WO2020159524A1 - Mrna display antibody library and methods - Google Patents
Mrna display antibody library and methods Download PDFInfo
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- WO2020159524A1 WO2020159524A1 PCT/US2019/016135 US2019016135W WO2020159524A1 WO 2020159524 A1 WO2020159524 A1 WO 2020159524A1 US 2019016135 W US2019016135 W US 2019016135W WO 2020159524 A1 WO2020159524 A1 WO 2020159524A1
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Definitions
- the field of the invention is compositions and methods for ultrahigh-diversity antibody libraries, especially as it relates to mRNA display libraries and use of mRNA display libraries for generating recombinant high-affinity binders.
- Targeting tumor antigens or neoepitopes with high-affinity, specific antibodies or binding molecules has been proven as effective methods for treating cancer patients. As more and more patient-specific and/or cancer specific tumor antigens and/or neoepitopes are identified via in vivo, in vitro, or in silico through omics data analysis, the demand of creating an antibody library or display library that provides high probabilities of selecting antibodies or binders that are stable, soluble, functional, and adaptable has grown.
- recombinant phage display libraries can be used. While such approach allows generation of libraries with reasonably high diversity, many rounds of enrichment for binders are often required, which is labor intensive and time consuming. Moreover, despite the relatively large diversity, the binders tend to have less than ideal affinities and stability. Still further, diversity is typically limited by practical considerations such as library volume, transfection efficiency, etc. Such and other approaches can be further optimized, for example, using multiple artificial selection pressures as is described in WO 2006/072773. While such methods may improve stability characteristics, significant amounts of library manipulation and time are required.
- mRNA display may be performed.
- mRNA sequences encoding candidate binding molecules typically scFv
- peptides encoded by the mRNA sequences are generated via in vitro translation to produce a fusion product that coupled the mRNA directly to the protein encoded by the mRNA.
- current mRNA display technology advantageously avoids problems associated with transfection limits and at least conceptually allows for higher diversity, problems with structural integrity or stability, relatively low affinity, and/or cross-reactivity still remain.
- VH-CDR3 spectratyping analysis was performed (see Protein Engineering, Design & Selection, 2015, vol. 28 no. 10, pp. 427-435). However, such process required iterative analysis and may not be productive for all antigens.
- inventive subject matter is directed to various compositions of, methods for, and use of a high-diversity nucleic acid library that encodes a plurality of antibodies or antibody fragments to allows for reliable and efficient identification of stable, soluble, and functional antibodies or binders to various biomolecules, and especially cancer antigens or neoepitope.
- one aspect of the subject matter includes a method of generating a high-diversity nucleic acid library that encodes a plurality of antibodies or antibody fragments.
- each member of the three sub-libraries comprises at least one random cassette that has a plurality of degenerate base positions. At least portions of at least two members of the three libraries are recombined to form an expression library member in an expression library, which has a plurality of expression library members. Each expression library member encoding a distinct antibody or antibody fragment. In a preferred embodiment, the expression library member is transcribed into an mRNA fragment, which then is coupled with a puromycin molecule at 3’-end.
- the inventors contemplate a composition having a plurality of nucleic acid libraries.
- the plurality of nucleic acid libraries includes (1) a V H -CDR.1/2 sub-library, (2) a plurality of V H -CDR.3 sub-libraries, and (3) a V L sub-library.
- Each of the sub-libraries (l)-(3) comprises a plurality of members and the each member of the sub-libraries comprises at least one random cassete that has a plurality of degenerate base positions.
- the inventors contemplate use of the composition above for generating a high-diversity nucleic acid library.
- the inventors contemplate a high-diversity nucleic acid library composition having a plurality of library members.
- the high-diversity nucleic acid library member includes a recombinant nucleic acid comprising a plurality of random cassetes, each having a plurality of degenerate base positions.
- the plurality of random cassetes is derived from at least two members from any of two libraries from the following: (1) a V H -CDR1/2 sub-library, (2) a plurality of V H -CDR3 sub-libraries, and (3) a V L sub-library.
- the inventors contemplate use of the high-diversity nucleic acid library for generating a therapeutic recombinant antibody against a cancer neoepitope.
- the inventors contemplate a method of generating a recombinant antibody.
- three sub-libraries (1) a V H - CDRI/2 sub-library, (2) a plurality of V H -CDR3 sub-libraries, and (3) a V L sub-library, each having a plurality of members are generated or provided.
- Each member of the three sub- libraries comprises at least one random cassette that has a plurality of degenerate base positions.
- At least portions of at least two members of the three libraries are recombined to form an expression library member in an expression library, which has a plurality of expression library members.
- Each expression library member encoding a distinct antibody or antibody fragment.
- the method continues with generating the recombinant antibody or fragment thereof using the expression library member.
- the inventors contemplate a method of isolating a high affinity binder having an affinity of equal or less than 100nM to an antigen, by contacting the antigen to a composition constructed by the methods described above.
- the inventors contemplates a recombinant nucleic acid fragment generated using an oligonucleotide selected from Table 1 or Table 2 provided below.
- the inventors contemplate a synthetic nucleic acid mixture having a nucleic acid sequence selected from Table 1 or Table 2 provided below.
- the inventors contemplate a recombinant virus.
- the recombinant virus comprises a recombinant nucleic acid that includes a member of an expression library that encodes a distinct antibody or antibody fragment.
- the member of the expression library is generated by generating or providing (1) a V H -CDR.1/2 sub-library, (2) a plurality of V H -CDR3 sub-libraries, and (3) a VL sub-library, wherein each of the sub-libraries (l)-(3) comprises a plurality of members, where each member of the sub libraries comprises at least one random cassette that has a plurality of degenerate base positions, and by recombining at least portions of at least two members of the V H -CDR1/2 sub-library, the plurality of V H -CDR3 sub-libraries, and the V L sub-library to form the expression library member in the expression library.
- the recombinant virus is a genetically modified, low immunogenic virus, which most preferably, can be a human adenovirus serotype 5 with a mutation in at least one of the following genes: El A, El B, E2B, E3.
- the plurality of members of the V H -CDR1/2 sub-library comprises a random cassette corresponding to at least one of a portion of V H CDRl and at a portion of V H CDR2.
- the plurality of members of the V H -CDR1/2 sub library comprises a plurality of random cassettes corresponding to at least the portion of V H CDR2
- the plurality of members of the V H -CDR1/2 sub-library comprises a plurality of random cassetes corresponding to at least a portion of V H CDRl and at a portion of V H CDR2.
