VEGFR-antibody light chain fusion proteins

Abstract

The present application provides an antibody fusion protein comprising a vascular endothelial growth factor receptor (VEGFR) fused to the C-terminus of an antibody light chain. Methods of making and using these antibody fusion proteins are also provided.

Description

[0001] This case is a divisional application of a case with an application date of October 11, 2018, an invention name of VEGFR-antibody light chain fusion protein, and an application number of 201880075518.8.

[0002] Cross-references to Related Patent Applications

[0003] This application claims the benefit of priority to U.S. patent application 62 / 571,773, filed October 12, 2017, the contents of which are incorporated herein by reference in their entirety.

[0004] Submit an ASCII text file of the sequence listing

[0005] The following, submitted as an ASCII text file, is incorporated herein by reference in its entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: 754392000240SEQLIST.TXT, Record Date: September 24, 2018, Size: 105 KB). Technical Field

[0006] The present invention relates to antibody fusion proteins comprising a vascular endothelial growth factor receptor (VEGFR) component fused to the C-terminus of an antibody light chain, and methods of making and using the same. Background Art

[0007] Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. The combination of VEGF and its tyrosine kinase receptor (VEGFR) expressed on vascular endothelial cells has triggered the cellular response involved in vascular endothelial cell proliferation and new blood vessel growth. However, the overexpression of VEGF causes disease. Tumor tissue tends to have a higher VEGF expression level, which stimulates tumor angiogenesis to provide nutrition for the solid tumors that grow. Angiogenesis also allows tumors to contact the host's vascular bed, which can provide a pathway for tumor cell metastasis. The overexpression of VEGF can also lead to non-neoplastic conditions, such as rheumatoid arthritis, psoriasis, atherosclerosis, hemangiomas, transplanted corneal tissue and other tissues' immune rejection, chronic inflammation and ocular neovascularization disorders (e.g., diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma and age-related macular degeneration (AMD)). Therefore, a possible mechanism for effectively treating neoplastic tumors and non-neoplastic disorders (such as ocular neovascularization disorders) is to suppress or substantially reduce the endothelial proliferation and angiogenesis activity of VEGF protein.

[0008] The interaction between a ligand and its receptor is typically of high affinity, reaching the nanomolar to picomolar range. The extracellular domain (ECD) of a receptor or a functional portion of the ECD has been successfully used as a "ligand trap" for therapeutic purposes (Claesson-Welsh L., Nat Med. 2008 Nov; 14(11): 1147-1148; Wiesmann C. et al., Cell. 1997 Nov 28; 91(5): 695-704; Harding TC et al., Sci Transl Med. 2013 Mar 27; 5(178): 178ra39).

[0009] Recombinant fusion proteins containing VEGFR components have been developed to treat diseases associated with VEGF expression, such as cancer and AMD. These fusion proteins are produced by fusing the VEGFR fragment to the N-terminus of the immunoglobulin Fc fragment or a portion of the immunoglobulin Fc fragment and are considered to be "VEGF traps." See, for example, US6100071, US7087411, and US7521049. The VEGFR fragment will bind to and inactivate endogenous VEGF, thereby providing a means for reducing or inhibiting endogenous VEGF activity, and subsequently reducing or inhibiting endothelial cell proliferation and angiogenesis. The VEGF trap (Aflibercept) can capture VEGF with a higher affinity (0.5 pM) than bevacizumab (VEGF neutralizing antibody, 50 pM).

[0010] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. Summary of the Invention

[0011] The present invention relates to antibody fusion proteins comprising a VEGFR component fused to the C-terminus of an antibody light chain, such as the C-terminus of an antibody light chain constant domain (CL domain), and methods of making and using the same.

[0012] One aspect of the present application provides an antibody fusion protein comprising: 1) an antibody comprising a light chain, and 2) a VEGFR component, wherein the VEGFR component is fused to the C-terminus of the antibody light chain (such as the antibody V L -C L In some embodiments, the antibody fusion protein comprises a first VEGFR component fused to the C-terminus of the first antibody light chain and a second VEGFR component fused to the C-terminus of the second antibody light chain. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different.

[0013] In some embodiments according to any of the above antibody fusion proteins, the VEGFR component comprises a first VEGFR Flt1 immunoglobulin-like (Ig-like) domain 2 (Flt1d2). In some embodiments, the VEGFR component further comprises a second VEGFR Flk1 Ig-like domain 3 (Flk1d3). In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component further comprises a third VEGFR Flk1 Ig-like domain 4 (Flk1d4).

[0014] In some embodiments according to any of the above antibody fusion proteins, the VEGFR component is at least about 4 kDa (such as at least about any of: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa).

[0015] In some embodiments according to any of the above antibody fusion proteins, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 - 12 M, about 10 -10 M to about 10 -12 M).

[0016] In some embodiments according to any of the above antibody fusion proteins, the VEGFR component and the C-terminus of the antibody light chain are connected by a linker. In some embodiments, the linker is a peptide linker, such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 6 or 7.

[0017] In some embodiments according to any of the above antibody fusion proteins, the antibody is selected from IgA, IgD, IgE, IgG, IgM, IgG-derived molecules, Fab, Fab', F(ab')2, Fab-scFv, F(ab')2-scFv2, Fab-scFv-Fc, Dock and Lock, scFv, di-scFv, diabody, diabody-Fc, diabody-CH3, and intrabody. In some embodiments, the antibody comprises a light chain constant domain (C L domain), and VEGFR components and antibody CL C-terminus of the domain (e.g., antibody V L -C L In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody, such as an IgG1 or IgG4 antibody, or a variant thereof.

[0018] In some embodiments according to any of the above antibody fusion proteins, the antibody is monospecific.

[0019] In some embodiments according to any of the above antibody fusion proteins, the antibody is multispecific (eg, bispecific).

[0020] In some embodiments according to any of the above-mentioned antibody fusion proteins, the antibody specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is a stimulatory immune checkpoint molecule. In some embodiments, the immune checkpoint molecule is an inhibitory immune checkpoint molecule. In some embodiments, the inhibitory immune checkpoint molecule is selected from PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 and CD73. In some embodiments, the inhibitory immune checkpoint molecule is PD-1. In some embodiments, the antibody comprises: heavy chain-CDR1 (HC-CDR1), HC-CDR2 and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and / or light chain-CDR1 (LC-CDR1), LC-CDR2 and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is nivolumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the inhibitory immune checkpoint molecule is PD-L1. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody is durvalumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the inhibitory immune checkpoint molecule is CTLA-4. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0021] In some embodiments according to any one of the above-mentioned antibody fusion proteins, the antibody specifically recognizes a tumor antigen. In some embodiments, the tumor antigen is HER2. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody is trastuzumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 26, 27 or 28. In some embodiments, the tumor antigen is EGFR (HER1). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody is cetuximab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 31.

[0022] In some embodiments according to any one of the above-mentioned antibody fusion proteins, the antibody specifically recognizes an angiogenic factor. In some embodiments, the angiogenic factor is angiopoietin-2 (Ang2). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody is Nesvacumab or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the angiogenic factor is TNFα. In some embodiments, the antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 32; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody is adalimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the angiogenic factor is IL-17α. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38, and a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody is ixekizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

[0023] Also provided is a pharmaceutical composition comprising any one of the above-mentioned antibody fusion proteins and optionally a pharmaceutically acceptable carrier.

[0024] Another aspect of the present application provides a method for treating an individual suffering from cancer, the method comprising administering to the individual any of the above-mentioned pharmaceutical composition or antibody fusion protein of an effective amount. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, liver cancer, skin cancer (e.g., melanoma), breast cancer, ovarian cancer, prostate cancer, colorectal cancer, or bladder cancer. In some embodiments, the method also comprises subjecting the individual to additional cancer treatment. In some embodiments, the pharmaceutical composition or antibody fusion protein is systemically administered, such as intravenously administered (iv). In some embodiments, the pharmaceutical composition or antibody fusion protein is locally administered, such as intratumorally administered. In some embodiments, the individual is a human.

[0025] Another aspect of the present application provides a method for treating an individual suffering from a non-neoplastic disorder, the method comprising administering to the individual an effective amount of any of the above-mentioned pharmaceutical compositions or antibody fusion proteins. In some embodiments, the non-neoplastic disorder is associated with VEGF overexpression. In some embodiments, the non-neoplastic disorder is selected from rheumatoid arthritis, psoriasis, atherosclerosis, hemangioma, immune rejection of transplanted corneal tissue and other tissues, chronic inflammation, or and ocular neovascularization disorder. In some embodiments, the pharmaceutical composition is administered systemically, such as intravenously (iv). In some embodiments, the non-neoplastic disorder is an ocular neovascularization disorder, such as AMD or diabetic retinopathy. In some embodiments, the ocular neovascularization disorder is associated with choroidal neovascularization, vascular leakage, and / or retinal edema. In some embodiments, the administration of the pharmaceutical composition or antibody fusion protein is selected from one of eye drops, subconjunctival injection, subconjunctival implantation, intravitreal injection, intravitreal implantation, sub-Tenon's fascia capsule injection, and sub-Tenon's fascia capsule implantation, such as intravitreal injection. In some embodiments, the individual is human.

[0026] Also provided are an isolated nucleic acid encoding any of the above-mentioned antibody fusion proteins; a vector comprising any of the above-mentioned isolated nucleic acids; an isolated host cell comprising any of the above-mentioned isolated nucleic acids or vectors; and a kit comprising any of the above-mentioned antibody fusion proteins, isolated nucleic acids, vectors, or isolated host cells.

[0027] Another aspect of the present application provides a method for preparing any of the above-mentioned antibody fusion proteins, the method comprising culturing a host cell comprising any of the above-mentioned isolated nucleic acids or vectors, or culturing any of the above-mentioned isolated host cells, under conditions effective to express the encoded antibody fusion protein; and obtaining the expressed antibody fusion protein from the host cell. In some embodiments, the method further comprises generating a host cell comprising any of the above-mentioned isolated nucleic acids or vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shown is an exemplary VEGFR-antibody light chain fusion protein comprising a monospecific full-length antibody and two VEGFR components, wherein each VEGFR component is fused to the C-terminus of each light chain of the antibody light chain. In an alternative form, the two VEGFR components can be different, can comprise three or more Ig-like domains, and / or can comprise any Ig-like domain of Flt-1 or Flk-1.

[0029] Figure 2 Shown is an exemplary VEGFR-antibody light chain fusion protein comprising a bispecific full-length antibody and two VEGFR components, wherein each VEGFR component is fused to the C-terminus of each light chain of the antibody light chain. In an alternative form, the two VEGFR components can be different, can comprise three or more Ig-like domains, and / or can comprise any Ig-like domain of Flt-1 or Flk-1.

[0030] Figure 3 Two exemplary VEGFR-antibody light chain fusion proteins are shown, each comprising a monospecific full-length antibody and a VEGFR component fused to the C-terminus of one light chain of the antibody. Binding to the VEGF dimer brings the two VEGFR-antibody light chain fusion proteins close. In an alternative form, the two VEGFR-antibody light chain fusion proteins can be different and can each comprise three or more Ig-like domains, and / or can comprise any Ig-like domain of Flt-1 or Flk-1.

[0031] Figure 4 Two exemplary VEGFR-antibody light chain fusion proteins are shown, each comprising a bispecific full-length antibody and a VEGFR component fused to the C-terminus of one light chain of the antibody. Binding to the VEGF dimer brings the two VEGFR-antibody light chain fusion proteins close. In an alternative form, the two VEGFR-antibody light chain fusion proteins can be different and can each comprise three or more Ig-like domains, and / or can comprise any Ig-like domain of Flt-1 or Flk-1.

[0032] Figure 5Two exemplary VEGFR-antibody light chain fusion proteins are shown, each comprising a VEGFR component fused to the C-terminus of a Fab and a light chain of the Fab. Two VEGFR-antibody light chain fusion proteins are brought into proximity with the VEGF dimer. In an alternative form, the two VEGFR-antibody light chain fusion proteins may be different and each may comprise three or more Ig-like domains and / or may comprise any Ig-like domain of Flt-1 or Flk-1. DETAILED DESCRIPTION

[0033] The present invention provides antibody fusion proteins comprising a VEGFR component fused to the C-terminus of an antibody light chain (hereinafter referred to as "VEGFR-antibody light chain fusion proteins"), methods for their preparation, and their use in treating diseases such as cancer and non-neoplastic disorders (such as ocular neovascularization disorders, e.g., diabetic retinopathy and AMD).

[0034] For example, compared to antibodies with C-terminal fusions on the heavy chain, the VEGFR-antibody light chain fusion proteins described herein can have substantially no aggregation of high stability, increased serum half-life and subcutaneous bioavailability, retain ADCC and CDC activity, which enables it to be more effective and deliver the antibody fusion protein at a lower dose. The VEGFR-antibody light chain fusion proteins described herein can also retain high binding affinity to both VEGF and the antigen recognized by the parent antibody.

[0035] Thus, one aspect of the present application provides an antibody fusion protein comprising: 1) an antibody comprising a light chain, and 2) a VEGFR component, wherein the VEGFR component is attached to the C-terminus of the antibody light chain (e.g., the antibody V L -C L C-terminal) fusion of the 5′-terminal domain.

[0036] Also provided are compositions (such as pharmaceutical compositions), kits, and articles of manufacture comprising the VEGFR-antibody light chain fusion proteins, methods for their preparation, and methods of using the VEGFR-antibody light chain fusion proteins to treat diseases (such as cancer, non-neoplastic disorders, e.g., ocular neovascular disorders).

[0037] I. Definition

[0038] As used herein, "angiogenesis," "angiogenic," or "neovascularization" refers to the formation, growth, and / or development of new blood vessels.

[0039] As used herein, the term "neovascular disorder" refers to a disorder characterized by altered or unregulated angiogenesis, rather than a disorder accompanied by oncogenic or neoplastic transformation, i.e., cancer. Examples of neovascular disorders include psoriasis, rheumatoid arthritis, and ocular neovascular disorders, such as diabetic retinopathy and AMD.

[0040] The term "ocular neovascular disorder" as used herein refers to a disorder characterized by altered or unregulated angiogenesis in the patient's eye. Exemplary ocular neovascular disorders include optic disc neovascularization, iris neovascularization, retinal neovascularization (RNV), choroidal neovascularization (CNV), corneal neovascularization, vitreous neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, diabetic macular edema, AMD, vascular retinopathy, retinal degeneration, uveitis, inflammatory diseases of the retina, and proliferative vitreoretinopathy.

[0041] "Choroidal neovascularization" (CNV) refers to the abnormal development, proliferation, and / or growth of blood vessels arising from the choriocapillaris. The blood vessels typically extend through Bruch's membrane, the RPE layer, and / or the subretinal space.

[0042] " macular degeneration associated conditions " refers to any one of the multiple obstacles and the disease of macular degeneration or loss of functional activity. The degeneration of functional activity or the loss can be due to the loss of cell proliferation and / or normal biological function of, for example, cell death, minimization. Macular degeneration can cause and / or show as the change of the structural integrity of macular cells and / or extracellular matrix, the change of normal cells and / or extracellular matrix structure and / or the loss of macular cell function. Cell can be any cell type normally present in the macula or near it, including RPE cells, photoreceptor cells and / or capillary endothelial cells. AMD is main macular degeneration associated conditions. Other macular degeneration associated conditions include Best macular dystrophy, fundus dystrophy, Mallatia Leventinese and Duoying's honeycomb retinal dystrophy.

[0043] "Ocular implant" refers to a device or structure of appropriate size, shape, and / or configuration and made of suitable materials so that it can be placed in the eye without causing unacceptable interference with the physiology or function of the eye. Preferably, the placement of the ocular implant does not significantly impair vision. Ocular implants are generally solid or semisolid articles and are generally macroscopic, i.e., visible to the naked eye.

[0044] The terms "angiogenesis inhibitor" and "anti-angiogenic agent" are used interchangeably herein to refer to an agent that inhibits or reduces one or more processes associated with angiogenesis, including but not limited to endothelial cell proliferation, endothelial cell survival, endothelial cell migration, differentiation of precursor cells into endothelial cells, and capillary blood vessel formation.

[0045] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that fully or partially decreases, inhibits, or interferes with one or more inhibitory immune checkpoint molecules, which can inhibit T-cell activation and function.

[0046] As used herein, the term "activator of a stimulatory immune checkpoint molecule" refers to a molecule that stimulates, activates, or increases the intensity of an immune response mediated by a stimulatory immune checkpoint molecule.

[0047] As used herein, "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of the present invention, favorable or desired clinical results include, but are not limited to, one or more of the following results: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying disease propagation (e.g., cancer cell metastasis), preventing or delaying disease recurrence, delaying or slowing disease progression, improving the disease state, providing disease relief (partial or complete), reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life and / or prolonging life. "Treatment" also encompasses the pathological consequences of reducing cancer or ocular neovascularization disorders. The method of the present invention contemplates any one or more of these aspects of treatment.

[0048] The term "prevent" and similar words such as "preventive," "prophylactic," etc., refer to methods for preventing, inhibiting, or reducing the likelihood of recurrence of a disease or condition (e.g., cancer, neovascular disorders (such as ocular neovascular disorders)). It also relates to delaying the recurrence of a disease or condition or delaying the recurrence of symptoms of a disease or condition. As used herein, "preventing" and similar words also include reducing the intensity, effects, symptoms, and / or burden of a disease or condition before the disease or condition recurs.

[0049] As used herein, "delaying" the development of cancer or neovascular disorders (such as ocular neovascular disorders) means delaying, hindering, slowing down, slowing down, stabilizing and / or delaying the development of the disease. The duration of this delaying effect may vary, depending on the medical history and / or the individual being treated. A method of "delaying" the development of cancer or neovascular disorders (such as ocular neovascular disorders) is a method of reducing the likelihood of disease development within a given time frame and / or reducing the extent of the disease within a given time frame when compared to not using the method. Such comparisons are generally based on clinical studies using statistically significant numbers of individuals. The development of cancer can be detected using standard methods, including but not limited to computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography or biopsy. Development can also refer to the progression of cancer or neovascular disorders (such as ocular neovascular disorders), which may initially be undetectable and include occurrence, recurrence and onset.

