CN118047868A - Anti-VEGF-A antibodies and uses thereof - Google Patents
Anti-VEGF-A antibodies and uses thereof Download PDFInfo
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Abstract
The present invention relates to vascular endothelial growth factor A (VEGF-A) binding molecules, in particular single domain antibodies to VEGF-A, to nucleic acids encoding said VEGF-A binding molecules, to expression vectors and host cells for expressing VEGF-A binding molecules, to methods of preparing said VEGF-A binding molecules, and to uses of said VEGF-A binding molecules.
Description
Technical Field
The present application relates to antibodies. More specifically, the present application relates to single domain antibodies that specifically bind to VEGF-A, methods of making the same, and uses thereof.
Background
Development of the vascular system is the basis of many physiological and pathological processes. Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is a group of growth factors with important pro-angiogenic activity, and has the functions of promoting endothelial cell mitosis, resisting cell apoptosis, increasing vascular permeability, promoting cell migration and the like. VEGF proteins are important signal transduction proteins involved in normal embryonic angiogenesis (de novo formation of the embryonic circulatory system) and abnormal angiogenesis (growth of blood vessels from the existing vasculature) (Ferrara et al, 1996, nature380:439-442; dvorak et al, 1995, am. J. Pathol. 146:1029-1039). VEGF has been implicated in the pathology of solid tumors and hematological malignancies, inter-ocular neovascular syndrome, inflammation and cerebral edema, and female genital tract (Ferrara et al 2003,Nature Medicine 9:669-676). VEGF mRNA is overexpressed in many human tumors including those of the lung, breast, gastrointestinal tract, kidney, pancreas and ovary (Berkman et al, 1993, J.Clin. Invest. 91:153-159). Since angiogenesis is involved in a variety of pathological conditions, including tumor growth, proliferative retinopathy, age-related macular degeneration, rheumatoid Arthritis (RA) and psoriasis, it has become an attractive therapeutic target (Folkman et al, 1992, J.biol. Chem. 267:10931-10934).
The VEGF gene family includes prototypes VEGFA, VEGFB, VEGFC, VEGFD and placental growth factor (PLGF). The human VEGFA gene is composed of: 8 exons separated by 7 introns. There are at least 6 different VEGF isoforms, VEGF121, VEGF145, VEGF162, VEGF165b, VEGF183, VEGF189 and VEGF206, where the subscripts refer to the number of amino acids remaining after cleavage of the signal. Natural VEGF is a 45kDa homodimeric hairpin binding glycoprotein (Ferrara et al 2003,Nature Medicine 9:669-676). There are three types of VEGF receptors: VEGFR1, VEGFR2, and VEGFR3. Binding of VEGF to the extracellular domain of the receptor triggers receptor dimerization, which promotes autophosphorylation of tyrosine residues in the intracellular domain, thereby activating downstream signals that promote cell proliferation, migration, anti-apoptosis, and increase vascular permeability. VEGFR1 and VEGFR2 are predominantly expressed in vascular endothelial cells, while VEGFR3 is predominantly expressed in lymphatic endothelial cells. Each receptor has 7 extracellular regions and 1 transmembrane region. VEGF also binds to neuropilin NRP1 (also known as vascular endothelial growth factor 165 receptor (VEGF 165R) or CD 304) and NRP2 (also known as vascular endothelial growth factor 165 receptor 2 (VEGF 165R 2)).
VEGFA is secreted by a variety of cells, such as endothelial cells and tumors, and was originally demonstrated to be a regulator of endothelial growth factor and vascular permeability (Peach C et al ,Molecular Pharmacology of VEGF-A Isoforms:Binding and Signalling at VEGFR2,2018,Int.J.Mol.Sci.19(4):1264;Claesson-Welsh,L. and Welsh, M, VEGFA and tumour angiogenesis,2012,Journal of Internal Medicine,273 (2)). Since VEGFA is a key regulator of angiogenesis in the growth process of solid tumors, therapeutic approaches targeting VEGFA have become innovative therapeutic approaches in oncology. The first VEGFA inhibitors were obtained in 2004 as a batch for first line treatment of metastatic colorectal cancer (Ferrara N and Adamis A.P.,Ten years of anti-vascular endothelial growth factor therapy,2016,Nature Reviews Drug Discovery,15:385-403).
Single domain antibodies (Single domain antibody, sdabs), which are a class of antibodies that lack the light chain but only the heavy chain variable region of the antibody, are also known as nanobodies (nanobodies) because of their small molecular weight. Single domain antibodies were first found in camelids in the 90 s of the 20 th century. The first single domain antibody is a (Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R(1993)Naturally occurring antibodies devoid of light chains.Nature 363(6428):446-448.) single domain antibody engineered from the heavy chain antibody found in camelids, and, although simple in structure, is capable of selectively binding to a specific antigen, as is the case with whole antibodies, achieving an affinity for specific antigen that is comparable to or even higher than that of conventional antibodies.
Compared with the traditional antibody, the single-domain antibody has the advantages of small molecular weight, strong stability, easy recombinant expression and the like. For example, they generally exhibit high solubility and stability and can also readily produce (Harmsen MM,De Haard HJ(2007)Properties,production,and applications of camelid single-domain antibody fragments.ApplMicrobiol Biotechnol 77(1):13-22.). in yeast, plant and mammalian cells and, furthermore, they have good thermal stability and good tissue penetration. But they are also cost-effective in production. The advantages of single domain antibodies make them suitable for various biotechnology and therapeutic applications. For example, they may be used to treat diseases including, but not limited to, cancer, infectious diseases, inflammatory diseases, and neurodegenerative diseases.
Although antibodies to VEGF-A have been developed, there remains Sub>A need for improved anti-VEGF-A antibodies as therapeutic agents. In addition, there are few single domain antibodies directed against VEGF-A at present, so the development of single domain antibodies targeting VEGF-A would have great significance in the treatment of related diseases.
Disclosure of Invention
The present invention meets the above-described need by providing antibodies, preferably single domain antibodies, that specifically bind VEGF-A.
In one aspect, the invention provides Sub>A vascular endothelial growth factor Sub>A (VEGF-Sub>A) binding molecule comprising Sub>A heavy chain variable region (VH) comprising:
1) HCDR1, HCDR2, HCDR3: which respectively comprises amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
2) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
3) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;
4) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively;
5) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively;
6) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 21 and SEQ ID NO. 3;
7) HCDR1, HCDR2, HCDR3: which respectively comprise the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 22 and SEQ ID NO. 3.
In some embodiments, the VEGF-Sub>A binding molecule comprises Sub>A heavy chain variable region (VH) comprising:
1) An amino acid sequence set forth in SEQ ID NO. 4, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 4;
2) An amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 8;
3) An amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 12;
4) The amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 16;
5) An amino acid sequence shown in SEQ ID NO. 20, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 20;
6) An amino acid sequence shown in SEQ ID NO. 23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 23;
7) The amino acid sequence shown in SEQ ID NO. 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 24;
8) An amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 25;
9) The amino acid sequence shown in SEQ ID NO. 26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 26;
10 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 27;
11 28 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 28;
12 29 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 29;
13 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 30;
14 31 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 31.
In some embodiments, the VEGF-Sub>A binding molecule is Sub>A heavy chain single domain antibody (VHH) comprising:
1) HCDR1, HCDR2, HCDR3: which respectively comprises amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
2) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
3) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;
4) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively;
5) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively;
6) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 21 and SEQ ID NO. 3;
7) HCDR1, HCDR2, HCDR3: which respectively comprise the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 22 and SEQ ID NO. 3.
In some embodiments, the VEGF-Sub>A binding molecule is Sub>A heavy chain single domain antibody (VHH) comprising:
1) An amino acid sequence set forth in SEQ ID NO. 4, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 4;
2) An amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 8;
3) An amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 12;
4) The amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 16;
5) An amino acid sequence shown in SEQ ID NO. 20, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 20;
6) An amino acid sequence shown in SEQ ID NO. 23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 23;
7) The amino acid sequence shown in SEQ ID NO. 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 24;
8) An amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 25;
9) The amino acid sequence shown in SEQ ID NO. 26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 26;
10 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 27;
11 28 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 28;
12 29 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 29;
13 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 30;
14 31 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 31.
In some embodiments, the VEGF-A binding molecule further comprises one or more amino acid residue mutations that remain specifically bound to VEGF-A.
In some embodiments, at least one of the mutations is in one or more VH sequences but not in any CDR sequence.
In some embodiments, the VEGF-A binding molecule is Sub>A VEGF antagonist, preferably an anti-VEGF-A antibody.
In some embodiments, the VEGF-A binding molecule is Sub>A chimeric antibody.
In some embodiments, the VEGF-A binding molecule is Sub>A humanized antibody.
In some embodiments, the VEGF-A binding molecule is Sub>A single domain antibody, preferably Sub>A heavy chain single domain antibody (VHH).
In some specific embodiments, the VHH is derived from a camelid comprising alpaca or llama.
In some embodiments, the VH is fused to an Fc domain of an IgG.
In some embodiments, the VH is fused to an Fc domain of a human IgG.
In some specific embodiments, the VEGF-Sub>A binding molecule is Sub>A chimeric antibody of VHH from Sub>A camelid with the Fc domain of human IgG.
In some embodiments, the VEGF-Sub>A binding molecule is Sub>A chimeric antibody of Sub>A VHH from Sub>A camelid with an Fc domain of human IgG1 or IgG 4.
In some embodiments, the Fc domain is the amino acid sequence shown as SEQ ID NO. 32, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 32.
In some embodiments, the VEGF-A binding molecule comprises:
1) The amino acid sequence set forth in SEQ ID NO. 33, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 33;
2) The amino acid sequence shown in SEQ ID NO. 34, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 34;
3) An amino acid sequence set forth in SEQ ID NO. 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 35;
4) The amino acid sequence shown in SEQ ID NO. 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 36;
5) The amino acid sequence shown in SEQ ID NO. 37, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 37;
6) The amino acid sequence set forth in SEQ ID NO. 38, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 38;
7) An amino acid sequence set forth in SEQ ID NO. 39, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 39;
8) An amino acid sequence set forth in SEQ ID NO. 40, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 40;
9) The amino acid sequence shown in SEQ ID NO. 41, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 41;
10 Amino acid sequence depicted as SEQ ID NO. 42, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence depicted as SEQ ID NO. 42;
11 Amino acid sequence depicted in SEQ ID NO. 43, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence depicted in SEQ ID NO. 43;
12 44 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 44;
13 An amino acid sequence shown as SEQ ID NO. 45, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 45;
14 The amino acid sequence shown as SEQ ID NO. 46, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 46.
In one aspect, the invention provides an antibody-drug conjugate comprising Sub>A VEGF-A binding molecule as described herein linked to one or more conjugate moieties.