- the plurality of the members of the V H -CDR3 sub-libraries comprises a random cassete corresponding to at least a portion of V H CDR3. It is contemplated that at least two random cassetes of members of the V H -CDR3 sub-libraries encodes peptides with different lengths.
- the plurality of the members of the V L sub-library comprises a random cassete at a portion of V L CDR3.
- the step of recombining comprises isolating the at least portions of the members of the V H -CDR1/2 sub-library and one of the plurality of V H -CDR3 sub-libraries and fusing together to form a V H domain library member in a V H domain library, wherein the V H domain library comprises a plurality of V H domain library members.
- the member of an expression library is generated by isolating at least a portion of the member of the V L sub-library and fusing the portion of the member of the V L sub-library with one of the V H domain library members to form the expression library member.
- the step of recombining comprises isolating the at least portions of the members of the V H -CDR1/2 sub-library and one of the plurality of V H -CDR3 sub-libraries and fusing together to form a first group of expression library members.
- the recombinant nucleic acid may further comprise a nucleic acid fragment encoding a signaling peptide facilitating a secretion of the distinct antibody or antibody fragment.
- the inventors contemplate a method of generating a recombinant antibody.
- a V H -CDR1/2 sub-library (2) a plurality of V H -CDR3 sub-libraries, and (3) a V L sub-library, wherein each of the sub- libraries (l)-(3) comprises a plurality of members are generated or provided.
- each member of the sub-libraries comprises at least one random cassete that has a plurality of degenerate base positions.
- the method continues with a step of recombining at least portions of at least two members of the V H -CDR1/2 sub-library, the plurality of V H -CDR3 sub-libraries, and the V L sub-library to form an expression library member in an expression library, wherein the expression library comprises a plurality of expression library members, each expression library member encoding a distinct antibody or antibody fragment. Then, recombinant viral vector comprising at least one expression library member can be generated.
- the recombinant virus is a genetically modified, low immunogenic virus, which most preferably, can be a human adenovirus serotype 5 with a mutation in at least one of the following genes: El A, El B, E2B, E3.
- the random cassete is generated using an oligonucleotide selected from SEQ ID NO: l - SEQ ID NO:25.
- the recombinant viral vector further comprises a nucleic acid fragment encoding a signaling peptide facilitating a secretion of the distinct antibody or antibody fragment.
- the method may further comprise a step of contacting a recombinant virus having the recombinant viral vector with a mammalian cell.
- the step of contacting comprises administering the recombinant virus to a mammal.
- the mammalian cell is an autologous cell of a patient having a tumor, and the step of contacting comprises co-incubating the autologous cell with the mammalian cell ex vivo.
- Fig. 1 illustrates one exemplary randomization strategy using VH3/Vkl pairs.
- Fig. 2 illustrates exemplary locations for sequence randomization in heavy chain CDR1 and CDR2.
- Fig. 3 illustrates exemplary sequence randomization in heavy chain CDR3.
- Fig. 4 illustrates exemplary sequence randomization in light chain CDR3 with nucleic acid sequences to the left and amino acid choices to the right.
- Fig. 5 illustrates an exemplary generation of hybrid nucleic acid elements by isolating and combining random cassetes of multiple recombinant nucleic acid segments.
- Fig. 6 shows a size exclusion chromatography result showing a single peak indicating a stable protein expression of aB7-H4 801 .
- Fig. 7 shows a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) data indicating similar molecular behavior of aB7-H4 801 compared to commercial antibodies.
- CE-SDS capillary electrophoresis sodium dodecyl sulfate
- Fig. 8 shows graphs indicating binding of in vitro selected aB7-H4 antibodies to B7- H4.
- Fig. 9 shows graphs of functional analysis of in vitro selected aB7-H4 binders and aPD-Ll binders.
- Fig. 10 shows graphs indicating binding affinities of aB7-H4 scFv and aB7-H4 IgGl.
- Fig. 11 shows an IL-8 activity assay and its result by measuring neutrophil size changes.
- Fig. 12 shows bar graphs indicating neutralization effect of aIL-8 antibody to IL-8 activity of increasing neutrophil size.
- Fig. 13 shows IL-8 activity assay and its results shown in bar graph indicating neutralization effect of aIL-8 antibody to IL-8 activity by inhibiting neutrophil migration.
- Fig. 14 shows exemplary results using mRNA display library compositions presented herein with respect to selected antigen targets.
- Fig. 15 shows an exemplary graph depicting affinities of selected binders configured as scFv versus IgG where the binders were identified using mRNA display library compositions presented herein.
- the inventors now discovered that specific and effective recombinant antibodies or fragments thereof can be generated or identified by constructing a high-diversity nucleic acid library using targeted diversification of selected domains of the antibodies or fragments thereof encoded by members of the high-diversity nucleic acid library.
- the inventors have now discovered that one or more domains or subdomains of antibody /binder can be pre-selected and a plurality of nucleic acid sub-libraries can be generated using random cassettes in a pre-selected domain or subdomain.
- the members of the sub-libraries can be recombined to construct the high-diversity nucleic acid library that allows high diversity among library members, yet provides higher probabilities of identifying antibodies/binders that are stable, soluble, functional, and adaptable when used in vivo against the cancer antigens or neoepitopes (preferably cancer-specific, patient-specific neoepitopes or neoantigens).
- the libraries presented herein allow for isolation of at least one binder to any arbitrary antigen, typically in a single or two-pass enrichment, where the binder has a K d of equal or less than 100nM, and more typically equal or less than lOnM.
- contemplated systems and methods allow for scFv libraries having a diversity of at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , at least 10 13 , at least 10 14 , at least 10 15 , or at least 10 16 distinct library members, all in a time frame that is significantly reduced as compared to conventional library construction.
- the speed of antibody discovery is substantially increased.
- tumor refers to, and is interchangeably used with one or more cancer cells, cancer tissues, malignant tumor cells, or malignant tumor tissue, that can be placed or found in one or more anatomical locations in a human body.
- the term“bind” refers to, and can be interchangeably used with a term “recognize” and/or“detect”, an interaction between two molecules with a high affinity with a K D of equal or less than 10 -6 M, or equal or less than 10 -7 M.
- the term“provide” or“providing” refers to and includes any acts of manufacturing, generating, placing, enabling to use, or making ready to use.
- variable domains in the heavy chain (V H ) and light chain (V L ) constitute, together, the epitope binding domain, which provides specificity to the antibodies.
- V H and VL includes three complementarity determining regions (CDRs, CDRl-3) with unique amino acid sequences based on their specificity to an antigen.
