138] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0139] LCDR1:DIGSKS(SEQ ID NO:10);

[0140] LCDR2: EDI (SEQ ID NO:11);

[0141] LCDR3: QVWDRDSDQY(SEQ ID NO:12);

[0142] (3) WH405

[0143] The amino acid sequences of the nine CDR regions of its second protein chain are as follows:

[0144] HCDR1: GGTFSSYA(SEQ ID NO:7);

[0145] HCDR2: IIPIFGTA (SEQ ID NO:8);

[0146] HCDR3: ARGLWEVRALPSVY(SEQ ID NO:9);

[0147] HCDR4: GYTFTSYVIH (SEQ ID NO: 1);

[0148] HCDR5: YINPYNDGTDYDEKFKG(SEQ ID NO:2);

[0149] HCDR6: EKDNYATGAWFA (SEQ ID NO:3);

[0150] LCDR1: KSSQSLLYSTNQKNYLA(SEQ ID NO:4);

[0151] LCDR2: WASTRES(SEQ ID NO:5);

[0152] LCDR3: QQYYSYRT (SEQ ID NO:6);

[0153] The amino acid sequences of the three CDR regions of its first protein chain are as follows:

[0154] LCDR1:DIGSKS(SEQ ID NO:10);

[0155] LCDR2: EDI (SEQ ID NO:11);

[0156] LCDR3: QVWDRDSDQY(SEQ ID NO:12);

[0157] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0158] As used in this article, targeting TGF-β1 / 2 refers to targeting both TGF-β1 and TGF-β2. Binding to TGF-β1 / 2 refers to binding to both TGF-β1 and TGF-β2.

[0159] As used herein, when referring to the amino acid sequence of the TGFβ1 protein (GenBank ID: NP_000651.3), it includes the full length of the TGFβ1 protein. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the TGFβ1 protein without affecting its biological function. Therefore, in this invention, the term "TGFβ1 protein" should include all such sequences, including their natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the TGFβ1 protein is as shown in SEQ ID NO:13 (excluding the last 6 His, totaling 112 amino acids).

[0160] As used herein, when referring to the amino acid sequence of the TGFβ2 protein (GenBank ID: NP_003229.1), it includes the full length of the TGFβ2 protein. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the TGFβ2 protein without affecting its biological function. Therefore, in this invention, the term "TGFβ2 protein" should include all such sequences, including their natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the TGFβ2 protein is as shown in SEQ ID NO:15 (excluding the last 6 His, totaling 112 amino acids).

[0161] As used herein, when referring to the amino acid sequence of the TGFβ3 protein (GenBank ID: NP_003230.1), it includes the full length of the TGFβ3 protein. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the TGFβ1 protein without affecting its biological function. Therefore, in this invention, the term "TGFβ3 protein" should include all such sequences, including their natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the TGFβ3 protein is as shown in SEQ ID NO:17 (excluding the last 6 His, totaling 112 amino acids).

[0162] As used herein, when referring to the amino acid sequence of the CD4 protein (cluster differentiation 4, NCBIGenBank: NP_000607.1), it includes the full-length CD4 protein, as well as the extracellular fragment of CD4 and soluble CD4 molecules (sCD4). However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CD4 protein without affecting its biological function. Therefore, in this invention, the term "CD4 protein" should include all such sequences, including their natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the extracellular fragment of CD4, sCD4, is as shown in SEQ ID NO:19 (excluding the last 6 His, totaling 365 amino acids).

[0163] As used herein, when referring to the amino acid sequence of TGFβRII protein (transforming growth factor betareceptor II, NCBI GenBank: NP_001020018.1), it includes the full-length TGFβRII protein and also the extracellular fragment of TGFβRII, namely TGFβRII-ECD. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of TGFβRII protein without affecting its biological function. Therefore, in this invention, the term "TGFβRII protein" should include all such sequences, including their natural or artificial variants. In one embodiment of the invention, the amino acid sequence of the extracellular fragment of TGFβRII, TGFβRII-ECD, is shown in SEQ ID NO:73.

[0164] As used in this article, the term EC 50 The half-maximal effect concentration (50% of maximal effect) refers to the concentration that can cause a 50% maximal effect.

[0165] As used in this article, the term IC 50 The half-maximal inhibition concentration (WMC) is the concentration at which a concentration can induce 50% maximum inhibition.

[0166] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in an antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883. In particular, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bifunctional antibody of this invention, the heavy chain may be the C-terminus of the heavy chain of an IgG antibody linked to the ScFv of another antibody, in which case the heavy chain contains nine CDRs. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0167] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0168] As used herein, the terms “antigen binding site” or “binding portion” refer to the portion of an antibody that allows antigen binding.

[0169] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and the term "F(ab')2 fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region. As used herein, "Fc region" refers to the entire portion from the hinge region to the CH2 and CH3 regions. The term "Fc fragment" specifically refers to the crystallizable fragment obtained by cleaving an intact IgG antibody with papain, which contains only the CH2 and CH3 constant regions of the two heavy chains.

[0170] As used herein, a “linker” or “connector fragment” refers to a portion capable of connecting two compounds, such as two polypeptides, for example, a polypeptide. Non-limiting examples of linkers include flexible linkers comprising a glycine-serine (e.g., (Gly4Ser)) repeat sequence, and linkers derived from: (a) an interdomain region of a transmembrane protein (e.g., a type I transmembrane protein); (b) a stem region of a type II C-lectin; or (c) an immunoglobulin hinge. As provided herein, a linker may refer to, for example, (1) a polypeptide region between the VH and VL regions of a single-chain Fv (scFv) or (2) a polypeptide region between an immunoglobulin constant region and an antigen-binding domain. In some embodiments, the linker consists of 5 to about 35 amino acids, for example, about 15 to about 25 amino acids. In some embodiments, the linker consists of at least 5 amino acids, at least 7 amino acids, or at least 9 amino acids.

[0171] In some cases, the antigen-binding fragment of the antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers as single polypeptide chains (see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other connectors that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0172] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0173] Antigen-binding fragments of an antibody (e.g., the antibody fragments described above) can be obtained from a given antibody using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be screened for specificity in the same manner as those used for intact antibodies.

[0174] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0175] As used herein, the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are usually obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see USP 4,816,567).

[0176] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), while the other portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP 4,816,567 to Cabillyetal.; Morrison et al., Proc.Natl.Acad.Sci.USA,81:6851 6855(1984)).

[0177] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature, 321:522 525 (1986); Reichmannetal., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).

[0178] As used herein, the term “epitope” refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. “Epitope” is also referred to in the art as an “antigenic determinant.” Epitopes or antigenic determinants typically consist of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation, which can be “linear” or “conformal.” See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0179] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0180] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0181] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0182] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8M, 10 -9 M or 10 -10 The antigen binds to M or a lower affinity (Kd). In some embodiments of the invention, the term "targeted" refers to specific binding.

[0183] As used herein, the term "Kd" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. A smaller equilibrium dissociation constant indicates a tighter antibody-antigen binding and a higher affinity between the antibody and the antigen. Typically, antibodies bind at a rate less than approximately 10-1. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 An antigen is bound by a dissociation equilibrium constant (Kd) of M or smaller, for example, as determined in a Biacore instrument using surface plasmon resonance (SPR).

[0184] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, glycine may be represented by G or Gly, and alanine may be represented by A or Ala.

[0185] As used herein, the term "pharmaceuticalally acceptable excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0186] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when administered to the body along with or before an antigen, can enhance the body's immune response to the antigen or alter the type of immune response. There are many types of adjuvants, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, and cytokines. Freund's adjuvant is currently the most commonly used adjuvant in animal studies. Aluminum hydroxide adjuvant is more frequently used in clinical trials.

[0187] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. An effective amount for treating a disease means an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0188] Beneficial effects of the invention:

[0189] The bispecific antibody WH405 of the present invention can specifically bind to CD4 and specifically bind to helper T cells, thereby activating Th2 cells, stimulating Th2 cell-mediated wound healing response, and promoting wound healing.

[0190] At the same time, WH405 can also bind to TGFβ2, systematically neutralize TGFβ2, inhibit the TGFβ2-mediated fibrosis process, and promote wound healing.

[0191] In addition, WH405 does not bind to TGFβ3, thus preserving TGFβ3's ability to promote wound healing. Attached Figure Description

[0192] Figure 1 Schematic diagram of the IgG-ScFv form (Morrison pattern) of the bifunctional antibody WH405.

[0193] Figure 2 The amino acid sequence of the bifunctional antibody WH405 in IgG-ScFv form (Morrison pattern) is shown, where the underlined amino acids are the CDR regions of the heavy or light chain of the antibody.

[0194] Figure 3 Indirect ELISA method for detecting the binding constant EC of bifunctional antibody WH405 and monoclonal antibody Ibalizumab to CD4 protein. 50 .

[0195] Figure 4The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1, TGFβ2, and TGFβ3 signaling activation by the bifunctional antibody WH405 and the monoclonal antibody NIS793.

[0196] Figure 5 Schematic diagram of the IgG-ScFv form (Morrison pattern) of bifunctional antibodies composed of different CD4 antibodies (Zanolimumab, Keliximab, Tregalizumab, Ibalizumab, and 13B8.2) and TGFβ1 / 2 antibodies.

[0197] Figure 6 Indirect ELISA method was used to detect the binding constants (ECs) of bispecific antibodies WH105, WH205, WH305, WH405, and WH505 to CD4 protein. 50 .

[0198] Figure 7 The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1, TGFβ2, and TGFβ3 signaling activation by bispecific antibodies WH105, WH205, WH305, WH405, and WH505.

[0199] Figure 8 Amino acid sequence alignment of CD4 and TGFβ1 proteins between different species.

[0200] Figure 9 Gene element composition of human CD4 transgenic mice and IL-4-IRES-tdTomato reporter mice, and gene element composition of hCD4 / +;IL-4-IRES-tdTomato transgenic mice obtained by mating the two mice.

[0201] Figure 10 The antibody content in the lymph nodes of human CD4 transgenic mice after injection of bispecific antibodies WH105, WH205, WH305, WH405 and WH505.

[0202] Figure 11 After intravenous injection, the bispecific antibody WH405 binds to CD4 molecules and enters the lymph nodes from the bloodstream. It specifically relieves the immunosuppression of CD4+ T cells by TGF-β1, promotes the differentiation of naïve CD4+ T cells into Th2 cells, and the differentiated Th2 cells can promote wound healing, promote tissue repair, improve fibrosis (upregulate collagen 3 and increase the ratio of collagen 3 to collagen 1), and inhibit scar formation.

[0203] Figure 12The activation status of Th2 cells in the paracortical region of lymph nodes three weeks after tail vein injection of control antibody, TGFβ1 / 2 antibody (NIS793), and different CD4 / TGFβ double antibodies (WH105, WH205, WH305, WH405, and WH505) into hCD4 / +;IL-4-IRES-tdTomato transgenic mice.

[0204] Figure 13 A human CD4 transgenic mouse model of wound healing and antibody dosing regimen for a circular full-thickness skin defect with a diameter of 1 cm on the back.

[0205] Figure 14 The size of scars in human CD4 transgenic mice with circular full-thickness skin defects on the back skin containing a 1cm diameter wound was determined after three weeks of intravenous injection of control antibody, TGFβ1 / 2 antibody, and different CD4 / TGFβ double antibodies (WH105, WH205, WH305, WH405, and WH505).

[0206] Figure 15 The size of scars in human CD4 transgenic mice with circular full-thickness skin defects on their backs, with a diameter of 1 cm, after three weeks of intravenous injection of control antibody, IL-4 neutralizing antibody, WH405 bispecific antibody, and WH405 combined with IL-4 neutralizing antibody.

[0207] Figure 16 The distribution of antigenic epitopes of different CD4 antibodies (Zanolimumab, Keliximab, Tregalizumab, Ibalizumab, and 13B8.2) binding to CD4 molecules, and the amino acid sequences of the extracellular regions D1, D2, D3, and D4 of the CD4 molecule.

[0208] Figure 17 The top image shows a schematic diagram of the complex structure of Ibalizumab's Fab and CD4 molecules, with the antigenic epitopes of Ibalizumab binding to CD4 molecules marked in the figure; the bottom image shows a surface diagram of the CD4 molecule structure, with the antigenic epitopes of Ibalizumab binding to CD4 molecules highlighted in black.

[0209] Figure 18 Schematic diagram of neutralizing antibodies that specifically inhibit different TGFβ isoforms.

[0210] Figure 19 Schematic diagrams of the structures of bifunctional antibodies WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101.

[0211] Figure 20Indirect ELISA method was used to detect the binding constant EC of bispecific antibodies WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101 to CD4 protein. 50 .

[0212] Figure 21 The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1, TGFβ2, and TGFβ3 signaling activation by bispecific antibodies WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101.

[0213] Figure 22 The activation status of Th2 cells in the paracortical region of lymph nodes three weeks after tail vein injection of control antibody, CD4 antibody (Ibalizumab), and different CD4 / TGFβ double antibodies (WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101) into hCD4 / +;IL-4-IRES-tdTomato transgenic mice.

[0214] Figure 23 The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1, TGFβ2, and TGFβ3 signaling activation by TGFβ1 antibody (CAT192), TGFβ2 antibody (MOR14797), and TGFβ3 antibody (2A10).

[0215] Figure 24 The size of scars in human CD4 transgenic mice with circular full-thickness skin defects on the back skin containing a 1 cm diameter wound was determined by intravenous injection of control antibody, TGFβ1 antibody (CAT192), TGFβ2 antibody (MOR14797), and TGFβ3 antibody (2A10) three weeks later.

[0216] Figure 25 The size of the scar in human CD4 transgenic mice with a circular full-thickness skin defect with a diameter of 1 cm on the back was measured three weeks after intravenous injection of control antibody, TGFβ1 antibody (CAT192), CD4 antibody (Ibalizumab), and double antibody (WH401).