In one aspect, the invention provides Sub>A nucleic acid encoding an anti-VEGF-A binding molecule described herein.
In some embodiments, the nucleic acid comprises:
1) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 4;
2) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 8;
3) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 12;
4) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 16;
5) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 20;
6) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 23;
7) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 24;
8) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 25;
9) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 26;
10 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 27;
11 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 28;
12 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 29;
13 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 30;
14 A nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 31; or (b)
Preferably, the nucleic acid further comprises a nucleotide coding sequence for the amino acid sequence shown in SEQ ID NO. 32.
In one aspect, the invention provides an expression vector comprising a nucleic acid as described herein.
In one aspect, the invention provides a host cell comprising a nucleic acid as described herein or an expression vector as described herein.
In one aspect, the invention provides Sub>A pharmaceutical composition comprising Sub>A VEGF-Sub>A binding molecule as described herein, or an antibody-drug conjugate as described herein, or Sub>A nucleic acid as described herein, or one or more of the expression vectors as described herein, and Sub>A pharmaceutically acceptable carrier.
In some embodiments, sub>A VEGF-Sub>A binding molecule described herein, or an antibody-drug conjugate described herein, or Sub>A pharmaceutical composition described herein, is used to treat or prevent Sub>A VEGF-Sub>A-related disease in Sub>A subject; or for use in Sub>A medicament for inhibiting or blocking VEGF-Sub>A binding to VEGFR in Sub>A subject.
In some embodiments, the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.
In some embodiments, the disease is a proliferative disorder or an ocular disease, the proliferative disease comprising cancer.
In some embodiments, the cancer is selected from: breast cancer, liver cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, lung cancer, thyroid cancer, nasopharyngeal cancer, renal cancer, head and neck cancer, and pancreatic cancer.
In some embodiments, the ocular disease is age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, and other ocular diseases involving neovasculature.
In one aspect, the invention provides Sub>A method of treating or preventing Sub>A VEGF-Sub>A-related disease in Sub>A subject; or Sub>A method of inhibiting or blocking VEGF-Sub>A binding to VEGFR in Sub>A subject, comprising administering to Sub>A subject in need thereof Sub>A therapeutically effective amount of Sub>A VEGF-Sub>A binding molecule described herein, or an antibody-drug conjugate described herein, or Sub>A pharmaceutical composition described herein.
In some embodiments, the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.
In some embodiments, the disease is a proliferative disorder or an ocular disease, the proliferative disease comprising cancer.
In some embodiments, the cancer is selected from: breast cancer, liver cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, lung cancer, thyroid cancer, nasopharyngeal cancer, renal cancer, head and neck cancer, and pancreatic cancer.
In some embodiments, the ocular disease is age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, and other ocular diseases involving neovasculature.
In one aspect, the invention provides Sub>A kit comprising Sub>A VEGF-A binding molecule as described herein, or an antibody-drug conjugate as described herein, or Sub>A pharmaceutical composition as described herein, for use in the treatment or diagnosis of Sub>A VEGF-A related disease.
In one aspect, the invention provides Sub>A kit comprising Sub>A pharmaceutical composition comprising Sub>A VEGF-Sub>A binding molecule as described herein, or an antibody-drug conjugate as described herein, or Sub>A nucleic acid as described herein, or one or more of the expression vectors as described herein, and Sub>A pharmaceutically acceptable carrier, prescription information, and Sub>A container.
Drawings
Fig. 1: blocking detection of crude single domain antibody extracts on human VEGFR2-Fc and VEGF-A 165 -his-Avi, wherein 5 clones P2-10, P2-23, P2-24, P2-26, P2-59 have significant blocking efficacy;
Fig. 2: the blocking efficacy detection of the IgG antibody on human VEGFR2 and VEGF-A 165 -his-Avi shows that each molecule has definite blocking efficacy and HY004001-1 has optimal blocking efficacy;
fig. 3: detection of blocking efficacy of engineered IgG antibodies against human VEGFR2 and VEGF-A 165 -his-Avi, blocking efficacy of HY004001-3 was relatively good;
Fig. 4: the humanized IgG antibodies were tested for blocking efficacy against human VEGFR2-Fc and VEGF-A 165 -his-Avi, with HY004001-3-hz1 through HY004001-3-hz3, with HY004001-3-hz1 having the best blocking efficacy.
Fig. 5: measurement of the blocking efficacy of HY004001-3-hz1 on VEGF-A 165 -VEGFR2 mediated intracellular signaling pathway it was seen that HY004001-3-hz1 had a better blocking efficacy than the Faricimab biological analogs.
Detailed Description
While this invention may be embodied in many different forms, there are disclosed herein specific illustrative embodiments thereof which embody the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Unless otherwise defined herein, scientific and technical terms used in connection with the present application will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (such as "include" and "contain") is not limiting. Furthermore, the scope provided in the specification and the appended claims includes all values between the endpoints and between the endpoints.
Generally, terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein, and techniques thereof, are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook J. & Russell d.molecular Cloning: A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2000); abbas et al Cellular and Molecular Immunology, 6 th edition ,W.B.Saunders Company(2010);Harlow and Lane Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.(1998);Ausubel et al ,Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Wiley,John&Sons,Inc.(2002); and cologan et al Short Protocols in Protein Science, wiley, john & Sons, inc. (2003). The terms relating to analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are well known and commonly used terms in the art. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
Definition of the definition
For a better understanding of the present invention, definitions and explanations of related terms are provided below.
As used herein, the term "VEGF" refers to vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) which is a group of growth factors having important pro-angiogenic activity, having the effects of promoting endothelial cell mitosis and anti-apoptosis, increasing vascular permeability, promoting cell migration, and the like. Since angiogenesis is involved in a variety of pathological conditions, including tumor growth, proliferative retinopathy, age-related macular degeneration, rheumatoid Arthritis (RA) and psoriasis, it has become an attractive therapeutic target (Folkman et al, 1992, J.biol. Chem. 267:10931-10934).
As used herein, the term "VEGF-Sub>A" means that vascular endothelial growth factor Sub>A (vegfSub>A) is Sub>A highly conserved 27 kdSub>A dimer glycoprotein, belonging to the VEGF family of VEGFB, VEGFC, VEGFD and placental growth factor (PIGF) (common subtypes produced by Nagy et al ,VEGF-A and the Induction of Pathological Angiogenesis,2007,Annual Review of Pathology Mechanisms of Disease 2:251-275). due to alternative splicing are vegfSub>A 121, vegfSub>A 165, vegfSub>A 189 and vegfSub>A 206, containing 121, 165, 189 and 206 amino acids, respectively (sSub>A-NguanraksSub>A D et al) ,The role of vascular endothelial growth factor apolymorphisms in breast cancer.2012,International journal of molecular sciences 13(11):14845–14864).
As used herein, the term "VEGFR" refers to vascular endothelial growth factor receptor, i.e., a receptor for VEGF action. VEGFR-1 (FLt-1), VEGFR-2 (KDR), VEGFR-3, 3 types of VEGFR subtypes constitute the VEGFR family. VEGFR-1 and VEGFR-2 are mainly expressed in vascular endothelial cells, stimulate endothelial cell proliferation and promote angiogenesis, occasionally also expressed in tumor cells, VEGFR-3 is expressed in fetal early venous endothelial cells in a transsexual manner, and endothelial cells in late fetal and postnatal stages are not expressed any more, so that VEGFR-3 of adults is distributed in lymphatic endothelial cells to regulate lymphatic production.
As used herein, the term "antibody" or "Ab" generally refers to any form of antibody that exhibits the desired biological or binding activity. It includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single domain antibodies. Antibodies may comprise a heavy chain and a light chain. Heavy chains can be divided into μ, δ, γ, α and ε, which define the isotypes of antibodies as IgM, igD, igG, igA and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). VH and VL regions can be further divided into hypervariable regions (known as Complementarity Determining Regions (CDRs)) separated by relatively conserved regions (known as Framework Regions (FR)). Each VH and VL consists of 3 CDRs and 4 FR in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminal to the C-terminal. The distribution of amino acids in various regions or domains follows Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and1991)) or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al, (1989) Nature 342:878-883. Antibodies may have different antibody isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subtypes), igA1, igA2, igD, igE, or IgM antibodies.
As used herein, the term "anti-VEGF-Sub>A antibody" refers to an antibody that is capable of specifically binding VEGF-Sub>A (e.g., human VEGF-Sub>A). Advantageously, the anti-VEGF-A antibodies specifically bind VEGF-A with an affinity sufficient to provide diagnostic and/or therapeutic uses.
As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds VEGF protein is substantially free of antigens other than VEGF protein). However, isolated antibodies that specifically bind to human VEGF proteins may have cross-reactivity with other antigens, such as VEGF proteins from other species. In addition, the isolated antibodies may be substantially free of other cellular material and/or chemicals.
The term "single domain antibody" as used herein refers to a class of heavy chain antibodies that naturally lack the light and heavy chain constant regions of antibodies found in camelids.
The term "chimeric antibody" as used herein refers to antibodies in which the variable region sequences are from one species and the constant region sequences are from another species, for example, in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
The term "humanized antibody" as used herein refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequence.
The term "specific binding" or "specifically binding to" as used herein refers to a non-random binding reaction between two molecules, e.g., between an antibody and an antigen.
As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide may be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. The vector may be transformed, transduced or transfected into a host cell to express the carried genetic material element in the host cell. Vectors are well known to those of skill in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as lambda phage or M13 phage and animal viruses. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40). The vector may contain a number of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may comprise an origin of replication.
As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as e.coli (e.coli) or bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus (Aspergillus), insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, COS cells, NSO cells, heLa cells, BHK cells, CHO cells, HEK293 cells or human cells. CHO cells refer to chinese hamster ovary cells that include a variety of commercially available subclones, such as CHO-K1, CHO-S, CHO-DXB11, CHO-DG44, and the like. Wherein CHO-K1 is typically used as an expression platform. CHO-K1 is commercially available from ATCC, ECACC, DSMZ and a number of other companies.
As used herein, the term "T cell" includes CD4 + T cells, CD8 + T cells, T helper type 1T cells, T helper type 2T cells, T helper type 17T cells, and suppressor T cells.