- V H andV L may provide great diversity to the library, it also creates inefficiency in generating all combinations of random sequences and screening all randomized combinations as not all randomized V H and V L can be soluble or stably expressed when it is recombined to form an antibody (e.g ., IgGl, etc.). Moreover, covering the entire diversity space is not practical due to the extremely large number of possible library members.
- subdomains of V H and VL can be divided into two categories: a framework region that are generally common among V H or V L of different antibodies (or genes encoding the antibodies) and a targeted diversification region that can be at least partially or completely randomized without significantly affecting the stability and/or solubility of the final peptide product (e.g., scFv, IgGl, etc.).
- the targeted diversification region of V H includes at least a portion of CDR1, CDR2-n (N-terminus side of CDR2), CDR2-c (C-terminus side of CDR2), and CDR3.
- the targeted diversification region of VL includes at least a portion of CDR3.
- a nucleic acid library can be created by generating recombinant nucleic acids that include one or more random sequence cassettes in one or more targeted diversification region of V H and/or V L .
- the inventors contemplate three different sub- libraries having different sets of random sequence cassettes in different targeted diversification region of V H and/or V L .
- each sub-library retains the diversity within randomized targeted diversification regions while avoiding too many randomized recombinant sequences in a single sub-library that may render the volume of the single sub-library impractical or inefficient to handle for quick or timely screenings. Furthermore, conserved areas between the targeted diversification regions are selected or designed for maximum stability and solubility.
- the sub-libraries include a V H -CDR1/2 sub-library.
- the V H - CDR1/2 sub-library comprises a plurality of recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to at least a portion of V H CDRl and/or at a portion of V H CDR2.
- the random cassette e.g., recombinant DNA
- V H CDRl corresponding to a portion of V H CDRl means that the random cassette is located in an area of the recombinant nucleic acid, in which sequences encoding CDRl portion should be present in order to encode a portion of V H domain which is at least structurally or functionally similar to V H domains of natural antibodies.
- recombinant nucleic acids in a V H - CDR1/2 sub-library may have a structure as below (randomized region is underlined, and fixed sequenced region is parenthesized):
- UTR refers to untranslated region and FW refers framework region ( e.g .,
- FW1 is the first framework region that may be distinct from the second framework region (FW2)).
- the random sequence cassettes can be inserted in areas of CDR1 or CDR2, or preferably, both CDR1 and CDR2.
- more than one random sequence cassettes, preferably two random sequence cassettes can be inserted in the area of CDR2: CDR2-n (for 5’-end side of CDR2) and CDR-c (for 3’-end side of CDR2).
- the sub-libraries can also include a plurality of V H -CDR3 sub-libraries.
- Each of V H - CDR3 sub-library comprises a plurality of recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to at least a portion of V H CDR3.
- a recombinant nucleic acids in V H -CDR1/2 sub-library may have a structure as below (randomized region is underlined, and fixed sequenced region is parenthesized):
- the fixed sequences e.g., Promoter - 5’ UTR - FW1 + CDR1 + FW2 + CDR2 + FW3, FW4
- the fixed sequences are selected to use the most common and/or conserved sequences among the natural antibodies (e.g., IgGls against various antigens) such that the fixed sequences are most expressable and adaptable to multiple formats including peptides expressed as a single chain variable fragment (scFv), a modified form of scFv, full length immunoglobulin, or a portion of immunoglobulin.
- the fixed sequences of the recombinant nucleic acids of V H -CDR1/2 sub-library and of the recombinant nucleic acids of V H -CDR3 sub-library are at least 70%, preferably at least 80%, more preferably at least 90% identical (shared) with each other.
- the sub-libraries can also include a V L sub-library.
- the V L sub-library comprises a plurality of recombinant nucleic acids (e.g., recombinant DNA) having one or more random sequence cassettes corresponding to at least a portion of V L CDR3. Similar to the V H - CDRl/2 sub-library, recombinant nucleic acids in V H -CDR1/2 sub-library may have a structure as below (randomized region is underlined, and fixed sequenced region is parenthesized):
- the fixed sequences of the recombinant nucleic acids of the VL sub-library are at least 70%, preferably at least 80%, more preferably at least 90% identical (shared) to those of recombinant nucleic acids of the V H -CDR1/2 sub-library or V H -CDR3 sub-library.
- the inventors contemplate that strategized random sequence cassettes for CDR1, CDR2, CDR3 of the V H and CDR3 of the V L domain would render a high complexity and large potential binding surface when expressed as a binding peptide ( e.g scFv, etc.).
- the strategized random sequence cassettes for CDR1, CDR2 of the V H -CDR1/2 sub library may be semi-random sequence cassettes having 3 or less, preferably 2 or less, or more preferably, one random sequence (encoding 3 or less, 2 or less, or one random amino acid per cassette) per cassette.
- the location of the random sequence in the random cassette may vary depending on the random amino acid in the cassette.
- the strategized random sequence cassettes for CDR3 of V H -CDR3 sub-library may include more randomized sequences such that 4 or more, preferably 5 or more, or more preferably 6 or more random sequences (encoding 4 or more, preferably 5 or more, or more preferably 6 or more random amino acids per cassette) are present per cassette.
- the strategized random sequence cassettes for CDR3 of V L sub-library may include more randomized sequences such that 4 or more, preferably 5 or more, or more preferably 6 or more random sequences (encoding 4 or more, preferably 5 or more, or more preferably 6 or more random amino acid per cassette) are present per cassette.
- each oligonucleotide includes a random sequences (highlighted) having degenerate code, shown as IUPAC ambiguity codes.
- one oligonucleotide for CDR1 random sequence cassette includes a random sequence“RVT”, which represents“A/G,A/C/G,T”, whose combination can encode one of threonine (T), alanine (A), asparagine (N), aspartic acid (D), serine (S) or glycine (G).
- RVT random sequence
- A threonine
- N alanine
- D aspartic acid
- S serine
- G serine
- G glycine
- the random sequence cassettes for V H -CDR3 sub-library may include nucleic acid sequences in different length.
- the random sequence cassettes for V H -CDR3 sub-library may be in any length between 10-30 amino acids, preferably between 10-25 amino acids, more preferably between 10-20 amino acids.
- the oligonucleotides for generating random sequence cassette for V H - CDR3 sub-library may include a various repeats (e.g., 4-10 repeats) of“NNK” (which represents G/A/T/C, G/A/T/C, G/T) between sequences encoding D/G-R/L and A/G (see also Fig. 3). Generation and diversity of light chain sequences are exemplarily shown in Fig. 4.