[0217] Figure 26 The size of the scar in human CD4 transgenic mice with a circular full-thickness skin defect with a diameter of 1 cm on the back was determined by intravenous injection of control antibody, TGFβ2 antibody (MOR14797), CD4 antibody (Ibalizumab), and double antibody (WH402) three weeks later.

[0218] Figure 27 The size of the scar in human CD4 transgenic mice with a circular full-thickness skin defect with a diameter of 1 cm on the back was determined by intravenous injection of control antibody, TGFβ3 antibody (2A10), CD4 antibody (Ibalizumab), and double antibody (WH403) three weeks later.

[0219] Figure 28 The size of scars in human CD4 transgenic mice with circular full-thickness skin defects on the back skin containing a 1 cm diameter wound was determined by intravenous injection of control antibodies and different CD4 / TGFβ double antibodies (WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101) three weeks later.

[0220] Figure 29 Different forms of dual antibodies for WH405: WH405, WH405-1, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH406-9, WH405-10, WH405-11, and WH405-12.

[0221] Figure 30 Indirect ELISA was used to detect the binding constants (ECs) of bispecific antibodies WH405, WH405-1, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH406-9, WH405-10, WH405-11, and WH405-12 to CD4 protein. 50 .

[0222] Figure 31 The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1 and TGFβ2 signaling activation by bispecific antibodies WH405, WH405-1, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH406-9, WH405-10, WH405-11 and WH405-12.

[0223] Figure 32The size of scars three weeks after intravenous injection of control antibody and different forms of WH405 bispecific antibody (WH405, WH405-1, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH406-9, WH405-10, WH405-11 and WH405-12) in human CD4 transgenic mice with circular full-thickness skin defects on the back.

[0224] Figure 33 Schematic diagram of the IgG-ScFv form (Morrison pattern) of bifunctional antibodies composed of different TGFβ1 / 2 antibodies NIS793 (XPA.42.089) and XPA.42.068 and CD4 antibody (Ibalizumab).

[0225] Figure 34 Indirect ELISA method for detecting the binding constant EC of bispecific antibodies WH405 and WH415 to CD4 protein. 50 .

[0226] Figure 35 The TGFβ reporter gene luciferase activity assay determined the inhibition of TGFβ1, TGFβ2, and TGFβ3 signaling activation by bispecific antibodies WH405 and WH415, TGFβ monoclonal antibody NIS793, and XPA.42.068.

[0227] Figure 36 Human CD4 transgenic mice with a circular full-thickness skin defect with a diameter of 1 cm on their backs were intravenously injected with control antibodies, TGFβ monoclonal antibodies NIS793 and XPA.42.068, and bispecific antibodies WH405 and WH415. The size of the scar area after three weeks was measured. Detailed Implementation

[0228] The specific embodiments of this application will be further described in detail below with reference to the accompanying drawings. These embodiments are only for illustrating this application and are not intended to limit the scope of the invention.

[0229] The embodiments of the present invention will now be described in detail with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased on the market.

[0230] Preparation Example 1: Preparation of Recombinant TGFβ1

[0231] 1. Constructing the pCMV-TGFβ1-His plasmid

[0232] TGFβ1 human cDNA (synthesized by Genewiz) was used as a template for PCR amplification, and the TGFβ1-His fragment was purified and recovered using a standard DNA product purification kit. The recovered TGFβ1-His fragment and the expression vector pCMV were digested with NheI and XbaI enzymes. The target gene fragment and linear expression vector were recovered by gel electrophoresis and ligated using T4 ligase. All ligation products were transformed into DH5α chemocompetent cells, plated on Agar plates containing ampicillin, and well-isolated single colonies were selected for colony PCR identification. Clones with positive PCR results were inoculated into LB medium and cultured, and the bacterial culture was sequenced for verification. Sequencing results showed that the inserted sequence in the positive recombinants was completely correct.

[0233] 2. Expression and purification of the fusion protein TGFβ1-His

[0234] HEK293F cells were transfected with the recombinant plasmid pCMV-TGFβ1-his. Five days later, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the medium was replaced with Binding Buffer A (20 mM HEPES, 150 mM NaCl, pH 7.4) before being loaded onto a HisTrap column. Proteins were linearly eluted with Elution Buffer (20 mM HEPES, 150 mM NaCl, 0.5 M Immidazole, pH 7.4). The purified sample was then replaced with Binding Buffer B (20 mM Tris-HCl, pH 9.0) using a HiTrap Desalting column and loaded onto a HiTrap Q column. Proteins were linearly eluted with Elution Buffer B (50 mM Tris-HCl, 1 M NaCl, pH 9.0), and the target sample was recovered and replaced with PBS. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE analysis.

[0235] The fusion protein TGFβ1-His was synthesized.

[0236] The amino acid sequence of TGFβ1-His is shown in SEQ ID NO:13, and the nucleic acid sequence encoding TGFβ1-His is shown in SEQ ID NO:14.

[0237] Preparation Example 2: Preparation of Recombinant TGFβ2

[0238] 1. Constructing the pCMV-TGFβ2-His plasmid

[0239] Using TGFβ2 human cDNA (synthesized by Genewiz) as a template, PCR amplification was performed, and the TGFβ2-His fragment was purified and recovered using a standard DNA product purification kit. The recovered TGFβ2-His fragment and the expression vector pCMV were digested with NheI and XbaI enzymes. The target gene fragment and linear expression vector were recovered by gel electrophoresis and ligated using T4 ligase. All ligation products were transformed into DH5α chemicompetent cells, plated on Agar plates containing ampicillin, and well-isolated single colonies were selected for colony PCR identification. Clones with positive PCR results were inoculated into LB medium and cultured, and the bacterial culture was sequenced for verification. Sequencing results showed that the inserted sequence in the positive recombinants was completely correct.

[0240] 2. Expression and purification of the fusion protein TGFβ2-His

[0241] HEK293F cells were transfected with the recombinant plasmid pCMV-TGFβ3-his. Five days later, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the medium was replaced with Binding Buffer A (20 mM HEPES, 150 mM NaCl, pH 7.4) before loading onto a HisTrap column. Proteins were linearly eluted with Elution Buffer (20 mM HEPES, 150 mM NaCl, 0.5 M Immidazole, pH 7.4). The purified sample was then replaced with Binding Buffer B (20 mM Tris-HCl, pH 9.0) on a HiTrap Desalting column and loaded onto a HiTrap Q column. Proteins were linearly eluted with Elution Buffer B (50 mM Tris-HCl, 1 M NaCl, pH 9.0), and the target sample was recovered and replaced with PBS. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE analysis.

[0242] The fusion protein TGFβ2-His was synthesized.

[0243] The amino acid sequence of TGFβ2-His is shown in SEQ ID NO:15, and the nucleic acid sequence encoding TGFβ1-His is shown in SEQ ID NO:16.

[0244] Preparation Example 3: Preparation of Recombinant TGFβ3

[0245] 1. Constructing the pCMV-TGFβ3-His plasmid

[0246] TGFβ3 human cDNA (synthesized by Genewiz) was used as a template for PCR amplification, and the TGFβ3-His fragment was purified and recovered using a standard DNA product purification kit. The recovered TGFβ3-His fragment and the expression vector pCMV were digested with NheI and XbaI enzymes. The target gene fragment and linear expression vector were recovered by gel electrophoresis and ligated using T4 ligase. All ligation products were transformed into DH5α chemocompetent cells, plated on Agar plates containing ampicillin, and well-isolated single colonies were selected for colony PCR identification. Clones with positive PCR results were inoculated into LB medium and cultured, and the bacterial culture was sequenced for verification. Sequencing results showed that the inserted sequence in the positive recombinants was completely correct.

[0247] 2. Expression and purification of the fusion protein TGFβ3-His

[0248] HEK293F cells were transfected with the recombinant plasmid pCMV-TGFβ3-his. Five days later, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the medium was replaced with Binding Buffer A (20 mM HEPES, 150 mM NaCl, pH 7.4) before loading onto a HisTrap column. Proteins were linearly eluted with Elution Buffer (20 mM HEPES, 150 mM NaCl, 0.5 M Immidazole, pH 7.4). The purified sample was then replaced with Binding Buffer B (20 mM Tris-HCl, pH 9.0) on a HiTrap Desalting column and loaded onto a HiTrap Q column. Proteins were linearly eluted with Elution Buffer B (50 mM Tris-HCl, 1 M NaCl, pH 9.0), and the target sample was recovered and replaced with PBS. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE analysis.

[0249] The fusion protein TGFβ3-His was synthesized.

[0250] The amino acid sequence of TGFβ3-His is shown in SEQ ID NO:17, and the nucleic acid sequence encoding TGFβ1-His is shown in SEQ ID NO:18.

[0251] Preparation Example 4: Expression and purification of recombinant protein CD4-His

[0252] 1. Constructing the pCMV-CD4-His plasmid

[0253] PCR amplification was performed using CD4 human cDNA (synthesized by Genewiz) as a template, and the CD4-His fragment was purified and recovered using a standard DNA product purification kit. The recovered CD4-His fragment and the expression vector pCMV were digested with NheI and XbaI enzymes. The target gene fragment and linear expression vector were recovered by gel electrophoresis and ligated using T4 ligase. All ligation products were transformed into DH5α chemocompetent cells, plated on Agar plates containing ampicillin, and well-isolated single colonies were selected for colony PCR identification. Clones with positive PCR results were inoculated into LB medium and cultured, and the bacterial culture was sequenced for verification. Sequencing results showed that the inserted sequence in the positive recombinants was completely correct.

[0254] 2. Expression and purification of recombinant protein CD4-His

[0255] HEK293F cells were transfected with the recombinant plasmid pCMV-CD4-his. Five days later, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the medium was replaced with Binding Buffer A (20 mM HEPES, 150 mM NaCl, pH 7.4) before being loaded onto a HisTrap column. Proteins were linearly eluted with Elution Buffer (20 mM HEPES, 150 mM NaCl, 0.5 M Immidazole, pH 7.4). The purified sample was then replaced with Binding Buffer B (20 mM Tris-HCl, pH 9.0) using a HiTrap Desalting column and loaded onto a HiTrap Q column. Proteins were linearly eluted with Elution Buffer B (50 mM Tris-HCl, 1 M NaCl, pH 9.0), and the target sample was recovered and replaced with PBS. The purified sample was then added to reduced protein electrophoresis loading buffer for SDS-PAGE analysis.

[0256] The fusion protein CD4-His was successfully synthesized.

[0257] The amino acid sequence of CD4-His is shown in SEQ ID NO:19, and the nucleic acid sequence encoding CD4-His is shown in SEQ ID NO:20.

[0258] Preparation Example 5: Preparation of anti-CD4 antibody Ibalizumab

[0259] The amino acid sequences of the heavy chain variable region and light chain variable region of the marketed CD4 monoclonal antibody Trogarzo (Ibalizumab) are based on US Patent Publication US005871732. The nucleic acid sequences encoding the heavy chain variable region and light chain variable region were synthesized by Genscript.

[0260] The constant region of the heavy chain was the Ig gamma-4 chain C region, ACCESSION: P01861; the constant region of the light chain was the Ig kappa chain C region, ACCESSION: P01834.

[0261] The heavy chain cDNA and light chain cDNA of Ibalizumab were cloned into the pCMV vector to obtain the recombinant expression plasmid of the antibody Ibalizumab.

[0262] The recombinant plasmid was transfected into HEK293F cells. The HEK293F cell culture medium was purified and then analyzed.

[0263] The anti-CD4 monoclonal antibody Ibalizumab was successfully prepared.

[0264] The amino acid sequence of the heavy chain variable region of Ibalizumab is shown in SEQ ID NO:21, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:22; the amino acid sequence of the light chain variable region of Ibalizumab is shown in SEQ ID NO:23, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:24.

[0265] Preparation Example 6: Preparation of Anti-TGFβ1 / 2 Antibody NIS793

[0266] The constant region of the heavy chain was the Ig gamma-2 chain C region, ACCESSION: P01859; the constant region of the light chain was the Ig lambda chain C region, ACCESSION: P0DOX8.

[0267] The heavy chain cDNA and light chain cDNA of NIS793 were cloned into the pCMV vector to obtain the recombinant expression plasmid of antibody NIS793.

[0268] The recombinant plasmid was transfected into HEK293F cells. The HEK293F cell culture medium was purified and then analyzed.

[0269] Antibody NIS793 against TGFβ1 / 2 was prepared.

[0270] The amino acid sequence of the heavy chain variable region of NIS793 is shown in SEQ ID NO:25, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:26; the amino acid sequence of the light chain variable region of NIS793 is shown in SEQ ID NO:27, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:28.

[0271] The amino acid sequence of the NIS793 heavy chain is shown in SEQ ID NO:29, and the nucleic acid sequence of the heavy chain is shown in SEQ ID NO:30; the amino acid sequence of the NIS793 light chain is shown in SEQ ID NO:31, and the nucleic acid sequence of the light chain is shown in SEQ ID NO:32.

[0272] Preparation Example 7: Preparation of Bispecific Antibody WH405

[0273] The heavy chain variable region and light chain variable region of the CD4 antibody Ibalizumab were linked together using a 3xGGGGS linker to construct an ScFv structure. Then, the ScFv was linked to the C-terminus of the NIS793 IgG2 antibody heavy chain using a 3xGGGGS linker to obtain the second protein chain cDNA plasmid of the bispecific antibody WH405.

[0274] The light chain cDNA of the NIS793 antibody, used as the first protein chain cDNA plasmid, was co-transfected with the second protein chain cDNA plasmid of WH405 into HEK293F cells for expression for 5 days. The HEK293F cell culture medium was then purified for detection.

[0275] A dual antibody against CD4 and TGFβ1 / 2, WH405, was obtained.

