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The most commonly used haloacetyl reagents contain an iodoacetyl group that reacts with sulfhydryl groups at physiological pH. In general, XTEN as monomers or as multimers with cumulative lengths longer that about residues incorporated into the conjugates result in longer half-life compared to shorter cumulative lengths, e. In the foregoing embodiment, the composition has the configuration of formula V: A chimeric protein may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
As used herein, the term “biologically active protein variant” includes proteins modified deliberately, as for example, by site directed mutagenesis, synthesis of the encoding gene, insertions, or accidentally through mutations and that retain 0401g. Due to the incorporation of a PABC self-eliminating linker, this dipeptide was rapidly degraded to liberate paclitaxel as a final cleavage product Elsadek B. In another embodiment, the helper sequence is selected from the group consisting of: The output current of each channel can be.
In one embodiment of the sdp composition, the first chromatography substrate is selected from the group consisting of HIC, cation exchange, anion exchange, and IMAC.
Unmarked lanes are molecular weight standards. In another embodiment, the XTEN sequenes of the compositions are substantially lacking secondary structure v measured by circular dichroism.
By “net charge density” of a protein or peptide herein is meant the net charge divided by the dssp number of amino acids in the protein. Determination of both the apparent molecular weight and apparent molecular weight factor for representative proteins is described in the Examples.
An “in-frame fusion” refers to the joining of two or more open reading frames ORFs to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
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Camera Side 4 1. The two intermediates are reacted resulting in the branched conjugate, 001b then two payload A molecules are added via click chemistry reaction resulting in the final product XTEN-payload conjugate. The leaving group in the reaction is water and no reducing agents hww. In the figure the following legend applies: Payload also includes a molecule that can be used for imaging or in vivo diagnostic purposes.
It has been shown that the acid-sensitive hydrazone bond is the actual cleavage site Jaracz S. However, protein polymers such as XTEN are unique in that, given their unstructured nature, generally lack three- dimensional structures. Thus, the invention contemplates that multiple XTEN gene constructs can be created with cysteines or lysines inserted at different locations within the XTEN sequence by the selection of restriction sites within the XTEN sequence and the design of oligonucleotides appropriate for the given location and that encode a cysteine or lysine, including use of designed oligonucleotides that result in multiple insertions in the csp XTEN sequence.
In general, a polypeptide made by recombinant means and expressed in a host cell is considered to be “isolated. Four bit Digital Counter More information. The polymer may be linear 00401b branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. EGS Ethylene glycol Z? In some embodiments of the dimeric XTEN-cross- linker-payload composition, the composition has the configuration of formula XI.
In some embodiments, where the XTEN serve primarily as a carrier, the invention encompasses XTEN conjugate compositions comprising two, three, four or more XTEN wherein the cumulative XTEN sequence length of the XTEN proteins is greater than about,,or to about amino acid residues, wherein the construct exhibits enhanced pharmacokinetic properties when administered to a subject compared to a payload not linked to the XTEN and administered at a comparable dose.
WOA2 – Xten conjugate compositions and methods of making same – Google Patents
As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. MacroCap Q elution fractionsrespectively. The overall net charge and net charge density is controlled by modifying the content of charged amino acids in the XTEN sequences, either positive or negative, with the net charge typically represented as the percentage of amino acids in the polypeptide contributing to a charged state beyond those residues that are cancelled by a residue with an opposing charge.
In another embodiment of the foregoing compositions, the composition further comprises a second cleavage sequence. Such agents may include peptides, proteins, carbohydrates, nucleic acids, nucleosides, oligonucleotides, and small molecule synthetic compounds, or analogs thereof. This enables one to adjust the relative potency or selectivity of the payloads in the resulting XTEN- folate conjugate.
The gel was stained with Coomassie Blue stain using standard methods. Most proteins have globular structure, which is the most compact three-dimensional structure a protein can have with the smallest hydrodynamic radius. In one embodiment, the bivalent configuration conjugate is created by reacting the engineered XTEN, such as those specifically provided in Table 1with a first XTEN-payload comprising a linker appropriate for reaction with the cysteine-engineered XTEN, followed by a second reaction with a second XTEN-payload comprising a linker appropriate for reaction with the lysine-engineered XTEN, resulting in the final product.
In other cases, a cysteine-motif can be created de novo and be of a pre-defined length and number of cysteine amino acids by linking nucleotides encoding cysteine to nucleotides encoding one or more amino acid residues used in XTEN e.
A host of physiologically relevant conditions for use in in vitro assays have been established. For an ADC to be effective and relatively nontoxic, the chemotherapeutic drug should remain in stable linkage with the mAb while in circulation but should be efficiently released on. A host 4001b physiologically relevant conditions for use in in vitro assays have been established. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine.
Items in search results. In other embodiments of the XTEN-crosslinker conjugate compositions, the compositions further comprise a single atom residue of a first payload conjugated to each firs t cross-linker wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen and sulfur. In the foregoing embodiments, the large hydrodynamic radius conferred by the XTEN in a XTEN-payload conjugate can lead to reduced clearance of the resulting conjugate, an increase in terminal half- life, and an increase in mean residence time.