- the oligonucleotides presented in Table 1 and 2 are provided in a single strand DNA, which can be converted using DNA polymerase I (Klenow fragment) into double-stranded DNA fragment to so be inserted into a backbone comprising the fixed sequenced region (e.g., 5’- (Promoter - 5’ UTR - FW1 + CDR1 + FW2 + CDR2 + FW3) - (FW4) for recombinant nucleic acids of V L sub-library, etc.).
- the oligonucleotides presented in Table 1 and 2 are also present with the complementary oligonucleotides to form a double stranded nucleic acids without using polymerase enzymes.
- the recombinant nucleic acids of sub-libraries also include a nucleic acid sequence encoding a protein tag such that the peptide encoded by the recombinant nucleic acids can be isolated using the binder against the protein tag.
- preferred proteins tag include a FLAG tag (with a sequence motif DYKDDDDK), a Myc tag (with a sequence motif EQKLISEEDL), and an HA-tag.
- the protein tags can be repeated to strengthen the signal or increase the detection (e.g., three repetitions of FLAG tag (3X FLAG), etc. )
- some random sequence cassettes inserted in the recombinant nucleic acids of sub-libraries may introduce frame shifts, nonsense mutations, and sequence(s) that are destabilizing the structure of the peptide encoded by the recombinant nucleic acids.
- the inventors contemplate that the recombinant nucleic acids of sub-libraries are in vitro tested so that any recombinant nucleic acids encoding unstable or misfolded peptides can be removed from the library.
- the recombinant nucleic acids of the V H -CDR3 sub-libraries or the V L sub-library can be tested for their binding affinity to protein A of Staphylococcus aureus or protein L of Finegoldia magna, which binds to structured epitopes of V H 3 domain or V L (VK) domain of
- any suitable methods to screen the recombinant nucleic acids by their binding affinities to protein A or protein L are contemplated.
- the recombinant nucleic acids of sub-libraries are transcribed into mRNAs by in vitro transcription and the 3’-end of the mRNAs are coupled (covalently linked) to puromycin.
- the puromycin-coupled mRNAs are in vitro translated such that the peptides transcribed from the puromycin-coupled mRNAs are coupled with the mRNAs via the puromycin.
- the peptides are contacted with protein A or protein L to identify peptides effectively binding to the protein A or protein L.
- peptides binding to protein A or protein L with an affinity with a K D of equal or less than 10 -6 M, preferably equal or less than 10 -7 M are selected and isolated.
- cDNAs of the isolated peptides can be generated via in vitro reverse-transcription of the mRNAs coupled with the puromycin and the peptides. The so generated cDNAs of the isolated peptides can be then inserted as random sequence cassettes to generate selected recombinant nucleic acids of V H -CDR3 sub-libraries or the V L sub-library.
- the recombinant nucleic acids of sub-libraries can be present in a form of mRNAs, which is optionally pre-coupled with puromycin molecule such that the in vitro transcription step for the recombinant nucleic acids (in DNA format) may not be needed.
- each of the at least two recombinant nucleic acids (members) of the sub-libraries can be recombined to form recombinant scFv nucleic acids.
- each of the at least two recombinant nucleic acids (members) is selected from different sub-libraries.
- one recombinant nucleic acid may be selected from each of the V H -CDR1/2 sub-library, the plurality of V H -CDR3 sub-libraries, and the V L sub-library.
- one recombinant nucleic acid may be selected from each of two of V H -CDR1/2 sub-library, the plurality of V H -CDR3 sub-libraries, and the V L sub-library.
- at least one of, more preferably all of, the recombinant nucleic acid(s) selected from the sub-libraries are pre-selected via affinity binding screening as described above.
- the recombinant scFv nucleic acids can be constructed by recombining a portion of the recombinant nucleic acids from sub-libraries.
- the portion of the recombinant nucleic acids includes the random sequence cassettes inserted into the recombinant nucleic acids.
- the portion of the recombinant nucleic acids of the V H -CDR.1/2 sub-library can be 5’-[CDRl + (FW2) + CDR21-3’ (random sequence cassettes are underlined), preferably 5’-(portion of FW1)-[CDR1 + (FW2) +
- the portion of the recombinant nucleic acids of the V H -CDR3 sub-libraries can be 5’- [CDR3] - 3’ (random sequence cassettes are underlined), preferably 5’- (portion of FW3) - CDR3 - (portion of FW4)-3’, more preferably, 5’- (portion of FW3) - CDR3 - (FW4)-3' or 5’- (a small linker) - CDR3 - (FW4)-3'
- the V H domain recombinant nucleic acid would be in a structure of 5’ - Promoter - 5’ UTR - FW1 + CDR1 + FW2 + CDR2 + FW3 - CDR3 - FW4 -3’(random sequence cassettes are underlined).
- the V H domain recombinant nucleic acid may also include a nucleic acid sequence encoding a protein tag (e.g., FLAG tag, Myc tag, HA tag, etc.) in its 3’-end as described above.
- a protein tag e.g., FLAG tag, Myc tag, HA tag, etc.
- such generated V H domain recombinant nucleic acids can be placed in a V H domain library as V H domain library members.
- V H domain recombinant nucleic acids can be further recombined with recombinant nucleic acids of the V L sub-library to form the recombinant scFv nucleic acids.
- Fig. 5 shows one exemplary method of recombining the sequences from sub-libraries. As shown, and also typically, a portion of the V H domain recombinant nucleic acid and a portion of the recombinant nucleic acid of the V L sub-library are fused into one the recombinant scFv nucleic acids.
- the portion of V H domain recombinant nucleic acid may include 5’- Promoter - [5’ UTR - FW1 + CDR1 + FW2 + CDR2 + FW3 - CDR3 -FW4 -3’ (preferably without any nucleic acid encoding a protein tag in its 3’-end), and the portion of the recombinant nucleic acid of the V L sub-library may include FW 1' + CDR1 + FW2’ + CDR2 + FW3’ - CDR3 - FW4’ (without promoter and 5’-UTR) such that the recombinant nucleic acid of the V L sub-library can be fused to the 3’-end of the portion of V H domain recombinant nucleic acid.
- the typical recombinant scFv nucleic acid would be in a structure of 5’- Promoter - [5’ UTR - FW1 + CDR1 + FW2 + CDR2 + FW3 - CDR3 - FW4]V h - [FWl’ + CDR1 + FW2’ + CDR2 + FW3’ - CDR3 - FW4’]V L -3' It is highly preferred that the portion of V H domain recombinant nucleic acid and the portion of the recombinant nucleic acid of the V L sub-library are placed in the same reading frame such that they encode a single polypeptide.