[0276] The amino acid sequence of the first protein chain of antibody WH405 is shown in SEQ ID NO:33, and the nucleic acid sequence is shown in SEQ ID NO:34; the amino acid sequence of the second protein chain of antibody WH405 is shown in SEQ ID NO:35, and the nucleic acid sequence is shown in SEQ ID NO:36. A schematic diagram of the structure of WH405 is shown below. Figure 1 As shown. The amino acid sequence and CDR region of heavy or light chain of WH405 are as follows. Figure 2 As shown.

[0277] Preparation Example 8: Preparation of bispecific antibodies WH105, WH205, WH305 and WH505

[0278] The heavy chain variable regions and light chain variable regions of CD4 antibodies (Zanolimumab, Keliximab, Tregalizumab, and 13B8.2) were linked together using a 3xGGGGS linker to construct an ScFv structure. The ScFv structure was then linked to the C-terminus of the NIS793 IgG2 antibody heavy chain using a 3xGGGGS linker to obtain the second protein chain cDNA plasmids of bispecific antibodies WH105, WH205, WH305, and WH505.

[0279] The distribution of antigenic epitopes binding to CD4 molecules by different CD4 antibodies (Zanolimumab, Keliximab, Tregalizumab, Ibalizumab, and 13B8.2) and the amino acid sequences of the extracellular regions D1, D2, D3, and D4 of the CD4 molecule are shown in the figure. Figure 16 As shown in the diagram. This is a schematic diagram of the complex structure of Ibalizumab's Fab and CD4 molecule. The diagram shows the antigenic epitope of Ibalizumab binding to the CD4 molecule and the surface structure of the CD4 molecule. Figure 17 As shown.

[0280] The light chain cDNA of the NIS793 antibody was used as the first protein chain cDNA plasmid and co-transfected into HEK293F cells with the second protein chain cDNA plasmids of WH105, WH205, WH305, and WH505 for expression for 5 days. The HEK293F cell culture medium was then purified for detection.

[0281] Bispecific antibodies WH105, WH205, WH305, and WH505 were successfully synthesized. Structural diagrams of the bispecific antibodies WH105, WH205, WH305, WH405, and WH505 are shown in the reference diagrams. Figure 5 .

[0282] The amino acid sequence of the heavy chain variable region of Zanolimumab is shown in SEQ ID NO:37, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:38; the amino acid sequence of the light chain variable region of Zanolimumab is shown in SEQ ID NO:39, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:40.

[0283] The amino acid sequence of the heavy chain of Keliximab is shown in SEQ ID NO:41, and the nucleic acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:42; the amino acid sequence of the variable region of the light chain of Keliximab is shown in SEQ ID NO:43, and the nucleic acid sequence of the variable region of the light chain is shown in SEQ ID NO:44.

[0284] The amino acid sequence of the heavy chain of Tregalizumab is shown in SEQ ID NO:45, and the nucleic acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:46; the amino acid sequence of the variable region of the light chain of Tregalizumab is shown in SEQ ID NO:47, and the nucleic acid sequence of the variable region of the light chain is shown in SEQ ID NO:48.

[0285] The amino acid sequence of the 13B8.2 heavy chain is shown in SEQ ID NO:49, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:50; the amino acid sequence of the 13B8.2 light chain variable region is shown in SEQ ID NO:51, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:52.

[0286] Preparation Example 9: Preparation of bispecific antibodies WH401, WH402, WH403, WH404, and WH406

[0287] The heavy chain variable region and light chain variable region of the CD4 antibody Ibalizumab were linked together using a 3xGGGGS linker to construct an ScFv structure. Then, the ScFv was linked to the C-terminus of the heavy chains of CAT192 IgG2, MOR14797 IgG2, 2A10 IgG2, XPA.42.681 IgG2, and 4A11 IgG2 using a 3xGGGGS linker to obtain the second protein chain cDNA plasmids of the bispecific antibodies WH401, WH402, WH403, WH404, and WH406.

[0288] The light chain cDNAs of antibodies CAT192, MOR14797, 2A10, XPA.42.681, and 4A11 were used as first-chain cDNA plasmids and co-transfected with second-chain cDNA plasmids of WH401, WH402, WH403, WH404, and WH406 into HEK293F cells for expression for 5 days. The HEK293F cell culture medium was purified and then analyzed.

[0289] The dual antibodies WH401, WH402, WH403, WH404 and WH406 were prepared.

[0290] The amino acid sequence of the heavy chain variable region of CAT192 is shown in SEQ ID NO:53, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:54; the amino acid sequence of the light chain variable region of CAT192 is shown in SEQ ID NO:55, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:56.

[0291] The amino acid sequence of the heavy chain variable region of MOR14797 is shown in SEQ ID NO:57, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:58; the amino acid sequence of the light chain variable region of MOR14797 is shown in SEQ ID NO:59, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:60.

[0292] The amino acid sequence of the heavy chain variable region of 2A10 is shown in SEQ ID NO:61, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:62; the amino acid sequence of the light chain variable region of 2A10 is shown in SEQ ID NO:63, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:64.

[0293] The amino acid sequence of the heavy chain variable region of XPA.42.681 is shown in SEQ ID NO:65, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:66; the amino acid sequence of the light chain variable region of XPA.42.681 is shown in SEQ ID NO:67, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:68.

[0294] The amino acid sequence of the 4A11 heavy chain variable region is shown in SEQ ID NO:69, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:70; the amino acid sequence of the 4A11 light chain variable region is shown in SEQ ID NO:71, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:72.

[0295] Preparation Example 10: Preparation of Bispecific Antibodies WH407 and CT101

[0296] The extracellular region of TGFβRII was linked to the C-terminus of the Ibalizumab heavy chain using a 3xGGGGS linker, resulting in the second protein chain cDNA plasmid of the bispecific antibody WH407.

[0297] The heavy chain variable region and light chain variable region of the CAT192 antibody were linked together using a 3xGGGGS linker to construct an ScFv structure. The ScFv was then linked to the C-terminus of the Ibalizumab heavy chain using a 3xGGGGS linker to obtain the second protein chain cDNA plasmid of the bispecific antibody CT101.

[0298] The light chain cDNA of Ibalizumab, used as the first protein chain cDNA plasmid, was co-transfected with the second protein chain cDNA plasmids of WH407 and CT101 into HEK293F cells for expression for 5 days. The HEK293F cell culture medium was then purified and analyzed.

[0299] Bispecific antibodies WH407 and CT101 were obtained.

[0300] The amino acid sequence of the extracellular region of TGFβRII is shown in SEQ ID NO:73, and the nucleic acid sequence is shown in SEQ ID NO:74.

[0301] Preparation Example 11: Bispecific antibodies WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8 Preparation of WH405-9, WH405-10, WH405-11 and WH405-12

[0302] The heavy chain variable region and light chain variable region of Ibalizumab and NIS793 are combined according to... Figure 29 They are assembled into various dual-antibody forms, with different functional areas connected together via 3xGGGGS linkers.

[0303] Different protein chain cDNA plasmids in various forms of bispecific antibodies were co-transfected into HEK293F cells for expression for 5 days. The HEK293F cell culture medium was then purified and analyzed.

[0304] The dual-antibiotic formulations WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11 and WH405-12 were prepared.

[0305] Preparation Example 12: Anti-TGFβ1 / 2 antibody XPA.42.068, anti-TGFβ1 antibody CAT192, and anti-TGFβ2 antibody Preparation of MOR14797, anti-TGFβ3 antibody 2A10

[0306] The constant region of the heavy chain was the Ig gamma-2 chain C region, ACCESSION: P01859; the constant region of the light chain was the Ig lambda chain C region, ACCESSION: P0DOX8.

[0307] The heavy chain cDNA and light chain cDNA of XPA.42.068, CAT192, MOR14797 and 2A10 were cloned into the pCMV vector to obtain recombinant expression plasmids of antibodies XPA.42.068, CAT192, MOR14797 and 2A10.

[0308] The recombinant plasmid was transfected into HEK293F cells. The HEK293F cell culture medium was purified and then analyzed.

[0309] Antibody XPA.42.068 against TGFβ1 / 2, antibody CAT192 against TGFβ1, antibody MOR14797 against TGFβ2, and antibody 2A10 against TGFβ3 were prepared.

[0310] The amino acid sequence of the heavy chain variable region of XPA.42.068 is shown in SEQ ID NO:75, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:76; the amino acid sequence of the light chain variable region of XPA.42.068 is shown in SEQ ID NO:77, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:78.

[0311] The amino acid sequence of the heavy chain variable region of CAT192 is shown in SEQ ID NO:53, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:54; the amino acid sequence of the light chain variable region of CAT192 is shown in SEQ ID NO:55, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:56.

[0312] The amino acid sequence of the heavy chain variable region of MOR14797 is shown in SEQ ID NO:57, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:58; the amino acid sequence of the light chain variable region of MOR14797 is shown in SEQ ID NO:59, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:60.

[0313] The amino acid sequence of the heavy chain variable region of 2A10 is shown in SEQ ID NO:61, and the nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:62; the amino acid sequence of the light chain variable region of 2A10 is shown in SEQ ID NO:63, and the nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:64.

[0314] Preparation Example 13: Preparation of Bispecific Antibody WH415

[0315] The heavy chain variable region and light chain variable region of the CD4 antibody Ibalizumab were linked together using a 3xGGGGS linker to construct an ScFv structure. The ScFv was then linked to the C-terminus of the XPA.42.068 IgG2 antibody heavy chain using the 3xGGGGS linker. Figure 32 The antibody was assembled into the form of IgG-(H)-ScFv bispecific antibody, and the second protein chain cDNA plasmid of bispecific antibody WH415 was obtained.

[0316] The light chain cDNA of the XPA.42.068 antibody, used as the first protein chain cDNA plasmid, was co-transfected with the second protein chain cDNA plasmid of WH415 into HEK293F cells for expression for 5 days. The HEK293F cell culture medium was then purified for detection.

[0317] A dual antibody against CD4 and TGFβ1 / 2, WH415, was prepared.

[0318] The neutralizing abilities of CAT192, MOR14797, 2A10, NIS793, 4A11, TGF-β decoy receptors, and XPA.42.681 are as follows: Figure 18As shown. CAT192 can neutralize TGF-β1, MOR14797 can neutralize TGF-β2, 2A10 can neutralize TGF-β3, NIS793 can neutralize both TGF-β1 and TGF-β2, 4A11 can neutralize both TGF-β2 and TGF-β3, the TGF-β decoy receptor can neutralize both TGF-β1 and TGF-β3, and XPA.42.681 can neutralize TGF-β1, TGF-β2, and TGF-β3. Schematic diagrams of the IgG-ScFv form (Morrison pattern) of bifunctional antibodies composed of different TGFβ1 / 2 antibodies NIS793 (XPA.42.089) and XPA.42.068 with CD4 antibody (Ibalizumab) are shown below. Figure 33 As shown.

[0319] The antibody forms, binding, and neutralizing abilities of WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 are as follows: Figure 19 , Figure 20 as well as Figure 21 As shown.

[0320] like Figure 20 and Figure 21 As shown, WH401 can bind CD4 and neutralize TGF-β1, WH402 can bind CD4 and neutralize TGF-β2, WH403 can bind CD4 and neutralize TGF-β3, WH404 can bind CD4 and neutralize TGF-β1, TGF-β2 and TGF-β3, WH405 can bind CD4 and neutralize TGF-β1 and TGF-β2, WH406 can bind CD4 and neutralize TGF-β2 and TGF-β3, WH407 can bind CD4 and neutralize TGF-β1 and TGF-β3, and CT101 can bind CD4 and neutralize TGF-β1.

[0321] Zanolimumab, Keliximab, Tregalizumab, 13B8.2, and Ibalizumab can bind to CD4. Zanolimumab, Keliximab, Tregalizumab, and 13B8.2 bind to the D1 domain of CD4. Ibalizumab binds to the D2 domain of CD4.

[0322] The amino acid sequence of the D1 domain of CD4 is shown in positions 1-100 of SEQ ID NO: 19, the amino acid sequence of the D2 domain of CD4 is shown in positions 101-178 of SEQ ID NO: 19, the amino acid sequence of the D3 domain of CD4 is shown in positions 179-292 of SEQ ID NO: 19, and the amino acid sequence of the D4 domain of CD4 is shown in positions 293-365 of SEQ ID NO: 19.

[0323] Example 1: ELISA method for detecting bispecific antibodies WH105, WH205, WH305, WH405, WH505, WH401, WH402, WH403, WH404, WH406, WH407, CT101, WH415, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, WH405-12, and the CD4 monoclonal antibody Ibalizumab with anti- Original CD4 binding activity

[0324] 1. The binding activity of the above antibodies to CD4 was determined by indirect ELISA.

[0325] The specific method is as follows:

[0326] The microplate was coated with CD4-His and incubated at 37°C for 3 hours. After washing, it was blocked with 1% BSA for 1 hour. After washing, serially diluted double antibody WH405 was added (specific concentrations are shown on the x-axis), and the plate was incubated at 37°C for 60 minutes. After washing, enzyme-labeled donkey anti-human IgG secondary antibody working solution was added, and the plate was incubated at 37°C for 30 minutes. After washing, TMB chromogenic solution was added, and the plate was developed in the dark for 5 minutes. The reaction was stopped by adding stop solution. The microplate was immediately placed in a microplate reader, and the OD values ​​of each well were read at 450 nm. The data were analyzed using SoftMax Pro software.

[0327] The results of detecting the binding of antibody WH405 and Ibalizumab to antigen CD4 are as follows: Figure 3 As shown, the binding results of WH105, WH205, WH305, WH405, and WH505 to antigen CD4 are as follows: Figure 6 As shown, the binding results of WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 to antigen CD4 are as follows: Figure 20 As shown, the binding results of WH405, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, and WH405-12 with antigen CD4 are as follows: Figure 30 As shown, the binding results of WH405 and WH415 to antigen CD4 are as follows: Figure 34 As shown. A curve was fitted with antibody concentration on the x-axis and absorbance value on the y-axis to calculate the antibody binding EC50. 50 The results are shown in Table 1.

