Patent for “Methods to optimize affinity-based antibody optimization using affinity maturation”

Therapeutic Antibodies – Vaughn Smider, Helen Hongyuan Mao, Taurus Biosciences LLC

Search Patent for “Methods to optimize affinity-based antibody optimization using affinity maturation”

Abstract for “Methods to optimize affinity-based antibody optimization using affinity maturation”

“Provided herein are rational methods of affinity maturation to evolve an antibody’s activity or a portion thereof based upon the structure/affinity/activity relationship of an antigen. The affinity matured antibodies are more or less binding to a target antibody.

Background for “Methods to optimize affinity-based antibody optimization using affinity maturation”

Numerous monoclonal therapeutic and diagnostic monoclonal antibody (MAbs), are used in clinical settings to diagnose and treat human diseases such as cancer and autoimmune disorders. Examples of therapeutic antibodies are Rituxan (Rituximab), Herceptin, Trastuzumab, Avastin and Remicade. It is important to design antibodies that can be used in therapeutic applications. This includes antibodies that have the ability to modulate a target’s functional activity and/or antibodies that are more bioavailable or stable in specific cellular and tissue environments. This article aims to offer methods for optimizing and enhancing the binding affiniites and selecting antibodies with desired affinity.

“This document describes methods for affinity maturation of antibodies and fragments of them based on structure/activity relation (SAR). These methods allow for the optimization of antibodies that have higher and better activity (e.g. The affinity matured starting antibody has a higher binding specificity and affinity than the target antigen.

“This method entails affinity maturation of a portion of a first or secondary antibody for a target protein. The method identifies a similar antibody or portion of an antibody that has a lower activity against the target antigen than its corresponding form. This means that the related antibody contains a related varible heavy chain, or a comparable variable light chains. J? or J? J? Or V?, or J?, germline segments for the nucleic acids molecule encoding variable light chain of first antibody. The method also compares the amino acids sequences of the variable heavy and variable light chains of the first antibody to those of the corresponding variable heavy or variable light chains of the related antibody. A target region within the variable light chain or heavy chain of the first antibody is then identified. This is where the target region refers to a portion of the first antibody that has at least one amino acids difference from the same area in the related antibody. Once a target area has been identified, modified antibodies are made that contain a variable heavy and variable light chains, or a portion thereof. Each modified antibody contains at least one variable heavy or variable light-chain in its target region. The target region is altered by replacing a single amino acids residue. This ensures that each modified antibody contains an amino acid replacement to a different amino Acid than the first. The activity of the resulting mutated antibodies is checked. Modified antibodies have a higher activity for the target antibody than the original antibody. One example of this method is to produce a plurality modified antibodies by producing nucleic acids molecules that encode modified forms or light chains of the first antibody. Each nucleic Acid molecule contains one codon that codes an amino acid in the target area that encodes a different type of variable heavy chain.

“The target region of the first antibody shows 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids differences to that in the related antibody. The method allows the comparison of the first antibody to 1, 2, 3, 4 or 5 related antibodies. The target region in the method is chosen from a CDR1, CDR2, CDR3, CDR4, FR1, CDR2, CDR3, CDR3, CDR4, FR2,, FR3 or FR4. The target region could be, for example, a CDR1, CDR2 and CDR3.

The activity to be assessed in the present method can include binding, signal transduction and differentiation, alteration or gene expression, cell proliferation, chemotaxis (cytotoxicity), cancer cell invasion, endothelial proliferation or tube formation. One example is that of binding. This activity can be assessed using immunoassays, whole cell panning, or surface plasmon resonances (SPR). For example, binding can be assessed by immunoassay such as by a radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or electrochemiluminescence assay. In particular, binding is assessed using an electrochemiluminescence assay such as meso scale discovery (MSD).”

“In the present method, the first antibody that has been affinity matured binds the target antigen with a binding affinity of at least 10?4 M. 10?5 M. 10?6 M. 10?7 M. 10?8 M, and lower, if the antibody is in a form that’s Fab.”

“An example of affinity maturation is comparing a similar antibody or portion thereof with the one that has 80%, 70% or 60%. It also includes comparing it to the antibody with the same activity. The related antibody may have the same level or similar activity as the target antigen, compared to a negative-control. Another example is when the binding affinity of the related antibody is lower than that of the first antibody. This means that the binding affinity in the Fab form is between 10?4 M and 10?5 M, 10,?6 M, 10,?7 M, or 10?8 M. The method described herein can be used to identify a target area within the variable heavy chains of the first antibody. Another example of a method described herein is to identify a target area within the variable heavy chain of the first anti-body. The method is then performed from there. A further example of this method is to identify a target area within the variable heavy chains of the first antibodies and perform the method therefrom. Separately and independently, a target area is identified within variable light chains of the first antibodies and the method then proceeds therefrom.

“A related antibody that has the related variable heavy chain is different from a similar antibody that has the corresponding variable light chains” A related antibody that has the related corresponding heavy chain is identical to a similar antibody that has the related variable light chain.

“In one example, the variable heavy or variable light chains of the first antibody show 80%, 85% and 90% respectively. Particularly, the variable heavy or variable light chains of the first antibody have at least 95% sequence similarity with the corresponding variable heavy or variable light chains of the related antibody.

“In another example, the second antibody contains a related variable heavy chain or variable lighter chain. This is one in which at minimum one of VH, DH, and JH germline sections of nucleic acids molecule encoding variable heavy chains of the first antibody are identical to one of VH, DH, and JH germline segment of nucleic acids molecule encoding variable heavy chains of the related antibody. J? J? J? or J?, or V?, and DH germline segments of nucleic acid molecules encoding the variable-light chain of the related antibodies. The related antibody may contain a similar variable heavy or variable light. This is because it contains at least one of VH, DH, and JH germline sections of nucleic acids molecule encoding variable heavy chains of the first antibody. J? J? J? or J? These examples show that the variable heavy or variable light chains of the first antibody have 60%, 70% and 90% respectively.