- the portion of V H domain recombinant nucleic acid and the portion of the recombinant nucleic acid of the V L sub-library are fused via a nucleic acid encoding a linker (a short peptide spacer fragment) between two portions.
- a linker a short peptide spacer fragment
- Any suitable length and order of peptide sequence for the linker or the spacer can be used.
- the length of the linker peptide is between 3-30 amino acids, preferably between 5-20 amino acids, more preferably between 5-15 amino acids.
- glycine-rich sequences e.g ., gly-gly-ser-gly-gly, etc.
- glycine-rich sequences e.g ., gly-gly-ser-gly-gly, etc.
- the recombinant scFv nucleic acids may also include a nucleic acid sequence encoding a protein tag (e.g., FLAG tag, Myc tag, HA tag, etc.) in its 3’-end as described above.
- a protein tag e.g., FLAG tag, Myc tag, HA tag, etc.
- such generated recombinant scFv nucleic acids can be placed in an expression library as expression library members.
- the so formed recombinant scFv nucleic acids are further screened and/or ranked based on their binding affinities to one or more ligands of interest (e.g., cancer antigens, neoepitopes, etc.), stability, pH sensitivity, and/or species cross reactivity.
- ligands of interest e.g., cancer antigens, neoepitopes, etc.
- stability of the scFv peptides encoded by the recombinant scFv nucleic acids can be analyzed by size exclusion chromatography measuring the size of the peptide over time.
- pH sensitivity and binding affinity of the scFv peptides encoded by the recombinant scFv nucleic acids can be analyzed by contacting the scFv peptides with one or more ligands in different buffer conditions (pH, temperature, etc.).
- the inventors contemplate that the recombinant scFv nucleic acids can be present in a form of mRNAs, which is optionally pre-coupled with puromycin molecule at the 3’-end of the mRNAs.
- the puromycin-coupled mRNAs can then be in vitro translated such that the peptides transcribed from the puromycin-coupled mRNAs are coupled with the mRNAs via the puromycin.
- the peptides are contacted with one or more ligands, optionally in different buffer conditions (pH, temperature, etc.) ⁇
- peptides binding to the ligand with an affinity with a K D of equal or less than 10 -6 M, preferably equal or less than 10 -7 M, between pH 5.0-8.0, preferably between pH 6.0-8.0, more preferably between pH 6.5 -8.0 are selected and isolated.
- cDNAs of the isolated peptides can be generated via in vitro reverse-transcription of the mRNAs coupled with the puromycin and the peptides.
- recombinant scFv nucleic acids can be grafted on and replaced the portion of the
- the so generated cDNA can be fused with the backbone of the immunoglobulin heavy chain constant region such that the variable region of heavy and light chain of the immunoglobulin can be replaced with the scFv formed by the isolated peptide.
- the inventors also contemplate that the V H portion (or derived from V H domain recombinant nucleic acid) and V L portion (or derived from of the recombinant scFv nucleic acid) of the recombinant scFv nucleic acids can be grafted on and replaced the portion of the immunoglobulin to form a recombinant immunoglobulin or fragments thereof.
- V H portion and V L portion (or derived from of the recombinant scFv nucleic acid) of the recombinant scFv nucleic acids are fused with the backbone of the immunoglobulin heavy chain constant region or light chain constant region, respectively, to form an immunoglobulin with variable regions specific to the desired ligand.
- the immunoglobulin can include any type (e.g IgG, IgE, IgM, IgD, IgA and IgY) and any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) of heavy chain or constant domain to constitute different types of immunoglobulin.
- the“antibody” can include, but not limited to a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody.
- contemplated systems and methods allow for the generation of species-specific antibodies by grafting the isolated V H and V L domains onto the remainder of the antibody of a desired species (e.g., human).
- the so generated cDNA can be fused with nucleic acids encoding other portion of the immunoglobulin to form a fragment of the immunoglobulin.
- the fragment of the immunoglobulin can be Fab fragments, Fab’ fragments, F(ab’)2, disulfide linked Fvs (sdFvs), and Fvs.
- the inventors further contemplate that a portion of the so generated cDNA can be fused with nucleic acids encoding other portion of the immunoglobulin to form any fragment comprising either V H segment and/or V L segment.
- the scFv portions may also be used as targeting entities for various proteins and non-protein molecules.
- the scFv portions may be coupled (typically as chimeric protein) to an ALT-803 type molecule to form a TxM entity that has specific targeting capability (see e.g., JBiol Chem. 2016 Nov.
- the scFv portion may be coupled to a carrier protein (e.g., albumin) to allow target specific delivery of one or more drugs to a specific location in a tumor microenvironment where the drugs are coupled to the carrier.
- a carrier protein e.g., albumin
- the inventors further contemplate that by construction the sub-libraries via targeted diversification of random sequences, and/or preselecting the members of the sub-libraries, the expression library can achieve approximately 10 12 complexity with minimal sacrifice of diversity by removing unstable, non-binding, or misfolded sequences.
- the above described approach to generate expression library provides meaningful size of sequence complexity, yet is practical to screen binders/antibodies in a small volume.
- the above described approach to generate expression library simplified the screening procedure of the binders/antibodies.
- nucleic acid sequences e.g., randomized sequences
- binding affinity e.g., Kd value
- pH sensitivity e.g., pH sensitivity
- species cross-reactivity e.g., via surface plasmon resonance assay, etc.
- binders e.g., with nano- and picomolar K d
- mRNA display techniques in which library members after in vitro translation are screened against a solid phase bound antigen.
- binders Once binders are identified, they can be further characterized by surface plasmon resonance spectroscopy with respect to affinity and K on /K off characteristics as is further described below.
- contemplated systems and methods allow for rapid detection of binders and generation of scFv or antibodies in a process that is entirely independent from an in vivo immune system.
- VH3/Vkl can be one of the good candidate regions for randomization among the various domains of immunoglobulin, VH3 is considered by far most stable and soluble VH domain, and Vkl of light chain is stable and soluble. Thus, it is contemplated that the VH3/Vkl randomized pairs would convert to a full size immunoglobulin more efficiently. Accordingly, the inventors developed pre-selection strategy using VH3 and Vkl frameworks. Fig. 1 shows one exemplary randomization strategy using VH3/Vkl pairs.
- Protein sequences of at least 14 immunoglobulin molecules specific to one antigen are compared and analyzed.
- the most stable and conserved sequences among 14 immunoglobulin molecules are used as frameworks and locus of variable sequences are analyzed to use as randomized sequences and the degree of randomization ( e.g complete random, partially random, etc.).