[0050] As used herein, the term "effective amount" refers to an amount of an agent or combination of agents sufficient to treat a particular disorder, condition, or disease, such as to improve, alleviate, mitigate, and / or delay one or more of its symptoms. Referring to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and / or reduce the tumor growth rate (such as inhibiting tumor growth) or prevent or delay other undesirable cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay progression. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of a drug or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, slow, slow, and preferably prevent cancer cells from infiltrating into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably prevent) tumor cancer cell metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. With reference to ocular neovascular disorders, an effective amount includes an amount sufficient to treat, inhibit, delay and / or prevent a neovascular disorder or a symptom thereof, for example, an amount sufficient to achieve one or more of the following: (i) inhibit or prevent drusen formation; (ii) cause a decrease in the number and / or size of drusen (drusen regression); (iii) cause a reduction or prevention of lipofuscin deposition; (iv) inhibit or prevent vision loss or slow the rate of vision loss; (v) inhibit choroidal neovascularization or slow the rate of choroidal neovascularization; (vi) cause ablation of choroidal neovascularization characterized by (vii) inhibit choroidal neovascularization or slow the rate of retinal neovascularization; (viii) cause a reduction in the size and / or number of ablation foci characterized by retinal neovascularization; (ix) improve visual acuity and / or contrast sensitivity; (x) reduce macular edema and / or reduce abnormal macular thickness; (xi) inhibit or prevent photoreceptor or RPE cell atrophy or apoptosis, or reduce the rate of photoreceptor or RPE cell atrophy or apoptosis; (xii) inhibit or prevent the progression of non-exudative macular degeneration to exudative macular degeneration.

[0051] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human.

[0052] The terms "antibody" or "antibody portion" are used in the broadest sense and encompass various antibody structures, including but not limited to monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0053] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of 5 basic heterotetrameric units together with an additional polypeptide called a J chain and contain 10 antigen binding sites, while IgA antibodies contain 2-5 basic 4-chain units, which can be polymerized to form a multivalent assembly with a J chain. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, but the two H chains are connected to each other by one or more disulfide bonds, which depends on the H chain isotype. Each H and L chain also has a regularly spaced intrachain disulfide bridge. Each H chain has a variable domain (V domain) at the N-terminus. H ), followed by three constant domains (C H ) and the four Cs of μ and ε isotypes H Each L chain has a variable domain (V L ), followed by a constant domain at its other end. L With V H Align, and C L With heavy chain (C H 1). Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. H and V L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and chapter 6. The L chains from any vertebrate species can be assigned to one of two clearly distinct types (called kappa and lambda) based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of its heavy chain (C H ) can be assigned to different classes or isotypes based on the amino acid sequence of the constant domain of the immunoglobulins. There are five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. H The gamma and alpha classes are further divided into subclasses due to relatively minor differences in sequence and function. For example, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl, and IgA2.

[0054] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from components of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free from association with all other components of its production environment. Contaminating components of its production environment, such as those obtained from recombinant transfected cells, are substances that may generally interfere with the research, diagnostic, or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified: (1) to greater than 95% by weight antibody, as determined, for example, by the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody (or construct) includes the antibody in situ within recombinant cells, as at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated polypeptide, antibody or construct will be prepared by at least one purification step.

[0055] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "V H ” and “V L These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site.

[0056] The term "variable" refers to the fact that certain segments of the variable domains between antibodies differ greatly in sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the entire span of the variable domain. Instead, it is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, which primarily adopt a β-sheet configuration and are connected by three CDRs, which form loops connecting and, in some cases, forming part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chains, contribute to the formation of the antigen binding site of the antibody (see, Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0057] As used herein, the term "monoclonal antibody" refers to an antibody derived from substantially homogeneous antibodies, i.e., except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in trace amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characterized in that it is derived from a substantially homogeneous antibody population and should not be construed as requiring antibody production by any ad hoc method. For example, the monoclonal antibodies to be used according to the present invention can be prepared by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Immunofluorescence Spectroscopy, 1996); Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004), and techniques for producing human antibodies or human-like antibodies in animals having part or all of a human immunoglobulin locus or gene encoding a human immunoglobulin sequence (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad.Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0058] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, a full-length 4-chain antibody includes an antibody having heavy and light chains that include an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0059] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding region and / or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; DARTs; linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules (such as scFv), and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fc" fragment, a name reflecting the ability to crystallize easily. The Fab fragment consists of the entire L chain together with the variable region domain (V H ) and a heavy chain (C H1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that the C H The constant domains have additional residues at the carboxyl terminus, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0060] The Fc fragment comprises the carboxyl-terminal portions of the two H chains held together by disulfide bonds.The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) present on specific cell types.

[0061] The term "constant domain" refers to the portion of an immunoglobulin molecule that has an amino acid sequence that is more conserved than the other portion of the immunoglobulin (i.e., the variable domain containing the antigen binding site). The constant domain comprises the C H 1. C H 2 and C H 3 domains (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

[0062] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0063] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. Six hypervariable loops (3 loops each from the H chain and the L chain) are emitted from the folding of these two domains, which contribute to the antigen binding of amino acid residues and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0064] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is a fragment comprising a V domain linked into a single polypeptide chain. H and V LPreferably, the sFv polypeptide further comprises an antibody fragment between V H and V L A polypeptide linker between domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0065] The "functional fragments" of the antibodies described herein comprise a portion of an intact antibody, generally including the antigen binding region or variable region of an intact antibody or an antibody Fc region that retains or has modified FcR binding ability. Examples of antibody fragments include linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0066] The term "diabody" refers to a diabody that is constructed by using a short linker (about 5-10 residues) at the V H and V L The sFv fragments are constructed between the domains (see previous paragraph) so that interchain pairing of the V domains is achieved rather than intrachain pairing, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific antibodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are bound to each other. H and V L The domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0067] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). "Humanized antibodies" are used as a subset of "chimeric antibodies."

[0068] Inhuman (for example, llama or Camelidae) antibody of " humanization " form is a chimeric antibody comprising the minimum sequence derived from non-human immunoglobulin.In some embodiments, humanized antibody is human immunoglobulin (receptor antibody), wherein the residue from the CDR (defined below) of receptor is replaced by the residue from non-human species (donor antibody) with desired specificity, affinity and / or ability, such as mouse, rat, rabbit, camel, llama, alpaca or non-human primate CDR.In some cases, the framework (" FR ") residues of human immunoglobulin are replaced by corresponding non-human residues.In addition, humanized antibody can comprise the residue not present in receptor antibody or donor antibody.These modifications can be carried out to further improve antibody performance, such as binding affinity. In general, a humanized antibody will comprise at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Patents 6,982,321 and 7,087,409.

[0069] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or prepared using any of the techniques for preparing human antibodies as disclosed herein. This definition of a human antibody explicitly excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be prepared using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also useful for preparing human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, such as immunizing a heterologous mouse (see, e.g., regarding XENOMOUSE). TM See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies generated via human B cell hybridoma technology.

[0070] As used herein, the term "hypervariable region," "HVR," or "HV" refers to a region of an antibody variable domain whose sequence is hypervariable and / or forms a structurally defined loop. Generally, a single-domain antibody comprises three HVRs (or CDRs): HVR1 (or CDR1), HVR2 (or CDR2), and HVR3 (or CDR3). HVR3 (or CDR3) exhibits the greatest diversity of the three HVRs and is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0071] The term "complementarity determining region" or "CDR" is used to refer to the hypervariable regions as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0072] The present invention relates to the invention of the present invention and the invention relates to the invention of the present invention.The present invention relates to the invention of the present invention and the invention relates to the present invention.The present invention relates to the invention of the present invention and the invention relates to the present invention.The present invention relates to the present invention and the invention relates to the present invention.The present invention relates to the present invention and the invention relates to the present invention.

[0073] Table 1: HVR Description

[0074]

[0075] The HVR may contain an "Extended HVR" as shown below: L 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3), and V H 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102 or 95-102 (H3) in. For each of these definitions, the variable domain residues are numbered according to Kabat et al. (supra).

[0076] The expression "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system for heavy chain variable domains or light chain variable domains used in antibody compilation in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of the FR or HVR of the variable domain or an insertion into the FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat numbering of residues in a given antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbering sequence in regions of homology.

[0077] Unless otherwise indicated, the numbering of residues in the immunoglobulin heavy chain is that of the EU index as in Kabat et al. (supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0078] "Framework" or "FR" residues are those variable domain residues other than the HVR residues herein defined.

[0079] "Human consensus framework" or "acceptor human framework" refers to the selection of human immunoglobulin V L or V H The framework sequence is the framework of the most frequently occurring amino acid residues. L or V H The sequence is selected from a subset of variable domain sequences. Generally, the subset of sequences is that in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Examples include sequences for V LFor VH, the subgroup may be subgroup κI, κII, κIII or κIV, as described in Kabat et al., supra. Additionally, for VH, the subgroup may be subgroup I, subgroup II, or subgroup III, as described in Kabat et al. Alternatively, a human consensus framework may be derived from the above subgroups, wherein specific residues (such as human framework residues) are selected based on their homology to the donor framework by aligning the donor framework sequence with a collection of various human framework sequences. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0080] An "affinity matured" antibody is one that has one or more alterations in one or more of its CDRs that result in an improvement in the antibody's affinity for the antigen compared to a parent antibody that does not have those alterations. In some embodiments, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are prepared by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity matured antibodies. H -domain and V L -Affinity maturation of domain shuffling. Random mutagenesis of CDR and / or framework residues is described, for example, by: Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0081] As used herein, the terms "specifically binds," "specifically recognizes," or "is specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antigen binding protein, which is determinative for the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antigen binding protein that specifically binds to a target (which may be an epitope) is an antigen binding protein that binds to that target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other targets. In some embodiments, the extent of binding of the antigen binding protein to an unrelated target is less than about 10% of the binding of the antigen binding protein to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antigen binding protein that specifically binds to a target or a VEGFR component that specifically binds to VEGF has a dissociation constant (K d ) is ≤10- 5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 M. In some embodiments, the antigen binding protein specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but does not require exclusive binding.

[0082] The term "specificity" refers to an antigen-binding protein that selectively recognizes a specific epitope of an antigen. For example, a natural antibody is monospecific. As used herein, the term "multispecific" means that an antigen-binding protein has multiple epitope specificities (i.e., it can specifically bind to two, three or more different epitopes on a biomolecule or can specifically bind to an epitope on two, three or more different biomolecules). As used herein, "bispecific" means that an antigen-binding protein has two different antigen-binding specificities. Unless otherwise specified, the order of the antigens that the bispecific antibodies are listed to bind is arbitrary. As used herein, the term "monospecific" means an antigen-binding protein with one or more binding sites that each bind to the same epitope of the same antigen.

[0083] As used herein, the term "valency" refers to the specified number of binding sites present in an antigen binding protein. For example, a native antibody or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites, respectively, in an antigen binding protein.

[0084] "Antibody effector function" refers to those biological activities attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. "Reducing or minimizing" an antibody effector function means that it is reduced by at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) from a wild-type or unmodified antibody. The determination of antibody effector function can be readily determined and measured by one of ordinary skill in the art. In a preferred embodiment, the antibody effector functions of complementary binding, complement-dependent cytotoxicity, and antibody-dependent cellular toxicity are affected. In some embodiments, the effector function is eliminated by a mutation in the constant region that eliminates glycosylation, such as a "no effector mutation." In one aspect, the null effect mutation is C H N297A or DANA mutation in region 2 (D265A+N297A). Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively, additional mutations that result in reduced or eliminated effector function include: K322A and L234A / L235A (LALA). Alternatively, effector function can be reduced or eliminated by production techniques, such as expression in host cells that do not glycosylate (e.g., E. coli) or that result in altered glycosylation patterns that are ineffective or less effective in promoting effector function (see, Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003).

[0085] "Antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are necessary for killing target cells by this mechanism. Primary cells used to mediate ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition thereto, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model, such as disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0086] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example, during the preparation or purification of the antibody, or can be removed by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the intact antibody can include antibody groups in which all K447 residues are removed, antibody groups in which the K447 residue is not removed, and antibody groups with a mixture of antibodies with or without the K447 residue. Native sequence Fc regions suitable for antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0087] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (Clq) to an antibody (of the appropriate subclass) bound to its cognate antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Antibody variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in U.S. Patent No. 6,194,551 Bl and WO99 / 51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0088] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody, VEGFR) and its binding partner (e.g., an antigen, VEGF). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. Binding affinity can be expressed as K d , K off , K on or K a As used herein, the term "K off " is intended to refer to the rate constant for the dissociation of an antibody (or antigen binding domain) from an antibody / antigen complex (or the dissociation of a VEGFR from a VEGF / VEGFR complex), as determined by a kinetic selection setup, expressed in s -1 As used herein, the term "K on " is intended to refer to the rate constant for the association of an antibody (or antigen binding domain) with an antigen to form an antibody / antigen complex (or the association of a VEGFR with VEGF to form a VEGF / VEGFR complex), expressed in M -1 s -1 As used herein, the term equilibrium dissociation constant "K D ” or “K d K refers to the dissociation constant for a specific antibody-antigen interaction (or VEGFR-VEGF interaction) and describes the concentration of antigen required to occupy half of all antibody binding domains present in a solution of antibody molecules at equilibrium (or the concentration of VEGF required to occupy half of all VEGFRs present in a solution of VEGFRs at equilibrium) and is equal to K off / K on , which is expressed in M. K dThe measurement of K assumes that all the binder is in solution. In cases where the antibody is tethered to the cell wall, such as in yeast expression systems, the corresponding equilibrium rate constant is expressed as EC50, which provides the K d A good approximation of the affinity constant, K a is the dissociation constant K d The reciprocal of M -1 Indicated as a unit.

[0089] Dissociation constant (K D or K d ) is used as an indicator showing the affinity of an antibody to an antigen (or VEGFR to VEGF). For example, simple analysis is possible by the following methods: using the Scatchard method using antibodies labeled with various labeling reagents, and using BiacoreX (manufactured by Amersham Biosciences) as an over-the-counter drug, a measurement kit, or a similar kit, according to the user manual and experimental operation method attached to the kit. K can be derived using these methods. D The values ​​are expressed in M ​​(Mol). An antibody or antigen-binding fragment thereof that specifically binds to a target (or a VEGFR component described herein that specifically binds to VEGF) may have, for example, a molecular weight of ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The dissociation constant (K d ).

[0090] The binding specificity of an antibody or antigen binding domain (or VEGFR components as described herein) can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Western blot, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore tests, and peptide scanning.

[0091] Half-maximal inhibitory concentration (IC 50 IC is a measure of the effectiveness of a substance (such as an antibody or VEGFR) in inhibiting a specific biological or biochemical function. It indicates how much of a particular drug or other substance (inhibitor, such as an antibody or VEGFR) is needed to inhibit half of a given biological process or component of a process, i.e., an enzyme, cell, cell receptor, or microorganism. This value is usually expressed as a molar concentration. 50 EC with agonist drugs or other substances (such as antibodies) 50 Equivalent. EC 50It also refers to the plasma concentration required to achieve 50% of the maximum effect in vivo. As used herein, "IC 50 " is used to indicate the effective concentration of an antibody required to neutralize 50% of the biological activity of an antigen in vitro (or the effective concentration of a VEGFR component required to neutralize 50% of the biological activity of VEGF). 50 or EC 50 Measurement can be performed by biological assays, such as inhibition of ligand binding by FACS analysis (competitive binding assay), cell-based cytokine release assay, or amplified luminescent proximity homogeneous assay (AlphaLISA).

[0092] "Percent (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a specified peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity, but not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN. TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0093] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule that is typically associated with the environment in which it is produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are in a form or arrangement that is not in which they occur in nature. Thus, isolated nucleic acid molecules are distinguished from nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules that are contained in cells that typically contain nucleic acid molecules, but that are present extrachromosomally or at a chromosomal location that is different from their natural chromosomal location.

[0094] The term "control sequence" refers to DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0095] When a nucleic acid is in functional association with another nucleic acid sequence, it is "operably linked". For example, if it is expressed as a pre-preprotein that participates in the secretion of a polypeptide, the DNA of the presequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if it affects the transcription of the sequence, the promoter or enhancer is operably linked to the coding sequence; or if it is positioned so as to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally speaking, "operably linked" means that the DNA sequences being connected are continuous, and in the case of a secretory leader sequence, are continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is accomplished by connecting at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used according to conventional practice.

[0096] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0097] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include the primary target cell and its progeny.

[0098] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of progeny may not be identical to that of the parent cell, but may contain mutations. Progeny with mutations that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0099] "Adjuvant setting" refers to a clinical setting in which an individual has a history of cancer and is generally (but not necessarily) responsive to therapy, including but not limited to surgery (e.g., surgical resection), radiation therapy, and chemotherapy. However, due to their history of cancer, these individuals are considered to be at risk for developing the disease. Treatment or administration in an "adjuvant setting" refers to a subsequent treatment modality. The degree of risk (e.g., when an individual in an adjuvant setting is considered "high risk" or "low risk") depends on a variety of factors, the most common of which is the extent of the disease at the time of initial treatment.

[0100] "Neoadjuvant setting" refers to the clinical setting in which the approach is performed prior to primary / definitive therapy.

[0101] "Biocompatible" refers to a material that is substantially nontoxic to the cells of the recipient in the amounts and at the location in which it is used, and does not induce or result in a significant deleterious or adverse effect on the body of the recipient at the location in which it is used, e.g., an unacceptable immune or inflammatory response, unacceptable scar tissue formation, etc. For example, a material that is biocompatible with the eye does not significantly interfere with the physiology or function of the eye.

[0102] "Bioerodeable" or "biodegradable" means that the material is capable of being physically and / or chemically broken down within a cell or within the body of a subject, for example, by hydrolysis under physiological conditions and / or by the action of natural biological processes, such as enzymes present within the cell or body, and / or by processes such as dissolution, dispersion, etc., to form smaller chemical substances that can generally be metabolized and optionally used, and / or excreted or otherwise disposed of by the body. Preferably, a biodegradable compound is biocompatible. A polymer whose molecular weight decreases over time in the body due to a decrease in the number of monomers is considered biodegradable.

[0103] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a formulation that is in such form as to permit the biological activity of the active ingredient to be effective and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered. Such formulations are sterile. A "sterile" formulation is sterile or free of all living microorganisms and their spores.

[0104] It is to be understood that embodiments of the invention described herein (such as "comprising" embodiments) include "consisting of and / or "consisting essentially of" embodiments.

[0105] Reference herein to a value or parameter that is "about" includes (and describes) variations involving that value or parameter itself. For example, description of "about X" includes description of "X."

[0106] As used herein, reference to "other than" a value or parameter generally refers to and describes a value or parameter "other than that value or parameter." For example, the method is not intended to treat type X cancer, which means that the method is intended to treat a type of cancer other than type X.

[0107] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0108] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0109] II. Antibody Fusion Proteins Comprising a VEGFR Component Fusion to the C-Terminus of an Antibody Light Chain

[0110] The antibody fusion proteins described herein comprise: 1) an antibody comprising a light chain, and 2) a VEGFR component, wherein the VEGFR component is attached to the C-terminus of the antibody light chain (e.g., the antibody V L -C L C-terminal) fusion of the 5′-terminal domain.