“In the present method, the first antibody is identified through screening a combinatorial library. The combinatorial library is created by combining a VH and a DH human germline segments or portions thereof in frame to create a sequence nucleic acid molecule encoding a VH chains or a portion thereof. A J? A J? A J? A germline segment, or portion thereof, is used to create a sequence of nucleic acids molecule encoding a part or all of the VL chains. Each of the VH DH, JH portions, V??, V??, V? or J? are sufficient to create an antibody or a portion thereof containing a VH/VL or a sufficient antigen binding area. To generate a variety of nucleic acids molecules, the steps of combinating are repeated many times. Two libraries are created from the nucleic acids molecules. The first library includes nucleic acids molecules that encode a VH or part thereof. The second library contains nucleic acids molecules that encode a VL or portion thereof. In order to create a library of cells, the nucleic acids molecules from both the first and the second libraries are introduced into each cell. Each cell has nucleic acids molecules that encode a different combination VH/VL from all other cells in the library. The cells are then grown to express the antibodies in the cells. This creates a library of antibodies, each of which contains a different combination VH/VL chains or sufficient to form an antigen binding spot from all the other antibodies and portions thereof in that library. The library is first screened by contacting an antigen or portion of an antibody with a target protein. This is followed by an assessment of binding of the antibody with the target proteins and/or whether it modulates the functional activity of the target proteins. Finally, an antibody is identified or a portion thereof that has activity for the target antibody. A similar antibody can also be identified by screening a combinatorial antigen library for the target protein to find a related one that has a lower activity than the first.

“An addressable library can be the combinatorial library that is being screened. An addressable library is where synthesized nucleic acids sequences are addressed individually. This creates a first addressed and second addressed nucleic Acid library. Each cell is addressed so that it contains nucleic acids molecules that encode a different combination VH/VL than any other cell in the addressable library. The plurality of antibodies, or portions thereof, are addressed so that each locus contains the same antibody, but is different from the others. The address of each antibody or portion of an antibody reveals its identity. A spatial array can be used to organize the addressable library. Each locus corresponds with a different member of the antibody. A multiwell plate can also be used as a spatial array. Another way is to attach the antibodies to the addressable library to a solid support, such as a slide, chip, slide or cellulose filter. The different antibody members are then immobilized on the surface of the support.

The target antigen in the affinity maturation process is a polypeptide or carbohydrate, lipid, nucleic acids, or small molecule. The target antigen may be expressed on the cell’s surface by bacteria, viruses, tumors, or any other cell. It can also be a recombinant protein, peptide, or both. The target antigen can be a protein that is targeted for therapeutic intervention. The target antigen may be involved in cell proliferation, differentiation, cell migration, or angiogenesis. Among the target antigens are, among others, a VEGFR-1 and VEGFR-2 (vascular endothelial grow factor receptors 1, 2 and 3), an epidermal growth factors receptor (EGFR), ErbB-2 and ErbB-3), IGF-R1, C?Met (also known hepatocyte growth hormone receptor; HGFR), and DLL4, DDR1 and KIT. origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB 1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-?R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-?, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.”

“A subset of amino acid residues in a target region are modified using amino acid replacement in the affinity maturation procedure described herein. Amino acid replacement is limited to amino acid residues that are different between the first and second antibodies in the target region. Amino acid replacement can also be used to modify only amino acid residues that differ between the target antibody and the related antibody. Amino acid replacement can be used to modify some amino acids in the target area. The amino acid replacement for an amino acid that has been modified can be applied to all 19 amino acid residues or a subset of them.

“In the method provided herein, that antibody is mutated by PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair. You can also mutate the antibody by NNK, NNS or NNY mutagenesis.

“Scanning mutagenesis is one aspect of the method. This allows for further identification of amino acid residues that can be mutagenized. This method involves producing multiple modified antibodies that contain a variable heavy and a volatile light chains, or a portion thereof. Each antibody contains an amino acid replacement in the target area. Each modified antibody is tested for activity against the target antigen. The second antibody chosen from the plurality of modified antibodies with the highest activity is the one that has retained or increased activity against the target antigen, and is used in lieu of the first in the affinity maturation process described above. The plurality can be made by producing nucleic acids molecules that encode modified forms or light chains of the first antibody that consists of the target antigen. Each nucleic Acid molecule contains one codon that encodes the target amino acid, and the codon of unmodified variable heavier or light chains that does not encode the neutral amin acid.

Further, in scanning mutagenesis, an antibody that has activity equal to or greater than the first antibody can be chosen. The amino acid position in the second antibody can be modified to have an amino acid substitute. This antibody will show a higher activity than the one that has lost its activity.

“The scanned amino acids can be either alanine or threonine in the examples of affinity maturation where scanning mutagenesis has been used. The scanned amino acid can be alanine, for example. A non-natural amino acids can also be included in the scanned amino acid.

“Further,” a subset the amino acids in the target area are modified by amino Acid Replacement to a scanned Amino Acid. Amino acid replacement to a scannable amino acid is used for amino acid residues that are different between the first and second antibodies in the target area. Amino acid replacement to a scannable amino acid is another way to modify amino acid residues. An additional example is that all amino acids in the target area are modified using amino acid replacement to a neutral amino acid.

The methods described herein allow you to determine the amino acid modifications in modified antibodies that differ from the ones without the replacements. The method of affinity maturation described herein can be used to select a modified antibody and then use it as the initial for affinity maturation. The methods herein also allow for one or more amino acids to be replaced in the target area of one or several variable heavy chains, or one or two variable light chains, of selected modified antigens. These modified antibodies are then screened for activity against the target antigen in order to identify an additional modified antibody with an enhanced activity relative to the first and selected modified antibodies.