- the inventors further generated targeted diversified sequences (randomized sequences, random oligos) for CDR1, CDR2-n, CDR2-C of V H domain (see Fig. 2) and for CDR3 of V H domain (see Fig. 3).
- the process of generating recombinant scFv nucleic acids using the random oligos of CDR1, CDR2-n, CDR2-C, CDR3 of V H domain, and CDR3 of VL domain is described above and also shown in the schematic diagram in Fig. 4.
- a high-diversity library was constructed as exemplarily shown in Fig. 5 and discussed in more detail above.
- a-B7-H4 801 (a-B7-H4, clone number 801) binder.
- the stability of the recombinant a-B7-H4 801 was determined by analytical size exclusion chromatography over 15 min to evaluate any degradation or deformation of the antibody. As shown in Fig.
- the eluate of a-B7-H4 801 shows a single peak without any significant smaller peaks, indicating the a-B7-H4 801 binder generated by methods described above could produce scFv or an antibody with high stability.
- the fragments of the recombinant a-B7-H4 801 and two commercially available a- B7-H4 antibodies (Rituxan®, LEAF®) were analyzed via Capillary electrophoresis sodium dodecyl sulfate (CE-SDS).
- CE-SDS separation of recombinant a-B7- H4 801 antibody and two commercially available a-B7-H4 antibodies (Rituxan®, LEAF®) fragments show two profound peaks, each corresponds to light chain (middle peak) and glycosylated heavy chain (right peak). Left peak indicates the location of a 10 Kd standard marker for the CE-SDS analysis.
- a-B7-H4 antibodies may show different binding characters (e.g., affinities, specificities, etc.) to the target ligand.
- Fig. 8 shows two recombinant a-B7-H4 antibodies, a-B7-H4 801 and a-B7-H4 817 that are tested for binding with B7-H4 expressing 293T cells, measured by mean fluorescence intensity (MFI).
- MFI mean fluorescence intensity
- the results show that a-B7-H4 801 antibodies have higher binding affinity to B7-H4 expressing 293T cells compared to a-B7-H4 817 antibodies, indicating differently randomized CDR domains may render different binding affinities to the ligand.
- the right most panels show the control experiment with nonspecific human IgGl (hlgGl).
- the recombinant a-B7-H4 antibodies were further tested to determine specific and effective binding to the ligands (B7-H4) expressed on the antigen presenting cells (APCs) using flow cytometry. As shown in Fig. 9, the recombinant a-B7-H4 antibodies could specifically bind to B7-H4 ligands (separating the peak out from nonspecific isotype binding), indicating that the recombinant a-B7-H4 antibodies are fully functional.
- scFv B7-H4 801 scFv B7-H4 801
- recombinant a-B7-H4 antibodies IgG a-B7-H4 801
- Flag-tagged scFv B7-H4 801 are immobilized on the surface via a-Flag biotinylated antibody, which is coupled with surface-linked neutravidin.
- scFv B7-H4 801 peptides are then contacted with analyte including B7-H4. Similar assay was performed with a-B7-H4 antibodies. As shown in Fig. 10 and Table 3, scFv B7-H4 801 and IgG a-B7-H4 801 shows substantially similar affinity and binding characteristics to B7-H4, indicating that they are functionally compatible. Further, as the binding affinity of in vitro translated peptide (scFv) can be directly measured without grafting the peptide into an antibody backbone, more recombinant scFv nucleic acids in the expression library can be screened efficiently.
- scFv binding affinity of in vitro translated peptide
- the inventors also generated a plurality of scFv peptides binding to interleukin-8 (IL- 8) (scFv IL-8) using the sub-libraries and expression library, and examined the affinity to IL- 8 in different conditions (temperatures and pH).
- scFv IL-8 peptides and their binding affinities measured in various conditions are shown in Table 6.
- Table 6 Exemplary scFv IL-8 peptides and their binding affinities measured in various conditions are shown in Table 6.
- clones 49-7, 49-1 and 49-12 contain similar V H CDR3 sequences
- clones 49-19, 49-37, and 49-25 contain similar V H CDR3 sequences.
- clones 49-3 and 43-2 contain similar V H CDR3 sequences.
- 49-18, 49-37, and 49-25 contain similar V H CDR3 sequences, the binding affinity (unit measured in K D X 10 -9 M) of those sequences varies between 0.894 X 10 -9 M and 25 X 10 -9 M.
- the inventors further tested whether the scFv IL-8 can effectively trap IL-8 to thereby neutralize the effect of IL-8 by measuring neutrophil size.
- neutrophils are enlarged ( e.g having a larger diameter, etc.) upon being stimulated by IL-8 (as shown in Fig. 11).
- the inventors found that such IL-8 effect on neutrophil enlargement could be largely abolished upon addition of the recombinant a-IL-8 antibody (mAh a!L-8 2 oi, as shown in Fig. 12, upper- left graph) or several scFv IL-8 peptides (aIL-8 #2 , aIL-8 49.3 , aIL-8 49.10 , as shown in Fig. 12, lower graphs), indicating that the scFv IL-8 peptides could effectively neutralize the effect of IL-8 by binding to free IL-8 in the media.
- IL-8 is a neutrophil chemotactic factor that causes neutrophils to migrate toward the site of IL-8 release ( e.g site of infection).
- neutrophils were placed on the bottom of the insert having a porous membrane and placed in the media including various concentration of IL-8 such that attracted neutrophils by IL-8 can trans -migrate out of the insert through the porous membrane toward the media.
- Fig. 13 number of migrated neutrophils increased by increasing IL-8 concentration in the media.
- IL-8 effect has almost completely abolished upon addition of the scFv IL-8 peptide (aIL-8 43-2 ) or the recombinant IL-8 antibody derived from a scFv IL-8 peptide (mAh aIL-8 201 ).
- Fig.14 depicts further experimental data for a variety of scFvs isolated using the mRNA display library as presented herein. More specifically, each data point represents an scFv for the target indicated at the bottom, and affinity values for each scFv was determined. As can be readily seen, the (same) library yielded multiple high-affinity binders for a variety of distinct targets, with all of the bonders in the sub-mi croM, and many in the sub-nanoM affinity range. Moreover, the inventors also studies whether the affinity of the scFvs could be preserved upon CDR grafting onto a human IgG. Fig.15 depicts exemplary results for 29 CDR grafting experiments for selected scFv that were grafted into a human IgGl scaffold.
- the humanized IgGl antibodies retained high specificity and affinity (typically within one order of magnitude).
- the recombinant scFv nucleic acids or recombinant nucleic acid encoding one or more antibody e.g., IgG, IgM, IgE, IgA, etc.