As used herein, the terms "identity", "similarity" refer to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by aligning and comparing the sequences. "percent identity" refers to the percentage of identical residues between amino acids or nucleotides in a comparison molecule and is calculated based on the size of the smallest molecule being compared. For these calculations, the gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, a.m.), 1988,New York:Oxford University Press; biocomputing Informaticsand Genome Projects, (Smith, d.w.), 1993,New York:Academic Press; computer Analysis of Sequence Data, part I, (Griffin, a.m. and Griffin, h.g. brains ),1994,New Jersey:Humana Press;von Heinje,G.,1987,Sequence Analysis in Molecular Biology,New York:Academic Press;Sequence Analysis Primer,(Gribskov,M. and deveux, j. Brains), 1991,New York:M.Stockton Press; and Carillo, et al, 1988,SIAMJ.Applied Math.48:1073.
As used herein, the term "immunogenicity" refers to the ability to stimulate the formation of specific antibodies or sensitized lymphocytes in an organism. It refers not only to the nature of antigens to stimulate the activation, proliferation and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the fact that specific immune responses of antibodies or sensitized T lymphocytes can develop in the immune system of an organism after stimulation of the organism with an antigen. Immunogenicity is the most important property of an antigen. Whether an antigen is able to successfully induce an immune response in a host depends on three factors: the nature of the antigen, the reactivity of the host and the means of immunization.
As used herein, the term "transfection" refers to the process of introducing nucleic acid into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipofection and chemical and physical methods such as electroporation. Numerous transfection techniques are well known in the art and are disclosed herein. See, e.g., graham et al 1973,Virology 52:456; sambrook et al 2001,Molecular Cloning:A Laboratory Manual, supra; davis et al 1986,Basic Methods in Molecular Biology,Elsevier; chu et al, 1981, gene 13:197.
As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted igs that bind to Fc receptors (fcrs) present on certain cytotoxic cells, such as Natural Killer (NK) cells, neutrophils, and macrophages, enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for such killing. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in Table 3 at pages 464 of Ravetch and Kinet, annu. Rev. Immunol9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay may be performed, for example as described in U.S. Pat. No. 5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model as disclosed in PNAS (USA) 95:652-656 (1998) by Clynes et al.
The term "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 (C1 q) to antibodies (appropriate subclasses) that bind to their cognate antigens. To assess complement activation, CDC assays may be performed, for example, as described in Gazzano-Santoro et al, J.Immunol. Methods202:163 (1996).
As used herein, the term "EC 50", also referred to as "half maximal effective concentration", refers to the concentration of a drug, antibody, or toxin that induces a 50% response between baseline and maximum after a particular exposure time. In the context of the present application, EC 50 is in units of "nM".
As used herein, the term "Kd" is intended to represent the rate of dissociation of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using well-established methods in the art. The preferred method of determining antibody Kd is by using surface plasmon resonance, preferably using a biosensor system such as a system.
As used herein, the term "VEGF-Sub>A associated disorder" or "VEGF-Sub>A associated disease" refers to any disorder caused, exacerbated, or otherwise associated with increased or decreased expression or activity of VEGF-Sub>A (e.g., human VEGF-Sub>A).
As used herein, the term "cancer" refers to solid and non-solid tumors (e.g., leukemia) mediated by growth, proliferation or metastasis of any tumor or malignant cell that is responsible for a medical disorder.
As used herein, the term "subject" includes any human or non-human animal, preferably a human.
As used herein, "ability to inhibit binding" or "block binding" refers to the ability of an antibody to inhibit the binding of two molecules (e.g., VEGFR and anti-VEGF-Sub>A antibodies) to any detectable extent. In some embodiments, an antibody that blocks binding between two molecules inhibits binding interactions between two molecules by at least 50%. In some embodiments, the inhibition may be greater than 60%, greater than 70%, greater than 80% or greater than 90%.
The term "treatment" as used herein in the context of treating a disease generally relates to treatment and therapy of a human or animal in which some desired therapeutic effect is achieved, e.g., inhibition of disease progression, including a decrease in the rate of progression, a arrest in the rate of progression, regression of the disease, improvement of the disease, and cure of the disease. Treatment as a prophylactic measure (i.e., prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a tumor or malignant cell, or some combination thereof. For a tumor, "treating" includes removing all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the progression of the tumor, or some combination thereof.
As used herein, the term "therapeutically effective amount" refers to an amount of an active compound or a material, composition or dosage form comprising an active compound that is effective for producing certain desired therapeutic effects commensurate with a reasonable benefit/risk ratio when administered according to a desired therapeutic regimen. In particular, sub>A "therapeutically effective amount" means an amount or concentration of an antibody effective to treat Sub>A human VEGF-A related disease or disorder.
As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient, and/or salt thereof is chemically and/or physically compatible with the other ingredients in the formulation, and physiologically compatible with the recipient.
As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active agent, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Modified by Gennaro AR, 19 th edition, pennsylvania: mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
As used herein, the term "adjuvant" refers to a non-specific immunopotentiator that, when delivered to an organism with an antigen or delivered to an organism in advance, can enhance the immune response to an antigen or alter the type of immune response in an organism. There are various adjuvants including, but not limited to, aluminum adjuvants (e.g., aluminum hydroxide), freund's adjuvants (e.g., freund's complete adjuvant and Freund's incomplete adjuvant), corynebacterium parvum, lipopolysaccharide, cytokines, and the like. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.
VEGF-A binding molecules
In some aspects, the invention comprises VEGF-A binding molecules.
In general, VEGF-A binding molecules may comprise any molecule that specifically binds VEGF-A. In some cases, sub>A "VEGF-A binding molecule" may comprise Sub>A "VEGF antagonist". By "VEGF antagonist" is meant any compound or biological molecule that blocks VEGF-A binding to VEGFR. The VEGF-A binding molecule or VEGF antagonist may be Sub>A polypeptide or protein, such as an antibody, more specifically an anti-VEGF-A antibody.
Antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or single domain antibodies. In particular embodiments, the VEGF-A binding molecule is Sub>A single domain antibody, which generally refers to an antibody consisting of Sub>A single monomer variable antibody domain. Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen.
More specifically, the VEGF-A binding molecule is Sub>A single domain antibody, preferably Sub>A heavy chain single domain antibody, which may be used interchangeably with the terms "VHH", "VHH antibody", "VHH domain", "VHH antibody fragment", "VHH" or "nanobody", etc. VHH molecules from camelidae antibodies are one of the smallest known complete antigen binding domains (about 15kDa, or 1/10 of conventional IgG) and are therefore well suited for delivery to dense tissues and into the limited space between macromolecules.
The single domain antibodies of the invention disclosed herein can be prepared by one of skill in the art according to methods known in the art or any future method. For example, VHH can be obtained using methods known in the art, for example by immunizing a camel and obtaining hybridomas therefrom, or by cloning a library of VHH of the invention using molecular biology techniques known in the art, followed by selection using phage display.
For example, single domain antibodies can be obtained by immunizing llama or alpaca with the desired antigen and subsequently isolating the mRNA encoding the heavy chain antibody. By reverse transcription and polymerase chain reaction, a gene library containing millions of cloned single domain antibodies was generated. Screening techniques such as phage display and ribosome display help identify antigen-binding clones. One technique is phage display, in which a library of (preferably human) antibodies is synthesized on phage, the library is screened with an antigen of interest or antibody binding portion thereof, and the antigen-binding phage is isolated from which immunoreactive fragments can be obtained. Methods for preparing and screening such libraries are well known in the art, and kits for generating phage display libraries are commercially available (e.g., pharmacia recombinant phage antibody system, catalog No. 27-9400-01; and STRATAGENE SURFZAPTM phage display kit, catalog No. 240612). Still other methods and reagents can be used to generate and screen antibody display libraries (see, e.g., barbas et al, proc. Natl. Acad. Sci. USA88:7978-7982 (1991)).
When the most potent clone is identified, its DNA sequence is optimized, for example, by affinity maturation or humanization. Humanization can prevent immune responses in humans against antibodies.
Thus, a single domain antibody can be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody; (2) By expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) By "humanization" of naturally occurring VHH domains (as described below) or by expression of nucleic acids encoding such humanized VHH domains; (4) By "camelization" of naturally occurring VH domains from any animal species, particularly mammalian species, e.g. from humans, or by expression of nucleic acids encoding such camelized VH domains; (5) "camelization" by "domain antibodies" or "dabs" as described by Ward et al (supra), or by expression of nucleic acids encoding such camelized VH domains; (6) Using synthetic or semi-synthetic techniques to prepare proteins, polypeptides or other amino acid sequences; (7) Preparing a nucleic acid encoding a VHH by using techniques for nucleic acid synthesis, and then expressing the nucleic acid thus obtained; and/or (8) by any combination of the foregoing. Suitable methods and techniques for performing the foregoing will be apparent to those skilled in the art based on the disclosure herein, and include, for example, the methods and techniques described in more detail below.
Single domain antibodies are typically generated by PCR cloning a library of variable domains into phage display vectors from blood, lymph node or spleen cdnas obtained from immunized animals. Antigen-specific single domain antibodies are typically selected by panning a library on immobilized antigen (e.g., antigen coated on the surface of tube plastic, biotinylated antigen immobilized on streptavidin beads or membrane proteins expressed on the cell surface). Affinity of adAb can be increased by in vitro modeling of this strategy, e.g.by site-directed mutagenesis of the CDR regions and further panning of immobilized antigen under increased stringency conditions (higher temperature, high or low salt concentration, high or low pH and low antigen concentration) (Wesolowski et al .,Single domain antibodies:promising experimental and therapeutic tools in infection and immunity.Med Microbiol Immunol(2009)198:157-174).
Methods for preparing VHHs that specifically bind to an antigen or epitope are described in the references, see for example: van DER LINDEN et al, journal of Immunological Methods,240 (2000) 185-195; li et al, J Biol chem, 287 (2012) 13713-13721; deffar et al, african Journal of Biotechnology vol.8 (12), pp.2645, 17june,2009 and WO94/04678.
In some embodiments, the VHH in the VEGF-Sub>A binding molecule is fused to an Fc domain of an antibody (e.g., an Fc domain of an IgG (e.g., igG4 or IgG 1)). In a specific embodiment, the Fc-domain is that of human IgG 4. By fusing VHH to Fc domains, effector functions can be more effectively recruited. Furthermore, fusion of VHH to Fc domains can help the VEGF-Sub>A binding molecules form dimers, and can also help to extend the in vivo half-life of the VEGF-Sub>A binding molecules.