[0111] In some embodiments, an antibody fusion protein is provided that comprises: 1) an antibody (such as a full-length antibody) comprising a light chain, and 2) a VEGFR component attached to the C-terminus of the antibody light chain (e.g., the antibody V L -C L In some embodiments, the VEGFR component comprises an immunoglobulin-like (Ig-like) domain 2 (Flt1d2) of a first VEGFR Flt-1. In some embodiments, the VEGFR component further comprises an Ig-like domain 3 (Flk1d3) of a second VEGFR Flk-1. In some embodiments, the VEGFR component further comprises an Ig-like domain 4 (Flk1d4) of a third VEGFR Flk1. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component consists essentially of, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component consists essentially of, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any one of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the KD of the binding between the VEGFR component and VEGF is 24 kDa. D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10-12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the VEGFR component and the C-terminus of the antibody light chain are connected by a linker (such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 6 or 7). In some embodiments, the antibody is selected from IgA, IgD, IgE, IgG, IgM, IgG-derived molecules, Fab, Fab', F(ab')2, Fab-scFv, F(ab')2-scFv2, Fab-scFv-Fc, Dock and Lock, scFv, di-scFv, diabody, diabody-Fc, diabody-CH3, and intrabody. In some embodiments, the antibody comprises a light chain constant domain (C L domain), and VEGFR components and antibody C L C-terminus of the domain (e.g., antibody V L -C L In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (such as bispecific). In some embodiments, the antibody specifically recognizes immune checkpoint molecules. In some embodiments, the immune checkpoint molecules are stimulatory immune checkpoint molecules. In some embodiments, the immune checkpoint molecules are inhibitory immune checkpoint molecules (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, and CD73). In some embodiments, the antibody specifically recognizes PD-1. In some embodiments, the antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with pembrolizumab (e.g., ) competitively binds to PD-1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is nivolumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with nivolumab (e.g., ) competitively binds to PD-1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody specifically recognizes PD-L1. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with atezolizumab (e.g., ) competitively binds to PD-L1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody is durvalumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with durvalumab (e.g., ) competitively binds to PD-L1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, the antibody specifically recognizes CTLA-4. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with ipilimumab (e.g., ) competitively binds to CTLA-4. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody specifically recognizes a tumor antigen. In some embodiments, the tumor antigen is HER2. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody is trastuzumab (e.g., ) or its antigen-binding fragment. In some embodiments, the antibody is combined with trastuzumab (e.g., ) competitively binds to HER2. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 26, 27 or 28. In some embodiments, the tumor antigen is EGFR (HER1). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody is cetuximab (e.g., ) or its antigen-binding fragment. In some embodiments, the antibody is combined with cetuximab (e.g., ) competitively binds to EGFR. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, the antibody specifically recognizes an angiogenic factor. In some embodiments, the angiogenic factor is Ang2. In some embodiments, the antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:35; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:35 and a light chain comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the antibody is nivolumab or an antigen-binding fragment thereof. In some embodiments, the antibody competes with nivolumab for Ang2 binding. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the angiogenic factor is TNFα. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 32; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody is adalimumab (e.g., ) or its antigen-binding fragment. In some embodiments, the antibody is combined with adalimumab (e.g., ) competitively binds to TNFα. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the angiogenic factor is IL-17A. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38, and a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody is ixekizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is combined with ixekizumab (e.g., ) competitively binds to IL-17A. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

[0112] In some embodiments, the VEGFR-antibody light chain fusion proteins described herein comprise only one VEGFR component.

[0113] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is attached to the C-terminus of the first antibody light chain (e.g., the antibody V L -C L domain), and the second VEGFR component is fused to the C-terminus of the second antibody light chain (e.g., antibody V L -C LIn some embodiments, the first and / or second VEGFR components are identical. In some embodiments, the first and / or second VEGFR components are different. In some embodiments, the first and / or second VEGFR components comprise Flt1d2. In some embodiments, the first and / or second VEGFR components further comprise Flk1d3. In some embodiments, the first and / or second VEGFR components further comprise Flk1d4. In some embodiments, the first and / or second VEGFR components comprise, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR components is fused to the C-terminus of the antibody light chain. In some embodiments, the first and / or second VEGFR components consist essentially of, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR components is fused to the C-terminus of the antibody light chain. In some embodiments, the first and / or second VEGFR components comprise the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first and / or second VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the first and / or second VEGFR component consists essentially of, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, at least one of the VEGFR components and the C-terminus of the antibody light chain are connected by a linker (such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 6 or 7). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody specifically recognizes an immune checkpoint molecule (e.g., an inhibitory immune checkpoint molecule). In some embodiments, the antibody specifically recognizes PD-1 (e.g., pembrolizumab or nivolumab). In some embodiments, the antibody specifically recognizes PD-L1 (e.g., atezolizumab or durvalumab). In some embodiments, the antibody specifically recognizes CTLA-4 (e.g., ipilimumab). In some embodiments, the antibody specifically recognizes a tumor antigen. In some embodiments, the antibody specifically recognizes HER2 (e.g., trastuzumab). In some embodiments, the antibody specifically recognizes EGFR (HER1) (e.g., cetuximab). In some embodiments, the antibody specifically recognizes angiogenic factors. In some embodiments, the antibody specifically recognizes Ang2 (e.g., nivolumab). In some embodiments, the antibody specifically recognizes TNFα (e.g., adalimumab). In some embodiments, the antibody specifically recognizes IL-17A (e.g., ixekizumab).

[0114] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is attached to the C-terminus of the first antibody light chain (e.g., the antibody V L -C L domain), and the second VEGFR component is fused to the C-terminus of the second antibody light chain (e.g., antibody V L -C L The VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus, the C-terminus of the VEGFR domain, wherein the first VEGFR component and the second VEGFR component each comprise the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: L -C L -L-Flr1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10-8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, at least one of the VEGFR components and the C-terminus of the antibody light chain are connected by a linker (such as a peptide linker comprising the amino acid sequence of SEQ ID NO: 6 or 7). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody specifically recognizes an immune checkpoint molecule (e.g., an inhibitory immune checkpoint molecule). In some embodiments, the antibody specifically recognizes PD-1 (e.g., pembrolizumab or nivolumab). In some embodiments, the antibody specifically recognizes PD-L1 (e.g., atezolizumab or durvalumab). In some embodiments, the antibody specifically recognizes CTLA-4 (e.g., ipilimumab). In some embodiments, the antibody specifically recognizes a tumor antigen. In some embodiments, the antibody specifically recognizes HER2 (e.g., trastuzumab). In some embodiments, the antibody specifically recognizes EGFR (HER1) (e.g., cetuximab). In some embodiments, the antibody specifically recognizes angiogenic factors. In some embodiments, the antibody specifically recognizes Ang2 (e.g., nivolumab). In some embodiments, the antibody specifically recognizes TNFα (e.g., adalimumab). In some embodiments, the antibody specifically recognizes IL-17A (e.g., ixekizumab).

[0115] In some embodiments, the C-terminus of the VEGFR component and the antibody light chain are connected by a linker. In some embodiments, the linker is a peptide linker (e.g., any one of SEQ ID NOs: 1-7 and 44). In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, at least one of the two VEGFR components and the two C-termini of the antibody light chain are connected by a linker. In some embodiments, both C-termini of the two VEGFR components and the antibody light chain are connected by a linker. In some embodiments, the two linkers are the same. In some embodiments, the two linkers are different.

[0116] In some embodiments, the VEGFR-antibody light chain fusion protein described herein comprises an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the antibody specifically recognizes a stimulatory immune checkpoint molecule. In some embodiments, the antibody specifically recognizes an inhibitory immune checkpoint molecule (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, and CD73) (the antibody is hereinafter referred to as "immune checkpoint inhibitor" or "inhibitor of inhibitory immune checkpoint molecules"). In some embodiments, the antibody specifically recognizes PD-1 (e.g., pembrolizumab or nivolumab). In some embodiments, the antibody specifically recognizes PD-L1 (e.g., atezolizumab or durvalumab). In some embodiments, the antibody specifically recognizes CTLA-4 (e.g., ipilimumab).

[0117] For example, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes an immune checkpoint molecule (such as PD-1, PD-L1, or CTLA-4), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C LIn some embodiments, the VEGFR component comprises Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 9, Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes an immune checkpoint molecule (such as PD-1, PD-L1, or CTLA-4), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the binding K between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10-13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody specifically recognizes PD-1 (e.g., pembrolizumab or nivolumab). In some embodiments, the antibody specifically recognizes PD-L1 (e.g., atezolizumab or durvalumab). In some embodiments, the antibody specifically recognizes CTLA-4 (e.g., ipilimumab).

[0118] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C LIn some embodiments, the VEGFR component comprises Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 9, Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 - 13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L-L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 13. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with pembrolizumab for binding to PD-1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, a HC-CDR1, a HC-CDR2, and a HC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is nivolumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with nivolumab for binding to PD-1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0119] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flr1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is nivolumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L domain) fused to the C-terminus of the VEGFR domain; wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with pembrolizumab for binding to PD-1. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C LThe C-terminus of the light chain of the second antibody (e.g., antibody V L -C L C-terminus of the VEGFR domain); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody is nivolumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with nivolumab for binding to PD-1. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17.

[0120] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C LIn some embodiments, the VEGFR component comprises Flt1d2 (or Flt1d2-Flk1d3) and the C-terminus of the antibody light chain (or the C-terminus of the VEGFR domain); in some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the VEGFR component comprises Flt1d2. In some embodiments, the VEGFR component further comprises Flk1d3. In some embodiments, the VEGFR component further comprises Flk1d4. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 19. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, a HC-CDR1, a HC-CDR2, and a HC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with atezolizumab for binding to PD-L1. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 22. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 22 (C-terminal end of the C ...

[0121] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody is durvalumab or an antigen-binding fragment thereof. In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with atezolizumab for binding to PD-L1. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes PD-L1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -CL C-terminus of the VEGFR domain); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody is durvalumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with durvalumab for binding to PD-L1. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

[0122] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes CTLA-4, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C LIn some embodiments, the VEGFR component comprises Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 9, Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 10. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes CTLA-4, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, a HC-CDR1, a HC-CDR2, and a HC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes for CTLA-4 binding with ipilimumab. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0123] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes CTLA-4, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the K of the binding between the VEGFR component and VEGF is D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with ipilimumab for binding to CTLA-4. In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes CTLA-4, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L The invention also provides a method for fusion of an antibody with a VEGFR component comprising the C-terminus of a VEGFR domain; wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with ipilimumab for binding to CTLA-4. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11.

[0124] In some embodiments, the VEGFR-antibody light chain fusion proteins described herein comprise antibodies that specifically recognize tumor antigens (such as HER2, EGFR).

[0125] Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes a tumor antigen (such as HER2, EGFR), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L In some embodiments, the VEGFR component comprises Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8, Flt1d2, Flk1d3, and Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10-9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the tumor antigen is HER2. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 25. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody VEGFR component) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C LThe antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody is trastuzumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with trastuzumab for binding to HER2. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 26, 27 or 28. In some embodiments, the tumor antigen is EGFR (HER1). Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the first antibody light chain (e.g., antibody V) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C L The C-terminus of the light chain of the second antibody (e.g., antibody V L -C L In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 30. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally linked to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the antibody VEGFR component) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7). L -C LThe C-terminus of the light chain of the second antibody (e.g., antibody V L -C L In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody is cetuximab or an antigen-binding fragment thereof. In some embodiments, the antibody competes with cetuximab for binding to EGFR. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 31.

[0126] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes a tumor antigen (such as HER2, EGFR), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the K of the binding between the VEGFR component and VEGF is DAbout 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the tumor antigen is HER2. Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 25. Accordingly, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER2, comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the light chain of the second antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; and wherein the antibody includes: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody is trastuzumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with trastuzumab for binding to HER2. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L-L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 26, 27, or 28. In some embodiments, the tumor antigen is EGFR (HER1). Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes HER1, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 30. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes EGFR, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the light chain of the second antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:29, and a light chain comprising the amino acid sequence of SEQ ID NO:30.In some embodiments, the antibody is cetuximab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with cetuximab for binding to EGFR. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 31.

[0127] In some embodiments, the VEGFR-antibody light chain fusion proteins described herein comprise antibodies that specifically recognize an angiogenic factor. In some embodiments, the antibody specifically recognizes Ang2. In some embodiments, the antibody specifically recognizes TNFα. In some embodiments, the antibody specifically recognizes IL-17A.

[0128] Therefore, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes an angiogenic factor (such as Ang2, TNFα, IL-17A), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the VEGFR component comprises Flt1d2. In some embodiments, the VEGFR component further comprises Flk1d3. In some embodiments, the VEGFR component further comprises Flk1d4. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any one of the following: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa). In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L-L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes an angiogenic factor (such as Ang2, TNFα, IL-17A), the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific).

[0129] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes Ang2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the VEGFR component comprises Flt1d2. In some embodiments, the VEGFR component further comprises Flk1d3. In some embodiments, the VEGFR component further comprises Flk1d4. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes Ang2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR components comprise, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa).In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V. L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 36. Accordingly, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes Ang2, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the light chain of the second antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody includes: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody is nivolumab or an antigen-binding fragment thereof. In some embodiments, the antibody competes with nivolumab for binding to Ang2. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 37.

[0130] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes TNFα, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (e.g., a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (e.g., a peptide linker of SEQ ID NO: 6 or 7). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the VEGFR component comprises Flt1d2. In some embodiments, the VEGFR component further comprises Flk1d3. In some embodiments, the VEGFR component further comprises Flk1d4. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes TNFα, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (e.g., a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (e.g., a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR components comprise, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa).In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V. L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 32; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 33. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes TNFα, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the light chain of the second antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 32; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody is adalimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes with adalimumab for binding to TNFα. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 34.

[0131] In some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes IL-17A, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7). In some embodiments, linker X and linker Y are the same. In some embodiments, linker X and linker Y are different. In some embodiments, the first VEGFR component and the second VEGFR component are the same. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the VEGFR component comprises Flt1d2. In some embodiments, the VEGFR component further comprises Flk1d3. In some embodiments, the VEGFR component further comprises Flk1d4. In some embodiments, the VEGFR component comprises, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes IL-17A, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR components comprise, from N-terminus to C-terminus: Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the VEGFR component is at least about 4 kDa (such as at least about any of: 4 kDa, 8 kDa, 12 kDa, 17 kDa, 20 kDa, 25 kDa, 27 kDa, or 30 kDa).In some embodiments, the VEGFR component-antibody light chain fusion polypeptide comprises, from N-terminus to C-terminus: V. L -C L -L-Flt1d2-Flk1d3 (L is an optional linker). In some embodiments, the K of the binding between the VEGFR component and VEGF D About 10 -8 M to about 10 -13 M (such as about 10 -8 M to about 10 -13 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12M). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgG antibody (such as an IgG1 or IgG4 antibody, or a variant thereof). In some embodiments, the antibody is monospecific. In some embodiments, the antibody is multispecific (e.g., bispecific). In some embodiments, the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 39. Thus, in some embodiments, the present invention provides an antibody fusion protein comprising: 1) an antibody (such as a full-length antibody) that specifically recognizes IL-17A, the antibody comprising a first light chain and a second light chain, and 2) a first VEGFR component and a second VEGFR component, wherein the first VEGFR component is optionally fused to the C-terminus of the light chain of the first antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker X (such as a peptide linker of SEQ ID NO: 6 or 7), and the second VEGFR component is optionally fused to the C-terminus of the light chain of the second antibody (e.g., the C-terminus of the VL-CL domain of the antibody) via a linker Y (such as a peptide linker of SEQ ID NO: 6 or 7); wherein each VEGFR component comprises the amino acid sequence of SEQ ID NO: 8; wherein the antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody is ixekizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the antibody competes for IL-17A binding with ixekizumab. In some embodiments, the antibody fusion protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 40.

[0132] Antibody fusion protein characteristics

[0133] Antibody fusion protein stability

[0134] The VEGFR-antibody light chain fusion proteins of the present invention can exhibit a high level of stability.

[0135] As used herein, the term "stable" with respect to a VEGFR-antibody light chain fusion protein means that after storage under defined conditions, the VEGFR-antibody light chain fusion protein retains an acceptable degree of chemical structure or biological function. Even if the VEGFR-antibody light chain fusion protein does not retain 100% of its chemical structure or biological function after a defined amount of storage time, it may still be stable. In some embodiments, after a defined amount of storage time, maintaining about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the structure or function of the VEGFR-antibody light chain fusion protein can be considered to be "stable."

[0136] Stability can be especially measured in the following manner: after storing the time of a defined amount at a defined temperature, the percentage of natural (non-aggregated or degraded) VEGFR-antibody light chain fusion proteins retained in the formulation (liquid or recombinant) is determined. The percentage of natural VEGFR-antibody light chain fusion proteins can especially be measured by size exclusion chromatography (for example, size exclusion high performance liquid chromatography [SE-HPLC]) so that natural means non-aggregated and undegraded. In some embodiments, after storing the time of a defined amount at a given temperature, at least about 90% (such as at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the native form of VEGFR-antibody light chain fusion proteins can be detected in the formulation. In some embodiments, at least about 90% (such as at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the native form of the VEGFR-antibody light chain fusion protein is detectable in the formulation after at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, or at least about 48 hours at room temperature (about 25°C).

[0137] Stability can be measured in particular by the following manner: after storing a defined amount of time at a defined temperature, the percentage of VEGFR-antibody light chain fusion proteins formed as aggregates in a determination formulation (liquid or reconstituted), wherein stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated VEGFR-antibody light chain fusion proteins can be measured in particular by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). In some embodiments, after storing a defined amount of time at a given temperature, less than about 10% (preferably less than about 5%) of the VEGFR-antibody light chain fusion proteins are present in the formulation as aggregates. In some embodiments, the VEGFR-antibody light chain fusion proteins described herein are substantially free of aggregates, for example, up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the VEGFR-antibody light chain fusion protein can be detected in the formulation in the form of aggregates after storage at a given temperature for a defined amount of time, for example, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, at least about 26 hours, at least about 28 hours, at least about 30 hours, at least about 32 hours, at least about 34 hours, at least about 36 hours, at least about 38 hours, at least about 40 hours, at least about 42 hours, at least about 44 hours, at least about 46 hours, or at least about 48 hours at room temperature (about 25°C).

[0138] Measuring the binding affinity of VEGFR-antibody light chain fusion protein and its target (antibody antigen and / or VEGF) can also be used to assess stability. For example, if after storing a defined amount of time (e.g., 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours) at room temperature (about 25°C), the antibody and VEGFR components included in the VEGFR-antibody light chain fusion protein both bind to the antibody antigen and VEGF with at least 84%, 90%, 95% or even greater affinity (respectively) of the binding affinity of the antibody before said storage, then the VEGFR-antibody light chain fusion protein of the present invention can be considered to be stable. Binding affinity can be measured by any method, such as ELISA or plasma resonance. Biological activity can be measured by antigen or VEGF activity assays, such as by contacting cells expressing VEGF with VEGFR-antibody light chain fusion protein. The combination of VEGFR-antibody light chain fusion protein and such cells can be directly measured, such as via FACS analysis. Alternatively, the downstream activity of the VEGF system can be measured in the presence of a VEGFR-antibody light chain fusion protein and compared with the activity of the VEGF system in the absence of a VEGFR-antibody light chain fusion protein. In some embodiments, VEGF can be endogenous to the cell. In other embodiments, VEGF can be ectopically expressed (i.e., heterologously expressed) in the cell.