- a recombinant entity e.g., bacterium, yeast, virus
- the recombinant scFv fragments can be produced by the recombinant entity or a cell infected by the recombinant entity.
- any suitable recombinant entity that can carry the recombinant nucleic acid encoding the recombinant scFv fragments and/or express the recombinant nucleic acid are contemplated.
- the recombinant entity may include any suitable virus including adenoviruses, adeno-associated viruses, alphaviruses, herpes viruses, lentiviruses, etc.
- adenoviruses are particularly preferred.
- the virus is a replication deficient and non-immunogenic virus, which is typically accomplished by targeted deletion of selected viral proteins (e.g ., El, E3 proteins).
- one desired viral vector may include a recombinant adenovirus genome with a deleted or non functional E2b gene.
- the recombinant entity can be a bacteria
- the expression vector can be a bacterial vector that can be expressed in a genetically-engineered bacterium, which expresses endotoxins at a level low enough not to cause an endotoxic response in human cells and/or insufficient to induce a CD- 14 mediated sepsis when introduced to the human body.
- One exemplary bacteria strain with modified lipopolysaccharides includes ClearColi® BL21(DE3) electrocompetent cells.
- This bacteria strain is BL21 with a genotype F- ompT hsdSB ( rB - mB-) gal dcm Ion l.(DE3 [lacl lacUV5-T7 gene 1 indl sam7 nin5 ]) msbA148 DgutQDkdsD DlpxLDlpxMDpagP DlpxP DeptA.
- DgutQDkdsD DlpxLDlpxMDpagP DlpxP DeptA encode the modification of LPS to Lipid IV A , while one additional compensating mutation
- msbA148 enables the cells to maintain viability in the presence of the LPS precursor lipid IVA.
- These mutations result in the deletion of the oligosaccharide chain from the LPS. More specifically, two of the six acyl chains are deleted.
- the six acyl chains of the LPS are the trigger which is recognized by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-kB and production of TLR4
- TLR4 Toll-like receptor 4
- MD-2 myeloid differentiation factor 2
- Lipid IV A which contains only four acyl chains, is not recognized by TLR4 and thus does not trigger the endotoxic response. While
- the recombinant entity is a yeast
- the expression vector can also be a yeast vector that can be expressed in yeast, preferably, in Saccharomyces cerevisiae (e.g., GI-400 series recombinant immunotherapeutic yeast strains, etc.).
- a plurality of recombinant scFv nucleic acids and/or recombinant nucleic acid encoding one or more antibody can be used to generate a set of recombinant entities (e.g., recombinant virus) to increase the diversity of the therapeutically effective recombinant entities.
- the plurality of recombinant scFv and/or nucleic acids recombinant nucleic acid encoding one or more antibody can be selected based on the affinity and/or binding characteristics (e.g., binding kinetics, etc.) of the scFv fragments or the antibody to an antigen, such that top 30%, top 20%, top 10%, or to 5% of the scFv fragments or antibodies with highest binding affinity or other binding characteristics can be selected from a pool of scFv fragments or antibodies.
- affinity and/or binding characteristics e.g., binding kinetics, etc.
- such selection process can include selection of high-pass fragments and enriching those through multiple rounds of selections.
- top 30% of the scFv fragments or antibodies with highest binding affinity can be selected in the first round of selection, and top 50% of the top 30% scFv fragments or antibodies (from the first round) with highest binding affinity in the second round of selection, and so on. While any suitable number of rounds of selections and pass-percentage (e.g., top 30%, top 20%, etc.) may be used, it is preferred that the final set of scFv fragments or antibodies may constitute top 30%, top 20%, top 10%, or to 5% of the scFv fragments with highest binding affinity in the entire pool of the scFv fragments or antibodies.
- Such obtained a set of recombinant scFv nucleic acids and/or recombinant nucleic acid encoding one or more antibody can be further used to generate a heterogeneous pool of recombinant entities (e.g., recombinant virus) that can further generate a plurality of different scFv fragments and/or antibodies binding to the same antigen.
- a heterogeneous pool of recombinant entities e.g., recombinant virus
- the recombinant scFv fragment or antibody with the highest binding affinity may not be the most therapeutically effective scFv fragment or antibody due to many variables in vivo (e.g., slight individual, structural variances in antigens among patients, different binding conditions (e.g., pH, other environmental obstructions, etc.), etc.), or because recombinant scFv fragment or antibody with the highest binding affinity may have an undesirable kinetic characteristics as a therapeutic antibody.
- a heterogeneous pool of recombinant entities using a set of nucleic acid encoding different scFv fragments or antibodies, a chance to identify a therapeutically effective scFv fragment (even if it is not the one with the highest affinity to the antigen).
- a heterogeneous pool of antibodies (generated from a heterogeneous pool of recombinant entities) binding to the same antigen may increase effectiveness to target the antigen in vivo compared to a homogeneous pool of antibodies that may lose effectiveness all together in specific in vivo conditions.
- a heterogeneous pool of recombinant entities especially recombinant virus carrying recombinant nucleic acid encoding various scFv fragments or antibodies targeting the same antigen can be more therapeutically beneficial and/or effective in treating a patient having a tumor.
- the expression vector may include a nucleic acid segment encoding signaling peptide for extracellular secretion such that the produced recombinant scFv or antibody can be secreted from the cell.
- signaling peptide any suitable signaling peptides are contemplated, exemplary signaling peptide may include 5-30 amino acid with a positively charged N-terminal region (n-region), a hydrophobic central region (h-region) and a neutral, polar C-terminal region (c-region), which may be cleavable during intracellular
- the nucleic acid segment encoding signaling peptide may preferably located at the N-terminus of the recombinant nucleic acid encoding the scFv, optionally via a short linker (e.g ., glycine-rich linker-encoding nucleic acid).
- a short linker e.g ., glycine-rich linker-encoding nucleic acid
- the expression vector may further include one or more element that can elicit or boost immune response against the tumor and/or boost the activity of the generated scFv fragments.
- the expression vector may include another nucleic acid segment encoding neoantigen(s), tumor-associated antigen(s), or tumor-specific patient-specific neoepitope, co-stimulatory molecules, immune stimulatory cytokines, a recombinant immunoglobulin protein complex, and/or checkpoint inhibitors.
- the cytokines any suitable cytokines that are capable of modulate the immune response (e.g., increase or decrease T cell activity, etc.) are contemplated.
- the contemplated co-stimulatory molecule may include B7.1 (CD80), B7.2 (CD86), CD30L, CD40, CD40L, CD48, CD70, CD112, CD155, ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, LFA3 (CD58), and members of the SLAM family.