[0328] Table 1. Binding constants (EC50) of each antibody to antigen CD4 50

[0329]

[0330] Example 2: Bispecific antibodies WH105, WH205, WH305, WH405, WH505, WH401, WH402, WH403, WH404, WH406, WH407, CT101, WH415, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, WH405-12, TGFβ1 / 2 monoclonal antibody NIS793, XAP.42.068, TGFβ 1. Monoclonal antibody CAT192, TGFβ2 monoclonal antibody MOR14797, and TGFβ3 monoclonal antibody 2A10 inhibited TGFβ1-induced TGFβ / TGFβ / TGFβ / TGFβ1 in HEK293 cells. Smad signaling pathway activation

[0331] HEK293 cells were used at 1 x 10⁻⁶ 5 The antibody was seeded at a concentration of 500 μL / well in 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, the plates were transfected with the TGFβ / Smad promoter active luciferase reporter plasmid pSMAD-Luc and the sea cucumber luciferase internal control plasmid pRL-TK. 24 h post-transfection, serially diluted versions of the antibody (final concentrations shown on the x-axis) and recombinant TGFβ1 (final concentration 2 ng / mL) were added, and the plates were cultured for another 12 h. After 12 h, the culture medium was discarded, and each well was lysed with 100 μL of 1x Passive Lysis Buffer (Promega). 70 μL of the lysis buffer was placed in a microplate, and 30 μL of substrate 1 solution was added. The microplate was immediately placed in a microplate reader, and the readings for each well were read using Luminescence. Add 30 μL of substrate 2 solution, and immediately place the microplate in a microplate reader. Select Luminescence to read the values ​​of each well as Bn (Dual Fluorescence Reporter System Kit, Promega, catalog number PR-E1910). Analyze the data using SoftMax Pro software. The An / Bn ratio represents the activation fold of the TGFβ / Smad signaling pathway.

[0332] The results of detecting the inhibition of TGFβ1 by WH405, NIS793, and XAP.42.068 are as follows: Figure 4 As shown, the inhibition results of WH105, WH205, WH305, WH405, and WH505 on TGFβ1 are as follows: Figure 7 As shown, the inhibition results of WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 on TGFβ1 are as follows: Figure 21 As shown, the inhibition results of CAT192, MOR14797, and 2A10 on TGFβ1 are as follows: Figure 23As shown, the inhibition results of WH405, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, and WH405-12 on TGFβ1 are as follows: Figure 31 As shown, the inhibition results of WH405 and WH415 on TGFβ1 are as follows: Figure 35 As shown. A curve was fitted with antibody concentration on the x-axis and the activation fold of the TGFβ / Smad signaling pathway on the y-axis to calculate the antibody's inhibitory constant IC50. 50 The results are shown in Table 2.

[0333] Table 2. Inhibition constants (IC50) of each antibody against antigen TGFβ1 50

[0334]

[0335] Example 3: Bispecific antibodies WH105, WH205, WH305, WH405, WH505, WH401, WH402, WH403, WH404, WH406, WH407, CT101, WH415, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, WH405-12, TGFβ1 / 2 monoclonal antibody NIS793, XAP.42.068, TGFβ1 Monoclonal antibodies CAT192, MOR14797 (TGFβ2), and 2A10 (TGFβ3) inhibited TGFβ2-induced TGFβ / β2 / β2 in HEK293 cells. Smad signaling pathway activation

[0336] HEK293 cells were used at 1 x 10⁻⁶ 5 Cells were seeded at a concentration of 500 μL / well in 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, the cells were transfected with the TGFβ / Smad promoter active luciferase reporter plasmid pSMAD-Luc and the sea cucumber luciferase internal control plasmid pRL-TK. 24 h post-transfection, serially diluted versions of the above antibodies (final antibody concentrations shown on the x-axis) and recombinant TGFβ2 (final concentration 2 ng / mL) were added, and the cells were cultured for another 12 h. After 12 h, the culture medium was discarded, and each well was lysed with 100 μL of 1x Passive Lysis Buffer (Promega). 70 μL of the lysis buffer was placed in a microplate, and 30 μL of substrate 1 solution was added. The microplate was immediately placed in a microplate reader, and the readings for each well were taken as An using Luminescence. Add 30 μL of substrate 2 solution, and immediately place the microplate in a microplate reader. Select Luminescence to read the values ​​of each well as Bn (Dual Fluorescence Reporter System Kit, Promega, catalog number PR-E1910). Analyze the data using SoftMax Pro software. The An / Bn ratio represents the activation fold of the TGFβ / Smad signaling pathway.

[0337] The results of detecting the inhibition of TGFβ2 by WH405 and NIS793 are as follows: Figure 4As shown, the inhibition results of WH105, WH205, WH305, WH405, and WH505 on TGFβ2 are as follows: Figure 7 As shown, the inhibition results of WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 on TGFβ2 are as follows: Figure 21 As shown, the inhibition results of CAT192, MOR14797, and 2A10 on TGFβ2 are as follows: Figure 23 As shown, the inhibition results of WH405, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, and WH405-12 on TGFβ2 are as follows: Figure 31 As shown, the inhibition results of WH405 and WH415 on TGFβ2 are as follows: Figure 35 As shown. A curve was fitted with antibody concentration on the x-axis and the activation fold of the TGFβ / Smad signaling pathway on the y-axis to calculate the antibody's inhibitory constant IC50. 50 The results are shown in Table 3.

[0338] Table 3. Inhibition constants (IC50) of each antibody against antigen TGFβ2 50

[0339]

[0340] Example 4: Bispecific antibodies WH405, WH401, WH402, WH403, WH404, WH406, WH407, CT101, WH415, TGF β1 / 2 monoclonal antibodies NIS793 and XAP.42.068, TGFβ1 monoclonal antibody CAT192, TGFβ2 monoclonal antibody MOR14797, and TGFβ3 monoclonal antibody 2A10 Inhibition of TGFβ3-induced activation of the TGFβ / Smad signaling pathway in HEK293 cells

[0341] HEK293 cells were used at 1 x 10⁻⁶ 5The antibody was seeded at a concentration of 500 μL / well in 24-well plates and incubated at 37°C with 5% CO2 for 24 h. Then, the plates were transfected with the TGFβ / Smad promoter-active luciferase reporter plasmid pSMAD-Luc and the sea cucumber luciferase internal control plasmid pRL-TK. 24 h after transfection, serially diluted versions of the antibody (final concentrations shown on the x-axis) and recombinant TGFβ3 (final concentration 2 ng / mL) were added, and the plates were cultured for another 12 h. After 12 h, the culture medium was discarded, and each well was lysed with 100 μL of 1x Passive Lysis Buffer (Promega). 70 μL of the lysis buffer was placed in a microplate, and 30 μL of substrate 1 solution was added. The microplate was immediately placed in a microplate reader, and the readings for each well were taken as An using Luminescence. Add 30 μL of substrate 2 solution, and immediately place the microplate in a microplate reader. Select Luminescence to read the values ​​of each well as Bn (Dual Fluorescence Reporter System Kit, Promega, catalog number PR-E1910). Analyze the data using SoftMax Pro software. The An / Bn ratio represents the activation fold of the TGFβ / Smad signaling pathway.

[0342] The results of detecting the inhibition of TGFβ3 by WH405 and NIS793 are as follows: Figure 4 As shown, the inhibition results of WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 on TGFβ3 are as follows: Figure 21 As shown, the inhibition results of CAT192, MOR14797, and 2A10 on TGFβ3 are as follows: Figure 23 As shown, the inhibition results of WH405 and WH415 on TGFβ3 are as follows: Figure 35 As shown. A curve was fitted with antibody concentration on the x-axis and the activation fold of the TGFβ / Smad signaling pathway on the y-axis to calculate the antibody's inhibitory constant IC50. 50 The results are shown in Table 4.

[0343] Table 4. Inhibition constants (IC50) of each antibody against antigen TGFβ3 50

[0344]

[0345] Example 5: Control antibody, TGFβ1 / 2 antibody (NIS793), different penicillin antibodies WH105, WH205, WH305, WH405 And a statistical analysis of drug entry into lymph nodes after intravenous infusion of WH505.

[0346] Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the mouse strain selected for this experiment was C57BL / 6J. Ibalizumab binds to human CD4 but not to mouse CD4, requiring humanization of the corresponding target site in C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, this experiment used humanized CD4 mice, i.e., replacing the mouse CD4 ORF with the human CD4 open reading frame (ORF).

[0347] Control antibody (non-reactive antibody, IgG2 isotype control), TGFβ1 / 2 antibody (NIS793), and different penicillin antibodies WH105, WH205, WH305, WH405, and WH505 were administered intravenously to 8-week-old humanized CD4 transgenic mice via tail vein injection (100 μg, dose 5 mg / kg). After a single administration, lymph nodes were harvested from the mice, placed in molds, embedded in OCT, and flash-frozen in liquid nitrogen. Freshly frozen tissue sections were prepared using a Reward Minux FS800 cryostat at -25 °C to -15 °C, with section thicknesses ranging from 10 to 14 μm. Tissues were fixed with 4% paraformaldehyde and then immunofluorescently stained with APC-conjugated anti-human IgG Fc secondary antibody (BioLegend, catalog number 647501), FITC-conjugated anti-mouse CD4 antibody (BioLegend, catalog number 100405), and DAPI. After dyeing, the images were scanned using a panoramic digital slicing scanning system (3DHISTECH, model Pannoracic MIDI). The scanned files were then analyzed using Slideviewer software.

[0348] Drugs that follow CD4+ T cells into lymph nodes can be represented by cells that are positive for APC-conjugated anti-human IgG Fc secondary antibody staining. The ratio of CD4+ T cells in the lymph node to the total number of cells can be used to detect the proportion of drugs entering the lymph node.

[0349] The results are as follows Figure 10 As shown. The results show that, Different dual antibodies WH105, WH205, WH305, WH405 and WH505 Following intravenous injection, both antibodies were able to enter the lymph nodes along with CD4+ T cells, while the control antibody and TGFβ monoclonal antibody were not. This result indicates that the bispecific antibody combination of TGFβ and CD4 antibody, after intravenous administration, can enter the lymph nodes along with CD4+ T cells, while neither the control antibody nor the TGFβ antibody can. The mechanism of action of the bispecific antibody WH405 after intravenous injection is as follows: Figure 11 As shown.

[0350] Example 6: Effects of intravenous infusion of different dual antibodies WH105, WH205, WH305, WH405, and WH505 on lymph node accessory lymph nodes Activation of Th2 cells in the cortex

[0351] Preparation of humanized hCD4 / + transgenic mice: Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the C57BL / 6J mouse strain was selected for this experiment; Ibalizumab can bind to human CD4 but not mouse CD4. Humanization of the corresponding target site needs to be performed on C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, this experiment used humanized CD4 mice, which were obtained by replacing the mouse CD4 ORF with the human CD4 open reading frame (ORF) to obtain humanized hCD4 / + transgenic mice.

[0352] By mating humanized hCD4 / + transgenic mice (Shanghai Southern Model Biotechnology, catalog number NM-HU-00115) with IL-4-IRES-tdTomato reporter mice (Shanghai Southern Model Biotechnology, catalog number NM-KI-200028), hCD4 / +;IL-4-IRES-tdTomato transgenic mice were obtained. These mice were capable of detecting the activation of Th2 cells in the paracortical region of lymph nodes after intravenous infusion of dual antibodies. The preparation process of hCD4 / +;IL-4-IRES-Tdtomato transgenic mice is as follows: Figure 9 As shown.

[0353] Control antibody (non-reactive antibody, IgG2 isotype control), NIS793 monoclonal antibody, and different bispecific antibodies WH105, WH205, WH305, WH405, and WH505 were administered intravenously to 6-8 week old humanized hCD4 / +;IL-4-IRES-tdTomato transgenic mice. The dose was 100 μg (5 mg / kg) administered via tail vein injection once a week for a total of three weeks. After three administrations, lymph nodes were harvested from the mice, placed in molds, embedded in OCT, and flash-frozen in liquid nitrogen. Fresh frozen tissue sections were prepared using a Reward Minux FS800 cryostat at -25°C to -15°C, with section thicknesses of 10-14 μm. The tissues were fixed with 4% paraformaldehyde and then immunofluorescently stained with APC-conjugated anti-mouse CD4 antibody (BioLegend, catalog number 100515) and DAPI. After dyeing, the images were scanned using a panoramic digital slicing scanning system (3DHISTECH, model Pannoracic MIDI). The scanned files were then analyzed using Slideviewer software.

[0354] Since Th2 cells are double positive for CD4 and IL-4, CD4 positive cells can be identified as Th2 cells by the combination of the FITC green fluorescence of the CD4-FITC antibody binding band and the tdTomato red fluorescence of IL-4 positive cells.

[0355] After maturation and differentiation, Th2 cells are mostly located in the paracortical region of lymph nodes. By analyzing the proportion of Th2 cells in this region among all CD4+ T cells, the activation status of Th2 cells in lymph nodes can be detected.

[0356] The results are as follows Figure 12 As shown in the figure. The results indicated that, compared with the control antibody, NIS793 antibody, and other bispecific antibodies WH105, WH205, WH305, and WH505, WH405 effectively activated lymph node Th2 cells. This result suggests that only a combination bispecific antibody such as ibalizumab (a CD4 antibody) and TGFβ1 / 2 antibody can effectively activate lymph node Th2 cells; other combinations of CD4 antibodies and TGFβ1 / 2 antibodies cannot activate lymph node Th2 cells.