[0139] Antibody fusion protein pharmacokinetics

[0140] In some embodiments, the serum half-life of the VEGFR-antibody light chain fusion protein of the present invention is longer than the half-life of the corresponding antibody. For example, the pK of the VEGFR-antibody light chain fusion protein is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the VEGFR-antibody light chain fusion protein is similar to the serum half-life of the corresponding antibody. In some embodiments, when administered to an organism, the serum half-life of the VEGFR-antibody light chain fusion protein is at least about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 24 hours, about 24 hours, about 20 hours, about 18 hours, about 16 hours, about 14 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 3 hours, about 2 hours or about 1 hour.

[0141] Clinical characteristics of antibody fusion proteins

[0142] In some embodiments, relative to the VEGFR-antibody fusion protein comprising an antibody and a VEGFR component fused to the C-terminal end of the heavy chain, and / or the VEGFR-antibody fusion protein comprising an antibody and a VEGFR component fused to the N-terminal end of the heavy chain, and / or the VEGFR-antibody fusion protein comprising an antibody and a VEGFR component fused to the N-terminal end of the light chain, VEGFR-antibody light chain fusion protein as described herein has improved clinical properties. In some embodiments, VEGFR-antibody light chain fusion protein as described herein retains the ADCC and CDC effector functions of the antibody, despite the presence of a bulky VEGFR component attached to the C-terminal end of the antibody light chain (for example, the C-terminal end of the antibody VL-CL domain). In some embodiments, VEGFR-antibody light chain fusion protein as described herein shows improved ADCC and / or CDC effector functions of the antibody compared to that of the corresponding antibody.

[0143] The ADCC of VEGFR-antibody light chain fusion protein can be tested as described in Example 2. In short, cancer cell lines (e.g., SKBR3 human breast cancer cell line, which express HER2 that can be recognized by the anti-HER2 antibody portion within the VEGFR-anti-HER2 light chain fusion protein) expressing an antigen that can be recognized by the VEGFR-antibody light chain fusion protein are mixed with effector cells (e.g., CD16+NK92 cells) in 96-well plates. Cancer cell lines can be engineered to express reporter genes, such as luciferase. VEGFR-antibody light chain fusion proteins of varying concentrations are added to each well. After incubation, reporter gene (e.g., luciferase) activity can be measured using a microplate reader. EC50 (representing ADCC activity) can then be calculated.

[0144] C-terminal heavy chain fusion sometimes changes the structure of the Fc region, even when using a flexible peptide linker. Therefore, antibody fusion proteins with heavy chain fusions exhibit low CDC relative to intact antibodies. In addition, heavy chain fusions are characterized by high Fc receptor (FcR) binding in the absence of antigen binding. This results in a relatively short half-life. These properties of heavy chain fusions can be changed, for example, by reducing and eliminating FcR binding by deglycosylation of the fusion moiety and / or preventing intracellular proteolysis by modifying the linker. However, deglycosylation results in the loss of ADCC and CDC, and linker-modified constructs still have relatively low CDC and suboptimal pharmacokinetic properties.

[0145] When a large portion (e.g., a VEGFR component) is fused to the C-terminus of an antibody light chain, the folding of the bulky portion as well as the antibody light chain itself can be affected, resulting in unpredictability in the 3D structure of the antibody fusion protein, which in turn can reduce or eliminate the binding specificity of the antibody to its one or more target antigens, as well as the biological activity of the bulky portion (e.g., the binding specificity of the VEGFR component to VEGF). However, it was surprisingly discovered in the present invention that a bulky VEGFR component (e.g., approximately 23.2 kDa) can be fused to the C-terminus of an antibody light chain while still maintaining the binding specificity of VEGFR to VEGF, as well as the binding specificity of the antibody to its one or more antigenic targets, even when two bulky VEGFR components are fused to the C-termini of two antibody light chains (e.g., the C-termini of the antibody VL-CL domains).

[0146] In some embodiments, the VEGFR-antibody light chain fusion proteins described herein can improve antibody cell specificity by reducing antibody activity in the absence of binding to target cells or targets (e.g., cancer cells or tissues that overexpress VEGF). This feature will be particularly useful for selectively masking or reducing the cytotoxicity of the antibody fusion protein, thereby protecting non-target cells or normal cells from toxic effects while exposing only target cells (e.g., cancer cells or tissues that overexpress VEGF) to toxic effects. In some embodiments, VEGFR-antibody light chain fusion proteins comprising VEGFR components can stabilize VEGF dimer binding within the same antibody fusion protein (see Figure 1 and Figure 2 In some embodiments, one VEGFR-antibody light chain fusion protein can pair with another nearby, forming a dimer of the VEGFR components from both parts and stabilizing VEGF binding (see Figure 3-5 ).

[0147] In some embodiments, VEGFR-antibody light chain fusion proteins as described herein retain the function (such as binding to VEGF) of complete antibody and VEGFR components with or without a linker peptide. In some embodiments, VEGFR-antibody light chain fusion proteins do not affect the structure of the antibody Fc region. In some embodiments, VEGFR-antibody light chain fusion proteins do not interfere with the binding of the Fc portion of the antibody to Fc receptors (such as FcγR and FcRn) or the binding of the antibody to one or more corresponding targets thereof. In some embodiments, VEGFR-antibody light chain fusion proteins do not affect the interaction (effector function) between the antibody portion and the immune system. In some embodiments, VEGFR-antibody light chain fusion proteins avoid heavy chain deformation, thereby resulting in reduced degradation after being taken up by cells carrying FcR, and then recirculating to leave the cell. In some embodiments, VEGFR-antibody light chain fusion proteins retain high binding affinity to both VEGF and the antigen recognized by parent IgG.

[0148] VEGFR components

[0149] VEGF and VEGFR

[0150] Many key factors in the process of neovascularization have been identified, and the VEGF family plays a major role. The human VEGF family consists of 6 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PIGF). In addition, multiple isoforms of VEGF-A, VEGF-B and PIGF are produced by alternative RNA splicing (Sullivan et al., MAbs, 2002, 2 (2): 165-75). VEGF-A is a major factor involved in angiogenesis; it binds both VEGFR-1 and VEGFR-2. The strategy of inhibiting angiogenesis by blocking VEGF-A signaling has established successful therapies for the treatment of certain cancers as well as retinal neovascular and ischemic diseases (Major et al., J Pharmacol Exp Then, 1997, 283(1):402-10; Willet et al., Nat. Med. 2004, 10:145-7; Papadopoulos et al., Angiogenesis, 2012, 15(2):171-85; Aiello et al., PNAS, 1995, 92:10457-61).

[0151] Compared to other growth factors that contribute to the angiogenesis process, VEGF is unique in its high specificity for endothelial cells lining the vascular system.

[0152] In addition to being an angiogenic factor for angiogenesis and vasculogenesis, VEGF also acts as a pleiotropic growth factor to exhibit a variety of biological effects in other physiological processes, such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx (Ferrara and Davis Smyth (1997) Endocrine Rev. 18: 4-25). In addition, VEGF has been reported to have mitogenic effects on some non-endothelial cell types (e.g., retinal pigment epithelial cells, pancreatic ductal cells, and Schwann cells). See, for example, Guerrin et al. (1995) J. Cell Physiol. 164: 385-394; Oberg-Welsh et al. (1997) Mol. Cell. Endocrinol. 126: 125-132; Arie et al. (1999) J. Neurosci. 19: 5731-5740.

[0153] A large body of evidence also suggests a key role for VEGF in the development of disorders or diseases involving pathological angiogenesis. VEGF mRNA is overexpressed by the majority of human tumors examined (Berkman et al., J Clin Invest 91: 153-159 (1993); Brown et al., Human Pathol. 26: 86-91 (1995); Brown et al., Cancer Res. 53: 4727-4735 (1993); Mattern et al., Brit. J. Cancer. 73: 931-934 (1996); and Dvorak et al., Am J. Pathol. 146: 1029-1039 (1995)). In addition, VEGF concentrations in ocular fluid are highly correlated with the presence of active proliferation of blood vessels in patients with diabetic and other ischemic retinopathy (Aiello et al., N. Engl. J. Med. 331: 1480-1487 (1994)). Furthermore, studies have demonstrated that the localization of VEGF in the choroidal neovascular membrane of patients is affected by AMD (Lopez et al., Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996)).

[0154] Anti-VEGF monoclonal antibodies are promising candidates for the treatment of solid tumors and various intraocular neovascular diseases. Although VEGF molecules are upregulated in tumor cells and their receptors are upregulated in tumor-infiltrating vascular endothelial cells, the expression of VEGF and its receptors remains low in normal cells not involved in angiogenesis. Anti-VEGF antibodies include, but are not limited to, bevacizumab. Brolucizumab, Vanucizumab (RG7221), and Ranibizumab

[0155] VEGF is a dimer with an apparent molecular weight of about 46 kDa, wherein each subunit has an apparent molecular weight of about 23 kDa. The endothelial cell proliferation activity of known VEGF is mediated by two high-affinity tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR / Flk-1), which are present only on the surface of vascular endothelial cells. DeVries et al., Science 225:989-991 (1992) and Terman et al., Oncogene 6:1677-1683 (1991). Both Flt-1 and KDR tyrosine kinase receptors have seven immunoglobulin-like (Ig-like) domains, which form the extracellular ligand binding region of the receptor, a transmembrane domain for anchoring the receptor on the cell surface where it is expressed, and an intracellular catalytic tyrosine kinase domain interspersed with a "kinase insertion sequence". While the KDR receptor binds only to the VEGF protein with high affinity, the Flt-1 receptor also binds to placental growth factor (PLGF), a molecule with significant structural homology to VEGF. An additional member of the receptor tyrosine kinase family, which has seven Ig-like domains in its extracellular ligand-binding region, is VEGFR-3 (Flt-4), which is not a receptor for VEGF or PLGF but instead binds to a different ligand: VH1.4.5. The VH1.4.5 ligand has been reported in the literature as VEGF-related protein (VRP) or VEGF-C.

[0156] In addition to being known as an endothelial cell-specific mitogen, VEGF is unique among angiogenic growth factors in its ability to induce a transient increase in vascular permeability to macromolecules (hence its original and alternative names, vascular permeability factor, VPF) (see Dvorak et al., (1979) J. Immunol. 122: 166-174; Senger et al., (1983) Science 219: 983-985; Senger et al., (1986) Cancer Res. 46: 5629-5632). Increased vascular permeability and the resulting deposition of plasma proteins in the extravascular space contribute to neovascularization by providing a temporary matrix for endothelial cell migration (Dvorak et al., (1995) Am. J. Pathol. 146: 1029-1039). Hyperpermeability is indeed a characteristic feature of new blood vessels, including those associated with tumors.

[0157] In some embodiments, the VEGFR components of the present invention comprise one or more Ig-like domains derived from the extracellular ligand binding region of the Flt-1 or KDR receptor (or the murine homolog of the KDR receptor, Flk-1) that mediate binding to the VEGF protein. In some embodiments, the VEGFR components of the present invention comprise Ig-like domains derived from the extracellular ligand binding region of both the Flt-1 and KDR receptors (or the murine homolog of the KDR receptor, Flk-1), or functional equivalents thereof, thereby forming a "chimeric VEGFR" component.

[0158] The term "Ig-like domain" of Flt-1, Flt-4, or Flk-1 is intended to encompass not only the entire wild-type domain, but also insertions, deletions, and / or substitutions thereof that substantially retain the functional properties of the intact domain. It will be apparent to those skilled in the art that many variants of the Ig domain are available that retain substantially the same functional properties as the wild-type domain.

[0159] When used in relation to an Ig-like domain "X", the term "functional equivalent" is intended to encompass an Ig-like domain "X" having at least one alteration (e.g., deletion, addition, and / or substitution) that retains substantially the same functional properties as the wild-type Ig domain "X", i.e., substantially equivalent binding to VEGF. It should be understood that various amino acid substitutions may be made in the Ig domain "X" with respect to the ability of these receptor components to bind to and inactivate VEGF without departing from the essence of the present invention. Thus, point mutations and other more extensive changes may be made in one or more Ig-like domains of the VEGFR components of the present invention in order to confer interesting properties that do not significantly affect the ability of the VEGFR component to bind to VEGF and inhibit VEGF activity. The functional properties of the VEGFR components of the present invention can be determined by any suitable screening assay known in the art for measuring the desired properties. Other assays, for example, changes in the ability to specifically bind to VEGF, can be measured by competitive VEGF binding assays. Modifications of protein properties (such as thermal stability, hydrophobicity, susceptibility to proteolytic degradation, or aggregation tendency) can be measured by methods known to those skilled in the art. In addition, see subsection "1. Amino Acid Sequence Variants" under section "V. Preparation Methods".

[0160] In some embodiments, the VEGFR components described herein comprise the Ig-like domain of Flt-1 1 or Flk-1. In some embodiments, the VEGFR components comprise the Ig-like domain 1 of Flt-1 (hereinafter referred to as Flt1d1), Flt1d2, Flt1d3, Flt1d4, Flt1d5, Flt1d6, Flt1d7, the Ig-like domain 1 of Flk-1 (hereinafter referred to as Flk1d1), Flk1d2, Flk1d3, Flk1d4, Flk1d5, Flk1d6, or Flk1d7.

[0161] In some embodiments, the VEGFR components described herein comprise 1, 2, 3, 4, 5, 6 or 7 Ig-like domains of Flt-1 1 or Flk-1. In some embodiments, the VEGFR components comprise 2, 3, 4, 5, 6 or 7 Ig-like domains, which comprise a mixture of Ig-like domains from Flt-1 and Flk-1. For example, in some embodiments, the VEGFR components comprise one Ig-like domain from Flk-1 and two Ig-like domains from Flt-1. In some embodiments, the VEGFR components comprise 2, 3, 4, 5, 6 or 7 Ig-like domains all from Flt-1. In some embodiments, the VEGFR components comprise 2, 3, 4, 5, 6 or 7 Ig-like domains all from Flk-1.

[0162] In some embodiments, the VEGFR component comprises 2, 3, 4, 5, 6, or 7 Ig-like domains from Flt-1 or Flt-1 that are directly connected to each other. In some embodiments, the VEGFR component comprises 2, 3, 4, 5, 6, or 7 Ig-like domains from Flt-1 or Flk-1 that are connected via domain linkers (such as those from Flt-1 or Flk-1, e.g., domain linkers connecting Flt1d2 and Flt1d3). In some embodiments, the Ig-like domains within the VEGFR component are linked by peptide linkers (see, e.g., the peptide linker section below), such as (GGGGS) n (SEQ ID NO: 4), wherein n is an integer between 1 and 8, e.g., (GGGGS)3 (SEQ ID NO: 6) or (GGGGS)6 (SEQ ID NO: 7). In some embodiments, the Ig-like domains within the VEGFR component are linked by a non-peptide linker (see, e.g., the Non-Peptide Linkers section below), such as a thioester.

[0163] In some embodiments, the VEGFR component consists of 1, 2, 3, 4, 5, 6, or 7 Ig-like domains of Flt-1 or Flk-1. In some embodiments, the VEGFR component consists essentially of 1, 2, 3, 4, 5, 6, or 7 Ig-like domains of Flt-1 or Flk-1. In some embodiments, the VEGFR component comprises a portion that is not an Ig-like domain from Flt-1 or Flk-1.

[0164] In some embodiments, the VEGFR components described herein comprise a first VEGFR Flt-1 Ig-like domain 2 (Flt1d2). In some embodiments, the VEGFR components further comprise a second VEGFR Flk-1 Ig-like domain 3 (Flk1d3). In some embodiments, the VEGFR components further comprise a third VEGFR Ig-like domain and / or a fourth VEGFR Ig-like domain, wherein the Ig-like domain is selected from the group consisting of Flk-1 Ig-like domain 4 (Flk1d4), Flk-1 Ig-like domain 1 (Flk1d1), Flk-1 Ig-like domain 5 (Flk1d5), Flk-1 Ig-like domain 4 (Flt1d4), and Flt-1 Ig-like domain 5 (Flt1d5). In some embodiments, the VEGFR components further comprise a third VEGFR Flk-1 Ig-like domain 4 (Flk1d4). In some embodiments, the VEGFR component comprises Flt1d2 and Flk1d3. In some embodiments, the VEGFR component consists essentially of Flt1d2 and Flk1d3. In some embodiments, the VEGFR component consists of Flt1d2 and Flk1d3. In some embodiments, the VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the VEGFR component comprises Flt1d2, Flk1d3, and Flk1d4. In some embodiments, the VEGFR component consists essentially of Flt1d2, Flk1d3, and Flk1d4. In some embodiments, the VEGFR component consists of Flt1d2, Flk1d3, and Flk1d4.

[0165] In some embodiments, the VEGFR-antibody light chain fusion proteins of the invention comprise (and in some embodiments consist of or consist essentially of) a light chain-VEGFR component fusion protein (from N-terminus to C-terminus) in the following configuration, wherein L is an optional linker (such as a non-peptide linker or a peptide linker):

[0166] FP1: Light chain-L-Flk1d3-Flt1d2

[0167] FP2: Light chain-L-Flk1d4-Flk1d3-Flt1d2

[0168] FP3: Light chain-L-Flk1d3-Flt1d2-Flk1d1

[0169] FP4: Light chain-L-Flt1d4-Flk1d3-Flt1d2

[0170] FP5: Light chain-L-Flk1d5-Flk1d4-Flk1d3-Flt1d2

[0171] FP6: Light chain-L-Flt1d5-Flt1d4-Flk1d3-Flt1d2

[0172] FP1': Light chain-L-Flt1d2-Flk1d3

[0173] FP2': Light chain-L-Flt1d2-Flk1d3-Flk1d4

[0174] FP3': Light chain-L-Flk1d1-Flt1d2-Flk1d3

[0175] FP4': Light chain-L-Flt1d2-Flk1d3-Flt1d4

[0176] FP5': Light chain-L-Flt1d2-Flk1d3-Flk1d4-Flk1d5

[0177] FP6': Light chain-L-Flt1d2-Flk1d3-Flt1d4-Flt1d5

[0178] In some embodiments, one or more Ig-like domains of Flt-1 or Flk-1 described herein are the sole Ig-like domains of a VEGFR component. In some embodiments, Flt1d2 is the sole Ig-like domain of a VEGFR component. In some embodiments, Flt1d2 and Flk1d3 are the sole Ig-like domains of a VEGFR component. In some embodiments, Flt1d2, Flk1d3, and Flk1d4 are the sole Ig-like domains of a VEGFR component. In some embodiments, Flk1d1, Flt1d2, and Flk1d3 are the sole Ig-like domains of a VEGFR component. In some embodiments, Flt1d2, Flk1d3, and Flt1d4 are the sole Ig-like domains of a VEGFR component. In some embodiments, Flt1d2, Flk1d3, and Flt1d4 are the sole Ig-like domains of a VEGFR component. In some embodiments, Flt1d2, Flk1d3, Flk1d4, and Flk1d5 are the only Ig-like domains of the VEGFR component. In some embodiments, Flt1d2, Flk1d3, Flt1d4, and Flt1d5 are the only Ig-like domains of the VEGFR component.