“Affinity maturation can be applied to the variable heavy chains of the first and second modified antibodies, each with an amino acid replacement in their target regions. You can then choose to perform the procedure on the variable heavy chain of the first antigen and select a second modified antibody each containing an amino acid replacement in its target region. To create a variety of third modified antibodies, the variable heavy chains of first modified antibodies can be combined with the variable lighter chain of second modified antibodies to produce a number of different combinations. Each of these modified antibodies will contain an amino acid replacement in target region of the variable high chain and variable low chain. These third antibodies can then be tested for activity against the target antigen and additional modified antibodies can also be chosen that have a higher activity than the first or second modified antibodies.

“It is possible to optimize other areas of an antibody using any of these methods. You can choose another region in the variable heavy or variable light chains of the modified antibody to further mutagenesis. A plurality of nucleic acids molecules can be made that encode modified forms the variable heavy chains or the variable light chains of the first modified antibodies. Each nucleic Acid molecule contains one codon that codes an amino acid in the chosen region. The variable heavy chain/variable light chain is then modified in the region by replacing a single amino amino acid residue. Each of the further modified antibodies is then produced. They each contain a variable heavy and a varible light chain or a portion thereof. The selected region of each antibody contains the replacement of an amino acids to create a new amino acid. Further modified antibodies are then screened for activity against the target antibody. Only those modified antibodies that show greater activity than the first are selected. The modified region can be CDR1, CDR2, CDR3, CDR3, or FR1., FR2, FR3, or FR4.

“Any of the affinity maturation techniques described herein can contain an antibody or a portion thereof. These antibodies could be F(ab), Fab?, or F(ab) )2, single-chain Fv(scFv), Fv. dsFv. diabody. Fd. Fd fragments and Fab fragments.

This method of affinity maturation is based on scanning mutation. The method involves producing nucleic acids molecules that encode modified forms or the variable heavy chains or the variable light chains of a first antigen. Each nucleic Acid molecule contains one codon that encodes an amino acid that is different from the unmodified variable heavier or lighter chain. Each of the modified antibodies is then made with a variable heavy and variable light chains, or a portion thereof. Each of these modifications contains the replacement of an amino acids position with another amino acid, as opposed to the original antibody. Each of the modified antibodies is tested for activity against the target antigen. The modified antibodies are screened for activity to the target antigen. A second antibody is chosen from those that have retained or increased activity. The second antibody can be further mutated by creating a number of nucleic acids molecules that encode modified forms a variable heavier chain or light chain of that second antibody. Each nucleic Acid molecule encodes an amino acid that is different from the scanned position in the second antigen. Each of the further modified antibodies is produced with a variable heavy and variable light chains, or a portion thereof that contains replacement for an amino acid from the second antibody. Further modified antibodies are tested for activity against the target antigen. A third antibody is chosen from the group of further modified antibodies that has a higher activity for the target antibody than the first or second.

“In one example, the scanning affinity maturation process described herein replaces every position in the variable heavy or variable lighter chain. A complementary determining area in the variable heavy or variable light chains can be selected. It could be CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 or CDRL3.

“The method herein selects a second antibody that contains a scanning mutation and exhibits increased binding or retained binding. The activity of the second antibody selected should be at least 75%, 80% or 90% of that of the first antibody.

“In the affinity maturation procedure provided herein, it is possible to determine the amino acid residue position modified in the second antibody to include an amino acid substituted.

“The scanning methods for affinity maturation described herein allow the scanning amino acids residue to be either an alanine, threonine or proline. An example of an amino acid is alanine. Another example is that the scanning amino acid can be a non-natural one. Each of the nucleic acids molecules encodes a variable chain or light chain. These are modified by replacing one amino acid residue with the same scanned amin acid. The method modifies the scanned amino acids position by substituting an amino acid to any other amino acid residues or to a subset of them.

“After a second antibody has been selected, further modification can be done to the antibody using the scanning methods for affinity maturation described herein. Modification does not include the addition of an amino acid to replace the scanned amino acids or the original amino acids at the same position in the first antibodies. The further modification of the second antibody can be effected by a method that is PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, and double-strand break repair. NNN, NNN or NNR mutagenesis can also be used to create further mutations.

“The activity assessed by the scanning methods for affinity maturation are binding, signal transduction and differentiation, alteration or gene expression, cell proliferation, chemotaxis (cytotoxicity), cancer cell invasion, endothelial proliferation, tube formation, and chemotaxis. If the activity is binding, immunoassay, whole-cell panning, and surface plasmon resonance are used to assess it. The immunoassay can be a radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or electrochemiluminescence assay. For example, the electrochemiluminescence assay can be meso scale discovery (MSD).”

The target antigen can be a polypeptide or carbohydrate, lipid, nucleic acids, or small molecule in the affinity maturation scanning methods. The target antigen may be expressed on the cell’s surface by bacteria, viruses, tumors, or any other cell. It can also be a recombinant protein, peptide, or both. A protein that can be targeted for therapeutic intervention may also be called the target antigen. The target antigen can be involved in cell proliferation, differentiation, cell migration or angiogenesis. Exemplary target antigen include a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d?origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-?R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R, EPO-R., a cadherin, an integrin, CD52, CD44, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF, EGF, HGF, TNF-?, LIGHT, BTLA, lymphotoxin (LT), IgE, G-CSF, GM-CSF and EPO.”

“In one aspect, scanning mutagenesis takes place within the variable heavy chains of the first antibody. The method is then performed from there. Another aspect of the method involves scanning mutagenesis within the variable heavy chain of the first antigen, and the steps are taken therefrom. Another aspect of the process is that scanning mutagenesis takes place within the variable heavy chains of the first antibodies. Steps of the procedure are also performed therefrom. Separately and independently, scanning mutagenesis takes place within the variable light chains of the first antibodies. Steps of the method are then performed therefrom.