In some embodiments, the antibodies provided herein further comprise one or more conjugate moieties. The conjugate moiety is a moiety that can be linked to an antibody. Such as a detectable label (e.g., luminescent label, fluorescent label, enzyme-substrate label), a modulator (e.g., a polymer such as PEG that extends half-life), or other therapeutic agent. It is contemplated that a variety of conjugate moieties may be linked to an antibody or antigen binding fragment thereof provided herein (see, e.g., "conjugate vaccine (Conjugate Vaccines)", contributions to microbiology and immunology (Contributions to Microbiology and Immunology), j.m.use and r.e.lewis, jr. (ed.), canager press (CARGER PRESS), (1989) of New York). These conjugate moieties may be attached to the antibody or antigen binding fragment thereof by covalent binding, affinity binding, intercalation, coordination binding, complexing, binding (association), blending (blending), or addition, among others.
Anti-VEGF-A antibodies comprising CDRs
In some embodiments, sub>A vascular endothelial growth factor Sub>A (VEGF-Sub>A) binding molecule of the invention, wherein the VEGF-Sub>A binding molecule comprises Sub>A heavy chain variable region (VH) comprising:
1) HCDR1, HCDR2, HCDR3: which respectively comprises amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
2) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
3) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;
4) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively;
5) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively;
6) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 21 and SEQ ID NO. 3;
7) HCDR1, HCDR2, HCDR3: which respectively comprise the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 22 and SEQ ID NO. 3.
In some specific embodiments, the VEGF-Sub>A binding molecule is Sub>A heavy chain single domain antibody (VHH) comprising:
1) HCDR1, HCDR2, HCDR3: which respectively comprises amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
2) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
3) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;
4) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively;
5) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively;
6) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 21 and SEQ ID NO. 3;
7) HCDR1, HCDR2, HCDR3: which respectively comprise the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 22 and SEQ ID NO. 3.
Unless otherwise indicated, the assignment of amino acids to each CDR may be according to one of the numbering schemes provided below: kabat et al (1991) Sequences of Proteins of Immunological Interest (5 th edition), US Dept.of HEALTH AND Human Services, PHS, NIH, NIH Publication No.91-3242; chothia et al, 1987, PMID:3681981; chothia et al, 1989, PMID:2687698; macCallum et al, 1996, PMID:8876650; or Dubel (2007) Handbook of Therapeutic Antibodies, 3 rd edition, wily-VCH VERLAG GmbH and co.
The variable regions and CDRs in an antibody sequence can be identified according to general rules that have been developed in the art (as described above, e.g., the Kabat numbering system) or by aligning the sequences with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel, inc., antibody Engineering, springer, new York, NY,2001 and Dinarello et al, current Protocols in Immunology, john Wiley and Sons Inc., hoboken, NJ, 2000. Exemplary databases of antibody sequences are described and available on www.bioinf.org.uk/abs under the "Abysis" website (maintained by a.c. martin, university of London biochemistry and molecular biology system (DEPARTMENT OF BIOCHEMISTRY & Molecular Biology University College London, london, england) of London, uk) and under the VBASE2 website www.vbase2.org, as described by Retter et al, nucleic acids res, 33 (Database issue): D671-D674 (2005). The sequences are preferably analysed using the Abysis database, which integrates sequence data from Kabat, IMGT and Protein Databases (PDB) with structural data from PDB, see Protein Sequence and Structure Analysis of Antibody Variable Domains.In:Antibody Engineering Lab Manual(Ed.:Duebel,S. and Kontermann, r., springer-Verlag, heidelberg, ISBN-13:978-3540413547, also available on the website bioin for. The Abysis database website also includes general rules that have been developed for identifying CDRs that can be used in accordance with the teachings herein. All CDRs described herein are obtained according to the Abysis database website of Kabat, unless otherwise indicated.
Anti-VEGF-A antibodies defined by VHH sequences
In some specific embodiments, the VEGF-Sub>A binding molecule is Sub>A heavy chain single domain antibody (VHH) comprising:
1) An amino acid sequence set forth in SEQ ID NO. 4, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 4;
2) An amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 8;
3) An amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 12;
4) The amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 16;
5) An amino acid sequence shown in SEQ ID NO. 20, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 20;
6) An amino acid sequence shown in SEQ ID NO. 23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 23;
7) The amino acid sequence shown in SEQ ID NO. 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 24;
8) An amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 25;
9) The amino acid sequence shown in SEQ ID NO. 26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 26;
10 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 27;
11 28 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 28;
12 29 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 29;
13 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 30;
14 31 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 31.
The percent identity between two amino acid sequences can be determined using the algorithm of e.meyers and w.miller (comp. Appl. Biosci.,4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined by the algorithms of Needleman and Wunsch (j.mol. Biol.48:444-453 (1970)), which have been incorporated into the GAP program in the GCG software package (available from http:// www.gcg.com) using either the Blossum 62 matrix or PAM250 matrix, with GAP weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6.
Antibody variants
In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.
A) Substitution, insertion, and deletion variants
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVR (Hyper-Variable Region) and FR. Conservative substitutions are shown in table a under the heading of "preferred substitutions". More substantial variations are provided in table a under the heading of "exemplary substitutions" and are described further below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
Table A
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Amino acids can be grouped according to common side chain characteristics as follows:
(1) Hydrophobic: norleucine, met, ala, val, leu, IIe;
(2) Neutral, hydrophilic: cys, ser, thr, asn, gin;
(3) Acidic: asp, glu;
(4) Alkaline: his, lys, arg;
(5) Residues that affect chain orientation: gly, pro;
(6) Aromatic: trp, tyr, phe.
Non-conservative substitutions may require replacement of one of these categories with a member of the other category.
One class of substitution variants involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further investigation will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).
Changes (e.g., substitutions) may be made to the HVR, for example, to improve antibody affinity. Such changes may be made to HVR "hot spots", i.e., residues encoded by codons that undergo mutations at high frequencies during the somatic maturation process (see, e.g., chowdhury, methods mol. Biol.207:179-196 (2008)), and/or residues that contact antigen, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described, for example, in Hoogenboom et al, methods in Molecular Biology 178:l_37 (code of O' Brien et al, human Press, totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by a variety of methods (e.g., error-prone PCR, strand 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 HVR-directed approaches in which several 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. In particular, CDR-H3 and CDR-L3 are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to the HVR that do not substantially reduce binding affinity. For example, such changes may be outside of antigen-contacting residues in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1,2, or 3 amino acid substitutions.
In some further embodiments, an anti-VEGF-Sub>A antibody may comprise conservative substitutions or modifications of amino acids in the variable regions of the heavy and/or light chains. It is understood in the art that certain conservative sequence modifications may be made that do not eliminate antigen binding (see, e.g., brummel et al (1993) Biochem 32:1180-8; de Wildt et al (1997) prot. Eng.10:835-41; komisarov et al (1997) J. Biol. Chem.272:26864-26870; hall et al (1992) J. Immunol.149:1605-12; kelley and O' Connell (1993) Biochem.32:6862-35; adib-Conquy et al (1998) int. Immunol.10:341-6 and beer et al (2000) Clin. Can. Res. 6:2835-43).
The term "conservative substitution" as used herein refers to an amino acid substitution that does not adversely affect or alter the basic properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions include substitutions in which an amino acid residue is substituted by another amino acid residue having a similar side chain, such as a substitution of a physically or functionally similar residue (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al, proteinEng.12 (10): 879-884 (1999); and Burks et al, proc. Natl. Acad. Sci. USA 94:412-417 (1997), which is incorporated herein by reference).
In some specific embodiments, the VHH consists of 1) the amino acid sequence shown in SEQ ID NO. 4; 2) The amino acid sequence shown in SEQ ID NO. 8; 3) The amino acid sequence shown in SEQ ID NO. 12; 4) The amino acid sequence shown in SEQ ID NO. 16; 5) The amino acid sequence shown in SEQ ID NO. 20; 6) The amino acid sequence shown in SEQ ID NO. 23; 7) The amino acid sequence shown in SEQ ID NO. 24; 8) The amino acid sequence shown in SEQ ID NO. 25; 9) The amino acid sequence shown in SEQ ID NO. 26; 10 Amino acid sequence shown in SEQ ID NO. 27; 11 28. SEQ ID NO. 28; 12 29. SEQ ID NO. 29; 13 SEQ ID NO. 30; 14 SEQ ID NO. 31.
B) Glycosylation variants
In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.
Where the antibody comprises an Fc region, the carbohydrate to which it is attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, double-antennary oligosaccharides, which are typically attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, e.g., wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GIcNAc in the "backbone" of the double-antennary oligosaccharide structure. In some embodiments, oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.
C) Variant Fc region
In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, igG2, igG3, or IgG4Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.
In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications in which the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to confirm reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus potentially ADCC activity), but retains FcRn binding capacity. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, annu.Rev.TmMunol "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. No.5,500,362 (see, e.g., hellstrom, I et al, proc.Nat 'I Acad.Sci USA83:7059-7063 (1986)) and Hellstrom, I et al, proc.Nat' I Acad.Sci.USA82:1499-1502 (1985); 5,821, 337 (see Bruggemann, m et al, J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays (see, e.g., ACT ITM non-radioactive cytotoxicity assays for flow cytometry (Cell Technology, inc. Mountain View, CA; and CytoTox96 non-radioactive cytotoxicity assays (Promega, madison, WI)). Effector cells useful for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells or/and in addition, ADCC activity of molecules of interest can be assessed in vivo, e.g., in animal models such as those disclosed in Clynes et al, proc na' I Acad Sci USA 95:652-656 (1998)) also Clq binding assays can be performed to confirm that antibodies cannot bind Clq and thus lack CDC activity see, e.g., clq and C3C binding elisa in WO2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J "immunol. Methods 202:163 (1996)), crag, MS. et al, blood 101:1045-1052 (2003), and" crag, M "S" and M "glen S" 103:103 (1998)) also see, e.g., in vivo clearance in Blood (2006. 27kov. 27.38, 17) and in vivo (17.175. 35) gastric 3C) methods.
In some specific embodiments, the Fc domain is the amino acid sequence shown as SEQ ID NO. 32, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 32.
D) Cysteine engineered antibody variants
In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residue is present at an accessible site of the antibody. By replacing those residues with cysteines, reactive thiol groups are thereby located at accessible sites of the antibody, and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as described further herein. Cysteine engineered antibodies may be generated as described, for example, in U.S. patent No.7,521,541.
E) Antibody derivatives
In certain embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers), and dextran or poly (eta-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary and if more than one polymer is attached they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular characteristics or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under specified conditions, and the like.
In other specific embodiments, the anti-VEGF-Sub>A antibody is Sub>A chimeric antibody comprising one VHH fused to an Fc domain of human IgG1 or IgG 4. In one embodiment, the anti-VEGF-A antibody is Sub>A chimeric antibody consisting of one VHH and the Fc domain of human IgG1, and this antibody is designated "HY004001-X, HY004002-X, HY004003-X, HY004004-X, HY004005-X" in the context of the present invention.