[0179] The linker sequence can be configured to reduce steric hindrance so that the VEGFR component and the antibody light chain can assume their optimal tertiary structure and / or interact appropriately with their target molecule. In some embodiments, the linker L is a peptide linker (see "Peptide Linker" section below). In some embodiments, the linker L comprises (GGGGS) n An amino acid sequence of the invention, wherein n is an integer between 1 and 8, for example (GGGGS)3 (SEQ ID NO: 6), or (GGGGS)6 (SEQ ID NO: 7).

[0180] In some embodiments, the VEGFR-antibody light chain fusion protein comprises a first VEGFR component and a second VEGFR component. In some embodiments, the first VEGFR component and the second VEGFR component are identical. In some embodiments, the first VEGFR component and the second VEGFR component each comprise a first VEGFR Flt1d2. In some embodiments, the first VEGFR component and the second VEGFR component each comprise a first VEGFR Flt1d2 and a second VEGFR Flk1d3. In some embodiments, the first VEGFR component and the second VEGFR component each comprise, from N-terminus to C-terminus: Flt1d2-Flk1d3, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the first and / or second VEGFR component comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first VEGFR component and the second VEGFR component each comprise a first VEGFR Flt1d2, a second VEGFR Flk1d3, and a third VEGFR Flk1d4. In some embodiments, the first VEGFR component and the second VEGFR component each comprise, from N-terminus to C-terminus, Flt1d2-Flk1d3-Flk1d4, wherein the N-terminus of the VEGFR component is fused to the C-terminus of the antibody light chain. In some embodiments, the first VEGFR component and the second VEGFR component are different. In some embodiments, the first VEGFR component comprises two Ig-like domains of Flt-1 or Flk-1, and the second VEGFR component comprises three Ig-like domains of Flt-1 or Flk-1. In some embodiments, the first VEGFR component comprises three Ig-like domains of Flt-1 or Flk-1, and the second VEGFR component comprises four Ig-like domains of Flt-1 or Flk-1. In some embodiments, the first VEGFR component comprises four Ig-like domains of Flt-1 or Flk-1, and the second VEGFR component comprises five Ig-like domains of Flt-1 or Flk-1. In some embodiments, the first VEGFR component comprises Flt1d2-Flk1d3 from the N-terminus to the C-terminus, and the second VEGFR component comprises Flt1d2-Flk1d3-Flk1d4 from the N-terminus to the C-terminus. In some embodiments, the first VEGFR component and the second VEGFR component comprise the same number of Ig-like domains with different sequences. For example, in some embodiments, the first VEGFR component comprises Flt1d2-Flk1d3-Flk1d4 from the N-terminus to the C-terminus, and the second VEGFR component comprises Flk1d1-Flt1d2-Flk1d3 from the N-terminus to the C-terminus.In some embodiments, the first VEGFR component comprises, from N-terminus to C-terminus, Flt1d2-Flk1d3-Flk1d4, and the second VEGFR component comprises, from N-terminus to C-terminus, Flt1d2-Flk1d3-Flt1d4.

[0181] VEGFR component size

[0182] The VEGFR component of the present invention is at least about 4 kDa. In some embodiments, the VEGFR component of the present invention is about 4 kDa to about 95 kDa, about 4 kDa to about 10 kDa, about 10 kDa to about 15 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 5 kDa, about 5 kDa to about 60 kDa, about 60 kDa to about 65 kDa, about 65 kDa to about 70 kDa, about 70 kDa to about 75 kDa, about 75 kDa to about 80 kDa, about 80 kDa to about 85 kDa, about 85 kDa to about 90 kDa, about 90 kDa to about 95 kDa, about 4 kDa to about 15 kDa, about 4 kDa to about 30 kDa, about 4 kDa to about 43 kDa, about 4 kDa to about 56 kDa, about 4 kDa to about 69 kDa, about 4 kDa to about 82 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 43 kDa, about 15 kDa to about 56 kDa, about 15 kDa to about 69 kDa, about 15 kDa to about 82 kDa, about 15 kDa to about 95 kDa, about 25 kDa to about 43 kDa, about 25 kDa to about 56 kDa, about 25 kDa to about 69 kDa, about 25 kDa to about 82 kDa, about 25 kDa to about 95 kDa, about 40 kDa to about In some embodiments, the VEGFR component is about 23.2 kDa.

[0183] The length of the VEGFR component of the present invention is at least about 36 amino acids (aa). In some embodiments, the length of the VEGFR component of the present invention is about 36 aa to about 864 aa, about 36 aa to about 91 aa, about 91 aa to about 136 aa, about 136 aa to about 182 aa, about 182 aa to about 227 aa, about 227 aa to about 273 aa, about 273 aa to about 318 aa, about 318 aa to about 364 aa, about 364 aa to about 409 aa, about 409 aa to about 455 aa, about 455 aa to about 500 aa, about 500 aa to about 545 aa, about 545 aa to about 591 aa, about 591 aa to about 636 aa, about 636 aa to about 682 aa, about 682 aa to about 727 aa, about 727 aa to about 773 aa, about 773 aa to about 818 aa, about 818 aa to about 864 aa, about 36 aa to about 136 aa, about 36 aa to about 273 aa, about 36 aa to about 391 aa, about 36 aa to about 509 aa, about 36 aa to about 627 aa, about 36 aa to about 745 aa, about 36 aa to about 745 aa, about 136 aa to about 273 aa, about 136 aa to about 391 aa, about 136 aa to about 509 aa, about 136 aa to about 627 aa, about 136 aa to about 745 aa, about 136 aa to about 864 aa, about 227 aa to about 391 aa, about 227 aa to about 509 aa, about 227 aa to about 627 aa, about 227 aa to about 745 aa, about 227 aa to about 864 aa, about 364 In some embodiments, the length of the VEGFR component is about 211 aa, about 364 aa to about 627 aa, about 364 aa to about 745 aa, about 364 aa to about 864 aa, about 455 aa to about 627 aa, about 455 aa to about 682 aa, about 455 aa to about 745 aa, about 455 aa to about 864 aa, about 591 aa to about 682 aa, about 591 aa to about 745 aa, about 591 aa to about 864 aa, about 682 aa to about 745 aa, or about 682 aa to about 864 aa.

[0184] VEGFR binding affinity

[0185] The binding specificity of the VEGFR components described herein to VEGF can be experimentally determined by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIAcore testing, and peptide scanning.

[0186] ELISA-based binding assays can be performed as described in Example 2. Briefly, an ELISA plate can be coated with VEGF (or the antigen to be tested, e.g., PD-1), and then different concentrations of VEGFR-antibody light chain fusion protein (or any antibody to be tested) are added to each well. After incubation and washing, a secondary antibody such as an HRP-conjugated anti-IgG Fc antibody can be added to detect VEGFR-antibody light chain fusion protein (or any antibody to be tested) bound to VEGF (or the antigen to be tested). HRP substrate is added to each well. The optical density (OD) of each well can be measured at 450 nm using a microplate reader. The EC can then be calculated. 50 .

[0187] In some embodiments, the K of the binding between the VEGFR components described herein and VEGF is D or EC 50 About 10 -5 M to about 10 -15 M. In some embodiments, the K of the binding between the VEGFR components described herein and VEGF is D About 10 -5 M to about 10 -6 M, about 10 -6 M to about 10 -7 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 - 10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -12 M to about 10 -13 M, about 10 -13 M to about 10 -14 M, about 10 -14 M to about 10 -15 M. Approx. 10-5 M to about 10 -15 M. Approx. 10-6M to about 10 -15 M, about 10 -7 M to about 10-15 M, about 10 -8 M to about 10 -15 M, about 10 -9 M to about 10 -15 M, about 10 -10 M to about 10 -15 M, about 10 -11 M to about 10 -15 M, about 10 -12 M to about 10 -15 M, about 10 -13 M to about 10 -15 M, about 10 -5 M to about 10 -14 M, about 10 -7 M to about 10 -14 M, about 10 -8 M to about 10 -14 M, about 10 -9 M to about 10 -14 M, about 10 -10 M to about 10 -14 M, about 10 -11 M to about 10 -14 M, about 10 -12 M to about 10 -14 M, about 10 -5 M to about 10 -13 M, about 10 -7 M to about 10 -13 M, about 10 -8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, about 10 -10 M to about 10 -13 M, about 10 -11 M to about 10 -13 M, about 10- 5 M to about 10 - 12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10- 9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -5 M to about 10 -11 M, about 10 -6 M to about 10 -11 M, about 10 -7 M to about 10 -11M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -5 M to about 10 -10 M, about 10 -6 M to about 10 -10 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M, about 10 -5 M to about 10 - 9 M, about 10 -6 M to about 10 -9 M, about 10 -7 M to about 10 -9 M, about 10 -5 M to about 10 -8 M,10 -6 M to about 10 -8 M, or 10 -5 M to about 10 - 7 In some embodiments, the antibody fusion protein binds to VEGF with an affinity of about 0.5 pM. In some embodiments, the antibody fusion protein binds to VEGF with an EC of about 10 nM to about 40 nM. 50 Binds to VEGF.

[0188] In some embodiments, the VEGFR-antibody light chain fusion protein comprises a first VEGFR component and a second VEGFR component fused to the C-terminus of two antibody light chains (e.g., the C-terminus of an antibody VL-CL domain). In some embodiments, the first VEGFR component and the second VEGFR component have similar binding affinities. In some embodiments, the first VEGFR component and the second EGFR component have different binding affinities.

[0189] In some embodiments, the K of binding between the second VEGFR component and VEGF is D Greater than the K for binding between the first VEGFR component and VEGF D For example, the K for binding between the second VEGFR component and VEGF is D The K of the binding between the first VEGFR component and VEGF can be Dabout 1-10 times, about 10-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times, about 100-500 times, or about 500-1000 times.

[0190] In some embodiments, the fusion of the VEGFR component to the C-terminus of the antibody light chain does not affect the binding affinity of the VEGFR component to VEGF. In some embodiments, the fusion of the VEGFR component to the C-terminus of the antibody light chain reduces the binding affinity of the VEGFR component to VEGF, such that the K of the binding between the fused VEGFR component and VEGF is less than 0. D The K of the binding between the unfused VEGFR component and VEGF D about 1-10 times, about 10-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times, about 100-500 times, or about 500-1000 times.

[0191] In some embodiments, the fusion of the VEGFR component to the C-terminus of the antibody light chain does not affect the binding affinity of the antibody to its target antigen. In some embodiments, the fusion of the VEGFR component to the C-terminus of the antibody light chain reduces the binding affinity of the antibody to its target antigen, such that the K of the binding between the VEGFR-fused antibody and its target antigen is less than 1%. D The K for binding between the unfused antibody and its target antigen can be D about 1-10 times, about 10-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times, about 100-500 times, or about 500-1000 times.

[0192] In some embodiments, fusion of a VEGFR component to the C-terminus of an antibody light chain increases the binding affinity of the antibody to its target antigen (e.g., by stabilizing antibody-antigen binding via VEGFR-VEGF binding at the same cell), such that the K of binding between the VEGFR-fused antibody and its target antigen is < 0. D Can be lower than the K for binding between the unfused antibody and its target antigen D about 1-10 times, about 10-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-60 times, about 60-70 times, about 70-80 times, about 80-90 times, about 90-100 times, about 100-500 times, or about 500-1000 times.

[0193] Thus, in some embodiments, the K of binding between a VEGFR-fused antibody and the antibody target antigen is D About 10 -5 M to about 10 -13 M. In some embodiments, the K of binding between a VEGFR-fused antibody and the antibody target antigen is D About 10 - 5 M to about 10 -6 M, about 10 -6 M to about 10 -7 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -12 M to about 10 -13 M, about 10 -5 M to about 10 -13 M, about 10 -6 M to about 10 -13 M, about 10 -7 M to about 10 -13 M, about 10 -8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, about 10 -10 M to about 10 -13 M, about 10 -11 M to about 10 -13 M, about 10 -5 M to about 10 -12 M, about 10 -6 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 - 12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -5 M to about 10 -11 M, about 10 -7 M to about 10 -11 M, about 10-8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -5 M to about 10 -10 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M, about 10 -5 M to about 10 -9 M, about 10 -7 M to about 10 -9 M, about 10 -5 M to about 10 -8 M, or about 10 -6 M to about 10 -8 M. In some embodiments, the K of binding between a VEGFR-fused antibody and the antibody target antigen is D About 10 -8 M to about 10 -16 M. In some embodiments, the K of binding between a VEGFR-fused antibody and the antibody target antigen is D About 10 -5 M to about 10 -6 M, about 10 -6 M to about 10 -7 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10- 9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -12 M to about 10 -13 M, about 10 -13 M to about 10 -14 M, about 10 -14 M to about 10 -15 M, about 10 -15 M to about 10 -16 M, about 10 -8 M to about 10 -16 M, about 10 -9 M to about 10 -16 M, about 10 -10 M to about 10 -16 M, about 10 -11 M to about 10 -16 M, about 10-12 M to about 10 -16 M, about 10 -13 M to about 10 -16 M, about 10 -14 M to about 10 -16 M, about 10 -8 M to about 10 -15 M, about 10 -9 M to about 10 -15 M, about 10 -10 M to about 10 -15 M, about 10- 11 M to about 10 -15 M, about 10 -12 M to about 10 -15 M, about 10 -13 M to about 10 -15 M, about 10 -8 M to about 10 -14 M, about 10 -9 M to about 10 -14 M, about 10 -10 M to about 10 -14 M, about 10 -11 M to about 10 -14 M, about 10 -12 M to about 10 -14 M, about 10 - 8 M to about 10 -13 M, about 10 -9 M to about 10 -13 M, about 10 -10 M to about 10 -13 M, about 10 -11 M to about 10 -13 M, about 10 -8 M to about 10 - 12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, or about 10 - 8 M to about 10 -10 M. In some embodiments, the EC binding between the VEGFR fused antibody and the antibody target antigen 50 From about 1 nM to about 30 nM.

[0194] Antibody Platform

[0195] The antibodies of the VEGFR-antibody light chain fusion protein of the present invention may have any possible form.

[0196] In some embodiments, the antibody comprises a single polypeptide chain. In some embodiments, the antibody comprises more than one (such as any one of 2, 3, 4 or more) polypeptide chains. One or more polypeptide chains can have any length, such as at least about 10, 20, 50, 100, 200, 300, 500 or more amino acids long. In the case of multi-chain antibodies, the nucleic acid sequence encoding the polypeptide chain can be operably connected to the same promoter or different promoters.

[0197] Natural antibodies, such as monoclonal antibodies, are immunoglobulin molecules that are immunoreactive with specific antigens. In some embodiments, the antibodies are agonist antibodies. In some embodiments, the antibodies are antagonist antibodies. In some embodiments, the antibodies are monoclonal antibodies. In some embodiments, the antibodies are full-length antibodies. In some embodiments, the antibodies are antigen-binding fragments selected from Fab, Fab', F(ab')2, miniantibodies, and other antigen-binding subsequences of full-length antibodies or their engineered combinations comprising light chain constant domains. In some embodiments, the antibodies are human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, the antibodies are monovalent antibodies. In some embodiments, the antibodies are multivalent antibodies, such as bivalent antibodies or tetravalent antibodies. In some embodiments, the antibodies are bispecific antibodies. In some embodiments, the antibodies are multispecific antibodies.

[0198] The antibodies of the present invention (such as anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-HER2, anti-EGFR, anti-Ang2 antibodies, anti-TNFα antibodies, or anti-IL-17A antibodies) comprise a heavy chain and a light chain. In some embodiments, the heavy chain comprises a V H In some embodiments, the heavy chain also comprises one or more constant domains, such as C H 1. C H 2. C H 3, or any combination thereof. In some embodiments, the light chain comprises V L In some embodiments, the light chain also comprises a constant domain, such as C L In some embodiments, the heavy chain and light chain are linked to each other via multiple disulfide bonds. In some embodiments, the antibody comprises an Fc fragment, such as an Fc fragment of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody does not comprise an Fc fragment. In some embodiments, the antibody is an antigen-binding fragment, which is a Fab.

[0199] Antibody format

[0200] The antibodies of the VEGFR-antibody light chain fusion proteins described herein can be in the form of any antibody or antibody fragment comprising a light chain (e.g., comprising a V L -C L In some embodiments, the light chain comprises a V domain, such as a full-length antibody, IgG-IgG, an IgG-derived molecule, Fab, Fab', F(ab')2, F(ab')2-scFv2, Fab-scFv-Fc, miniantibodies, docking and locking, scFv, di-scFv, diabodies, diabodies-Fc, diabodies-CH3, intrabodies, and other antigen-binding subsequences of full-length antibodies or engineered combinations thereof. L and C L In some embodiments, the antibody fragment comprising the light chain comprises V L -LC L (L is an optional linker). In some embodiments, the antibody fragment comprising the light chain comprises V H -L1-V L -L2-C L (L1 and L2 are optional linkers). In some embodiments, the antibody fragment comprising the light chain comprises V L1 -L1-C L -L2-V L2 -L3-V H2 , or V L1 -L1-C L -L2-V H2 -L3-V L2, (L1, L2, L3 are optional linkers). For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9: 129-134 (2003). For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life, see U.S. Patent 5,869,046. For a review of multispecific antibodies, see Weidle et al., Cancer Genomics Proteomics, 10(1): 1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2): 65-79, 2015; Stamova et al., Antibodies, 1(2): 172-198, 2012. Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and preparation by recombinant host cells (e.g., E. coli or phage), as described herein.

[0201] In some embodiments, the antibodies of the present invention are full-length antibodies (e.g., having human immunoglobulin constant regions), such as IgA, IgD, IgE, IgG, IgM, or immunoglobulin derivatives. In some embodiments, the antibodies are monospecific. In some embodiments, the antibodies are multispecific (such as bispecific). Multispecific antibodies have binding specificity for at least two different antigens or epitopes (e.g., bispecific antibodies have binding specificity for two antigens or epitopes).