[0357] Example 7: Effects of intravenous infusion of different bispecific antibodies WH105, WH205, WH305, WH405, and WH505 on mouse wounds Effects of healing

[0358] Preparation of humanized hCD4 / + transgenic mice: Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the C57BL / 6J strain was selected in this embodiment. Ibalizumab binds to human CD4 but not mouse CD4, requiring humanization of the corresponding target site in C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, humanized CD4 mice were used in this experiment, i.e., the open reading frame (ORF) of human CD4 was replaced with the ORF of mouse CD4, resulting in humanized hCD4 / + transgenic mice.

[0359] Skin defect wound creation: This embodiment uses a mouse full-thickness skin defect model. The method involves surgically removing the hair on the mouse's back to create a circular full-thickness skin defect wound with a diameter of 1 cm. The wound is exposed and kept in a sterile environment. After the wound scabs over (2-3 days), a scar gradually forms, and the model can last for more than 3 weeks.

[0360] After creating skin defects on the backs of 6-8 week old humanized hCD4 / + transgenic mice, control antibodies (non-reactive antibodies, IgG2 isotype control), NIS793 monoclonal antibody, and different bispecific antibodies WH105, WH205, WH305, WH405, and WH505 were administered intravenously via tail vein at a dose of 100 μg (5 mg / kg) once a week for a total of 3 administrations. Twenty-one days after administration, the scar area on the backs of the mice was measured (details are as follows). Figure 13 (As shown).

[0361] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0362] The results are as follows Figure 14 As shown in the figure. The results showed that, compared with the control antibody, TGFβ1 / 2 monoclonal antibody (NIS793), and bispecific antibodies WH105, WH205, WH305, and WH505, the scar area after WH405 treatment was significantly reduced, indicating that WH405 has a significant wound healing promoting ability. This significant wound healing promoting ability is related to WH405's activation of Th2 cells and induction of their tissue repair ability.

[0363] In addition, TGFβ1 / 2 monoclonal antibody (NIS793), bispecific antibodies WH105, WH205, WH305 and WH505 showed a relative reduction in scar area after treatment compared with the control antibody, indicating that these antibodies all have a certain degree of wound healing promotion effect, but this effect does not depend on the tissue repair capacity of Th2 cells.

[0364] Example 8: Control antibody, WH405 bispecific antibody, IL-4 neutralizing antibody, and WH405 combined with IL-4 neutralizing antibody, intravenously. Effects of infusion on wound healing in mice.

[0365] Preparation of humanized hCD4 / + transgenic mice: Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the C57BL / 6J strain was selected in this embodiment. Ibalizumab binds to human CD4 but not mouse CD4, requiring humanization of the corresponding target site in C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, humanized CD4 mice were used in this experiment, i.e., the open reading frame (ORF) of human CD4 was replaced with the ORF of mouse CD4, resulting in humanized hCD4 / + transgenic mice.

[0366] Skin defect wound creation: This embodiment uses a mouse full-thickness skin defect model. The method involves surgically removing the hair on the mouse's back to create a circular full-thickness skin defect wound with a diameter of 1 cm. The wound is exposed and kept in a sterile environment. After the wound scabs over (2-3 days), a scar gradually forms, and the model can last for more than 3 weeks.

[0367] After creating skin defects on the backs of 6-8 week old humanized hCD4 / + transgenic mice, control antibodies (non-reactive antibodies, IgG2 isotype control), NIS793 monoclonal antibody, and different bispecific antibodies WH105, WH205, WH305, WH405, and WH505 were administered intravenously via tail vein at a dose of 100 μg (5 mg / kg) once a week for a total of 3 administrations. Twenty-one days after administration, the scar area on the backs of the mice was measured (details are as follows). Figure 13 (As shown).

[0368] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0369] The results are as follows Figure 15 As shown in the figure, the results indicated that compared to the control antibody and IL-4 neutralizing antibody, the scar area after dual antibody WH405 treatment was significantly reduced. However, when WH405 was treated in combination with the IL-4 neutralizing antibody, the wound-healing ability of WH405 completely returned to the level of the control antibody. Since the IL-4 neutralizing antibody can neutralize the Th2 immune response, neutralizing IL-4 inhibits the Th2 immune response, thereby suppressing the wound-healing ability of WH405. These results suggest that the wound-healing effect of WH405 is achieved through WH405 activating Th2 cells in lymph nodes and inducing Th2 cell-mediated tissue repair.

[0370] Example 9: Different dual antibodies WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101 intravenous... Activation of Th2 cells in the paracortical area of ​​lymph nodes after intravenous infusion

[0371] Preparation of humanized hCD4 / + transgenic mice: Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the C57BL / 6J strain was selected for this experiment. Ibalizumab binds to human CD4 but not mouse CD4, requiring humanization of the corresponding target site in C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, humanized CD4 mice were used, i.e., the open reading frame (ORF) of human CD4 was replaced with the ORF of mouse CD4, resulting in humanized hCD4 / + transgenic mice.

[0372] By mating humanized hCD4 / + transgenic mice with IL-4-IRES-tdTomato reporter mice, hCD4 / +;IL-4-IRES-tdTomato transgenic mice were obtained. These mice were able to detect the activation of Th2 cells in the paracortical area of ​​lymph nodes after intravenous infusion of dual antibodies.

[0373] Control antibodies (non-reactive antibodies, IgG2 isotype control), CD4 antibody (Ibalizumab), and different penicillin antibodies WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 were administered intravenously to 6-8 week old humanized hCD4 / +;IL-4-IRES-tdTomato transgenic mice. The dose was 100 μg (5 mg / kg) administered via tail vein injection once a week for a total of three administrations. After three administrations, lymph nodes were collected from the mice, embedded in OCT scans, and then frozen sections were prepared. The sections were stained with immunofluorescence using FITC-conjugated anti-mouse CD4 antibody and DAPI, following the specific procedures outlined in Example 6.

[0374] (Th2 cells / total cells)% = Th2 cells / total cells;

[0375] Th2 cell detection method: Since Th2 cells are double positive for CD4 and IL-4, CD4 positive cells are identified as Th2 cells by the combination of the FITC green fluorescence of the CD4-TITC antibody binding band and the tdTomato red fluorescence of IL-4 positive cells.

[0376] Total cell detection method: CD4 single positive cells (i.e., cells with FITC green fluorescence but no tdTomato red fluorescence).

[0377] The results are as follows Figure 22 As shown in the figure, the results indicated that, compared with the control antibody, CD4 antibody (Ibalizumab), and other bispecific antibodies WH402, WH403, and WH406, bispecific antibodies WH401, WH404, WH405, WH407, and CT101 effectively activated lymph node Th2 cells. This result suggests that only bispecific antibodies combining TGFβ antibodies (which neutralize TGFβ1) with Ibalizumab can effectively activate lymph node Th2 cells; bispecific antibodies combining TGFβ2 or TGFβ3 antibodies with Ibalizumab cannot activate lymph node Th2 cells.

[0378] Example 10: Intravenous infusion of control antibody, TGFβ1 antibody, TGFβ2 antibody, and TGFβ3 antibody in mice with injury Effects of oral healing

[0379] Preparation of humanized hCD4 / + transgenic mice: Since C57BL / 6J mice are widely used as the background strain for transgenic mice, the C57BL / 6J strain was selected in this embodiment. Ibalizumab binds to human CD4 but not mouse CD4, requiring humanization of the corresponding target site in C57BL / 6J mice. To test the CD4 / TGFβ bispecific antibodies of different CD4 antibodies, humanized CD4 mice were used in this experiment, i.e., the open reading frame (ORF) of human CD4 was replaced with the ORF of mouse CD4, resulting in humanized hCD4 / + transgenic mice.

[0380] Skin defect wound creation: This embodiment uses a mouse full-thickness skin defect model. The method involves surgically removing the hair on the mouse's back to create a circular full-thickness skin defect wound with a diameter of 1 cm. The wound is exposed and kept in a sterile environment. After the wound scabs over (2-3 days), a scar gradually forms, and the model can last for more than 3 weeks.

[0381] After creating skin defects on the backs of 6-8 week old humanized hCD4 / + transgenic mice, the mice were intravenously injected with control antibodies (non-reactive antibodies, IgG2 isotype control), TGFβ1 antibody (CAT192), TGFβ2 antibody (MOR14797), and TGFβ3 antibody (2A10), 100 μg (5 mg / kg) each time via tail vein injection, once a week for a total of 3 times. The scar area on the backs of the mice was measured 21 days after administration.

[0382] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0383] The results are as follows Figure 24 As shown in the figure. The results showed that, compared with the control antibody, the scar area after treatment with TGFβ1 monoclonal antibody (CAT192) did not change, indicating that TGFβ1 monoclonal antibody did not affect wound healing; compared with the control antibody, the scar area after treatment with TGFβ2 antibody (MOR14797) decreased, indicating that TGFβ2 monoclonal antibody could promote wound healing; compared with the control antibody, the scar area after treatment with TGFβ3 antibody (2A10) increased, indicating that TGFβ3 monoclonal antibody inhibited wound healing.

[0384] Example 11: In mice, after intravenous infusion of control antibody, TGFβ1 antibody, CD4 antibody, and CD4 / TGFβ1 dual antibody... Impact on wound healing

[0385] After the skin defect wounds on the backs of 6-8 week old humanized hCD4 / + transgenic mice were created (the specific method for creating the skin defect wounds is shown in Example 8), the mice were intravenously injected with control antibody (non-reactive antibody, IgG2 isotype control), TGFβ1 antibody (CAT192), CD4 monomer (Ibalizumab), and CD4 / TGFβ1 bispecific antibody (WH401), 100 μg (5 mg / kg) each time via tail vein injection, once a week for a total of 3 times. The scar area on the backs of the mice was measured 21 days after administration.

[0386] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0387] The results are as follows Figure 25 As shown in the figure, the results indicated that, compared with the control antibody, the scar area remained unchanged after treatment with TGFβ1 antibody (CAT192) and CD4 monoclonal antibody (Ibalizumab), indicating that TGFβ1 monoclonal antibody and CD4 antibody do not affect wound healing. Compared with the control antibody, TGFβ1 monoclonal antibody, and CD4 antibody, the scar area after treatment with CD4 / TGFβ1 bispecific antibody (WH401) was significantly reduced. TGFβ1 monoclonal antibody did not promote wound healing, while CD4 / TGFβ1 bispecific antibody did. These results suggest that TGFβ1 monoclonal antibody promotes wound healing only when combined with CD4 antibody (Ibalizumab) to form a bispecific antibody.

[0388] Example 12: In mice, after intravenous infusion of control antibody, TGFβ2 antibody, CD4 monomer, and CD4 / TGFβ2 bispecific antibody... Impact on wound healing

[0389] After the skin defect wounds on the backs of 6-8 week old humanized hCD4 / + transgenic mice were created (the specific method for creating the skin defect wounds is shown in Example 8), the mice were intravenously injected with control antibody (non-reactive antibody, IgG2 isotype control), TGFβ2 antibody (MOR14797), CD4 monomer (Ibalizumab), and CD4 / TGFβ2 bispecific antibody (WH402), 100 μg (5 mg / kg) each time via tail vein injection, once a week for a total of 3 times. The scar area on the backs of the mice was measured 21 days after administration.

[0390] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0391] The results are as follows Figure 26As shown in the figure, the results indicated that, compared with the control antibody, CD4 monoclonal antibody (Ibalizumab), the scar area treated with TGFβ2 antibody (MOR14797) and CD4 / TGFβ2 bispecific antibody (WH402) was significantly reduced, and the degree of scar reduction was consistent between the two, indicating that both TGFβ2 antibody and CD4 / TGFβ2 bispecific antibody can promote wound healing, and their effects are consistent. This result suggests that the wound healing-promoting ability of TGFβ2 antibody is independent of its combination with CD4 monoclonal antibody (Ibalizumab) to form a bispecific antibody.

[0392] Example 13: In mice, after intravenous infusion of control antibody, TGFβ3 antibody, CD4 antibody, and CD4 / TGFβ3 bispecific antibody... Impact on wound healing

[0393] After creating skin defects on the backs of 6-8 week old humanized hCD4 / + transgenic mice, the mice were intravenously injected with control antibody (non-reactive antibody, IgG2 isotype control), TGFβ3 antibody (2A10), CD4 monomer (Ibalizumab), and CD4 / TGFβ3 bispecific antibody (WH403), 100 μg (5 mg / kg) each time, once a week for a total of 3 times. The scar area on the backs of the mice was measured 21 days after administration.

[0394] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0395] The results are as follows Figure 27 As shown in the figure. The results showed that, compared with the control antibody, CD4 monomer (Ibalizumab), the scar area after treatment with TGFβ3 monoclonal antibody (2A10) and CD4 / TGFβ3 bispecific antibody (WH403) was significantly increased, and the degree of scar increase was consistent between the two, indicating that both TGFβ3 monoclonal antibody and CD4 / TGFβ3 bispecific antibody can inhibit wound healing, and their inhibitory effects are consistent.

[0396] Example 14: Intravenous infusion of WH401, WH402, WH403, WH404, WH405, WH406, WH407 and CT101 Afterwards, the effect on wound healing in mice

[0397] After the skin defect wounds on the backs of 6-8 week old humanized hCD4 / + transgenic mice were created (the specific method for creating the skin defect wounds is shown in Example 8), control antibodies (non-reactive antibodies, IgG2 isotype control), WH401, WH402, WH403, WH404, WH405, WH406, WH407, and CT101 were injected intravenously, 100 μg (dose 5 mg / kg) each time, once a week, for a total of 3 administrations. The scar area on the backs of the mice was measured 21 days after administration.

[0398] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0399] The results are as follows Figure 28 As shown in the figure. The results showed that, compared with the control antibody, WH401, WH402, WH404, and WH407 had a certain effect on promoting wound healing; while compared with the control antibody and all other bispecific antibodies, WH405 had the best effect on promoting wound healing.