Nucleic acid molecules encoding antibodies of the invention
In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence encoding a VHH as disclosed herein.
The nucleic acids of the invention may be obtained using standard molecular biology techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), nucleic acids encoding such antibodies can be recovered from the gene library.
Exemplary nucleic acid molecules of the invention are: 1) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 4; 2) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 8; 3) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 12; 4) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 16; 5) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 20; 6) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 23; 7) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 24; 8) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 25; 9) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 26; 10 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 27; 11 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 28; 12 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 29; 13 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 30; 14 A nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 31; or preferably, the nucleic acid further comprises a nucleotide coding sequence for the amino acid sequence shown in SEQ ID NO. 32. In some embodiments, the nucleic acid has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a nucleotide coding sequence of the above amino acid sequence. In some embodiments, the percent identity results from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.
Carrier body
Nucleic acid molecules encoding anti-VEGF-A antibodies may be inserted into vectors for further cloning (amplification of DNA) or for expression using recombinant techniques known in the art. In another embodiment, the antibodies may be produced by homologous recombination as known in the art. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy chain of the antibody). Many vectors are available. The carrier component generally includes, but is not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters (e.g., SV40, CMV, EF-1 a), and transcription termination sequences. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216;4,634,665 and 5,179,017). For example, selectable marker genes typically confer resistance to drugs (e.g., G418, hygromycin or methotrexate) on the host cell into which the vector has been introduced. Selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for DHFR-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
In some embodiments, the vector system includes mammalian, bacterial, yeast systems, and the like, and includes plasmids such as, but not limited to ,pALTER,pBAD,pcDNA,pCal,pL,pET,pGEMEX,pGEX,pCI,pCMV,pEGFP,pEGFT,pSV2,pFUSE,pVITRO,pVIVO,pMAL,pMONO,pSELECT,pUNO,pDUO,Psg5L,pBABE,pWPXL,pBI,p15TV-L,pPro18,pTD,pRS420,pLexA,pACT2.2, and the like, as well as other laboratory and commercially available vectors. Suitable vectors may include plasmids or viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses). In one embodiment of the invention, the vector may be pET, for example pETbac containing a hexahistidine tag and a c-Myc-tag gene.
Host cells
Vectors comprising nucleic acid sequences encoding VEGF-A binding molecules may be introduced into host cells for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose include eubacteria, e.g., gram-negative or gram-positive organisms, e.g., enterobacteriaceae such as escherichia (e.g., escherichia coli), enterobacteriaceae, erwinia, klebsiella, proteus, salmonella such as salmonella typhimurium, serratia such as serratia marcescens, and shigella, and bacillus such as bacillus subtilis and bacillus licheniformis, pseudomonas such as pseudomonas aeruginosa and streptomyces.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-VEGF-A antibody encoding vectors. Saccharomyces cerevisiae or Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and can be used in the present invention, such as schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as Kluyveromyces lactis (K.lactis), kluyveromyces fragilis (K.fragilis) (ATCC 12,424), kluyveromyces bulgaricus (K.bulgaricus) (ATCC 16,045), kluyveromyces wikiwi (K.winkeramii) (ATCC 24,178), K.walti (ATCC 56,500), kluyveromyces drosophila (K.drosophila) (ATCC 36,906), kluyveromyces thermotolerans (K.thermostolerans) and Kluyveromyces marxianus (K.marxianus); achillea millefolium (yarrowia) (EP 402,226); pichia pastoris (EP 183,070); candida (Candida); trichoderma reesia (EP 244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces) such as Schwannomyces western (Schwanniomyces occidentalis); and filamentous fungi, such as Neurospora (Neurospora), penicillium (Penicillium), tolypocladium, and Aspergillus hosts such as Aspergillus nidulans (A. Nidulans) and Aspergillus niger (A. Niger).
Other suitable host cells for expressing the anti-VEGF-A antibodies provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants and corresponding permissive insect host cells have been identified from the following hosts: spodoptera frugiperda (Spodoptera Frugiperda, carpenterworm), aedes aegypti (AEDES AEGYPTI, mosquito), aedes albopictus (Aedes albopictus, mosquito), drosophilamelanogaster (drosophila) and Bombyx mori (Bombyx mori). Various strains for transfection are publicly available, for example the L-1 variant of the NPV of Spodoptera frugiperda (Autographa californica) and the Bm-5 strain of the NPV of Bombyx mori, and according to the invention these viruses can be used as viruses herein, in particular for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.
Host cells are transformed with the expression or cloning vectors described above for anti-VEGF-A antibody production and cultured in conventional nutrient mediSub>A modified as necessary to induce promoters, select transformants, or amplify genes encoding the desired sequences.
The host cells used to produce the anti-VEGF-A antibodies provided herein may be cultured in Sub>A variety of mediSub>A. Commercially available media such as Ham's F (Sigma), minimal essential media (MINIMAL ESSENTIAL Medium, MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (DMEM, sigma) are suitable for culturing host cells. Furthermore, ham et al, meth.Enz.58:44 (1979); barnes et al, anal. Biochem.102:255 (1980); U.S. patent No. 4,767,704;4,657,866;4,927,762;4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or any of the media described in U.S. Pat. re.30,985 may be used as a medium for the host cells. Any of these media may be supplemented with hormones and/or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin (GENTAMYCINTM) drug), trace elements (defined as inorganic compounds, typically present at final concentrations in the micromolar range), and glucose or equivalent energy sources, as desired. Any other necessary supplements may also be included in suitable concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those used with the host cells previously selected for expression and will be apparent to the ordinarily skilled artisan.
When recombinant techniques are used, the antibodies may be produced intracellularly, in the periplasmic space, or secreted directly into the medium. If antibodies are produced intracellularly, as a first step, the particulate debris (host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al, bio/Technology 10:163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (ph 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) within about 30 minutes. Cell debris can be removed by centrifugation. In the case of antibody secretion into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.
Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.
After any one or more preliminary purification steps, the mixture comprising the antibody of interest and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5 and 4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).
Pharmaceutical composition
In some aspects, the invention relates to Sub>A pharmaceutical composition comprising at least one VEGF-Sub>A binding molecule as disclosed herein and Sub>A pharmaceutically acceptable carrier.
The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-VEGF-Sub>A antibodies enhance the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, combinations of various components known in the art, or more.
Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethylanisole, butylated hydroxytoluene and/or propyl arsenate. As disclosed herein, the antibody-containing compositions of the present invention comprise one or more antioxidants, such as methionine, to reduce oxidation of the antibody. The reduction in oxidation may prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the invention provides compositions comprising one or more antibodies and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibodies are admixed with one or more antioxidants, such as methionine, so that the antibodies are protected from oxidation to extend their shelf life and/or increase activity.
To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous carriers such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactated ringer's injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed, corn, sesame or peanut oil, antimicrobial or fungistatic concentrations of antimicrobial agents, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), sequestering or chelating agents such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent used as a carrier may be added to a pharmaceutical composition in a multi-dose container containing phenol or cresol, a mercuric preparation, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.
Administration, formulation and dosage
The pharmaceutical compositions of the invention may be administered to a subject in need thereof in vivo by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal and intrathecal, or by implantation or inhalation. The compositions of the present invention may be formulated as solid, semi-solid, liquid or gaseous forms of formulation; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants and aerosols. The appropriate formulation and route of administration may be selected depending upon the intended application and treatment regimen.
Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalants and controlled release dosage forms thereof.
Formulations suitable for parenteral administration (e.g., by injection) include aqueous or nonaqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable ingredients such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents and solutes which render the formulation isotonic with the blood (or other relevant body fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerin, vegetable oils, and the like. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, ringer's solution or lactated ringer's injection. Similarly, the particular dosage regimen (i.e., dosage, time and repetition) will depend on the particular individual and medical history of the individual, and empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).
The frequency of administration can be determined and adjusted during treatment and based on reducing the number of proliferating or tumorigenic cells, maintaining such a reduction in tumor cells, reducing proliferation of tumor cells or delaying the development of metastasis. In some embodiments, the administered dose may be adjusted or reduced to control potential side effects and/or toxicity. Alternatively, sustained-release formulations of the therapeutic compositions of the present invention may be suitable.
Those skilled in the art will appreciate that the appropriate dosage may vary from patient to patient. Determining the optimal dose generally involves balancing the level of therapeutic benefit with any risk or adverse side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of clearance of the compound, the duration of treatment, other co-administered drugs, compounds and/or materials, the severity of the disease, as well as the species, sex, age, weight, disease, general health and previous medical history of the patient. The amount of the compound and the route of administration are ultimately at the discretion of the physician, veterinarian or clinician, but the dosage is typically selected to achieve the local concentration at the site of action of the desired effect without causing substantial adverse or adverse side effects.
In general, VEGF-A binding molecules may be administered in Sub>A variety of ranges. In some embodiments, VEGF-A binding molecules provided herein may be administered at Sub>A therapeutically effective dose of about 0.01mg/kg to about 100mg/kg (e.g., about 0.01mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, or about 100 mg/kg). In some of these embodiments, the antibody is administered at a dose of about 50mg/kg or less, and in some of these embodiments, the dose is 10mg/kg or less, 5mg/kg or less, 1mg/kg or less, 0.5mg/kg or less, or 0.1mg/kg or less. In certain embodiments, the dosage administered may vary during the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequent administered dose. In certain embodiments, the dosage administered may vary during the course of treatment depending on the subject's response.
In any event, the antibodies of the invention are preferably administered to a subject in need thereof as desired. The frequency of administration can be determined by one of skill in the art, for example, based on considerations by the treating physician, the age of the subject being treated, the severity of the disease being treated, the general health of the subject being treated, and the like.
In certain preferred embodiments, the course of treatment involving the antibodies of the invention will comprise multiple doses of the selected drug product administered over a period of weeks or months. More specifically, the antibodies of the invention may be administered once per day, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage may be varied or the time interval adjusted based on patient response and clinical practice.
The dosage and regimen of the disclosed therapeutic compositions can also be determined empirically in individuals administered one or more administrations. For example, an incremental dose of a therapeutic composition produced as described herein may be administered to an individual. In selected embodiments, the dosage may be gradually increased or decreased or reduced in side effects or toxicity, respectively, as determined empirically or observed. To assess the efficacy of a selected composition, a particular disease, disorder, or marker of disease may be tracked as described previously. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopy of tumor samples; measuring the reduction of pain or paralysis of an indirect tumor marker (e.g., PSA for prostate cancer) or tumorigenic antigen identified according to the methods described herein; improvement of speech, vision, respiration or other disability associated with tumors; appetite increases; or an improvement in quality of life or an increase in survival as measured by the accepted test. It will be appreciated by those skilled in the art that the dosage will vary depending on the individual, the type of neoplastic disease, the stage of neoplastic disease, whether neoplastic disease has begun to metastasize to other locations in the individual, and the treatments used in the past and in parallel.