[0202] Multivalent and / or multispecific antibodies

[0203] The antibodies of the VEGFR-antibody light chain fusion proteins described herein can be in any format known in the art (see, for example, Weidle et al., Cancer Genomics Proteomics, 10(1): 1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33(2): 65-79, 2015; Stamova et al., Antibodies, 1(2): 172-198, 2012; Spiess et al., Mol. Immunol., 2015 Oct; 67(2Pt A): 95-106). The antibodies can be in the form of an "IgG-derived molecule" comprising an Fc region. For example, the antibodies can be in the form of, but are not limited to, IgG-IgG, Cov-X-Body, Common LC (light chain), DAF (dual-acting Fab, which comprises evolved Fvs with dual specificity), CrossMab, DutaMab TM 、 LUZ-Y, Fcab, kappa-lambda antibody, Orthogonal Fab, DT-IgG (dual-targeting IgG), IgG-dsscFv2 (disulfide-stabilized scFv2), IgG(H)-scFv, scFv-(H)IgG, scFv-(L)IgG, IgG(L)-scFv, DVD (dual variable domain), IgG-dsFv (disulfide-stabilized Fv), processed IgG-dsFv, IgG(L,H)-Fv, V(H)-IgG, V(L)-IgG, IgG(H)-V, IgG(L)-V, Knobs-into-holes, Charge pair, Fab-arm exchange, SEEDbody (chain exchange engineered domain), IgG-scFab (single-chain Fab), scFab-dsscFv, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody TM , or DVI-IgG. Knob-in-hole technology can be used for heterodimerization of different H-chains in, for example, common LC, CrossMab, IgG-dsFv or IgG-scFab. Antibodies can also be of the "Fc-less bispecific" format class, which typically comprises single Fabs with different specificities fused together via a linker. For example, antibodies can be in the form of, but not limited to, F(ab')2, F(ab')2-scFv2, Fab-scFv2, Fab-scFv, Fab-scFv-Fc, DNL-F(ab)3 (docking and locking trivalent Fab), scFv, scFv-scFv (e.g., ), diabodies, scBsDb (single-chain bispecific diabodies), DARTs (dual affinity retargeting molecules), TandAbs (tetravalent tandem antibodies), scBsTaFv (single-chain bispecific tandem variable domains), diabodies-CH3, Fab-scFv. Divalent- or trivalent Fab-scFv or Fab-scFv2 formats are produced by fusion of VH-CH1 and / or L chains with scFvs. The antibodies to be used according to the present disclosure may be chemically modified derivatives of any of the aforementioned antibody forms, or they may comprise ligands, peptides, or combinations thereof. The antibodies to be used according to the present disclosure may also be further modified using conventional techniques known in the art, for example, by using one or more amino acid deletions, insertions, substitutions, additions, and / or recombination and / or one or more other modifications known in the art (such as post-translational modifications and chemical modifications, such as glycosylation and phosphorylation), alone or in combination. Chemical / biochemical or molecular biological methods for such modifications are known in the art and are described, inter alia, in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition, 1989 and 3rd edition, 2001; Gerhardt et al.; Methods for General and Molecular Bacteriology; ASM Press, 1994; Lefkovits; Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2002).

[0204] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to prepare bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992)); and preparing trispecific antibodies, as described, e.g., in Tutt et al., J. Immunol. 147: 60 (1991). Also included herein are engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies" (see, e.g., US 2006 / 0025576A1).

[0205] connector

[0206] In some embodiments, the VEGFR-antibody light chain fusion protein may include a linker between the C-terminus of the VEGFR component and the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain). The length, flexibility, and / or other properties of the one or more linkers used in the VEGFR-antibody light chain fusion protein may have a certain impact on the following properties, including but not limited to affinity, specificity, or avidity for one or more specific antigens or epitopes and VEGFR components. For example, a longer linker can be selected to ensure that the two adjacent domains do not interfere with each other spatially. In some embodiments, the linker (such as a peptide linker) includes flexible residues (such as glycine and serine) so that the adjacent domains can move freely relative to each other. For example, a glycine-serine doublet can be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a cleavable linker.

[0207] Other linker considerations include effects on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and planned to degrade), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be incorporated into micelles or liposomes, etc.

[0208] In some embodiments, the VEGFR-antibody light chain fusion protein comprises two VEGFR components, wherein the first VEGFR component and the C-terminus of the first antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) are connected by a first linker, and the second VEGFR component and the C-terminus of the second antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) are connected by a second linker. In some embodiments, the first linker and the second linker are identical. In some embodiments, the first linker and the second linker are different. In some embodiments, the VEGFR-antibody light chain fusion protein comprises two VEGFR components, wherein only one VEGFR component and one C-terminus of the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) are connected by a linker.

[0209] Non-peptide linkers

[0210] The coupling of the VEGFR component and the C-terminus of the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain) can be accomplished by any chemical reaction that will bind the two molecules, as long as the antibody and VEGFR component retain their corresponding activities, i.e., binding to the specific antigen and VEGF, respectively. This connection can include many chemical mechanisms, such as covalent bonding, affinity binding, embedding, coordinate binding, and complexation. In some embodiments, the binding is covalent bonding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used to couple protein molecules (such as VEGFR components) to the antibodies of the present invention. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various types of coupling agents known in the art, but is exemplary of the more common coupling agents (see, Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987)).

[0211] Linkers that can be used in the present application are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984)), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). In some embodiments, the non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (Pierce Chem. Co., catalog number (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., catalog number #21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propionamido] hexanoate (Pierce Chem. Co., catalog number #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co.) conjugated to EDC. Chem. Co., catalog #24510).

[0212] The above-mentioned linkers contain components with different properties, resulting in VEGFR-antibody fusion proteins with different physiochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Linkers containing NHS-esters are less soluble than sulfo-NHS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond and can form an antibody fusion protein with increased stability. Generally speaking, disulfide bonds are less stable than other bonds because disulfide bonds are cleaved in vitro, resulting in less available antibody fusion protein. Specifically, sulfo-NHS can enhance the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide conjugates (such as EDC) form esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0213] Peptide linker

[0214] The peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived only from the hinge region of an antibody heavy chain can be used as a linker. See, for example, WO 1996 / 34103.

[0215] In some embodiments, the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids long. In some embodiments, the peptide linker is no more than about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or less amino acids long. In some embodiments, the length of the peptide linker is any of: about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0216] The basic technical feature of such peptide linkers is that they do not contain any polymerization activity. The properties of peptide linkers, including the lack of promotion of secondary structure, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9 (1), 73-80). A particularly preferred amino acid in the context of "peptide linkers" is Gly. In addition, peptide linkers that do not promote any secondary structure are preferred. The connection of domains to each other can be provided, for example, by genetic engineering. Methods for preparing fused and operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0217] The peptide linker may be a stable linker that is not cleavable by proteases, in particular by matrix metalloproteinases (MMPs).

[0218] The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymers (G) n (SEQ ID NO: 1), glycine-serine polymers (including, for example, (GS) n (SEQ ID NO: 2), (GSGGS) n (SEQ ID NO:3), (GGGGS) n (SEQ ID NO:4) and (GGGS) n (SEQ ID NO: 5), wherein n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine polymers and glycine-serine polymers are relatively unstructured and therefore may be able to act as neutral tethers between components. Glycine even enters significantly more phi-psi space than alanine and is less restricted than residues with longer side chains (see, Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those of ordinary skill will recognize that the design of the antibody fusion protein may include a fully or partially flexible linker, such that the linker may include a flexible linker portion and one or more portions that impart a less flexible structure to provide the desired antibody fusion protein structure.

[0219] In some embodiments, the C-terminus of the VEGFR component and the antibody light chain are linked together by a linker of sufficient length to enable the VEGFR component-light chain to fold in a manner that allows binding to VEGF and the antigen specifically recognized by the antibody. In addition to this embodiment, such linkers may also include, for example, (GGGGS) n wherein n is an integer between 1 and 8, for example, (GGGGS)3 (SEQ ID NO: 6; hereinafter referred to as "(G4S)3" or "GS3"), or (GGGGS)6 (SEQ ID NO: 7; hereinafter referred to as "(G4S)6" or "GS6"). In some embodiments, the peptide linker comprises (GSTSGSGKPGSGEGS) n The amino acid sequence of (SEQ ID NO: 44), wherein n is an integer between 1 and 3.

[0220] Antibodies that specifically recognize immune checkpoint molecules

[0221] In some embodiments, the antibody fusion protein of the present invention comprises an antibody that specifically recognizes an immune checkpoint molecule (such as, anti-PD-1, anti-PD-L1, or anti-CTLA-4 full-length antibody), and a VEGFR component fused to the C-terminus of the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain). Antibodies that specifically recognize immune checkpoint molecules are interchangeably referred to herein as "immune checkpoint regulators."

[0222] Immune checkpoints are molecules that turn on (stimulatory molecules) or off (inhibitory molecules) signals in the immune system. Immune checkpoint proteins regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Stimulatory checkpoint molecules include, but are not limited to, CD27, CD40, OX40, GITR, and CD137, which belong to the tumor necrosis factor (TNF) receptor superfamily, and CD28 and ICOS, which belong to the B7-CD28 superfamily. Inhibitory checkpoint molecules include, but are not limited to, programmed death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3, HAVCR2), V-domain Ig inhibitor of T cell activation (VISTA, B7-H5), B7-H3, B7-H4 (VTCN1), HHLA2 (B7-H7), B lymphocyte and T lymphocyte attenuator (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), adenosine A2A receptor (A2AR), T cell immunoreceptor with Ig and ITIM domains (TIGIT), 2B4 (CD244) and its ligands. Many checkpoint proteins have been extensively studied, such as CTLA-4 and its ligands CD86 (B7-1) and CD86, and PD-1 (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) (see, e.g., Pardoll, Nature Reviews Cancer 12:252-264 (2012)).

[0223] The antibody that specifically recognizes immune checkpoint molecules or " immune checkpoint regulator " of the present invention can be immune checkpoint inhibitor (inhibitor of inhibitory immune checkpoint molecules) or activator of stimulatory immune checkpoint molecules.Immune checkpoint inhibitor (inhibitor of inhibitory immune checkpoint molecules) is particularly concerned in the present invention, such as PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC, CD273), LAG-3, TIM-3 (HAVCR2), BTLA, CTLA-4, TIGIT, VISTA (B7-H5), B7-H4 (VTCN1), CD160 (BY55), HHLA2 (B7-H7), 2B4 (CD244), CD73, B7-1 (CD80), B7-H3 (CD276), KIR or IDO inhibitor.

[0224] In some embodiments, the antibody that specifically recognizes an immune checkpoint molecule is an activator of a stimulatory immune checkpoint molecule, such as an agonist antibody, for example, anti-CD28, anti-OX40, anti-ICOS, anti-GITR, anti-4-1BB, anti-CD27, anti-CD40, anti-CD3, and anti-HVEM.

[0225] In some embodiments, antibodies that specifically recognize immune checkpoint molecules are immune checkpoint inhibitors. In some embodiments, immune checkpoint inhibitors target T cells. In some embodiments, immune checkpoint inhibitors target tumor cells. For example, in some cases, when tumor cells are connected to specific T cell receptors, they can turn off activated T cells. However, immune checkpoint inhibitors can prevent tumor cells from attaching to T cells, so that T cells remain activated (see, for example, Howard West, JAMA Oncol. 1 (1): 115 (2015)). In some embodiments, immune checkpoint inhibitors are antibodies (such as antagonist antibodies) targeting inhibitory immune checkpoint proteins, including but not limited to anti-CTLA-4, anti-TIM-3, anti-LAG-3, anti-KIR, anti-PD-1, anti-PD-L1, anti-CD73, anti-B7-H3, anti-CD47, anti-BTLA, anti-VISTA, anti-A2AR, anti-B7-1, anti-B7-H4, anti-CD52, anti-IL-10, anti-IL-35, and anti-TGF-β. In some embodiments, immune checkpoint inhibitors are inhibitors of inhibitory checkpoint molecules selected from the following: PD-1, PD-L1, LAG-3, TIM-3, HHLA2, CD160, CD73, BLTA, B7-H4, TIGIT and VISTA. In some embodiments, immune checkpoint inhibitors are antibodies that specifically recognize PD-1. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically recognizes PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically recognizes CTLA-4. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically recognizes at least two different inhibitory immune checkpoint molecules (e.g., a bispecific antibody).

[0226] CTLA-4

[0227] Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CD152) is a homolog of CD28 and is known as an inhibitory immune checkpoint molecule that is upregulated on activated T cells. CTLA-4 also binds to B7-1 and B7-2, but with greater affinity than CD28. The interaction between B7 and CTLA-4 inhibits T cell activation, which constitutes an important mechanism of tumor immune escape. Anti-CTLA-4 antibody therapy (such as ipilimumab, for example, ) have shown promise in various cancers, such as melanoma.

[0228] Exemplary anti-CTLA-4 antibodies that can be used in the present application include, but are not limited to, ipilimumab (e.g., ) and temirizumab (formerly temirizumab, CP-675,206).

[0229] Ipilimumab (eg, ) is a fully human anti-CTLA-4 immunoglobulin G1 (IgG1) monoclonal antibody (mAb) that blocks the downregulation of T cell activation. Ipilimumab is a CTLA-4 immune checkpoint inhibitor that blocks T cell inhibitory signals induced by the CTLA-4 pathway and increases the number of tumor-reactive T effector cells.

[0230] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes CTLA-4 (hereinafter referred to as an "anti-CTLA-4 antibody"). In some embodiments, the anti-CTLA-4 antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CTLA-4 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, and a light chain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-CTLA-4 antibody is ipilimumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the anti-CTLA-4 antibody competes for binding to CTLA-4 with any of the anti-CTLA-4 antibodies described herein.

[0231] PD-1

[0232] PD-1 is part of the B7 / CD28 family of co-stimulatory molecules that regulate T cell activation and tolerance, so antagonistic anti-PD-1 antibodies can be used to overcome tolerance. PD-1 has been defined as a receptor for B7-4. B7-4 can inhibit immune cell activation when bound to inhibitory receptors on immune cells. Engagement of the PD-1 / PD-L1 pathway leads to inhibition of T cell effector function, cytokine secretion, and proliferation. (Turnis et al., OncoImmunology 1(7):1172-1174, 2012). High levels of PD-1 are associated with exhausted or chronically stimulated T cells. In addition, increased PD-1 expression is associated with decreased survival in cancer patients. Reagents for downregulating PD-1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signals in immune cells can lead to enhanced immune responses. Exemplary anti-PD-1 antibodies that can be applied in the present application include, but are not limited to (pembrolizumab, MK-3475) and (nivolumab).

[0233] Pembrolizumab (e.g., ) is a humanized antibody used in cancer immunotherapy. It targets the programmed cell death 1 (PD-1) receptor. The drug was initially used to treat metastatic melanoma. On September 4, 2014, the U.S. Food and Drug Administration (FDA) approved the drug under the FDA Fast Track Development Program. It has been approved for the treatment of advanced melanoma. On October 2, 2015, the U.S. FDA approved For the treatment of metastatic non-small cell lung cancer in patients whose tumors express PD-L1 and who have failed other chemotherapy agents.

[0234] Nivolumab (eg, ) is a human IgG4 anti-PD-1 monoclonal antibody. It is similar to ipilimumab (e.g., ) in combination to study the effects of simultaneous inhibition of PD-1 and CTLA-4 receptors in non-human primates. It has demonstrated clinical efficacy as a monotherapy or in combination with ipilimumab in the treatment of multiple tumor types, including renal cell carcinoma, melanoma, NSCLC, and certain lymphomas. BMS recently announced its immunotherapy combination therapy. and ipilimumab for the treatment of melanoma.

[0235] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes PD-1 (hereinafter referred to as an "anti-PD-1 antibody"). In some embodiments, the anti-PD-1 antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 12, and a light chain comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the anti-PD-1 antibody is pembrolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 15, and a light chain comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody is nivolumab (e.g., In some embodiments, the anti-PD-1 antibody competes with any of the anti-PD-1 antibodies described herein for specific binding to PD-1.

[0236] PD-L1

[0237] PPD-L1 (Programmed cell death ligand 1) is also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 serves as a ligand for PD-1 to play a major role in suppressing the immune system in specific events such as pregnancy, tissue allergies, autoimmune diseases, and other disease states such as hepatitis and cancer. The formation of the PD-1 receptor / PD-L1 ligand complex transmits an inhibitory signal that reduces the proliferation of CD8+ T cells in the lymph nodes. Exemplary anti-PD-L1 antibodies that can be applied to the present application include, but are not limited to, atezolizumab (e.g., ) and durvalumab (eg, MEDI4736, IMFINZI TM ).

[0238] Atezolizumab (e.g., ) is a fully humanized, engineered monoclonal antibody of the IgG1 isotype against PD-L1. It is an FDA-approved immunotherapy for patients with progressive advanced urothelial carcinoma (e.g., bladder cancer) and non-small cell lung cancer (NSCL) after platinum-containing chemotherapy.

[0239] Durvalumab (eg, IMFINZI TM ) is a human immunoglobulin G1κ (IgG1κ) monoclonal antibody that blocks the interaction of PD-L1 with PD-1 and CD80 (B7.1) molecules. It is an FDA-approved immunotherapy for locally advanced or metastatic urothelial carcinoma.

[0240] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes PD-L1 (hereinafter referred to as an "anti-PD-L1 antibody"). In some embodiments, the anti-PD-L1 antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-PD-L1 antibody is atezolizumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-PD-L1 antibody is durvalumab or an antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody competes with any of the anti-PD-L1 antibodies described herein for specific binding to PD-L1.

[0241] Antibodies that specifically recognize tumor antigens

[0242] In some embodiments, the antibody fusion protein of the present invention comprises an antibody that specifically recognizes a tumor antigen (such as an anti-HER2 or anti-EGFR full-length antibody) and a VEGFR component fused to the C-terminus of the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain).

[0243] In some embodiments, the antigen specifically recognized by the antibody is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, the TAA or TSA is expressed on cancer cells. In some embodiments, the TAA or TSA is expressed on blood cancer cells. In some embodiments, the TAA or TSA is expressed on cells of solid tumors. As non-limiting examples, certain forms of solid tumor cancer include glioblastoma, non-small cell lung cancer, lung cancer that is not non-small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, colorectal cancer, gastric cancer, spleen cancer, skin cancer (such as melanoma), brain cancer that is not glioblastoma, kidney cancer, thyroid cancer, head and neck tumors, bladder cancer, esophageal cancer, etc. In some embodiments, the TAA or TSA is one or more of the following: EphA2, HER2, GD2, Glypican-3, 5T4, 8H9, α v β6 integrin, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor α, GD2, GD3, HLA-AIMAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, λ, Lewis-Y, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGFR2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, and other exemplary antigens, which are antigens present in the extracellular matrix of tumor cells, such as fibronectin, tenascin, or tumor fetal variants in areas of tumor necrosis.

[0244] In some embodiments, the tumor antigen specifically recognized by the antibody is HER2 or EGFR (HER1).