[0400] Example 15: Different dual-antibody forms of WH405 (WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, Intravenous infusion of WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, and WH405-12 promoted wound healing in mice. The impact of combination

[0401] After the skin defect wounds on the backs of 6-8 week old humanized hCD4 / + transgenic mice were created (the specific method for creating the skin defect wounds is shown in Example 8), control antibodies (non-reactive antibodies, IgG2 isotype control) and different penicillin antibodies WH405, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11 and WH405-12 were administered intravenously via the tail vein, 100 μg each time (dose 5 mg / kg), once a week for a total of 3 administrations. Twenty-one days after administration, the scar area on the backs of the mice was measured.

[0402] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0403] The results are as follows Figure 32 As shown in the figure. The results showed that, compared with the control antibody, WH405, WH405-2, WH405-3, WH405-4, WH405-5, WH405-6, WH405-7, WH405-8, WH405-9, WH405-10, WH405-11, and WH405-12 all had significant wound healing promoting effects, and their wound healing promoting effects were comparable.

[0404] Example 16: Control antibody, TGFβ1 / 2 antibody (NIS793, XPA.42.068) and penicillin antibodies WH405 and WH415 intravenously Effect of intravenous infusion on wound healing in mice

[0405] After the skin defect wounds on the backs of 6-8 week old humanized hCD4 / + transgenic mice were created (the specific method for creating the skin defect wounds is shown in Example 8), control antibodies (non-reactive antibodies, IgG2 isotype control), TGFβ1 / 2 antibodies (NIS793, XPA.42.068), and bispecific antibodies WH405 and WH415 were injected intravenously, 100 μg (dose 5 mg / kg) each time via tail vein injection, once a week, for a total of 3 administrations. The scar area on the backs of the mice was measured 21 days after administration.

[0406] Scar area: When calculating scar area, most scars are elliptical in shape. The formula for calculating the area of ​​an ellipse is S = πab / 4, where S represents the area of ​​the ellipse, and a and b represent the length and width of the scar, respectively.

[0407] The results are as follows Figure 36 As shown in the figure. The results showed that, compared with the control antibody, the TGFβ1 / 2 antibody NIS793 and XPA.42.068 had a certain wound healing ability due to the inhibition of TGFβ2 activity. The bispecific antibodies WH405 and WH415 showed significantly better wound healing response than the TGFβ1 / 2 antibody NIS793 and XPA.42.068, firstly because the CD4 / TGFβ1 bispecific antibody specifically activated the Th2 cell-mediated tissue repair response, and secondly because it inhibited the activity of TGFβ2.

[0408] Although the present invention has been fully and clearly described with respect to the specific embodiments disclosed herein, it is not limited thereto.

[0409] For those skilled in the art, modifications and substitutions to the invention may be made based on the guidance of these descriptions, and such modifications and substitutions are included within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

[0410] The sequence involved in this invention is as follows:

[0411] The amino acid sequences of the three CDR regions of the variable region of the Ibalizumab heavy chain are as follows:

[0412] HCDR1: GYTFTSYVIH (SEQ ID NO: 1);

[0413] HCDR2:YINPYNDGTDYDEKFKG(SEQ ID NO:2);

[0414] HCDR3:EKDNYATGAWFA (SEQ ID NO:3);

[0415] The amino acid sequences of the three CDR regions in the variable region of the ibalizumab light chain are as follows:

[0416] LCDR1:KSSQSLLYSTNQKNYLA(SEQ ID NO:4);

[0417] LCDR2:WASTRES(SEQ ID NO:5);

[0418] LCDR3:QQYYSYRT (SEQ ID NO:6);

[0419] The amino acid sequences of the three CDR regions in the variable region of the NIS793 heavy chain are as follows:

[0420] HCDR1:GGTFSSYA(SEQ ID NO:7);

[0421] HCDR2:IIPIFGTA (SEQ ID NO:8);

[0422] HCDR3:ARGLWEVRALPSVY(SEQ ID NO:9);

[0423] The amino acid sequences of the three CDR regions in the NIS793 light chain variable region are as follows:

[0424] LCDR1:DIGSKS (SEQ ID NO:10);

[0425] LCDR2:EDI(SEQ ID NO:11);

[0426] LCDR3:QVWDRDSDQY(SEQ ID NO:12);

[0427] The amino acid sequence of TGFβ1-His is as follows (118 aa):

[0428] ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSHHHHHH (SEQ ID NO:13)

[0429] The nucleic acid sequence encoding TGFβ1-His (354 bp);

[0430] GCCCTGGATACCAACTATTGCTTCAGCTCCACAGAGAAGAACTGCTGTGTGCGGCAGCTGTACATTGACTTTAGGAAGGACCTGGGTTGGAAGTGGATCCACGAGCCCAAGGGCTACCATGCCAACTTCTGCCTCGGGCCCTGCCCCTACATTTGGAGCCTGGACACGCAGTACAGCAAGGTCCTGGCCCTGTACAACCAGCATAACCCGGGCGCCTCGGCGGCGCCGTGCTGCGTGCCGCAGGCGCTGGAGCCGCTGCCCATCGTGTACTACGTGGGCCGCAAGCCCAAGGTGGAGCAGCTGTCCAACATGATCGTGCGCTCCTGCAAGTGCAGCCATCATCATCATCATCAT (SEQ ID NO:14);

[0431] The amino acid sequence of TGFβ2-His is as follows (118aa):

[0432] ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCSHHHHHH (SEQ ID NO:15)

[0433] The nucleic acid sequence encoding TGFβ2-His (354 bp):

[0434] GCTTTGGATGCGGCCTATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCTAGGGTGGAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTATGGAGTTCAGACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTTCTCCTTGCTGCGTGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGATTGAACAGCTTTCTAATATGATTGTAAAGTCTTGCAAATGCAGCCATCATCATCATCATCAT (SEQ ID NO:16);

[0435] The amino acid sequence of TGFβ3-His is as follows (118 aa):

[0436] ALDTNYCFRNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCSHHHHHH (SEQ ID NO:17)

[0437] The nucleic acid sequence encoding TGFβ3-His (354 bp):

[0438] GCTTTGGACACCAATTACTGCTTCCGCAACTTGGAGGAGAACTGCTGTGTGCGCCCCCTCTACATTGACTTCCGACAGGATCTGGGCTGGAAGTGGGTCCATGAACCTAAGGGCTACTATGCCAACTTCTGCTCAGGCCCTTGCCCATACCTCCGCAGTGCAGACACAACCCACAGCACGGTGCTGGGACTGTACAACACTCTGAACCCTGAAGCATCTGCCTCGCCTTGCTGCGTGCCCCAGGACCTGGAGCCCCTGACCATCCTGTACTATGTTGGGAGGACCCCCAAAGTGGAGCAGCTCTCCAACATGGTGGTGAAGTCTTGTAAATGTAGCCATCATCATCATCATCAT (SEQ ID NO:18);

[0439] The amino acid sequence of CD4-His is as follows (371 aa):

[0440] KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWHHHHHH (SEQ ID NO:19);

[0441] The nucleic acid sequence encoding CD4-His (1113 bp):

[0442]

[0443] The amino acid sequence of the variable region of the Ibalizumab heavy chain is: (122 aa):

[0444] QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS (SEQ ID NO:21);

[0445] Nucleic acid sequence encoding the variable region of the Ibalizumab heavy chain: (366 bp):

[0446] CAGGTGCAACTGCAACAGTCCGGACCCGAAGTCGTGAAACCAGGAGCTTCCGTGAAGATGAGCTGCAAAGCATCCGGATACACCTTCACCAGCTACGTGATCCACTGGGTGAGGCAGAAACCTGGCCAGGGCCTGGACTGGATCGGCTACATCAACCCTTACAACGACGGAACCGACTACGAC GAGAAATTCAAAGGCAAAGCTACCCTGACCAGCGACACCAGCACCTCCACTGCTTACATGGAGCTGTCCAGCCTGAGGTCCGAAGACACCGCTGTGTACTACTGCGCTAGGGAGAAGGACAACTACGCTACCGGCGCTTGGTTCGCCTACTGGGGCCAGGGAACCCTGGTGACCGTGTCCAGC (SEQ ID NO:22);

[0447] The amino acid sequence of the variable region of the light chain of Ibalizumab is (112 aa):

[0448] DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK (SEQ ID NO:23);

[0449] Nucleic acid sequence encoding the variable region of the Ibalizumab light chain: (336 bp):

[0450] GACATCGTGATGACCCAGTCCCCTGACTCCCTGGCTGTGTCCCTGGGCGAACGGGTCACCATGAACTGCAAATCTTCCCAGTCCCTGCTGTACTCCACCAACCAGAAGAACTACCTGGCTTGGTACCAGCAGAAACCTGGCCAGTCCCCCAAACTGCTCATCTACTGG GCTTCCACCAGGGAAAGCGGCGTGCCTGACAGATTCTCCGGAAGCGGCAGCGGAACCGACTTCACCCTGACCATCTCCAGCGTGCAGGCTGAAGACGTGGCTGTCTACTACTGCCAGCAGTACTACAGCTACAGGACCTTCGGCGGAGGCACCAAGCTGGAGATCAAG (SEQ ID NO:24)

[0451] The amino acid sequence of the variable region of the heavy chain of antibody NIS793: (121 aa)

[0452] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLWEVRALPSVYWGQGTLVTVSS (SEQ ID NO:25)

[0453] Nucleic acid sequence of the variable region of the heavy chain of antibody NIS793: (363 bp)

[0454] CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCGGCACCTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCGGCATTATTCCGATTTTTGGCACCGCGAACTATG CGCAGAAATTTCAGGGCCGCGTGACCATTACCGCGGATGAAAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCGGCCTGTGGGAAGTGCGCGCTGCCGAGCGTGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC (SEQ IDNO:26)

[0455] The amino acid sequence of the variable region of the light chain of antibody NIS793: (109 aa)

[0456] SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTVLG (SEQ ID NO:27)

[0457] Nucleic acid sequence of the variable region of the light chain of antibody NIS793: (327 bp)

[0458] AGCTATGAACTGACCCAGCCGCCGAGCGTGAGCGTGGCGCCGGGCCAGACCGCGCGCATTACCTGCGGCGCGAACGATATTGGCAGCAAAAGCGTGCATTGGTATCAGCAGAAAGCGGGCCAGGCGCCGGTGCTGGTGGTGAGCGAAGATATTATTCGCCCGAGCGGCATTCCGGAACGCATTAGCGGCAGCAACAGCGGCAACACCGCGACCCTGACCATTAGCCGCGTGGAAGCGGGCGATGAAGCGGATTATTATTGCCAGGTGTGGGATCGCGATAGCGATCAGTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTGGGC (SEQ ID NO:28)

[0459] Amino acid sequence of the heavy chain of antibody NIS793: (447 aa)

[0460] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLWEVRALPSVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:29)

[0461] Nucleic acid sequence of the heavy chain of antibody NIS793: (1341 bp)

[0462]

[0463] The amino acid sequence of the light chain of antibody NIS793: (214 aa)

[0464] SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTV LGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:31)

[0465] Nucleic acid sequence of the light chain of antibody NIS793: (642 bp)

[0466] AGCTATGAACTGACCCAGCCGCCGAGCGTGAGCGTGGCGCCGGGCCAGACCGCGCGCATTACCTGCGGCGCGAACGATATTGGCAGCAAAAGCGTGCATTGGTATCAGCAGAAAGCGGGCCAGGCGCCGGTGCTGGTGGTGAGCGAAGATATTATTCGCCCGAGCGGCATTCCGGAACGCATTAGCGGCAGCAACAGCGGCAACACCGCGACCCTGACCATTAGCCGCGTGGAAGCGGGCGATGAAGCGGATTATTATTGCCAGGTGTGGGATCGCGATAGCGATCAGTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTGGGCCAGCCGAAAGCGAACCCGACCGTGACCCTGTTTCCGCCGAGCAGCGAAGAACTGCAGGCGAACAAAGCGACCCTGGTGTGCCTGATTAGCGATTTTTATCCGGGCGCGGTGACCGTGGCGTGGAAAGCGGATGGCAGCCCGGTGAAAGCGGGCGTGGAAACCACCAAACCGAGCAAACAGAGCAACAACAAATATGCGGCGAGCAGCTATCTGAGCCTGACCCCGGAACAGTGGAAAAGCCATCGCAGCTATAGCTGCCAGGTGACCCATGAAGGCAGCACCGTGGAAAAAACCGTGGCGCCGACCGAATGCAGC (SEQ ID NO:32)

[0467] Amino acid sequence of the first protein chain of antibody WH405: (214 aa)

[0468] SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQID NO:33)

[0469] Nucleic acid sequence of the first protein chain of antibody WH405: (642 bp)

[0470] AGCTATGAACTGACCCAGCCGCCGAGCGTGAGCGTGGCGCCGGGCCAGACCGCGCGCATTACCTGCGGCGCGAACGATATTGGCAGCAAAAGCGTGCATTGGTATCAGCAGAAAGCGGGCCAGGCGCCGGTGCTGGTGGTGAGCGAAGATATTATTCGCCCGAGCGGCATTCCGGAACGCATTAGCGGCAGCAACAGCGGCAACACCGCGACCCTGACCATTAGCCGCGTGGAAGCGGGCGATGAAGCGGATTATTATTGCCAGGTGTGGGATCGCGATAGCGATCAGTATGTGTTTGGCACCGGCACCAAAGTGACCGTGCTGGGCCAGCCGAAAGCGAACCCGACCGTGACCCTGTTTCCGCCGAGCAGCGAAGAACTGCAGGCGAACAAAGCGACCCTGGTGTGCCTGATTAGCGATTTTTATCCGGGCGCGGTGACCGTGGCGTGGAAAGCGGATGGCAGCCCGGTGAAAGCGGGCGTGGAAACCACCAAACCGAGCAAACAGAGCAACAACAAATATGCGGCGAGCAGCTATCTGAGCCTGACCCCGGAACAGTGGAAAAGCCATCGCAGCTATAGCTGCCAGGTGACCCATGAAGGCAGCACCGTGGAAAAAACCGTGGCGCCGACCGAATGCAGC (SEQ ID NO:34)