Compatible formulations for parenteral administration (e.g., intravenous injection) may comprise VEGF-A binding molecules as provided herein at Sub>A concentration of about 10 μg/ml to about 100mg/ml. In some embodiments, the concentration of VEGF-A binding molecules may comprise 20μg/ml、40μg/ml、60μg/ml、80μg/ml、100μg/ml、200μg/ml、300μg/ml、400μg/ml、500μg/ml、600μg/ml、700μg/ml、800μg/ml、900μg/ml or 1mg/ml. In other preferred embodiments, the concentration of VEGF-A binding molecules will comprise 2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、8mg/ml、10mg/ml、12mg/ml、14mg/ml、16mg/ml、18mg/ml、20mg/ml、25mg/ml、30mg/ml、35mg/ml、40mg/ml、45mg/ml、50mg/ml、60mg/ml、70mg/ml、80mg/ml、90mg/ml or 100mg/ml.
Application of the invention
The VEGF-A binding molecules of the invention have Sub>A number of in vitro and in vivo uses. For example, these molecules may be administered to cultured cells in vitro or ex vivo, or to a human subject in vivo, for example, to inhibit angiogenesis.
Preferred subjects include human patients in need of inhibition of angiogenesis. The methods are particularly useful for treating human patients suffering from conditions treatable by the specific binding of VEGF (VEGFR) thereby inhibiting downstream signaling pathways for the purpose of inhibiting angiogenesis. In a specific embodiment, the method is particularly suitable for treating cancer in vivo. For the purpose of inhibiting angiogenesis, an anti-VEGF-Sub>A antibody may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g. Sub>A subject carrying Sub>A tumor or virus). When an anti-VEGF-Sub>A antibody is administered with another agent, the two may be administered in any order or simultaneously.
The invention further provides Sub>A method for detecting the presence of VEGF-A antigen in Sub>A sample or measuring the amount of human VEGF-A antigen, comprising contacting the sample and Sub>A control sample with Sub>A VEGF-A binding molecule under conditions that allow Sub>A complex to form between the VEGF-A binding molecule and VEGF. Complex formation is then detected, wherein differential complex formation between samples as compared to Sub>A control sample indicates the presence of VEGF-Sub>A antigen in the sample. Furthermore, the VEGF-A binding molecules of the invention may be used to purify human VEGF-A by immunoaffinity purification.
Treatment of cancer
The diseases and conditions associated with VEGF-A may be immune related diseases or conditions.
In some embodiments, the VEGF-Sub>A related disorders and conditions include tumors and cancers, including but not limited to: breast cancer, liver cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, lung cancer, thyroid cancer, nasopharyngeal cancer, renal cancer, head and neck cancer, and pancreatic cancer.
In certain embodiments, the tumor and cancer are metastatic, particularly metastatic tumors that express VEGF-A.
Combined use of chemotherapy
The antibodies may be used in combination with chemotherapy or radiation therapy. The antibodies may be used in combination with an anticancer agent, a cytotoxic agent, or a chemotherapeutic agent.
The term "anti-cancer agent" or "antiproliferative agent" means any agent that can be used to treat cell proliferative disorders such as cancer, and includes, but is not limited to: cytotoxic agents, cytostatic agents, anti-angiogenic agents, deconvolution agents (debulking agents), chemotherapeutic agents, radiation therapy and radiotherapy agents, targeted anti-cancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormonal therapies, radiation therapy and anti-metastatic agents and immunotherapeutic agents. It will be appreciated that in selected embodiments as described above, such anti-cancer agents may comprise conjugates and may be conjugated to the disclosed site-specific antibodies prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to a unpaired cysteine of an engineered antibody to provide an engineered conjugate as described herein. Accordingly, such engineered conjugates are expressly included within the scope of the present invention. In other embodiments, the disclosed anti-cancer agents will be administered in combination with site-specific conjugates comprising different therapeutic agents as described above.
As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cellular function and/or causes cell destruction. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, bacterial (e.g., diphtheria toxin, pseudomonas endotoxin and exotoxin, staphylococcal enterotoxin a), fungal (e.g., α -sarcina, restrictocin), plant (abrin, ricin, pristimerin, mistletoe, pokeweed antiviral protein, saporin, gelonin, momoridin, trichosanthin, barley toxin, aleurites (Aleurites fordii) protein, caryophyllin protein, phytolacca mericana protein (PAPI, PAPII and PAP-S), balsam pear inhibitors, jatrophin, croton toxin, lycopodium inhibitors, gelonin, mitegellin, restrictocin, phenol mycin, neomycin and trichothecene compounds) or animal (e.g., cytotoxic rnases, such as exopancreatic rnases; dnase I, including fragments and/or variants thereof).
For the purposes of the present invention, "chemotherapeutic agent" includes chemical compounds (e.g., cytotoxic or cytostatic agents) that non-specifically reduce or inhibit the growth, proliferation and/or survival of cancer cells. These chemicals are generally directed to intracellular processes required for cell growth or division and are therefore particularly effective for cancer cells that are typically fast growing and dividing. For example, vincristine depolymerizes microtubules, thereby inhibiting the entry of cells into mitosis. In general, a chemotherapeutic agent may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic offspring (e.g., TICs). These agents are typically used in combination and are typically the most effective, for example, in a regimen such as CHOP or FOLFIRI.
Examples of anticancer agents that may be used in combination with the site-specific constructs of the invention (as a component of the site-specific conjugate or in the unconjugated state) include, but are not limited to, alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methyl melamines, polyacetyls (acetogenins), camptothecins, bryostatin, calistatin (callystatin), CC-1065, kerithin (cryptophycins), dolastatin, docarpium, eleutherobin, hydrocine, sha Kedi factor (sarcodictyin), spongosine (spongistatin), nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, epothilones, chromoprotein enediyne antibiotic chromophores aclacinomycins (aclacinomysins), actinomycin, amphotericin, azoserine, bleomycin, actinomycin C, carboxin (carabicin), carminomycin, amphotericin, chromomycins (chromomycinis), dactinomycin, daunorubicin, dithiin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, exenatide, idarubicin, doxycycline, mitomycin, mycophenolic acid, norgamycin, olivomycin, perlecithromycin (potfiromycin), puromycin, tri-iron doxorubicin, rodubicin, streptozocin, streptozotocin, tubercidin, ubemest, net stavudine, zorubicin; anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-epinephrine, folic acid supplements such as furin acid (frolinic acid), aceglucurolactone, aldehyde phosphoramide glycosides, aminolevulinic acid, enimine, amsacrine, bei Sibu (bestrabucil), bisacodyl, idazoxamine, dif-famine (defofamine), colchicine, diaquinone, ifenesin (elfornithine), iri-ammonium acetate, elpaspalon, etodol, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoid (maytansinoids), mitoguazone, mitoxantrone, mo Danma mole (mopidanmol), nitlin (nitraerine), jetstretin, amantadine, pirarubicin, loxoprotein, podophyllonic acid, 2-ethyl, procarba, polysaccharide complex (JHS Natural Products, eugenol, OR alternatively; rhizopus extract; a sirzopyran; germanium spiroamine; temozolomide; triiminoquinone; 2,2',2 "-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verakulin A (verracurin A), cyclosporin a and serpentine; uratam; vindesine; dacarbazine; mannitol; dibromomannitol; dibromodulcitol; pipobromine; -casitoxin (gacytosine); arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes; chlorambucil (chloranbucil); gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; a platinum analog; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine, vinorelbine; norxiaoling; teniposide; eda traxas; daunorubicin; aminopterin; hilded; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine; a retinoid; capecitabine; combretastatin; leucovorin; oxaliplatin; inhibitors of PKC-Sub>A, raf, H-Ras, EGFR and VEGF-Sub>A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Also included in this definition are anti-hormonal agents used to modulate or inhibit hormonal effects on tumors, such as antiestrogens and selective estrogen receptor modulators, aromatase inhibitors which inhibit aromatase which modulates estrogen production in the adrenal gland, and anti-androgens; troxacitabine (1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccine, rIL-2; topoisomerase 1 inhibitors; rmRH; vinorelbine and epothilone, and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing.
Used in combination with radiotherapy
The invention also provides a combination of antibodies and radiation therapy (i.e., any mechanism for locally inducing DNA damage in tumor cells, such as gamma-irradiation, X-rays, UV-irradiation, microwaves, electron emission, etc.). Combination therapies using targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed conjugates can be used in combination with targeted anticancer agents or other targeting means. Typically, radiation therapy is administered in pulses over a period of about 1 week to about 2 weeks. Radiation therapy may be administered to a subject with head and neck cancer for about 6 to 7 weeks. Optionally, radiation therapy may be administered as a single dose or as multiple sequential doses.
Diagnosis of
The present invention provides in vitro and in vivo methods for detecting, diagnosing or monitoring proliferative disorders and methods of screening cells from a patient to identify tumor cells, including tumorigenic cells. Such methods comprise identifying an individual having cancer for treatment or monitoring progression of cancer, comprising contacting a patient or a sample obtained from the patient (in vivo or in vitro) with an antibody described herein, and detecting the presence or absence or level of binding of the bound antibody to a bound or free target molecule in the sample. In some embodiments, the antibody will comprise a detectable label or reporter as described herein.
In some embodiments, binding of an antibody to a particular cell in a sample may indicate that the sample may contain tumorigenic cells, thereby indicating that an individual with cancer may be effectively treated with an antibody as described herein.
Samples may be analyzed by a variety of assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), competitive binding assays, fluorescent immunoassays, immunoblot assays, western blot analysis, and flow cytometry assays. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computerized tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, and the like, as known to those skilled in the art.
Pharmaceutical package and kit
Pharmaceutical packages and kits comprising one or more containers containing one or more doses of the antibodies are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody, with or without one or more other agents. For other embodiments, such unit doses are supplied in single use, pre-filled syringes. In other embodiments, the compositions contained in the unit dose may comprise saline, sucrose, or the like; buffers such as phosphates and the like; and/or formulated in a stable and effective pH range. Alternatively, in some embodiments, the conjugate composition may be provided as a lyophilized powder, which may be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. Any label on or associated with the container indicates that the encapsulated conjugate composition is to be used to treat the selected neoplastic disease condition.