Further optimization can be achieved using the method described herein. This method may include the determination of the amino acids modifications in the third antibody that have been modified compared to the one without the amino acid substitutions. You can also create combination mutants. The method described herein also provides a way to generate combination mutants. This involves selecting the third antibody from the list and using it as the first one for maturation. Another example of optimization is when one or more amino acids are replaced in one or several variable heavy chains, or one or multiple variable light chains, of selected third antibody. The further modified antibodies are then screened for activity to the target antibody to identify an additional modified antibody with a higher activity than the first, second, or selected third antibodies. The steps can be applied to the variable heavy chains of the first and third antibodies, which each contain an amino acid replacement in their variable heavy chains. This is different from the first antibody’s corresponding variable heavy-chain. The steps of this method can be performed independently or separately on the variable heavy chain of first antibody. Third modified antibodies with an amino replacement in the varible light chain are then selected. A third antibody’s variable heavy chains can be combined with a different third antibody’s variable light chains to create a variety of further modified antibodies. Each one contains an amino acid replacement for the variable heavy and variable light chains. These are different from the first antibody’s variable heavy and variable light chains. Further modified antibodies can be tested for activity (e.g. The fourth antibody that exhibits increased activity against the target antibody is selected.

Another example is that a different area of the variable heavy or variable light chains of the third antibody can be selected for further mutation. A plurality of nucleic Acid molecules are created that encode modified forms the variable heavy chains or variable lights chains of the third antibody. Each nucleic Acid molecule in this plurality encodes an amino acid that differs from the first modified variable hard or variable light chains. The modified variable heavy chain/variable light chain is then modified in the chosen region by replacing one amino acid residue. A plurality further modified antibodies are then produced, each with a variable heavy and a varible light chain or a portion thereof. The selected region of each antibody contains the replacement of an amino acids to a different amino Acid than the third. Further modified antibodies are then screened for activity (e.g. The activity of the further modified antibodies is screened (e.g., binding) to the target antibody. Only those additional modified antibodies with increased activity are selected. The CDR1, CDR2, CDR3, CDR3, CDR3 and CDR4 regions that are subject to further mutation can be used in this example.

“The antibody may be a full-length antibody, a fragment thereof, or a combination of both a variable heavy and variable light chain. The antibody could be either a full-length or fragment of an antibody that contains Fab, Fab?, F(ab)?. )2, single chain Fv (scFv), Fab, Fab?, F(ab?) Fd fragments and Fab fragments.

“A method of antibody conversion is also provided. This involves the selection of an antibody that has an activity that is different or inverted to that of the reference or first antibody against the same target antigen. An antibody’s activity can be converted from being an antagonist to becoming an activator in one example. The method selects an antagonist antibody, or a fragment of it, as the first antibody. This antibody blocks the target antigen’s functional activity. Each modified antibody is made with a variable heavy and variable light chains, or a small portion of them that can bind to antigen. At least one variable heavy or variable light chain has been modified so it contains at most one amino acid modification relative to the first. Amino acid modification can be defined as the substitution of at least one amino acid residue. Each of the modified antibodies will contain a plurality of amino acids that have been altered to replace the original antibody. One example of this method is to produce a plurality or modified antibodies by making nucleic acids molecules that encode modified forms or light chains of the first antibody. Each nucleic Acid molecule in the plurality will encode a variable or light chain modified by replacing one amino acid residue. Each modified antibody is tested for activity against the target antigen after mutagenesis. The first antibody is converted to an activator if it has been identified or selected with antibodies that have a higher functional activity.

“In some cases of the conversion of an antagonist antibody into an activator, the functional activity of each antibody is determined before they are screened. Antibodies with a greater binding affinity than the corresponding antibody for the target protein are selected or identified. Next, the selected subset of antibodies is further screened for functional activity in order to identify or select those with converted activator activities.

“Another example of the method for antibody conversion is when an antibody’s activity is transformed from an activator into an antagonist. The method involves selecting an antibody, or a fragment thereof, that is an activator antibody. This allows the antibody to increase its functional activity in relation to the target antigen. Each modified antibody is made with a variable heavy and variable light chains, or a small portion of them that can bind to antigen. At least one variable heavy or variable light chain has been modified so it contains at most one amino acid modification relative to the first. Amino acid modification can be defined as the substitution of at least one amino acid residue. Each of the modified antibodies will contain a plurality of amino acids that have been altered to replace the original antibody. One example of this method is to produce a plurality or modified antibodies by producing nucleic acids molecules that encode modified forms or light chains of the first antibody. Each nucleic Acid molecule in the plurality will encode a variable or light chain modified by replacing one amino acid residue. Each modified antibody is tested for activity against the target antigen after mutagenesis. The first antibody is converted to an antagonist if it has an antibodiy that results in a decrease of the target antigen’s functional activity.

“In some cases of the conversion of an activator antibody into an antagonist, the functional activity of each antibody is determined before they are screen for its functional activity. Antibodies with a lower binding affinity than the corresponding antibody for the target protein are selected or identified. Next, the selected subset of antibodies is further screened for functional activity in order to identify or select those with converted antagonist activity.

“In each of the conversion methods above, the target antigen is a VEGFR-1, VEGFR-2, VEGFR-3 (vascular endothelial growth factor receptors 1, 2, and 3), a epidermal growth factor receptor (EGFR), ErbB-2, ErbB-b3, IGF-R1, C-Met (also known as hepatocyte growth factor receptor; HGFR), DLL4, DDR1 (discoidin domain receptor), KIT (receptor for c-kit), FGFR1, FGFR2, FGFR4 (fibroblast growth factor receptors 1, 2, and 4), RON (recepteur d?origine nantais; also known as macrophage stimulating 1 receptor), TEK (endothelial-specific receptor tyrosine kinase), TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor), CSF1R (colony stimulating factor 1 receptor), PDGFRB (platelet-derived growth factor receptor B), EPHA1, EPHA2, EPHB1 (erythropoietin-producing hepatocellular receptor A1, A2 and B1), TNF-R1, TNF-R2, HVEM, LT-?R, CD20, CD3, CD25, NOTCH, G-CSF-R, GM-CSF-R or EPO-R.”