[0471] Amino acid sequence of the second protein chain of antibody WH405: (711 aa)

[0472] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLWEVRALPSVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQGLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRSEDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQQYYSYRTFGGGTKLEIK (SEQ ID NO:35)

[0473] Nucleic acid sequence of the second protein chain of antibody WH405: (2133 bp)

[0474]

[0475] The amino acid sequence of the variable region of the Zanolimumab heavy chain is: (115 aa)

[0476] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARVINWFDPWGQGTLVTVSS (SEQ ID NO:37)

[0477] The nucleic acid sequence of the variable region of the Zanolimumab heavy chain: (345 bp)

[0478] CAGGTGCAGCTGCAGCAGTGGGGCGCGGGCCTGCTGAAACCGAGCGAAACCCTGAGCCTGACCTGCGCGGTGTATGGCGGCAGCTTTAGCGGCTATTATTGGAGCTGGATTCGCCAGCCGCCGGGCAAAGGCCTGGAATGGATTGGCGAAATTAACCATAGCGGCAGCACCA ACTATAACCCGAGCCTGAAAAGCCGCGTGACCATTAGCGTGGATACCAGCAAAAACCAGTTTAGCCTGAAACTGAGCAGCGTGACCGCGGCGGATACCGCGGTGTATTATTGCGCGCGCGTGATTAACTGGTTTTGATCCGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC (SEQ ID NO:38)

[0479] The amino acid sequence of the variable region of the Zanolimumab light chain is: (107 aa)

[0480] DIQMTQSPSSVSASVGDRVTITCRASQDISSWIAWYQHKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPYTFGQGTKLEIK (SEQ ID NO:39)

[0481] The nucleic acid sequence of the variable region of the Zanolimumab light chain is: (321 bp)

[0482] GATATTCAGATGACCCAGAGCCCGAGCAGCGTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATATTAGCAGCTGGATTGCGTGGTATCAGCATAAACCGGGCAAAGCGCCGAAACTGCTGATTTATGCGGCGAGCAGCCTGCAGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGGCGAACAGCTTTCCGTATACCTTTGGCCAGGGCACCAAACTGGAAATTAAA (SEQ ID NO:40)

[0483] Amino acid sequence of the variable region of the heavy chain of Keliximab: (121 aa)

[0484] QVQLQEAGPGLVKPSETLSLTCSVSGGSISGDYYWFWIRQSPGKGLEWIGYIYGSGGGTNYNPSLNNRVSISIDTSKNLFSLKLRSVTAADTAVYYCASNILKYLHWLLYWGQGVLVTVSS (SEQ ID NO:41)

[0485] Nucleic acid sequence of the variable region of the heavy chain of Keliximab: (363 bp)

[0486] CAGGTGCAGCTGCAGGAAGCGGGCCCGGGCCTGGTGAAACCGAGCGAAACCCTGAGCCTGACCTGCAGCGTGAGCGGCGGCAGCATTAGCGGCGATTATTATTGGTTTTGGATTCGCCAGAGCCCGGGCAAAGGCCTGGAATGGATTGGCTATATTTATGGCAGCGGCGGCGGCACCAACTATAACCCGAGCCTGAACAACCGCGTGAGCATTAGCATTGATACCAGCAAAAACCTGTTTAGCCTGAAACTGCGCAGCGTGACCGCGGCGGATACCGCGGTGTATTATTGCGCGAGCAACATTCTGAAATATCTGCATTGGCTGCTGTATTGGGGCCAGGGCGTGCTGGTGACCGTGAGCAGC (SEQ IDNO:42)

[0487] Amino acid sequence of the variable region of the Keliximab light chain: (109 aa)

[0488] SYELSQPRSVSVSPGQTAGFTCGGDNVGRKSVQWYQQKPPQAPVLVIYADSERPSGIPARFSGSNSGNTATLTISGVEAGDEADYYCQVWDSTADHWVFGGGTRLTVLG (SEQ ID NO:43)

[0489] Nucleic acid sequence of the variable region of the Keliximab light chain: (327 bp)

[0490] AGCTATGAACTGAGCCAGCCGCGCAGCGTGAGCGTGAGCCCGGGCCAGACCGCGGGCTTTACCTGCGGCGGCGATAACGTGGGCCGCAAAAGCGTGCAGTGGTATCAGCAGAAACCGCCGCAGGCGCCGGTGCTGGTGATTTATGCGGATAGCGAACGCCCGAGCGGCATTCCGGCGCGCTTTAGCGGCAGCAACAGCGGCAACACCGCGACCCTGACCATTAGCGGCGTGGAAGCGGGCGATGAAGCGGATTATTATTGCCAGGTGTGGGATAGCACCGCGGATCATTGGGTGTTTGGCGGCGGCACCCGCCTGACCGTGCTGGGC (SEQ ID NO:44)

[0491] Amino acid sequence of the variable region of the heavy chain of Tregalizumab: (124 aa)

[0492] EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWVRQAPGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFAYWGQGTLVTVSS (SEQ ID NO:45)

[0493] Nucleic acid sequence of the variable region of the heavy chain of Tregalizumab: (372 bp)

[0494] GAAGAACAGCTGGTGGAAAGCGGCGGCGGCCTGGTGAAACCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAGCTTTAGCGATTGCCGCATGTATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGATTGGCGTGATTAGCGTGAAAAGCGAAAACTATGGCGCGAACTATGC GGAAAGCGTGCGCGGCCGCTTTACCATTAGCCGCGATGATAGCAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAAACCGAAGATACCGCGGTGTATTATTGCAGCGCGAGCTATTATCGCTATGATGTGGGCGCGTGGTTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:46)

[0495] The amino acid sequence of the variable region of the Tregalizumab light chain is: (111 aa)

[0496] DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPWTFGQGTKVEIK (SEQ ID NO:47)

[0497] The nucleic acid sequence of the variable region of the Tregalizumab light chain: (333 bp)

[0498] GATATTGTGATGACCCAGAGCCCGGATAGCCTGGCGGTGAGCCTGGGCGAACGCGCGACCATTAACTGCCGCGCGAGCAAAAGCGTGAGCACCAGCGGCTATAGCTATATTTATTGGTATCAGCAGAAACCGGGCCAGCCGCCGAAACTGCTGATTTATCTGGCGAGCATTCTGGAAAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGGCGGAAGATGTGGCGGTGTATTATTGCCAGCATAGCCGCGAACTGCCGTGGACCTTTGGCCAGGGCACCAAAGTGGAAATTAAA (SEQ ID NO:48)

[0499] Amino acid sequence of the heavy chain variable region of 13B8.2: (116 aa)

[0500] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTFGVHWVRQSPGKGLEWLGVIWRSGITDYNVPFMSRLSITKDNSKSQVFFKLNSLQPDDTAIYYCAKNDPGTGFAYWGQGTLVTVS (SEQ ID NO:49)

[0501] Nucleic acid sequence of the heavy chain variable region of 13B8.2: (348 bp)

[0502] CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCACCTTTGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGCGCAGCGGCATTACCGATTATAACGTGCCGTTTATGAGCCGCCTGAGCATTACCAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAACTGAACAGCCTGCAGCCGGATGATACCGCGATTTATTATTGCGCGAAAAACGATCCGGGCACCGGCTTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGC (SEQ ID NO:50)

[0503] The amino acid sequence of the variable region of the 13B8.2 light chain is: (107 aa)

[0504] DIQMTQSPASSLSASVGETVTFTCRASENIYSYLAWYQQKQGKSPQLLVHDAKTLAEGVPSRFSGGGSGTQFSLKINTLQPEDFGTYYCQHHYGNPPTFGGGTKLEIK (SEQ ID NO:51)

[0505] Nucleic acid sequence of the variable region of the 13B8.2 light chain: (321 bp)

[0506] GATATTCAGATGACCCAGAGCCCGGCGAGCCTGAGCGCGAGCGTGGGCGAAACCGTGACCTTTACCTGCCGCGCGAGCGAAAACATTTATAGCTATCTGGCGTGGTATCAGCAGAAACAGGGCAAAAGCCCGCAGCTGCTGGTGCATGATGCGAAAACCC TGGCGGAAGGCGTGCCGAGCCGCTTTAGCGGCGGCGGCAGCGGCACCCAGTTTAGCCTGAAAATTAACACCCTGCAGCCGGAAGATTTTGGCACCTATTATTGCCAGCATCATTATGGCAACCCGCCGACCTTTGGCGGCGGCACCAAACTGGAAATTAAA (SEQ ID NO:52)

[0507] The amino acid sequence of the variable region of the CAT192 heavy chain: (123 aa)

[0508] EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKELEWVAVISYDGSIKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGEYSGYDTDPQYSWGQGTTVTVSS (SEQ ID NO:53)

[0509] Nucleic acid sequence of the variable region of the CAT192 heavy chain: (369 bp)

[0510] GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCGTGGTGCAGCCGGGCCGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATGGCATGCATTGGGTGCGCCAGGCGCCGGGCAAAGAACTGGAATGGGTGGCGGTGATTAGCTATGATGGCAGCATTAAATATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGCGCACCGGCGAATATAGCGGCTATGATACCGATCCGCAGTATAGCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO:54)

[0511] Amino acid sequence of the variable region of the CAT192 light chain: (107 aa)

[0512] EIVLTQSPSSLSASVGDRVTITCRSSQGIGDDLGWYQQKPGKAPILLIYGTSTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCLQDSNYPLTFGGGTRLEIK (SEQ ID NO:55)

[0513] Nucleic acid sequence of the variable region of the CAT192 light chain: (321 bp)

[0514] GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCGTGGTGCAGCCGGGCCGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATGGCATGCATTGGGTGCGCCAGGCGCCGGGCAAAGAACTGGAATGGGTGGCGGTGATTAGCTATGATGGCAGCATTAAATATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCGCGCGCACCGGCGAATATAGCGGCTATGATACCGATCCGCAGTATAGCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO:56)

[0515] Amino acid sequence of the variable region of the MOR14797 heavy chain: (120 aa)

[0516] QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIYWNDDKSYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDFYYSGYFDSWGQGTLVTVSS (SEQ ID NO:57)

[0517] Nucleic acid sequence of the variable region of the MOR14797 heavy chain: (360 bp)

[0518] CAGGTGACACTGAGAGAGTCAGGCCCTGCCCTGGTGAAACCTACTCAGACCCTGACCCTGACCTGCACCTTTAGCGGCTTTAGCCTGAGCACTAGCGGAATGGGCGTGGGCTGGATTAGACAGCCCCCTGGCAAGGCCCTGGAGTGGCTGGCTCACATCTACTGGAACGACGATAAGTCCTACTCTACTAGCCTGAAAACTAGGCTGACTATCTCTAAGGACACCTCTAAGAATCAGGTGGTGCTGACTATGACTAATATGGACCCCGTGGACACCGCTACCTACTACTGCGCTAGAGACTTCTACTATAGCGGCTACTTCGATAGCTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGC (SEQ ID NO:58)

[0519] Amino acid sequence of the variable region of MOR14797 light chain: (106 aa)

[0520] DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTNTMNTFGQGTKVEIK (SEQ ID NO:59)

[0521] Nucleic acid sequence of the variable region of MOR14797 light chain: (318 bp)

[0522] GATATTCAGATGACTCAGTCACCTAGTAGCCTGAGCGCTAGTGTGGGCGATAGAGTGACTATCACCTGTAGAGCCTCTCAGGATATCTCTAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTTCGGCGCCTCTAGCCTGCAGTCAGGCGTGCCCTCTAGGTTTAGCGGCTCAGGCTCAGGCACCGACTTCACCCTGACTATTAGTAGCCTGCAGCCCGAGGACTTCGCTACCTACTACTGTCAGCAGACTAACACTATGAACACCTTCGGCCAGGGCACTAAGGTGGAGATTAAG (SEQ ID NO:60)

[0523] The amino acid sequence of the variable region of the 2A10 heavy chain is: (119 aa)

[0524] EVQLLESGGGLVQPGGSLRLSCAASGFDFNSYGMSWVRQAPGKGLELVSDIVSKTYNYATYYSDSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCTVAPGGSFDYWGQGTLVTVSS (SEQ ID NO:61)

[0525] Nucleic acid sequence of the variable region of the 2A10 heavy chain: (357 bp)

[0526] GAAGTGCAGCTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTGATTTTAACAGCTATGGCATGAGCTTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAACTGGTGAGCGATATTGTGAGCAAAACCTATAACTATGCGA CCTATTATAGCGATAGCGTGAAAGATCGCTTTACCATTAGCCGCGATGATAGCAAAAACACCCTGTATCTGCAGATGAACAGCCTGCGCCGGAAGATACCGCGGTGTATTATTGCACCGTGGCGCCGGGCGGCAGCTTTGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC (SEQ ID NO:62)

[0527] The amino acid sequence of the variable region of the 2A10 light chain is: (111 aa)

[0528] DIQLTQSPSSSLSASVGDRVTITCRASQSVSISRFNLMHWYQQKPGKAPKLLIYRASNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHSRESPWTFGGGTKVEIK (SEQ ID NO:63)

[0529] Nucleic acid sequence of the variable region of the 2A10 light chain: (333 bp)

[0530] GATATTCAGCTGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGAGCGTGAGCATTAGCCGCTTTAACCTGATGCATTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATCGCGCGAGCAACCTGGCGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCATAGCCGCGAAAGCCCGTGGACCTTTGGCGGCGGCACCAAAGTGGAAATTAAA (SEQ ID NO:64)

[0531] Amino acid sequence of the heavy chain variable region of XPA.42.681: (122 aa)

[0532] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSFLWLVPSDAFDIWGQGTMVTVSS (SEQ ID NO:65)