The invention also provides kits for producing single or multi-dose administration units of the site-specific conjugates and optionally one or more anticancer agents. The kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugate in conjugated or unconjugated form. In other preferred embodiments, the container includes a sterile access port (e.g., the container may be an intravenous bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits typically comprise a pharmaceutically acceptable formulation of the engineered conjugate in a suitable container, and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, such a kit may contain, in addition to an antibody of the invention, any one or more anti-cancer agents, such as chemotherapeutic agents or radiotherapeutic agents; an anti-angiogenic agent; an anti-metastatic agent; targeting anticancer agents; a cytotoxic agent; and/or other anticancer agents.
More specifically, the kits may have a single container containing the disclosed antibodies, with or without additional components, or they may have different containers for each desired reagent. Where provided for combined therapeutic agents in combination, a single solution may be pre-mixed in molar equivalent combination or with one component being more than the other. Alternatively, the conjugate of the kit and any optional anticancer agent may be stored separately in separate containers prior to administration to the patient. The kit may further comprise a second/third container means for holding a sterile pharmaceutically acceptable buffer or other diluent, such as bacteriostatic water for injection (BWFI), phosphate Buffered Saline (PBS), ringer's solution and dextrose solution.
When the components of the kit are provided in one or more liquid solutions, the liquid solution is preferably an aqueous solution, particularly preferably a sterile aqueous solution or a saline solution. However, the components of the kit may be provided as a dry powder. When the reagents or components are provided in dry powder form, the powder may be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.
As briefly described above, the kit may also contain means for administering the antibody and any optional components to the patient, such as one or more needles, intravenous (i.v.) injection bags or syringes, or even eye drops, pipettes or other similar devices, by which the formulation may be injected or introduced into the animal body or administered to the affected area of the body. The kits of the present invention also typically include means for holding vials or the like, as well as other tightly closed components for commercial sale, such as injection molded or blow molded plastic containers in which the desired vials and other devices are placed and held.
Summary of the sequence Listing
The application is accompanied by a sequence listing comprising a plurality of nucleic acid and amino acid sequences. Table B below provides an overview of the sequences involved.
Table B
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Examples
The invention described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. These examples are not intended to be an indication that the experiments below are all or only experiments performed.
Example 1 preparation and screening of VEGF-A Single-Domain antibodies
1.1 Immunization of alpaca
Two llamas were immunized with human VEGF-A 165 protein (purchased from Beijing Yiqiao Shenzhou, 11066-HNAH) (Alpaca). The primary immunization dose was 0.4mg with complete adjuvant, followed by 0.2mg each time with incomplete adjuvant, each time with an inter-immunization interval of 2 weeks for a total of eight immunizations.
1.2 Serum immunotiter assay
Starting from the second immunization, 5ml of blood was taken 1 week after each injection for serum immunotiter determination. I.e. 1.1 VEGF-A 165 antigen protein coat on ELISA plate, 200ng per well, after blocking and washing, serum samples starting from a 1:2000 ratio were added, and after incubation binding and washing, development was performed using a secondary antibody against camel monodomain antibody (gold Style, A01861).
1.3 Phage library construction
Blood was taken 10ml after six and eight exemptions per camel, and pooled for construction of phage display libraries of single domain antibodies: firstly, performing RNA extraction, namely separating lymphocytes by using lymphocyte separating liquid (Soxhoba, P8610), treating and cracking the lymphocytes by using an RNA extraction reagent (TaKaRa, 9109), removing impurities by using chloroform and precipitating RNA by using isopropanol, washing the precipitate by using 75% ethanol, drying at room temperature, and dissolving by using water for injection; then reverse transcription is carried out on the RNA by using a reverse transcription kit (Thermo Scientific, K1622) to prepare cDNA; amplifying DNA containing single-domain antibody variable region genes by using a nested PCR method, performing Sfi I digestion on the DNA, and then performing T 4 DNA ligase connection with a display carrier subjected to Sfi I digestion; the ligation products were purified and then electrotransformed into TG1 competent cells, and phage display libraries were prepared using M13KO7 helper phage infection. The final constructed libraries (HYA 1-6,8 and HYA2-6, 8) had a library capacity of 8.1E+09 and 2.04E+09, respectively.
1.4 Panning of phage libraries
Antigen (VEGF-A 165 -Fc, sanyou, PHA 129) was coated on a high adsorption 96 well ELISA plate, 2. Mu.g per well, 6.45E+11 and 6.4E+11cfu library phage from 1.3 above were added, respectively, incubated and bound by shaking at room temperature, washed with PBST wash containing 0.05% Tween-20, and eluted, the titer of the eluted phage was determined, and wells coated with irrelevant target proteins served as parallel controls.
The results are shown in Table 1, and after two rounds of panning, the eluted phage from VEGF-A 165 -Fc wells were enriched relative to the control wells by 12.4 fold and 12.6 fold, respectively.
Table 1: phage library panning data
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1.5 Screening of monoclonal phages
Using the enriched phage library eluted from 1.4, log-phase TG1 bacteria were infected, diluted and plated, and then cloned into 2YT medium containing Carb and M13KO7 helper phage overnight for culture. The following day, the culture plate was centrifuged at 4200rpm, the supernatant was subjected to phage ELISA detection, VEGF-A 165 -coated wells were used as measurement wells, BSA (bovine serum albumin) -coated wells were used as control wells, HRP (horseradish peroxidase) -conjugated anti-M13 antibody (11973-MM 05T-H) was used as secondary antibody, and the bacterial solution of clones with measurement well read (OD 450 nm) of 1 or more and control well read of 0.1 or less was selected for plasmid extraction and gene sequencing, the sequence obtained by sequencing was translated into amino acid sequence, sequence alignment was performed, and the repetitive sequence or similar sequence with CDR3 differing by 1 amino acid was removed to obtain unique sequence. A total of 10 unique sequences were obtained.
Example 2 detection of blocking efficacy based on crude extract of Single Domain antibody Zhou Zhiqiang
The plasmid with unique sequence is transformed into BL21 Rosetta (DE 3) competent bacteria, transferred into 2.5ml 2 XYT culture medium containing Carb antibiotics, and cultured until OD 600 reaches 0.4, and then added with 2 XYT culture medium containing IPTG and Carb, and the final concentration of IPTG is 0.1mM, and cultured overnight at 28 ℃. The next day, the bacterial solution was centrifuged at 4200rpm at 4℃for 15 minutes, the bacterial cells were resuspended in 1ml PBS, and then subjected to three freeze-thawing cycles at-80℃to 37℃to release the single-domain antibodies in the periplasmic chamber. After centrifugation at 4200rpm and 4℃for 30 minutes, the supernatant was taken to prepare a crude extract of the periplasmic cavity single domain antibody.
Then, a blocking ELISA assay of VEGFR2 and VEGF-A 165 -his-Avi (Beepusex, VE5-H82Q 0) was performed using a single domain antibody crude extract, i.e., human VEGFR2-Fc (100-H02H, siemens) was coated on plates overnight at 4℃for 300ng per well, the plates were blocked for 2 hours at room temperature on the next day with 1% BSA (bovine serum albumin), the crude extract was mixed with 0.25. Mu.g/ml of VEGF-A 165 -his-Avi in equal volumes, the crude extract was diluted 1:2 gradient starting from stock solution, total stock solution (1X), 1:2, 1:4, 0 four concentration points, the crude extract was incubated with VEGF-A 165 -his-Avi for 30min at room temperature, incubated at room temperature, the next day with plates were incubated at room temperature, streptavidin-HRP (Shanghai-HRP) (1:3000 ratio) was added, and a single domain control was performed using a similar single domain antibody as the initial target (control, 3234). Absorbance values at 450nm were read using a microplate reader (Biotek, synergy H1 MF).
As a result, as shown in FIG. 1, it was found that 5 clones (P2-10, P2-23, P2-24, P2-26, P2-59) among 10 clones had significant blocking efficacy against VEGFR2-VEGF-A 165.
Example 3 expression and blocking Activity determination of IgG antibodies
The sequences corresponding to clones P2-10, P2-23, P2-24, P2-26, and P2-59 were constructed on top of human IgG1 Fc (containing the N297A mutation) and transiently transfected and expression purified in HEK293 cells, and the expressed molecules were designated HY004001-1, HY004002-1, HY004003-1, HY004004-1, HY004005-1 (SEQ ID NO:33 to SEQ ID NO: 37). Blocking assays for VEGFR2-Fc and VEGF-a 165 were then performed as in example 2 using these molecules, using Eylea biosimilar as positive control, using single domain antibodies targeting unrelated targets as negative control.
As Sub>A result, as shown in FIG. 2, it can be seen that each molecule has Sub>A clear blocking potency, which is weaker than the EyleSub>A biological analog with extremely high affinity for VEGF-A. Of these, HY004001-1 had the best blocking efficacy, and had an EC 50 value of 0.084. Mu.g/ml, which was approximately doubled weaker than Eylea biological analog (EC 50 value of 0.04. Mu.g/ml).
Example 4 modification of post-translational modification sites of antibodies
The sequence of antibody HY004001-1 (SEQ ID NO: 33) was analyzed and found to have a potential deamidation site "NG" in CDR2, which was mutated and engineered to "QG" and "NA", respectively, designated HY004001-2 (SEQ ID NO: 38) and HY004001-3 (SEQ ID NO: 39), respectively, HEK293 was transiently transfected and purified as well, and the purified IgG was tested for blocking efficacy against VEGFR2 and VEGF-A 165 as positive control by Faricimab biological analogues (Baiying organism, B936701) according to the procedure described in example 2.
As a result, as shown in FIG. 3, it was seen that the blocking efficacy of the engineered molecules was slightly reduced and that the blocking efficacy of HY004001-3 was relatively better, but that the blocking efficacy of these molecules was better than that of the control Faricimab biological analog.
Example 5 humanization and Activity determination of antibodies
The antibody HY004001-3 was subjected to structural simulation using SWISS-MODEL, and humanized mutation was evaluated at camel-derived amino acid sites within the non-CDR regions of the sequence based on the resulting structure, and human germline genes V 3-30 x 01 and J 1 x 01 were used as target sequences for humanized mutation, with preferential mutation at amino acid sites on the surface of the antibody structure and not adjacent to the CDRs, postposition mutation at sites within the antibody structure (buried) and adjacent to the CDRs. According to the principle, several humanized antibody sequences were designed: HY004001-3-hz1 to HY004001-3-hz7 (shown as SEQ ID NO:40 to SEQ ID NO: 46), followed by transient transfection expression in HEK 293. After expression was complete, the blocking efficacy of each variant molecule against human VEGFR2-VEGF-A 165 was tested using the method described in example 2.