“Provided herein are anti-DLL4 antibody monomers that have a binding affinity for DLL4 of 10?8 M (or lower) as measured by surfaceplasmon resonance (SPR). This monomeric Ig fragment is an activator and activator of DLL4 activities. An example of the binding affinity range is 10?6 M to10?8 M. A scFv dimer or a full-length antibody can be used. An antibody multimer can be a full-length antibody containing a constant area from an IgG1 constant region, IgG2, IgG3, IgA, or IgM. The constant region can be an IgG1 constant area, or a modified version thereof.

“An example is that the antibody multimer has a heavy-chain CDR1(CDRH1), a heavy-chain CDR2(CDRH2), and a heavy-chain CDR3 [CDRH3] set forth by SEQID NO:2909. A heavy chain CDR1/CDRL1 set forth SEQID NO:2911. A light chain CDR2/CDRL2 set forth SEQID NO:2911. And a light CDR3 set forth SEQID NO:2908 to any of the 2908 to activate DLL4 and DLL4 activator of the antibody. The antibody multimer, for example, contains a heavy-chain with a variable area set forth by SEQ ID No: 88 and a lighter chain consisting of a variable area set forth by SEQ ID no:107.

“Another example is that the antibody multimer has a heavy-chain CDR1(CDRH1), a heavy-chain CDR2(CDRH2), and a heavy-chain CDR3 [CDRH3] set forth by SEQID NO:2915. A light chain CDR1/CDRL1 set forth SEQID NO:2917. A light chain CDR2/CDRL2 set forth SEQID NO:2918. And a light CDR3 (CDRL3 (CDRL3). The antibody binds to the SEQID NOS:2914 to DLL4 activator of the DLL4 and activates. The antibody multimer, for example, contains a heavy-chain with a variable area set forth by SEQ ID No: 89 and a lighter chain consisting of a variable area set forth by SEQ ID no:108.

“In the examples of antibody multimers given herein, the heavy chain may contain an IgG1 constant area (e.g. SEQ ID NO. 2922), a light-chain constant region, lambda and kappa (e.g. ”

“Provided herein are methods for treating aberrant angiogenesis due to an angiogenic condition or disease. This includes administering any of these antibody multimers to a subject. The DLL4 receptor activity is then increased. The Notch-1 and Notch-4 DLL4 receptors are examples. An angiogenic condition or disease can include cancer, diabetes retinopathies, and other diabetic complications, inflammation diseases, endometriosis, and age-related macular degradation.

“Outline”

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“Unless otherwise stated, technical and scientific terms used in this disclosure have the same meanings as those commonly understood by those skilled in the art to whom the invention(s). All patents, publications, patent applications, Genbank sequences and databases, Genbank sequences, websites, and other published materials mentioned throughout this disclosure are incorporated herein by reference in its entirety, unless otherwise noted. If there are multiple definitions of terms in this section, the ones found here will prevail. It is understood that URLs and other identifiers or addresses can change, and that information on the internet may come and go. However, you can find equivalent information by searching the internet. This demonstrates the availability and public dissemination thereof.

“An antibody” is an expression that refers to immunoglobulins or immunoglobulin parts, natural or synthetic, as well as any portion thereof, which contains at least a portion the variable region of an immunoglobulin molecule sufficient to form an antigen-binding site. An antibody or a portion of an antibody includes any protein that has a binding domain that is substantially or homologous with an immunoglobulin-antigen binding site. An antibody can refer to an antibody that has two heavy chains (which may be denoted H or H? two heavy chains (which can be designated H and H?) and two light chain(which can be identified L and L). Each heavy chain can either be a full-length immunoglobulin chain or a portion sufficient to form an antigen binding spot (e.g. Heavy chains can include VH chains VHCH1 chains and VHCH1-CH2-CH3 chain, and each light chains can be either a full-length light chains or a portion sufficient to form an Antigen Binding Site (e.g. Light chains include VL chains and L-CL chains. Each heavy chain (H or H?) Each heavy chain (H and H?) pairs with one of the light chains (L or L?, respectively). Antibodies typically include only a small portion (or none at all) of the variable heavy chain (VH) and/or variable light chain (VL). An antibody can also include all or part of the constant area.

“Although full-length antibodies are included, as well as portions thereof, including fragments such as F(ab?) and Fab, Fab? for purposes of this document, the term antibody also includes partial-length antibodies. )2, single-chain Fvs, Fv, dsFv and diabody Fd. Fragments Fab fragments Fd fragments and scFv fractions. Other fragments known include, but not limited to, Fd fragments and scFab fragments. (Hust, et al. BMC Biotechnology (2007) 7:14). Antibodies can be members of any immunoglobulin type, including IgG and IgM.

A full-length antibody, as used herein is one that has two full-length heavy chain (e.g. VH-CH1?CH2-CH3 orVH-CH1?CH2-CH3?CH4), two full-length heavy chains (VL?CL), and two hinge regions (VL?CL), such as antibodies made by antibody secreting cells and synthetic antibodies with the same domains.

“Also used herein, antibody fragment/antibody portion with reference to a?portion thereof?” or a?fragment of? An antibody fragment is any part of a full-length antigen that is shorter than its full length, but still contains at least some of the variable area of the antibody enough to form an antigen binding website (e.g. One or more CDRs and retains the binding specificity and/or activity of the full length antibody. Antibody fragments can also include full-length antibody derivatives made by enzymatic treatment, synthetically, e.g. recombinantly made derivatives. You can find examples of fragments from antibodies such as F(ab), Fab, and Fab?. ?2, single-chain Fvs, Fv, and dsFv, as well as Fv, Fab?, F(ab?) Fragments (see Methods in Molecular Biology Vol 207: Recombinant Immunobodies for Cancer Therapy Methods & Protocols (2003) Chapter 1, pp 3-25, Kipriyanov). Multiple chains can be linked together by disulfide bridges or peptide linkers. A fragment of antibody usually contains 50 to 200 amino acids.