[0245] HER2

[0246] The HER family of receptor tyrosine kinases is an important mediator of cell growth, differentiation, and survival. The receptor family includes four different members, including epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2 (ErbB2 or p185 neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

[0247] HER2 (human epidermal growth factor receptor 2, receptor tyrosine protein kinase erbB-2, CD340, p185 neu ) is a protein encoded by the proto-oncogene ERBB2 in humans. It comprises an extracellular ligand binding domain, a transmembrane domain and an intracellular domain, which can interact with numerous signal transduction molecules and exhibit ligand-dependent and ligand-independent activities. HER2 can heterodimerize with any of the other three receptors ErbB1, ErbB3 and ErbB4, and is considered to be a preferred dimerization partner of other ErbB receptors. Dimerization results in autophosphorylation of tyrosine residues in the receptor cytoplasmic domain and triggers a variety of signal transduction pathways that promote cell proliferation and counteract apoptosis, such as mitogen-activated protein kinases (MAPK), signal transducers and activators of transcription (STAT), protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K / Akt) pathways.

[0248] p185 neuInitially identified as the product of the transforming gene from the neuroblastoma of chemically treated rats. The activated form of the neu proto-oncogene is produced by a point mutation (valine to glutamic acid) in the transmembrane region of the coded protein, resulting in the constitutive dimerization of the protein in the absence of a ligand. Amplification of the human homologue of neu was observed in breast and ovarian cancer, and was associated with poor prognosis (Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989); Referring to U.S. Patent No. 4,968,603). So far, point mutations similar to the neu proto-oncogene have not been reported for human tumors. The overexpression of HER2 (due to gene amplification frequently but not uniformly) has also been observed in other cancers, including gastric cancer, endometrial cancer, salivary gland cancer, lung cancer, kidney cancer, colon cancer, thyroid cancer, pancreatic cancer and bladder cancer. See, King et al., Science, 229:974 (1985); Yokota et al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol., 6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen et al., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034 (1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al., Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184 (1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol. Carcinog., 3:254-257 (1990); Aasland et al., Br. J. Cancer 57:358-363 (1988); Williams et al., Pathobiology 59:46-52 (1991); and McCann et al., Cancer, 65:88-92 (1990), etc. HER2 can be overexpressed in prostate cancer (Gu et al., Cancer Lett. 99:185-9 (1996); Ross et al., Hum. Pathol. 28:827-33 (1997); Ross et al., Cancer 79:2162-70 (1997); and Sadasivan et al., J. Urol. 150:126-31 (1993)).

[0249] Exemplary HER2 antibodies that can be used in the present application include, but are not limited to, trastuzumab Trastuzumab-emtansine conjugate Ertuinomab and pertuzumab

[0250] Trastuzumab It is one of the five best-selling therapeutic antibodies and is a humanized anti-HER2 receptor monoclonal antibody that can significantly increase the survival rate of patients with HER2-positive breast cancer. HER receptors are proteins embedded in the cell membrane and transmit molecular signals from outside the cell (molecules called EGF) to the inside of the cell and turn genes on and off. The HER protein (human epidermal growth factor receptor (EGFR)) binds to human epidermal growth factor and stimulates cell proliferation. In some cancers, especially certain types of breast cancer, HER2 is overexpressed and causes cancer cells to multiply uncontrollably.

[0251] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes HER2 (hereinafter referred to as an "anti-HER2 antibody"). In some embodiments, the anti-HER2 antibody comprises: HC-CDR1, HC-CDR2, and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2, and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-HER2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-HER2 antibody is trastuzumab (e.g., ) or an antigen-binding fragment thereof. In some embodiments, the anti-HER2 antibody specifically binds to HER2 competitively with any of the anti-HER2 antibodies described herein.

[0252] EGFR (HER1)

[0253] Epidermal growth factor receptor (EGFR, HER1 or ErbB-1) is a transmembrane receptor for members of the epidermal growth factor family (EGF family). EGFR is a receptor for EGFR (ErbB1 or HER1), HER2 (ErbB2 or p185 neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2), are members of the ErbB family, which are receptor tyrosine kinases that mediate cell growth, differentiation and survival. Mutations that affect EGFR expression or activity can lead to cancer. EGFR can be activated by the binding of its specific ligands, such as EGF (transforming growth factor α (TGFα)). Upon activation, EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity, and autophosphorylation at the C-terminal domain of EGFR leads to a series of signal transduction cascades, mainly MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation.

[0254] Exemplary anti-EGFR antibodies that can be used in the present application include, but are not limited to, cetuximab (e.g., ) and panitumumab (eg, ABX-EGF, ).

[0255] Cetuximab (eg, Cetuximab is a recombinant human / mouse chimeric monoclonal antibody that specifically binds to the extracellular domain of human EGFR. Cetuximab is composed of the Fv region of a mouse anti-EGFR antibody and the human IgG1 heavy chain and kappa light chain constant regions. Indicated for the initial treatment of locally or regionally advanced squamous cell carcinoma of the head and neck in combination with radiation therapy. It is also indicated for the treatment of K-Ras wild-type, EGFR-expressing, metastatic colorectal cancer (mCRC).

[0256] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes EGFR (hereinafter referred to as an "anti-EGFR antibody"). In some embodiments, the anti-EGFR antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 29; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-EGFR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29, and a light chain comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the anti-EGFR antibody is cetuximab or an antigen-binding fragment thereof. In some embodiments, the anti-EGFR antibody specifically binds to EGFR competitively with any of the anti-EGFR antibodies described herein.

[0257] Antibodies that specifically recognize angiogenic factors

[0258] In some embodiments, the antibody fusion protein of the present invention comprises an antibody that specifically recognizes an angiogenic factor (such as, anti-Ang2, anti-TNFα, or anti-IL-17A full-length antibody), and a VEGFR component fused to the C-terminus of the antibody light chain (e.g., the C-terminus of the antibody VL-CL domain). In some embodiments, the antibody that specifically recognizes angiogenic factors inhibits or antagonizes angiogenic factor function and is interchangeably referred to herein as an "angiogenesis inhibitor."

[0259] As previously mentioned, angiogenesis is the process of growing new blood vessels from existing vasculature. It plays an important role in multiple physiological processes including embryonic development and tissue and wound repair (Folkman J et al., Angiogenic factors. Science 1987; 235: 442-7). The physiological steps of angiogenesis have been well characterized and involve proteolysis of the extracellular matrix, proliferation, migration and assembly of endothelial cells into tubular channels, recruitment and differentiation of mural cells and production of extracellular matrix (Carmeliet P et al., Nature. 2011; 473: 298-307). Pathological angiogenesis can occur in tumor formation, ocular disorders (e.g., diabetic retinopathy, diabetic macular edema or macular degeneration), arthritis, psoriasis, fibrotic diseases, inflammatory diseases and arteriosclerosis (Polverini PJ. Crit Rev Oral Biol Med. 1995; 6 (3): 230-47).

[0260] In some embodiments, the angiogenic factors of the present invention include angiopoietin (ANG, such as Ang1, Ang2, Ang3), ephrin (Eph), fibroblast growth factor (FGF, such as aFGF, bFGF), neuropilin (NRP), plasminogen activator (such as uPA, tPA), angiogenin, platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (PD-ECGF), tumor growth factor beta (TGF-β), TGF-α, vascular endothelial growth factor (VEGF), vascular endothelial cadherin (VE-cadherin), epidermal growth factor (EGF), nerve growth factor (NGF), hypoxia-inducible factor (HIF), connective tissue growth factor (CTGF), granulocyte macrophage colony stimulating factor (GM-CSF ), insulin-like growth factor (IGF), Del-1, follistatin, granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor / scatter factor (HGF / SF), leptin, midkine, placental growth factor, pleiotrophin (PTN), progranulin, proliferative protein, tumor necrosis factor-α (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 17 (IL-17), interleukin 18 (IL-18), interleukin 20 (IL-20), interleukin 23 (IL-23), chemoattractants such as CC motif ligands (CCL28, CCL21), CXC motif ligands (CXCL1, CXCL5), macrophage migration inhibitory factor (MIF), immune cell surface proteins such as cluster of differentiation (CD) and its receptors. These factors have been reported to be overexpressed and play a key role in angiogenesis-related diseases (Elshabrawy et al., Angiogenesis (2015) 18:433-448; Brian P. Eliceiri, Circ. Res. 2001 Nov 7;89(12):1104-10).

[0261] Angiogenesis is a complex biological process that involves various growth factors and signaling receptors, and targeting a single molecule in the signaling cascade may not provide an effective clinical treatment for uncontrolled angiogenesis in diseases such as cancer. Therefore, there is an increasing need to develop innovative therapies that can combine multiple key angiogenic factors in a synergistic manner to effectively inhibit angiogenesis and disease progression, as provided in this application.

[0262] In some embodiments, the antibody or angiogenesis inhibitor that specifically recognizes an angiogenesis factor can be an anti-VEGF antibody that binds to a VEGF ligand (see, e.g., U.S. Patents 5,730,977, 6,884,879, and 8,975,381). In some embodiments, the angiogenesis inhibitor can be an anti-Ang2 antibody (see, e.g., U.S. Patents 6,166,185, 7,5210,53, and 7,973,140).

[0263] Angiopoietin-2 (Ang2)

[0264] Angiopoietin-2 (Ang2) is an antagonistic ligand of the receptor Tie2 present in vascular endothelial cells. It is believed that by competing with angiopoietin-1 (Ang1), an agonist of Tie2, for binding to Tie2, signal transduction through Tie2 is inhibited. Ang1, a ligand that activates the Tie2 receptor, serves as a key regulator for maintaining vascular stability by maintaining the barrier function of vascular endothelial cells. Vascular endothelial cells are activated, as demonstrated by overexpression of VEGF or inflammation, and vascular permeability is increased. It is believed that Ang1 induces the stability of vascular endothelial cells and reduces vascular permeability by accelerating the junctional integrity of vascular endothelial cells, wherein the increased Ang2 in activated vascular endothelial cells serves to inhibit the stability of vascular endothelial cells obtained by Ang1 by competing with Ang1. Therefore, Ang2 inhibits Ang1-Tie2 binding, which maintains the stability of vascular endothelial cells and therefore maintains signal transduction, thereby ultimately accelerating angiogenesis via the dynamic rearrangement of blood vessels.

[0265] In normal adults, the three major sites of angiogenesis are the ovary, placenta, and uterus, which are the main normal (i.e., non-cancerous) tissues in which Ang2 mRNA has been detected. It is stated that many published studies have demonstrated vessel-selective Ang2 expression in disease states associated with angiogenesis. These pathological conditions include, for example, psoriasis, macular degeneration, and cancer (Bunone et al., American Journal of Pathology, 155:1967-1976 (1999); Etoh, T. et al., Cancer Research, 61:2145-2153 (2001); Hangai, M. et al., Investigative Ophthalmology & Visual Science, 42:1617-1625 (2001); Holash, J. et al., Investigative Ophthalmology & Visual Science, 42:1617-1625 (1999); Kuroda, K. et al., Journal of Investigative Dermatology, 116:713-720 (2001); Otani, A. et al., Investigative Ophthalmology & Visual Science, 40:1912-1920 (1999); Stratmann, A. et al., American Journal of Pathology, 153: 1459-1466 (1998); Tanaka, S. et al., J Clin Invest, 103: 34-345 (1999); Yoshida, Y. et al., International Journal of Oncology, 15: 1221-1225 (1999); Yuan, K. et al., Journal of Periodontal Research, 35: 165-171 (2000); Zagzag, D. et al., Experimental Neurology, 159: 391-400 (1999). Most of these studies have focused on cancer, where many tumor types appear to show vascular Ang-2 expression.Unlike its expression in pathological angiogenesis, Ang-2 expression in normal tissues is extremely limited (Maisonpierre, PC et al., Science, 277: 55-60 (1997); Mezquita, J. et al., Biochemical and Biophysical Research Communications, 260: 492-498 (1999)). Various publications have proposed Ang-1, Ang-2 and / or Tie-2 as possible targets for anti-cancer therapy.

[0266] Exemplary anti-Ang2 antibodies that can be used in the present application include, but are not limited to, Nivolumab and Vanucizumab (RG7221).

[0267] Nivolumab (e.g., REGN910) is an experimental monoclonal antibody originally designed for the treatment of cancer that targets Ang2. As of May 2017, it is in Phase II clinical trials for the treatment of diabetic macular edema.

[0268] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes Ang2 (hereinafter referred to as an "anti-Ang2 antibody"). In some embodiments, the anti-Ang2 antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-Ang2 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the anti-Ang2 antibody is nivolumab or an antigen-binding fragment thereof. In some embodiments, the anti-Ang2 antibody specifically binds to Ang2 competitively with any of the anti-Ang2 antibodies described herein.

[0269] TNFα

[0270] Tumor necrosis factor (TNF, TNFα, cachexia or cachexia) is a cytokine involved in systemic inflammation and is one of the cytokines that constitute the acute phase reaction. It is mainly produced by activated macrophages and is also produced by other cell types, such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils and neurons. TNFα is mainly involved in the regulation of immune cells. It can cause fever, apoptotic cell death, cachexia, inflammation, and inhibit tumorigenesis and viral replication, and responds to sepsis via cells that produce IL1 and IL6. TNFα can promote angiogenesis in the body. In the cornea, TNFα appears to stimulate blood vessel growth.

[0271] Exemplary anti-TNFα antibodies that can be used in the present application include, but are not limited to, adalimumab (e.g., ), infliximab (e.g., ), certolizumab (e.g., ), golimumab (e.g., CNTO148, ), and etanercept (e.g., ).

[0272] Adalimumab (eg, ) is a recombinant DNA-derived human IgG1 monoclonal antibody specific for TNFα, which is used to treat rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), ulcerative colitis, plaque psoriasis (Ps), hidradenitis suppurativa, and juvenile idiopathic arthritis (JIA).

[0273] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes TNFα (hereinafter referred to as an "anti-TNFα antibody"). In some embodiments, the anti-TNFα antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 32; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-TNFα antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 32, and a light chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the anti-TNFα antibody is adalimumab or an antigen-binding fragment thereof. In some embodiments, the anti-TNFα antibody specifically binds to TNFα competitively with any of the anti-TNFα antibodies described herein.

[0274] IL-17A

[0275] Interleukin-17A (IL-17, CTLA8) is a proinflammatory cytokine produced by activated T cells and regulates the activity of NF-κB and mitogen-activated protein kinase (MAPK). High levels of IL-17A are associated with a variety of chronic inflammatory diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis. A positive correlation between IL-17A production and asthma severity has also been found. IL-17A can enhance tumor growth in vivo by inducing IL-6, which in turn activates the oncogenic transcription factor STAT3 and promotes tumor survival and angiogenesis. For example, IL-17A appears to favor the development of colorectal cancer by promoting the production of VEGF from cancer cells, thereby favoring angiogenesis.

[0276] Exemplary anti-IL-17A antibodies that can be used in the present application include, but are not limited to, ixekizumab (e.g., ), and secukinumab (e.g., ).

[0277] Ixezumab (eg, ) is a humanized monoclonal antibody that blocks IL-17A and reduces inflammation. It has been approved for the treatment of moderate to severe plaque psoriasis.

[0278] In some embodiments, the VEGFR-antibody light chain fusion protein comprises an antibody (such as a full-length antibody) that specifically recognizes IL-17A (hereinafter referred to as an "anti-IL-17A antibody"). In some embodiments, the anti-IL-17A antibody comprises: a heavy chain HC-CDR1, HC-CDR2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38; and / or a light chain LC-CDR1, LC-CDR2, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-IL-17A antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-IL-17A antibody is ixekizumab or an antigen-binding fragment thereof. In some embodiments, the anti-IL-17A antibody competes for specific binding to IL-17A with any of the anti-IL-17A antibodies described herein.

[0279] Antibody variants

[0280] In some embodiments, it is envisioned that the amino acid sequence variants of the antibodies provided herein. For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. In order to obtain the final construct, any combination of deletions, insertions and substitutions can be performed, provided that the final construct has the desired characteristics, for example, antigen binding.

[0281] a) Substitutions, insertions, deletions and variants

[0282] In some embodiments, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More significant changes are provided under the heading "Exemplary Substitutions" and are further described below with respect to amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened to obtain desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. In addition, see subsection "1. Amino Acid Sequence Variants" under section "V. Preparation Methods."

[0283] Table 1: Amino acid substitutions

[0284] Original residue Exemplary Substitutions Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn:Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg lie(I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln: Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Tip; Phe; Thr; Ser Phe Val(V) Ile; Leu:Met; Phe:Ala; norleucine Leu

[0285] Amino acids can be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0286] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0287] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally speaking, one or more resulting variants selected for further study will have modifications (e.g., improvements) of certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or certain biological properties of the parent antibody that will be substantially retained. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0288] Changes (e.g., substitutions) can be made in HVR, for example to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that are mutated at high frequency during somatic cell maturation (see, e.g., Chowdhury, Methods Mol.Biol.207:179-196 (2008)) and / or SDR (a-CDR)), and the resulting variant VH or VL binding affinity is tested. Affinity maturation by construction and reselection from a secondary library has been described in, for example, Hoogenboom et al., Methods in Molecular Biology178:1-37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-directed approach, in which multiple HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are typically targeted.

[0289] In some embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, as long as such changes do not significantly reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in an HVR. Such changes may be outside of an HVR "hotspot" or CDR.

[0290] A useful method for identifying residues or regions of antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or residue group (e.g., charged residues, such as Arg, Asp, His, Lys, and Glu) of the target residue is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at the amino acid position to demonstrate functional sensitivity to the initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex can identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain desired properties.

[0291] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides comprising one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody with an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0292] b) Glycosylation variants

[0293] In some embodiments, the antibody within the VEGFR-antibody light chain fusion protein provided herein is altered to increase or decrease the degree of antibody glycosylation. Addition or deletion of glycosylation sites of an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0294] In the case where the antibody comprises an Fc district, the carbohydrate attached thereto can be changed. The natural antibody produced by mammalian cells generally comprises the biantennary oligosaccharide of side chain, which is generally attached by the N-key of the Asn297 of the CH2 domain to the Fc district. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharide can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the " stem " of biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the antibody of the present application can be modified to produce antibody variants with some improved characteristics.

[0295] In some embodiments, antibody variants are provided that have a carbohydrate structure that does not contain fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry, as described in, for example, WO 2008 / 077546. Asn297 refers to the asparagine residue at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations within the antibody, Asn297 may also be located at approximately ±3 amino acids upstream or downstream of site 297, i.e., between sites 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications involving "defucosylated" or "fucose-deficient" antibody variants include: US2003 / 0157108; WO 2000 / 61739; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells that lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0296] Antibody variants also have bisecting oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharides attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0297] c) Fc region variants

[0298] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody within a VEGFR-antibody light chain fusion protein provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0299] In some embodiments, the present application contemplates an antibody variant that has some but not all effector functions, making it a desirable candidate for applications where the half-life of the VEGFR-antibody light chain fusion protein in vivo is important but certain effector functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells used to mediate ADCC, NK cells express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be employed (see, e.g., ACTI for flow cytometry). TMNon-radioactive cytotoxicity assays (Cell Technology, Inc. Mountain View, CA). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0300] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ).