As a result, as shown in FIG. 4, it was found that humanized variant molecules HY004001-3-hz1 to HY004001-3-hz3 retained the blocking efficacy of parent molecule HY004001-3, while each of HY004001-3-hz4 to HY004001-3-hz7 lost the blocking efficacy due to the design of the variant molecules with higher level of humanization, which suggests that several camel-derived amino acid sites are non-mutable. Of HY004001-3-hz1 to HY004001-3-hz3, the blocking efficacy of HY004001-3-hz1 is best, obviously due to the control antibodies Faricimab, their EC 50 values are 0.081 and 0.335 μg/ml, respectively.
EXAMPLE 6 determination of binding molecule kinetics of antibodies to VEGF-A 165
Binding KD values of HY004001-3-hz1 and Faricimab to VEGF-A 165 (11066-HNAH, yizhushen) were measured using Fortibio methods (Saidoles, octet RH 16), antibodies were captured using an AHC probe, VEGF-A 165 was placed in PBST buffer containing 0.05% Tween-20, 5 concentrations were set, binding time was 180s, dissociation time was 300s, and HY004001-3-hz1 had a KD value significantly better than Faricimab as shown in Table 2.
Table 2. Binding molecule kinetics test results.
Example 7 determination of the blocking efficacy of antibodies against VEGF-A 165 -VEGFR2 mediated intracellular signaling pathway
The inhibition of VEGF-A 165 -initiated VEGFR2 intracellular signaling was tested using a luciferase reporter cell line (Sanyou organism, XHA 007) of the VEGFR2 downstream signaling pathway, the antibodies were mixed with VEGF-A 165 after gradient dilution, incubated for 30 minutes at room temperature, then added to the reporter cells at a final VEGF-A 165 concentration of 10ng/ml, the cells were 4E+04 per well, incubated for 6 hours in a 37℃incubator, and then Bright Light (Nuo Wei Zan, DD 1204-01) was added and the fluorescent signal intensity was measured. Faricimab biological analogs were used as positive controls, and antibodies targeting unrelated targets were used as negative controls.
As shown in FIG. 5, it can be seen that antibody HY004001-3-hz1 can effectively block VEGF-A 165 -VEGFR2 mediated activation of intracellular signaling pathway and has significantly better blocking potency than positive control Faricimab biological analogs, with blocking EC 50 values of 0.012 and 0.031 μg/ml, respectively.
It should be understood that while the present invention has been described by way of example in terms of its preferred embodiments, it is not limited to the above embodiments, but is capable of numerous modifications and variations by those skilled in the art. The selection and use of a particular antibody may be adapted and altered accordingly to the particular needs. It will thus be appreciated that those skilled in the art will be able to devise numerous alternative arrangements which, although not explicitly described herein, embody the principles of the invention and are included within its spirit and scope.
Claims (32)
1. Sub>A vascular endothelial growth factor Sub>A (VEGF-Sub>A) binding molecule, wherein the VEGF-Sub>A binding molecule comprises Sub>A heavy chain variable region (VH), the VH comprising:
1) HCDR1, HCDR2, HCDR3: which respectively comprises amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;
2) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
3) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11;
4) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15 respectively;
5) HCDR1, HCDR2, HCDR3: which comprises the amino acid sequences shown in SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19 respectively;
6) HCDR1, HCDR2, HCDR3: which respectively comprises the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 21 and SEQ ID NO. 3;
7) HCDR1, HCDR2, HCDR3: which respectively comprise the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO. 22 and SEQ ID NO. 3.
2. Sub>A VEGF-Sub>A binding molecule according to claim 1, wherein the VH comprises:
1) An amino acid sequence set forth in SEQ ID NO. 4, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 4;
2) An amino acid sequence set forth in SEQ ID NO. 8, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 8;
3) An amino acid sequence shown in SEQ ID NO. 12, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 12;
4) The amino acid sequence set forth in SEQ ID NO. 16, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 16;
5) An amino acid sequence shown in SEQ ID NO. 20, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 20;
6) An amino acid sequence shown in SEQ ID NO. 23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 23;
7) The amino acid sequence shown in SEQ ID NO. 24, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 24;
8) An amino acid sequence set forth in SEQ ID NO. 25, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 25;
9) The amino acid sequence shown in SEQ ID NO. 26, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 26;
10 27 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 27;
11 28 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 28;
12 29 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 29;
13 30 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 30;
14 31 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 31; .
3. Sub>A VEGF-Sub>A binding molecule according to claim 1 or 2, further comprising one or more amino acid residue mutations but which remain specifically bound to VEGF-Sub>A.
4. Sub>A VEGF-Sub>A binding molecule according to claim 3, wherein at least one of the mutations is in the VH sequence but not in any of the CDR sequences.
5. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 4, wherein the VEGF-Sub>A binding molecule is Sub>A VEGF antagonist, preferably an anti-VEGF-Sub>A antibody.
6. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 5, wherein the VEGF-Sub>A binding molecule is Sub>A chimeric antibody.
7. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 5, wherein the VEGF-Sub>A binding molecule is Sub>A humanized antibody.
8. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 5, wherein the VEGF-Sub>A binding molecule is Sub>A single domain antibody, preferably Sub>A heavy chain single domain antibody (VHH).
9. Sub>A VEGF-Sub>A binding molecule according to any one of claim 8, wherein the VHH is derived from Sub>A camelid comprising alpacSub>A or llamSub>A.
10. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 9, wherein the VH is fused to the Fc domain of IgG.
11. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 10, wherein the VH is fused to the Fc domain of human IgG.
12. Sub>A VEGF-Sub>A binding molecule according to claim 9, wherein the VEGF-Sub>A binding molecule is Sub>A chimeric antibody of VHH from Sub>A camelid with the Fc domain of human IgG.
13. Sub>A VEGF-Sub>A binding molecule according to claim 12, wherein the VEGF-Sub>A binding molecule is Sub>A chimeric antibody of Sub>A VHH from Sub>A camelid with an Fc domain of human IgG1 or IgG 4.
14. Sub>A VEGF-Sub>A binding molecule as claimed in claims 1 to 13 wherein the Fc domain is the amino acid sequence shown in SEQ ID No. 32 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID No. 32.
15. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 13, which comprises:
1) The amino acid sequence set forth in SEQ ID NO. 33, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 33;
2) The amino acid sequence shown in SEQ ID NO. 34, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 34;
3) An amino acid sequence set forth in SEQ ID NO. 35, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 35;
4) The amino acid sequence shown in SEQ ID NO. 36, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 36;
5) The amino acid sequence shown in SEQ ID NO. 37, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 37;
6) The amino acid sequence set forth in SEQ ID NO. 38, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 38;
7) An amino acid sequence set forth in SEQ ID NO. 39, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 39;
8) An amino acid sequence set forth in SEQ ID NO. 40, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence set forth in SEQ ID NO. 40;
9) The amino acid sequence shown in SEQ ID NO. 41, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 41;
10 Amino acid sequence depicted as SEQ ID NO. 42, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence depicted as SEQ ID NO. 42;
11 Amino acid sequence depicted in SEQ ID NO. 43, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence depicted in SEQ ID NO. 43;
12 44 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown in SEQ ID NO. 44;
13 An amino acid sequence shown as SEQ ID NO. 45, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 45;
14 The amino acid sequence shown as SEQ ID NO. 46, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence shown as SEQ ID NO. 46.
16. An antibody-drug conjugate comprising the VEGF-Sub>A binding molecule of any one of claims 1-15 linked to one or more conjugate moieties.
17. Sub>A nucleic acid encoding the anti-VEGF-Sub>A binding molecule of any one of claims 1-15.
18. The nucleic acid of claim 17, comprising:
1) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 4;
2) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 8;
3) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 12;
4) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 16;
5) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 20;
6) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 23;
7) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 24;
8) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 25;
9) A nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 26;
10 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 27;
11 Nucleotide coding sequence of the amino acid sequence shown in SEQ ID NO. 28;
12 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 29;
13 Nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 30;
14 A nucleotide coding sequence of the amino acid sequence shown as SEQ ID NO. 31; or (b)
Preferably, the nucleic acid further comprises a nucleotide coding sequence for the amino acid sequence shown in SEQ ID NO. 32.
19. An expression vector comprising the nucleic acid of claim 17 or 18.
20. A host cell comprising the nucleic acid of claim 17 or 18 or the expression vector of claim 18.
21. Sub>A pharmaceutical composition comprising Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 16, or Sub>A nucleic acid according to claim 17 or 18, or one or more of the expression vectors according to claim 19, and Sub>A pharmaceutically acceptable carrier.
22. Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 16, or Sub>A pharmaceutical composition according to claim 21, for use in the preparation of Sub>A medicament for treating or preventing Sub>A VEGF-Sub>A-related disease in Sub>A subject; or for use in Sub>A medicament for inhibiting or blocking VEGF-Sub>A binding to VEGFR in Sub>A subject.
23. The use of claim 22, wherein the medicament is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.
24. The use of claim 22, wherein the disease is a proliferative disorder or an ocular disease, the proliferative disease comprising cancer.
25. The use of claim 24, wherein the cancer is selected from the group consisting of: breast cancer, liver cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, lung cancer, thyroid cancer, nasopharyngeal cancer, renal cancer, head and neck cancer, and pancreatic cancer.
26. The use of claim 22, wherein the ocular disease comprises age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, and other ocular diseases involving neovasculature.
27. Treating or preventing Sub>A VEGF-Sub>A-related disease in Sub>A subject; or Sub>A method of inhibiting or blocking VEGF-Sub>A binding to VEGFR in Sub>A subject, comprising administering to Sub>A subject in need thereof Sub>A therapeutically effective amount of Sub>A VEGF-Sub>A binding molecule according to any one of claims 1-15, or an antibody-drug conjugate according to claim 16, or Sub>A pharmaceutical composition according to claim 21.
28. The method of claim 27, wherein the drug is administered orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.
29. The method of claim 27, wherein the disease is a proliferative disorder or an ocular disease, the proliferative disease comprising cancer.
30. The method of claim 29, wherein the cancer is selected from the group consisting of: breast cancer, liver cancer, esophageal cancer, gastric cancer, carcinoma of large intestine, lung cancer, thyroid cancer, nasopharyngeal cancer, renal cancer, head and neck cancer, and pancreatic cancer.
31. The use of claim 29, wherein the ocular disease comprises age-related macular degeneration, diabetic retinopathy, retinal vein occlusion, pathological myopia, neovascular glaucoma, and other ocular diseases involving neovasculature.
32. Sub>A kit comprising Sub>A VEGF-Sub>A binding molecule according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 16, or Sub>A pharmaceutical composition according to claim 21, for use in the treatment or diagnosis of Sub>A VEGF-Sub>A related disease.
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