Referring to an “antibody” or a portion of it that is sufficient for formation an antigen binding site. This means the antibody or portion thereof must contain at least one or two of the following: typically, 3, 4, 5, or all 6 CDRs from the VH or VL. It should be sufficient to retain at most a portion the binding specificity the full-length antibody that contains all 6 CDRs. A sufficient antigen binding site requires at minimum CDR3 (CDRH3). You will also need the CDR3 for the light chain (CDRL3). One of the skills in the art can identify CDRs based upon kabat numbering or Chothia numbering. (see e.g. Kabat, E. A. et. al. (1991). Sequences of Proteins of Immunological Interet, Fifth Edition, U.S. Department of Health and Human Services. NIH Publication Number. 91-3242, Chothia, C. and al. (1987) J. Mol. Biol. 196:901-917). Based on Kabat numbering CDR?LI corresponds with residues L24-L34; L50-L56 CDR?L2 corresponds, L50?L56; L89-L97 CDR?L3 corresponds residues L89?L97; H31-H35a, 35a, or 35b depending upon the length; H50-H65 CDR?H2 corresponds residues; H95-H102 CDR?H3 corresponds residues H95-H102

“As used herein, ?antigen-binding site? The interface formed by one or several complementary determining regions, also known as hypervariable region, is called?antigen-binding site? Three CDRs are found in each antigen binding site. The heavy chain variable region has three CDRs, and the light chain variable regions has three. Two antigen combining sites are common in antibody molecules. Each site contains portions of the heavy chain variable regions and portions of the light chain variable regions. Other portions of variable region domains can be found in the antigen combining sites, as well as the CDRs.

“A Fv antibody fragment, as used herein is composed of one variable heavy (VH), and one variable light(VL) domain linked with noncovalent interactions.”

“A dsFv, as used herein, refers to an Fv that has an engineered intermolecular diulfide bond which stabilizes VH-VL pairs.”

“Fd fragment” is an antibody fragment containing a variable region domain (VH), and one constant domain domain (CH1) from an antibody heavy chain.

“As used herein, ?Fab fragment? An antibody fragment is an antibody that contains the full-length of the antibody. It is either a portion of the full length antibody that results in digestion of a full length immunoglobulin with Papain or a fragment that has the same structure as that produced synthetically. recombinantly. A Fab fragment is composed of a light chain (containing a CL and VL portions) and another chain that contains a variable domain of an heavy chain (VH), and one constant domain portion (CH1) of the heavy chains (CH1). It can be recombinantly made.

“A F(ab) fragment is, as used herein. A F(ab)2 fragment is an antibody fragment formed by digestion of an immunoglobulin using pepsin at pH 4.2-4.5. The same structure was produced recombinantly. F(ab) The F(ab)2 fragment contains two Fab pieces, but each heavy chain portion has a few additional amino acids. This allows for recombinant production.

“A Fab? “A Fab? fragment is a fragment that contains one half (one heavy and one light) of the F(ab?) )2 fragment.”

“Fd” is the acronym for Fd. “Fd? fragment” is an antibody fragment containing one heavy-chain portion of F(ab) )2 fragment.”

“An Fv is a fragment containing only the VH and VL domains of an antibody molecule.” A fragment that contains only the VH or VL domains of an anti-body molecule is called a fragment.

A scFv fragment is an antibody fragment that has a variable light chain and a variable heavy chain, covalently linked by a polypeptide linking agent in any order. The linker must be sufficiently long to bridge the two domains without significant interference. Exemplary linkers include (Gly-Serr)n residues with some Glu and Lys residues scattered throughout to increase solubility.

“Diabdies” is a dimeric scFv. They are typically shorter than scFvs’ peptide linkers and preferentially dimerize.

“HsFv” refers to antibody fragments where the constant domains that are normally found in a Fabfragment have been replaced with a heterodimeric coil-coil domain (see Arndt and al. J Mol Biol. 7:312:221-228).”

“An ‘antibody multimer’ is defined herein. An antibody that contains at least two antigen-binding spots. Dimers, trimers, tetramers pentamers and higher-order oligomers are all examples of antibody multimers. A multimer antibody can be formed by a skilled person who has knowledge in the art. Multimeric forms, for example, include antibody oligomers formed via a multimerization domain. This coordinates or facilitates interaction of at most two polypeptides.

“A multimerizationdomain is a sequence or amino acids that promotes stable interaction between a polypeptide molecule and one or more additional molecules. Each multimerizationdomain can be the same multimerization domain or different to form stable multimers with the first domain. A polypeptide can be joined to the multimerization domain either directly or indirectly. Multimerization domains that are examples include immunoglobulin sequences and portions thereof, leucine zips, hydrophobic areas, hydrophilic region, and compatible protein?protein interaction domains. For example, the multimerization domain can be an immunoglobulin constant area or domain. This could include IgG1, IgG2, IgG3 and subtypes IgG4 or IgG1.

“A monospecific?” is defined as a protein that is specific to a single epitope. An antibody that has two or more antigen binding sites. Each antigen-binding site immunospecifically binds the same epitope.

“A?multispecific? is defined herein. An antibody is one that has two or more antigen binding sites. At least two antigen-binding site immunospecifically binds to different epitopes.

“A?bispecific? is defined herein. A multispecific antibody is one that has two or more antigen binding sites. It can immunospecifically bind two epitopes. A?trispecific? A?trispecific antibody is a multispecific antibody with three or more antigen binding sites. It can immunospecifically bind three epitopes. A multispecific antibody with four or more antigen binding sites can immunospecifically bind four epitopes.

“A monomeric Ig fragment is referred to herein. An antibody portion that only contains one antigen-binding area. A monomeric Ig fragment, for instance, may contain a Fv, Fab or scFv.