[0301] Certain antibody variants with improved or decreased binding to FcRs have been described. (See, e.g., U.S. Patent 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001))

[0302] In some embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0303] In some embodiments, alterations are made in the Fc region that result in altered (ie, improved or decreased) CIq binding and / or CDC, e.g., as described in U.S. Patent 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0304] In some embodiments, the antibody variant comprises a variant Fc region comprising one or more amino acid substitutions that increase half-life and / or improve binding to FcRn. Antibodies with increased half-life and improved binding to FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Patent 7,371,826 ).

[0305] See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent 5,648,260; U.S. Patent 5,624,821; and WO 94 / 29351 for additional examples of Fc region variants.

[0306] VEGFR-antibody light chain fusion proteins (such as a VEGFR component fused to a full-length antibody) are contemplated that comprise any one or a combination of the Fc variants described herein.

[0307] d) Cysteine ​​engineered antibody variants

[0308] In some embodiments, it is desirable to produce cysteine ​​engineered antibodies, e.g., "thio Mabs," wherein one or more residues of an antibody are substituted with cysteine ​​residues. In a specific embodiment, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker drug moieties, to produce immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues may be substituted with cysteine: A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described, for example, in U.S. Patent No. 7,521,541.

[0309] III. Pharmaceutical Compositions

[0310] The application also provides a pharmaceutical composition comprising any one of the antibody fusion proteins as described herein, the antibody fusion protein comprising a VEGFR component (such as VEGFR-anti-PD-1, VEGFR-anti-PD-L1, VEGFR-anti-CTLA-4, VEGFR-anti-HER2, VEGFR-anti-EGFR, VEGFR-anti-Ang2, VEGFR-anti-TNFα, or VEGFR-anti-IL-17A antibody light chain fusion protein) fused to the C-terminus of the antibody light chain (such as the C-terminus of the antibody CL domain), and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared by mixing the VEGFR-antibody light chain fusion protein described herein with a desired purity and an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution.

[0311] The pharmaceutical composition is preferably stable, wherein the VEGFR-antibody light chain fusion protein described herein substantially retains its physical and chemical stability and integrity upon storage. A variety of analytical techniques that can be used to measure protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and for a selected period of time. For rapid screening, the formulation can be kept at 40°C for 2 weeks to 1 month, at which point stability is measured. When the formulation is to be stored at 2-8°C, generally speaking, the formulation should be stable at 30°C or 40°C for at least 1 month and / or stable at 2-8°C for at least 2 years. When the formulation is to be stored at 30°C, generally speaking, the formulation should be stable at 30°C for at least 2 years and / or stable at 40°C for at least 6 months. For example, the degree of aggregation during storage can be used as an indicator of protein stability. In some embodiments, the stable formulations of the VEGFR-antibody light chain fusion proteins described herein may comprise less than about 10% (preferably less than about 5%) of the VEGFR-antibody light chain fusion proteins present in the formulation as aggregates.

[0312] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonic agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., zinc-protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0313] Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins , such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM , polyethylene glycol (PEG), and PLURONICS TM or polyethylene glycol (PEG).

[0314] Buffers are used to control pH within a range that optimizes the therapeutic effect, particularly when stability is pH-dependent. Buffers are preferably present at a concentration in the range of about 50 mM to about 250 mM. Suitable buffers for use herein include organic and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. Additionally, the buffer may comprise histidine and trimethylamine salts, such as Tris.

[0315] Preservatives are added to slow the growth of microorganisms and are typically present in the range of 0.2% to 1.0% by weight / volume. Adding preservatives can, for example, help prepare multi-purpose (multiple dose) formulations. Suitable preservatives for use in the present application include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., benzalkonium chloride, benzalkonium bromide, benzalkonium iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and meta-cresol.

[0316] There is a tonicity agent, sometimes referred to as a "stabilizer", to regulate or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, it is generally referred to as a "stabilizer" because it can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. Taking into account the relative amounts of other ingredients, the tonicity agent can be between 0.1% by weight and 25% by weight, preferably any amount between 1% by weight and 5% by weight. Preferred tonicity agents include polyols, preferably trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0317] Additional excipients include agents that can act as one or more of the following: (1) fillers, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container walls. Such excipients include: polyols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, inositol, galactose, galactitol, glycerol, cyclic alcohols (such as inositol), polyethylene glycol, PEG-100; alcohols; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-thioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0318] A nonionic surfactant or detergent (also known as a "wetting agent") is present to aid in solubilizing the therapeutic agent and protecting the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in a range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0319] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), polyols, Polyoxyethylene sorbitan monoether ( Examples of the detergents include lauroyl alcohol, lauric acid polyol, glyceryl stearate, sucrose fatty acid ester, methylcellulose, and carboxymethylcellulose. Examples of the anionic detergents include sodium lauryl sulfate, sodium dioctylsulfosuccinate, and sodium dioctylsulfonate. Examples of the cationic detergents include benzalkonium chloride and benzethonium chloride.

[0320] In order for the pharmaceutical composition to be used for in vivo administration, it must be sterile. The pharmaceutical composition can be sterilized by filtering through a sterile filtration membrane. The pharmaceutical composition herein is typically placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle.

[0321] The route of administration is according to known and acceptable methods, for example, by single or multiple boluses or infusion over a long period of time in a suitable manner, for example, by injection or infusion subcutaneously, intravenously, intraperitoneally, intramuscularly, intraarterially, intralesionally or intraarticularly, topically, by inhalation, or by sustained release or extended release. In some embodiments, the pharmaceutical composition is administered topically, such as intratumorally or intravitreally.

[0322] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antagonist in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT®. TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

[0323] The pharmaceutical compositions herein may also contain more than one active compound necessary for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may contain a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, or a growth inhibitory agent. These molecules are suitably present in combination in amounts effective for the intended purpose.

[0324] The active ingredient can also be embedded in microcapsules, for example, prepared by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition.

[0325] The antibody fusion proteins disclosed herein can be formulated as immunoliposomes. Liposomes containing the antibody fusion proteins are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Patents 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent 5,013,556.

[0326] Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).The liposomes are extruded through filters of defined pore size to produce liposomes with the desired diameter.

[0327] In some embodiments, the pharmaceutical composition is contained in a disposable vial (such as a disposable sealed vial). In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in bulk in a container. In some embodiments, the pharmaceutical composition is stored frozen.

[0328] Pharmaceutical composition for treating ocular neovascularization disorders

[0329] Specifically, to treat ocular neovascular disorders such as diabetic retinopathy and AMD, a pharmaceutical composition comprising a VEGFR-antibody light chain fusion protein (such as a VEGFR-anti-TNFα, a VEGFR-anti-TNFα, or a VEGFR-anti-IL-17A antibody light chain fusion protein) is administered directly to the eye, for example, topically. Topical administration methods include, but are not limited to, eye drops, subconjunctival injection, subconjunctival implantation, intravitreal injection, intravitreal implantation, sub-Tenon's sac injection, or sub-Tenon's sac implantation. In some embodiments, a pharmaceutical composition comprising a VEGFR-antibody light chain fusion protein and, optionally, a pharmaceutically acceptable carrier, is administered by intravitreal injection.

[0330] Compositions suitable for topical administration are known in the art (see, for example, U.S. Patent Application 2005 / 0059639). In various embodiments, the compositions of the present invention may comprise a liquid comprising an active agent in the form of a solution, a suspension, or both. As used herein, liquid compositions include gels. Preferably, the liquid composition is aqueous. Alternatively, the composition may take the form of an ointment. In a preferred embodiment, the composition is an aqueous composition that can gel in situ, more preferably an aqueous solution that can gel in situ. Such compositions may comprise a gelling agent in a concentration that effectively promotes gelation upon contact with tears from the eye or outside of the eye. The aqueous composition of the present invention has an ophthalmologically compatible pH and osmolality. The composition may comprise an ophthalmic depot formulation for subconjunctival administration, comprising an active agent. The microparticles comprising the active agent may be embedded in a biocompatible, pharmaceutically acceptable polymer or lipid encapsulating agent. The depot formulation may be suitable for releasing all or substantially all of the active material over an extended period of time. Polymer or lipid matrix (if present) can be suitable for fully degrading, to be transported from the application site after releasing all or substantially all active agents. Depot formulations can be liquid preparations, which include pharmaceutically acceptable polymers and dissolved or dispersed active agents. During injection, polymers, for example, form depots at the injection site by gelling or precipitation. The composition can include solid products, which can be inserted into the appropriate position in the eye, such as between the eye and the eyelid or in the conjunctival sac, inserted into the chamber of the eye, such as the anterior chamber or posterior chamber, or can be implanted in or on the avascular area outside the sclera, choroidal space or vitreous body, wherein the product releases the active agent. The solid products suitable for implanting in the eye in this way generally include polymers, and can be bioerodible or non-bioerodible. Implants can be permeable or impermeable to active agents. In some embodiments, implants can be positioned on avascular areas, such as on the sclera, to allow the drug to diffuse across the sclera to the desired treatment site, such as the intraocular space and the macula of the eye. Furthermore, the site of transscleral diffusion may be proximal to a site of neovascularization, such as a site proximal to the macula.

[0331] A variety of polymer delivery vehicles for providing sustained release have been used in the ocular environment and can be used to administer the compositions of the present invention. Various biodegradable polymers, such as biocompatible polymers, can be used. The polymer can be a homopolymer, a copolymer (including a block copolymer), linear, branched, or cross-linked. Available polymers include, but are not limited to, polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), poly(phosphazene), poly(phosphate), polycaprolactone, polyanhydride, ethylene-vinyl acetate, polyorthoester, polyether, and poly(β-amino ester). Peptides, proteins such as collagen or albumin, polysaccharides such as chitosan, alginate, hyaluronic acid (or derivatives of any of these), and dendrimers (e.g., PAMAM dendrimers) can also be used. Methods for preparing such formulations will be apparent to those skilled in the art. Certain materials can also be commercially available, for example, from Alza Corporation. Any of these polymers or combinations thereof can be used in various embodiments of the present invention.

[0332] Additional exemplary polymers include cellulose derivatives such as carboxymethyl cellulose, polycarbonate or polyurea, cross-linked poly(vinyl acetate), and the like, ethylene-vinyl ester copolymers having an ester content of 4% to 80% such as ethylene-vinyl acetate (EVA) copolymers, ethylene-vinyl hexanoate copolymers, ethylene-vinyl propionate copolymers, ethylene-vinyl butyrate copolymers, ethylene-vinyl valerate copolymers, ethylene-trimethylvinyl acetate copolymers, ethylene-diethylvinyl acetate copolymers, ethylene-3-methylbutyrate copolymers, ethylene-3-3-dimethylbutyrate copolymers, and ethylene-vinyl benzoate copolymers, or mixtures thereof.

[0333] Poly(orthoesters) have been introduced into the eye and exhibit favorable properties for sustained-release ophthalmic drug delivery (Einmahl, S., Invest. Opthalmol. Vis. Sci., 43(5), 2002). Polylactide particles have been used to target agents to the retina and RPE following intravitreal injection of a suspension of such particles (Bourges, JL et al., Invest. Opthalmol. Vis. Sci., 44(8), 2003).

[0334] The method for preparing sustained-release formulations involves combining or mixing VEGFR-antibody light chain fusion protein with polymer components to form a mixture. The mixture can then be extruded, compressed, molded, etc. to form a single composition. Optionally, heat and / or pressure can be used. The single composition can then be processed to form a single implant or particle suitable for being placed in the patient's eye. Additional methods for incorporating therapeutically active agents into polymer matrices are known in the art. The polymer matrix can be formed into various shapes, such as rods, disks, wafers, etc., which can have a series of different sizes (e.g., length, width, etc.) and volumes. Exemplary shapes include spherical, cylindrical, spiral, disc-shaped or spiral, spiral, cubic, conical, elliptical, biconvex, hemispherical or nearly hemispherical, etc.

[0335] IV. Methods of treating diseases

[0336] While vascular endothelial cell proliferation is desirable in certain circumstances, vascular endothelial cell proliferation and angiogenesis are also important components of a variety of diseases and disorders, including tumor growth and cancer cell metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, AMD, hemangiomas, immune rejection of transplanted corneal and other tissues, and chronic inflammation.

[0337] VEGFR-antibody light chain fusion proteins as described herein (such as VEGFR-anti-PD-1, VEGFR-anti-PD-L1, VEGFR-anti-CTLA-4, VEGFR-anti-HER2, VEGFR-anti-EGFR, VEGFR-anti-Ang2, VEGFR-anti-TNFα or VEGFR-anti-IL-17A antibody light chain fusion proteins), and compositions thereof (such as pharmaceutical compositions) can be used in a variety of applications, such as diagnosis, molecular assays and therapy.

[0338] One aspect of the present invention provides a method for treating a disease or condition in an individual in need thereof, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising a VEGFR-antibody light chain fusion protein as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the disease is cancer. In some embodiments, the disease is a non-neoplastic disease or a neovascular disorder. In some embodiments, the disease is an ocular neovascular disorder, such as AMD or diabetic retinopathy.

[0339] The present invention contemplates, in part, VEGFR-antibody light chain fusion proteins, nucleic acid molecules encoding them and / or vectors, host cells comprising nucleic acid molecules encoding them and / or vectors, which can be administered alone or in any combination with another therapy, and in at least some aspects administered together with a pharmaceutically acceptable carrier or excipient. In some embodiments, prior to administering the VEGFR-antibody light chain fusion protein, it can be mixed with suitable pharmaceutical carriers and excipients well known in the art. Compositions prepared according to the present disclosure can be used to treat or delay the deterioration of cancer or ocular neovascularization disorders.

[0340] cancer

[0341] In some embodiments, a method for treating cancer (such as a solid tumor, or a cancer with abnor...

Claims

1. An antibody fusion protein comprising 1) an antibody comprising a light chain (antibody light chain), and 2) a vascular endothelial growth factor receptor (VEGFR) component, wherein the VEGFR component is fused to the C-terminus of the antibody light chain.

2. The antibody fusion protein according to claim 1, wherein The antibody comprises a first light chain and a second light chain, wherein the antibody fusion protein comprises a first VEGFR component and a second VEGFR component; and wherein the first VEGFR component is fused to the C-terminus of the first antibody light chain, and the second VEGFR component is fused to the C-terminus of the second antibody light chain.

3. The antibody fusion protein of claim 2, wherein the first VEGFR component and the second VEGFR component are the same or different.

4. The antibody fusion protein according to any one of claims 1 to 3, wherein the VEGFR component comprises the immunoglobulin-like (Ig-like) domain 2 (Flt1d2) of a first VEGFR Flt1; preferably, the VEGFR component further comprises the Ig-like domain 3 (Flk1d3) of a second VEGFR Flk1; more preferably, the VEGFR component comprises the amino acid sequence of SEQ ID NO:

8.

5. The antibody fusion protein according to any one of claims 1-4, wherein the VEGFR component and the C-terminus of the antibody light chain are connected via a linker; preferably, the linker comprises the amino acid sequence of SEQ ID NO: 6 or 7.

6. The antibody fusion protein according to any one of claims 1-5, wherein the antibody comprises a light chain constant domain (CL domain), and the VEGFR component is fused to the C-terminus of the antibody CL domain; preferably, the antibody is a full-length antibody.

7. The antibody fusion protein according to any one of claims 1-6, wherein the antibody specifically recognizes an immune checkpoint molecule, a tumor antigen or an angiogenic factor; preferably, the immune checkpoint molecule is PD-1, PD-L1 or CTLA-4, the tumor antigen is EGFR (HER1) or Her2, and the angiogenic factor is angiopoietin-2 (Ang2), TNFα or IL-17A.

8. The antibody fusion protein according to claim 7, wherein: (1) The antibody specifically recognizes PD-1, which comprises: (i) heavy chain-CDR1 (HC-CDR1), HC-CDR2 and HC-CDR3 of a heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and / or light chain-CDR1 (LC-CDR1), LC-CDR2 and LC-CDR3 of a light chain comprising the amino acid sequence of SEQ ID NO: 13; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 12 and a light chain comprising the amino acid sequence of SEQ ID NO: 13; (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 15; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 16; or, (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and a light chain comprising the amino acid sequence of SEQ ID NO: 16; (2) The antibody specifically recognizes PD-L1, which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 18; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 19; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 18 and a light chain comprising the amino acid sequence of SEQ ID NO: 19; (iii) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 21; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 22; or, (iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a light chain comprising the amino acid sequence of SEQ ID NO: 22; (3) The antibody specifically recognizes CTLA-4, which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 9; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 10; or, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10; (4) The antibody specifically recognizes HER2, which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 24; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 25; (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 24 and a light chain comprising the amino acid sequence of SEQ ID NO: 25; (5) The antibody specifically recognizes EGFR (HER1), which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 29; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 30; or, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 29 and a light chain comprising the amino acid sequence of SEQ ID NO: 30; (6) The antibody specifically recognizes angiopoietin-2 (Ang2), comprising: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 35; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 36; or, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and a light chain comprising the amino acid sequence of SEQ ID NO: 36; (7) The antibody specifically recognizes TNFα, which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 32; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 33; or, (ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a light chain comprising the amino acid sequence of SEQ ID NO: 33; or (8) The antibody specifically recognizes IL-17A, which comprises: (i) HC-CDR1, HC-CDR2 and HC-CDR3 of the heavy chain comprising the amino acid sequence of SEQ ID NO: 38; and / or LC-CDR1, LC-CDR2 and LC-CDR3 of the light chain comprising the amino acid sequence of SEQ ID NO: 39; or (ii) wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO:

39.

9. The fusion protein according to claim 8, wherein The antibody fusion protein comprises: (1) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 12 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 14; (2) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 15 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 17; (3) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 18 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 20; (4) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 21 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 23; (5) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 9 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11; (6) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 24 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 26-28; (7) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 29 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 31; (8) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 35 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 37; (9) a heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 32 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 34; (10) A heavy chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO:38 and a light chain fusion polypeptide comprising the amino acid sequence of SEQ ID NO:

40.

10. A pharmaceutical composition comprising the antibody fusion protein according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. A method for treating an individual suffering from cancer, the method comprising administering to the individual an effective amount of the pharmaceutical composition according to claim 10; preferably, the cancer is lung cancer, liver cancer, skin cancer (e.g., melanoma), breast cancer, ovarian cancer, prostate cancer, colorectal cancer or bladder cancer.

12. A method for treating an individual suffering from a non-neoplastic disorder, the method comprising administering to the individual an effective amount of a pharmaceutical composition according to claim 10, wherein the non-neoplastic disorder is associated with VEGF overexpression; preferably, the non-neoplastic disorder is selected from rheumatoid arthritis, psoriasis, atherosclerosis, hemangioma, immune rejection of transplanted corneal tissue and other tissues, chronic inflammation or ocular neovascular disorder; more preferably, the non-neoplastic disorder is an ocular neovascular disorder; most preferably, the ocular neovascular disorder is age-related macular degeneration (AMD) or diabetic retinopathy.

13. The method of claim 12, wherein the pharmaceutical composition is administered by intravitreal injection when treating the ocular neovascular disorder.

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