[0533] Nucleic acid sequence of the heavy chain variable region of XPA.42.681: (366 bp)

[0534] CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACACTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGATCATTCCTGTGGCTGGTTCCCTCTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA (SEQID NO:66)

[0535] Amino acid sequence of the light chain variable region of XPA.42.681: (109 aa)

[0536] SYVLTQPPSVSVAPGKTARITCGGNNIGFRSVHWYQQKSGQAPVLVIYFDRARPSGIPERFSASNSENTATLTIRRVEAGDEADYYCQVWDSDSDDLVFGGGTQLTVLG (SEQ ID NO:67)

[0537] Nucleic acid sequence of the light chain variable region of XPA.42.681: (326 bp)

[0538] TCCTATGTGCTGACTCAGCCACCCTCAGTGTCCGTGGCCCCAGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGATTTAGAAGTGTGCACTGGTACCAACAGAAGTCAGGCCAGGCCCCTGTCCTGGTCATCTATTTTGATCGCGCCCGGCCCTCAGGGATCCCGAGCGATTCTCTGCCTCCAACTCTGAGAACACGGCCACCCTGACCATCAGGAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTGACAGTGATGATCTAGTCTTCGGCGGAGGCACCCAGCTGACCGTCCTAGGT (SEQ ID NO:68)

[0539] Amino acid sequence of the variable region of the 4A11 heavy chain: (125 aa)

[0540] EQQLVESGGGLVQPGGSLRLSCAVSGFSLSSYTVNWVRQAPGKGLEWIGYISYGGSAYYASWANGRFTISKDSAKNSVYLQMNSLRAEDTAVYFCARHMQVGGAPTGSMAAFDPWGPGTLVTVSS (SEQ ID NO:69)

[0541] Nucleic acid sequence of the variable region of the 4A11 heavy chain: (375 bp)

[0542] GAACAGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGTGAGCGGCTTTAGCCTGAGCAGCTATACCGTGAACTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGATTGGCTATATTAGCTATGGCGGCAGCGCGTATTATGCGAGCTGGGCGAACGGCCGCTTTACCATTAGCAAAGATAGCGCGAAAAACAGCGTGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTTTTGCGCGCGCCATATGCAGGTGGGCGGCGCGCCGACCGGCAGCATGGCGGCGTTTGATCCGTGGGGCCCGGGCACCCTGGTGACCGTGAGCAGC (SEQ ID NO:70)

[0543] Amino acid sequence of the variable region of the 4A11 light chain: (112 aa)

[0544] DAQLTQSPSSLSASVGDRVTITCQSSQSVYNNNYLSWFQQKPGKPPKLLIYGASTLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAGGYSGSSDKYAFGGGTKVEIK (SEQ ID NO:71)

[0545] Nucleic acid sequence of the variable region of the 4A11 light chain: (336 bp)

[0546] GATGCGCAGCTGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCAGAGCAGCCAGGCGTGTATAACAACTATCTGAGCTGGTTTCAGCAGAAACCGGGCAAACCGCCGAAACTGCTGATTTATGGCGAGCACCCTG ACCAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTGCGCGGGCGGCTATAGCGGCAGCAGCGATAAATATGCGTTTGGCGGCGGCACCAAAGTGGAAATTAAA (SEQ ID NO:72)

[0547] TGFβRII residue:( 137 aa)

[0548] TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITCICECPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKPKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ IDNO:73)

[0549] TGFβRII transcription factor:( 411 bp)

[0550] ACGATCCCACCGCACGTTCAGAAGTCGGTTAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGAC (SEQ ID NO:74)

[0551] Amino acid sequence of the heavy chain variable region of XPA.42.068: (122 aa)

[0552] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNTGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSFLWLVPSDAFDIWGQGTMVTVSS (SEQ ID NO:75)

[0553] Nucleic acid sequence of the heavy chain variable region of XPA.42.068: (366 bp)

[0554] CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACCTTTACCGGCTATTATATGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGCTGGATTAACCCGAACACCGGCGGCACCAACTATGCGCAGAAATTTCAGGGCCGCGTGACCATGACCCGCGATACCAGCATTAGCACCGCGTATATGGAACTGAGCCGCCTGCGCAGCGATGATACCGCGGTGTATTATTGCGCGCGCAGCTTTCTGTGGCTGGTGCCGAGCGATGCGTTTGATATTTGGGGCCAGGGCACCATGGTGACCGTGAGCAGC (SEQID NO:76)

[0555] Amino acid sequence of the light chain variable region of XPA.42.068: (109 aa)

[0556] SSELTQPPSVSVAPGEKARITCGGNNIGRKSVHWYQQRPGQAPVVVLYYDRVRPSGIPERFSGSNSGNTATLTITRVEAGDEADYFCQVWDNTSEHVVFGGGTQLTVLG (SEQ ID NO:77)

[0557] Nucleic acid sequence of the light chain variable region of XPA.42.068: (326 bp)

[0558] AGCAGCGAACTGACCCAGCCGCCGAGCGTGAGCGTGGCGCCGGGCGAAAAAGCGCGCATTACCTGCGGCGGCAACAACATTGGCCGCAAAAGCGTGCATTGGTATCAGCAGCGCCCGGGCCAGGCGCCGGTGGTGGTGCTGTATTATGATCGCGTGCGCCCGA GCGGCATTCCGGAACGCTTTAGCGGCAGCAACAGCGGCAACACCGCGACCCTGACCATTACCCGCGTGGAAGCGGGCGATGAAGCGGATTATTTTTTGCCAGGTGTGGGATAACACCAGCGAACATGTGGTGTTTGGCGGCGGCACCCAGCTGACCGTGCTGGGC (SEQ ID NO:78)

[0559] Connecting fragments: (15 aa)

[0560] GGGGSGGGGSGGGGS (SEQ ID NO:79)

[0561] Connecting segments to form units: (5 aa)

[0562] GGGGS (SEQ ID NO:80)

[0563] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A bispecific antibody that binds to human CD4 and TGF-β1 / 2 but not TGF-β3, characterized in that, The bispecific antibody comprises: The first protein functional region includes a first antigen binding site that targets CD4; The second protein functional region includes a second antigen-binding site that targets TGF-β1 / 2.

2. The bispecific antibody as described in claim 1, characterized in that, The antibody also contains an Fc region.

3. The bispecific antibody as described in claim 1 or 2, characterized in that, The first antigen binding site can bind amino acids at positions 77, 79, 96, 121-124, 127-134 and 163 of the polypeptide shown in SEQ ID NO:

19. And / or, The second antigen binding site contains HCDR1-HCDR3 as shown in SEQ ID:7-SED ID NO:9 and LCDR1-LCDR3 as shown in SEQ ID:10-SED ID NO:

12. And / or, The bispecific antibody can reduce scar area after wound healing and / or increase the ratio of Th2 cells / CD4+ T cells.

4. The bispecific antibody according to any one of claims 1-3, characterized in that, The first antigen binding site contains HCDR1-HCDR3 as shown in SEQ ID:1-SED ID NO:3 and LCDR1-LCDR3 as shown in SEQ ID:4-SED ID NO:

6.

5. The bispecific antibody according to any one of claims 1-4, characterized in that, The first protein functional region comprises a heavy chain variable region consisting of an amino acid sequence as shown in SEQ ID NO:21 and a light chain variable region consisting of an amino acid sequence as shown in SEQ ID NO:23; And / or, the second protein functional region comprises a heavy chain variable region consisting of an amino acid sequence as shown in SEQ ID NO:25 and a light chain variable region consisting of an amino acid sequence as shown in SEQ ID NO:

27.

6. The bispecific antibody according to any one of claims 1-5, characterized in that, The first protein functional region is an anti-CD4 antibody or its antigen-binding fragment; preferably, the antigen-binding fragment is selected from Fab, Fab', Fd, Fv, dAb, ScFv, and complementarity-determining region fragments; preferably, the antibody is selected from antibodies, humanized antibodies, or chimeric antibodies; And / or, the second protein functional region is an antibody against TGF-β1 / 2 or its antigen-binding fragment; preferably, the antigen-binding fragment is selected from Fab, Fab', Fd, Fv, dAb, ScFv, and complementarity-determining region fragments; preferably, the antibody is selected from antibodies, humanized antibodies, or chimeric antibodies.

7. The bispecific antibody according to any one of claims 1-6, characterized in that, The bispecific antibody is any one of the following: (1) The first protein functional region is a single-chain antibody, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-ScFv; (2) The first protein functional region is a variable region fragment, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-Fv; (3) The first protein functional region is a variable region fragment, the second protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of Fab-Fv-Fc; (4) The second protein functional region is a variable region fragment, the first protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of Fab-Fv-Fc; (5) The first protein functional region is an antigen-binding fragment, the second protein functional region is an immunoglobulin, and the bispecific antibody exists in the form of IgG-Fab; (6) The first protein functional region is an antigen-binding fragment, the second protein functional region is a single-chain antibody, and the bispecific antibody exists in the form of DVD-Ig (Fab-ScFv-Fc). (7) The first protein functional region is a single-chain antibody, the second protein functional region is an antigen-binding fragment, and the bispecific antibody exists in the form of DVD-Ig (Fab-ScFv-Fc). (8) The first protein functional region is a single-chain antibody, the second protein functional region is a single-chain antibody, and the bispecific antibody exists in the form of ScFv-Fc-ScFv or ScFv-ScFv-Fc.

8. The bispecific antibody according to any one of claims 1-7, characterized in that, The bispecific antibody is any one of the following: (1) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the second protein functional region through a linker; (2) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the second protein functional region through a linker; (3) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker; (4) The first protein functional region is ScFv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker; (5) The first protein functional region is Fv, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker; (6) The first protein functional region is Fv, the second protein functional region is Fab, the C end of the first protein functional region is hinged to the Fc region, and the second protein functional region is connected to the N end of the first protein functional region through a connector; (7) The first protein functional region is Fab, the second protein functional region is Fv, the C end of the second protein functional region is hinged to the Fc region, and the first protein functional region is connected to the N end of the second protein functional region through a connector; (8) The first protein functional region is Fab, the second protein functional region is IgG, and the first protein functional region is connected to the C-terminus of the second protein functional region through a linker; (9) The first protein functional region is Fab, the second protein functional region is IgG, and the first protein functional region is connected to the N-terminus of the second protein functional region through a linker; (10) The first protein functional region is Fab, the second protein functional region is ScFv, the first protein functional region is connected to the C end of the Fc region through a linker; the second protein functional region is hinged to the Fc region; (11) The first protein functional region is ScFv, the second protein functional region is ScFv, the first protein functional region is connected to the C end of the Fc region through a connector, and the second protein functional region is hinged to the Fc region; (12) The first protein functional region is ScFv, the second protein functional region is ScFv, the second protein functional region is hinged to the Fc region, and the second protein functional region is connected to the C end of the first protein functional region through a connector.

9. The bispecific antibody according to any one of claims 1-10, characterized in that, The bispecific antibody comprises a first protein chain and a second protein chain, wherein the first protein chain has the amino acid sequence shown in SEQ ID NO:33; and the second protein chain has the amino acid sequence shown in SEQ ID NO:

35.

10. The bispecific antibody according to any one of claims 1-9, characterized in that, The bispecific antibody is characterized in that the first protein functional region is connected to the second protein functional region through a linker, wherein the linker is (GGGGS)m, and m is an integer; And / or, the first or second protein functional region is connected to the FC via a connector, wherein the connector is (GGGGS)m or a hinge, where m is an integer.

11. The bispecific antibody according to any one of claims 1-10, characterized in that, The constant region of the immunoglobulin is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.

12. The bispecific antibody according to any one of claims 1-11, characterized in that, The bispecific antibody can selectively bind to human CD4 molecules, human TGF-β1 molecules, and human TGF-β2 molecules; And / or, the bispecific antibody does not bind to TGF-β3 or human TGF-β3.

13. The bispecific antibody according to any one of claims 1-12, characterized in that, The bispecific antibody is less than 10 -9 M's EC 50 The EC binds to human CD4 protein. 50 Detected by ELISA.

14. The bispecific antibody according to any one of claims 1-12, characterized in that, The bispecific antibody exhibits an IC50 of less than 100 pM against human TGFβ1 in a TGFβ reporter luciferase activity assay. 50 .

15. The bispecific antibody according to any one of claims 1-12, characterized in that, The bispecific antibody exhibits an IC50 of less than 5 nM against human TGFβ2 in a TGFβ reporter luciferase activity assay. 50 .

16. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a bispecific antibody as claimed in any one of claims 1-15.

17. A carrier, characterized in that, It includes the isolated nucleic acid molecule as described in claim 16.

18. A host cell, characterized in that, It comprises the isolated nucleic acid molecule of claim 16, or the vector of claim 17.

19. A method for preparing the bispecific antibody according to any one of claims 1-15, characterized in that, The steps of culturing the host cells of claim 18 under suitable conditions and recovering the bispecific antibody from the cell culture.

20. A coupling, characterized in that, The conjugate comprises a bispecific antibody and a conjugation portion, wherein the bispecific antibody is the bispecific antibody as described in any one of claims 1-15, and the conjugation portion is a detectable label; preferably, the conjugation portion is a small chemical molecule, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

21. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-15 or the conjugate of claim 20; optionally, it further comprising pharmaceutically acceptable excipients.

22. Use of the bispecific antibody of any one of claims 1-15, the nucleic acid molecule of claim 16, the vector of claim 17, the host cell of claim 18, the bispecific antibody prepared by the method of claim 19, or the conjugate of claim 20 in the preparation of a drug for promoting wound healing or reducing wound scar area.

23. Use of anti-CD4 antibody and anti-TGFβ1 antibody in combination in the preparation of drugs that promote wound healing or reduce wound scar area.

24. Use of anti-CD4 antibody and anti-TGFβ1 / β2 antibody in combination in the preparation of drugs that promote wound healing or reduce wound scar area.

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