A polypeptide domain, as used herein, is a portion of a polypeptide (a sequence consisting of 3 or more amino acids, usually 5 or 7), that can form an independently folded structure within a polypeptide that is structurally or functionally distinctable or definable. A polypeptide domain can be a part that forms an independent folded structure within a polypeptide composed of one or more structural motif (e.g. Combinations of alpha helices or beta strands connected with loop regions) and/or is recognized by a specific functional activity such as antigen binding or enzyme activity. One domain of a polypeptide may be more than one. A polypeptide may have one or more structural and/or functional domains. One domain of a polypeptide can be distinguished on the basis of its structure and function. A domain may contain a contiguous linear sequence amino acids. A domain may also include a number of non-contiguous amino acids portions that are not connected along the linear sequence. A polypeptide typically contains multiple domains. Each domain of an antibody molecule, for example, has a number of immunoglobulin (Ig), heavy chains and light chains. They are each approximately 110 amino acids long.

“An Ig domain, as used herein, is a domain that has been recognized by those skilled in the art. It is distinguished by a structure called the Immunoglobulin fold (Ig) fold), which contains two beta-pleated sheet, each with anti-parallel beta amino acids connected by loops. Hydrophobic interactions and an intra-chain disulfide link sandwich the two beta sheets of the Ig fold. Function can further distinguish individual immunoglobulin domains in an antibody chain. A light chain may contain one variable domain (VL), one constant domain domain (CL), and a heavy chain one variable domain (VH), and three to four constant domains (CH). An example of an immunoglobulindomain is each VL, CL and VH domains.

“A?variable domain’ is a term used herein. Referring to an antibody, a?variable domain? is an Ig domain of an antibody heavy chain or light chain. It contains a sequence amino acids that differs among different antibodies. Each heavy and light chain have one variable region domain (VL and VH). Variable domains are responsible for antigen recognition and antigen specificity. CDRs are found in each variable region and they belong to the antigen binding sites domain and framework regions.

“As used in this document, the reference to a VH chain or VL chain (also known as VH domain or VLdomain) refers the polypeptide chains that form the variable domain of an anti-body.”

“A?region? is a term used herein. An antibody is a region that contains a specific function or structure. In an antibody, regions of an antibody include the complementarity-determining region, the framework region, and/or the constant region. A region of an antibody, as used herein, is generally a complementarity-determining region CDR1, CDR2 or CDR3 of the variable heavy chain or variable light chain (CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, CDRH3, CDRH1, CDRH2, CDRH3, CDRH3, CDRH1, CDRH2, CDRH3, CDRH1, CDRH2, CDRH3, CDRH1), or is a frame region FR2, FR2 or FR3 or the variable heavy chains or the variable heavy to be either the variable heavy or the variable heavy chain.

“As used herein, ?hypervariable region,? ?HV,? ?complementarity-determining region? CDR? CDR? (antibody CDR) They are interchangeable to mean one of several portions in each variable region that form an antigen binding spot for an antibody. CDR1 through CDR2 are the three CDRs that make up each variable region domain. CDR3 is the third. These CDRs do not lie along the linear amino acids sequence but are located in the fold polypeptide. CDRs can be found within the loops connecting the parallel strands in the variable domain’s beta sheets.

“Framework regions (FRs), as used herein, are regions within the antibody variable regions domains that are located within beta sheets. The FR regions are comparatively less conserved in terms of their amino acids sequences than the hypervariable areas.

A constant region domain, as used herein is a domain within an antibody heavy or lighter chain that contains a sequence amino acids that is comparatively less conserved among antibodies than that of the variable domain. Each light chain has one light chain constant area (CL) domain, while each heavy chain contains one to several heavy chain constant regions (CH) domains. These domains include CH1, CH2, and CH3. IgA, IgD, and IgG are full-length IgA, IgD, and IgG isotypes. They contain CH1,CH2 CH3 as well as a hinge region. IgE, IgM, and IgG contain CH1,CH2 CH3 AND CH4. The Fab arm of an antibody molecule is extended by the CH1 and CL domains, which contribute to antigen interaction and rotation. Antibody constant areas can perform effector functions such as clearance of pathogens and antigens to which the antibody binds. Through interactions with different cells, biomolecules, and tissues.

Humanized antibodies are antibodies that have been modified to include human? It is possible to use different sequences of amino acid so that an injection to a person does not trigger an immune reaction. These antibodies can be prepared using a variety of methods. Human antibodies can be used to determine the amino acid composition in the non-variable areas. These regions can be identified by computer programs.

“As used herein, ?antibody conversion? “Antibody conversion” is a process where the functional activity of an antigen-specific antibody, or fragment thereof, for a target protein or substrate is altered, usually by changing one or more amino acids residues. This results in an antibody that has an inverse functional activity to the reference or starting antibody. If the reference or starting antibody has antagonist activity against a target antigen then antibody coversion transforms the antibody into an activator/modulator or agonist activity. Another example is when the starting or reference antibodies exhibit activator/modulator activity against a target antigen. In this case, antibody conversion transforms the antibody to an antagonist.

“Affinity maturation” is the term used herein. An antibody is created from a reference antibody (also known as a template or parent antibodies). This process involves mutation of one or more amino acids to increase activity against a target antibody than the corresponding form of the reference. The evolved antibody has a higher activity for a target antigen than the reference or template.

An affinity matured antibody is an antibody with a higher activity for a target antibody than a reference antibody. The affinity matured antibody binds more strongly to the target antigen than the parent or reference antibody. The affinity matured antibody usually binds to the same epitope that the reference antibody.

An optimized antibody is an antibody or a portion thereof that exhibits increased activity against a target or antigen relative to a reference antibody. This could include a higher binding affinity or functional activity, and/or improved binding affinity to a target proteins. The antibody is usually optimized through one or more amino acids modifications (amino acids deletion, replacement, or insertion), compared to a non-modified parent antibody. An activity such as binding affinity is generally increased by 1.5-fold to 1000fold. This is usually at least or around 2-fold to 40-fold. Germline antibody Hit does not contain the modification(s ).

“As used herein:?structure affinity/activity relation? SAR refers to the relationship between structure and activity (e.g. (SAR) refers to the relationship between structure (e.g. Knowing the SAR allows us to identify a specific region in a sequence that is responsible for the activity of an anti-body. These methods are used to determine SAR.

“Activity towards a target proteins or target antigens, as used herein, refers to binding selectivity or binding affinity, and/or modulation a functional activity. Or other measurements that reflect the activity of an antibody, or a portion thereof, towards a target Protein. Activity of an antibody can be measured using a binding or affinity based assay, such as an ELISA, electrochemiluminescence assay (e.g. Meso Scale Discovery, or surface plasmon resonance can be used to measure the activity of an antibody. Cell-based assays are also available as described in this article.

“As used herein as?functional activity?” Refers to the activities of a protein (e.g. A target protein is a portion of a full-length protein that is associated with a complete protein. Functional activities can include biological activity, catalytic, enzymatic activity, and antigenicity. They also include immunogenicity, ability form multimers, signaling and downstream effects, and the ability to bind specifically to receptors or ligands for the polypeptide. For purposes herein, modulation (i.e. Modulation (i.e. activation or inhibition of a function of a polypeptide using an antibody or portion of it herein) means that the functional activity of the protein is altered or changed in the presence or absence of the antibody.

“Binding activity” is a characteristic of a molecule (e.g. A polypeptide that determines whether or not it binds to one or more binding partner(s). The ability to bind the binding partners, the affinity it has with them (e.g. High affinity, avidity with binding partners, strength of binding relationship with binding partners and specificity in binding with them.

“As used herein, ?affinity? or ?binding affinity? An antibody molecule, or a portion thereof, is measured by the strength of binding to an epitope on a target antigen or protein. The equilibrium association constant (KA), or equilibrium dissociation constant(KD) are often used to measure affinity. Low affinity antibody-antigen interaction can cause molecules to dissociate quickly. High affinity antibody-antigen binding, on the other hand, is stronger and lasts longer. An antibody’s affinity to a target protein will be determined by its equilibrium association constant (KA). This is usually greater than or equal than about 106M or 107M or greater that or equals about 108M or greater than/equal to about109M or 109M or more than 1010M or 1011M or 1012M. An equilibrium dissociation constant (KD), of 10’4M, 10’6M to 10?7M, or 10??8M to 10??11M or 10??12M, can also be used to characterize antibodies. A lower dissociation constant is indicative of a greater binding affinity. High affinity antibodies are generally those with a sub-nanomolar or nanomolar dissociation constant. These affinities can easily be determined by conventional methods, such as equilibrium dialysis, radioimmunoassay with radiolabeled target antibody, or any other method that is known to skilled artisans. Analyzing affinity data can be done, for instance, using the method of Scatchard and al., Ann N.Y. Acad. ScL, 51:660 (1949).”

“As used herein, ?epitope? An antibody recognizes a specific region on the antigen- or protein’s surface. There are two types of peptidide epitopes: those that are either continuous epitopes and those that have discontinuous epitopes. The three-dimensional structure of a protein is used to determine an epitope, rather than the linear sequence of amino acids.

“binds to the same epitope” is the definition herein. Referring to more than one antibody means that they compete for binding with an antigen. They bind to the same segments of amino acids, either continuous or discontinuous. The phrase “binds to the same epitope?” is well-known by those skilled in the art. This does not necessarily mean the antibodies will bind to the exact same amino acids. Different amino acids can be bound by antibodies. A first antibody may bind to an amino acid segment that is not completely covered by the second antibody. Another example is when a first antibody binds to one or more segments that overlap significantly with the segments bound by the second antibodies. These antibodies are considered to “bind to the same epitope” for the purposes of this article.

Antibody competition assays can help determine if an antibody binds to the same epitope. As another antibody. These assays are well-known in the art. It is common for antibodies to bind to the same epitope to compete with each other 70% to 72%, 73% to 73%, 73% to 74%, 74% to 74%, 74% to 74%, 74% to 74%, 74% to 74%, 74% to 75%, 75% to 85%, 90%, 95% and more. Radioimmunoassays and assays with other labels can be used to determine the degree of competition between two antibodies. A DLL4 antigen may be incubated in the presence of an equal amount of unlabeled anti?DLL4 antibodies. It is then possible to compare the amount of unlabeled anti-DLL4 antibody binding to the antigen with the amount bound in the absence of the blocking antibody. The percentage difference in binding signals between the unlabeled and blocking antibodies is used to determine competition. If there is a 70% inhibition in binding of the labeled antigen in the presence or absence of the blocking antibodies, there will be competition between the 70% and 70% antibodies. Referring to competition between the first and second antibodies of 70% or greater, for example 70%, 72%/73%, 74%/75%, 74%/75%, 85%/85%, 95% or less, then the first antibody inhibits binding to the antigen (or vice versa) by 70%, 71%/72%/73%, 74%/74%, 74%/74%, 75%/80%, 85%/95% or more (compared with binding to the antigen in the absence the first antibody). The second antibody can inhibit the binding of the first antibody to an antigen by 70%, 72% 73%, 74% 74%, 74% 75%, 80% 85%, 95% or more.

“Surface plasmon resonance” is the term used herein. This optical phenomenon allows the analysis of real time interactions by detections of changes in protein concentrations within a biosensor mat (GE Healthcare Life Sciences).

“As used herein, ?epitope mapping? It is the identification of molecular determinants that are necessary for the recognition of antibody-antigens.

“A?target protein?” is the term used herein. or ?target antigen? This refers to candidate proteins and peptides that are recognized by an antibodies or a portion thereof, and/or whose activity can be modulated by an antigen or part thereof. Any peptide, peptide, or protein that contains an epitope to allow for antibody recognition is considered a target protein. Target proteins are proteins that play a role in the etiology or development of a disorder or disease by virtue of their expression or activity. Here are some examples of target proteins.

Summary for “Methods to optimize affinity-based antibody optimization using affinity maturation”