- the cytokine may be an IL-15 super agonist (IL-15N72D), and/or an IL-15 superagonist/IL-15RaSushi-Fc fusion complex, e.g., ALT-803) that is coupled with at least one of IL-7, IL-15, IL-18, IL-21, and IL-22, or preferably both IL-7 and IL-21.
- IL-15N72D an IL-15 super agonist
- IL-15RaSushi-Fc fusion complex e.g., ALT-803
- the contemplated co-stimulatory molecule may include B7.1 (CD80), B7.2 (CD86), CD30L, CD40, CD40L, CD48, CD70, CD112, CD155, ICOS-L, 4-1BB, GITR-L, LIGHT, TIM3, TIM4, ICAM-1, LFA3 (CD58), and members of the SLAM family.
- Exemplary checkpoint inhibitor includes antibodies or binding molecules to CTLA-4 (especially for CD8 + cells), PD-1 (especially for CD4 + cells), TIM1 receptor,
- CD 160 such as ipilimumab, nivolumab.
- the recombinant entity can be used to produce the scFv fragment and/or an antibody in vitro.
- the recombinant bacteria or recombinant yeast can be induced and/or cultured to express the scFv fragment protein, and such expressed scFv fragment protein can be further purified and/or isolated with any suitable methods (e.g., affinity binding purification, etc.) for further use.
- the recombinant entity can be used to infect a mammalian cell in vivo and/or ex vivo.
- the recombinant virus having the recombinant nucleic acid encoding the scFv fragment can infect mammalian cells in vitro (or ex vivo) by co-incubating the recombinant virus with the mammalian cells to produce the scFv fragments in the mammalian cells.
- the mammalian cells can be any suitable mammalian cells that can produce proteins from exogenous nucleic acid secret the recombinant scFv fragments, and may include any stablished cell lines (human or non-human, e.g., HEK-293 cells, CHO cells, etc.) or autologous cells obtained from a patient having a tumor (e.g., autologous B cells isolated and/or expanded ex vivo, etc.).
- stablished cell lines human or non-human, e.g., HEK-293 cells, CHO cells, etc.
- autologous cells obtained from a patient having a tumor e.g., autologous B cells isolated and/or expanded ex vivo, etc.
- the recombinant virus having the recombinant nucleic acid encoding the scFv fragment and/or an antibody can be administered to a person or a mammal (having a tumor) such that the scFv fragment and/or an antibody can be produced and secreted in vivo.
- the recombinant virus can be formulated in in any pharmaceutically acceptable carrier, and preferably formulated as a sterile injectable composition with a virus titer of between 10 4 -10 12 virus particles per dosage unit.
- alternative formulations are also deemed suitable for use herein, and all known routes and modes of administration are contemplated herein.
- the term“administering” a recombinant virus formulation refers to both direct and indirect administration of the recombinant virus formulation, wherein direct administration of the recombinant virus formulation is typically performed by a health care professional (e.g., physician, nurse, etc.), and wherein indirect administration includes a step of providing or making available the recombinant virus formulation to the health care professional for direct administration (e.g., via injection, infusion, oral delivery, topical delivery, etc.) ⁇
- the recombinant virus formulation is administered via systemic injection including subcutaneous, subdermal injection, or intravenous injection.
- systemic injection may not be efficient (e.g., for brain tumors, etc.)
- the recombinant virus formulation is administered via intratumoral injection.
- the dose and/or schedule may vary depending on the tumor type, size, location, patient’s health status (e.g., including age, gender, etc.), and any other relevant conditions. While it may vary, the dose and schedule may be selected and regulated so that the recombinant virus does not provide any significant toxic effect to the host normal cells, yet sufficient to be effective to induce production of the recombinant scFv fragment to so treat the tumor.
- the contemplated dose of the recombinant virus formulation is at least 10 6 virus particles/day, or at least 10 8 virus particles/day, or at least 10 10 virus particles/day, or at least 10 11 virus particles/day.
- a single dose of recombinant virus formulation can be administered at least once a day or twice a day (half dose per administration) for at least a day, at least 3 days, at least a week, at least 2 weeks, at least a month, or any other desired schedule.
- the dose of the recombinant virus formulation can be gradually increased during the schedule, or gradually decreased during the schedule.
- several series of administration of recombinant virus formulation can be separated by an interval (e.g., one administration each for 3 consecutive days and one administration each for another 3 consecutive days with an interval of 7 days, etc.).
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Priority Applications (8)
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JP2021544556A JP2022521147A (en) | 2019-01-31 | 2019-01-31 | mRNA display antibody library and method |
DE19913048.5T DE19913048T1 (en) | 2019-01-31 | 2019-01-31 | MRNA DISPLAY ANTIBODY LIBRARY AND METHODS |
KR1020217025365A KR20210106001A (en) | 2019-01-31 | 2019-01-31 | mRNA Display Antibody Libraries and Methods |
CN201980090819.2A CN113366104B (en) | 2019-01-31 | 2019-01-31 | mRNA display antibody libraries and methods |
EP19913048.5A EP3918063A4 (en) | 2019-01-31 | 2019-01-31 | Mrna display antibody library and methods |
CA3124941A CA3124941A1 (en) | 2019-01-31 | 2019-01-31 | Mrna display antibody library and methods |
PCT/US2019/016135 WO2020159524A1 (en) | 2019-01-31 | 2019-01-31 | Mrna display antibody library and methods |
AU2019427773A AU2019427773A1 (en) | 2019-01-31 | 2019-01-31 | mRNA display antibody library and methods |
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PCT/US2019/016135 WO2020159524A1 (en) | 2019-01-31 | 2019-01-31 | Mrna display antibody library and methods |
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EP (1) | EP3918063A4 (en) |
JP (1) | JP2022521147A (en) |
KR (1) | KR20210106001A (en) |
CN (1) | CN113366104B (en) |
AU (1) | AU2019427773A1 (en) |
CA (1) | CA3124941A1 (en) |
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US11912756B2 (en) | 2017-11-20 | 2024-02-27 | Nantbio, Inc. | MRNA display antibody library and methods |
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- 2019-01-31 JP JP2021544556A patent/JP2022521147A/en active Pending
- 2019-01-31 WO PCT/US2019/016135 patent/WO2020159524A1/en unknown
- 2019-01-31 DE DE19913048.5T patent/DE19913048T1/en active Pending
- 2019-01-31 AU AU2019427773A patent/AU2019427773A1/en active Pending
- 2019-01-31 CN CN201980090819.2A patent/CN113366104B/en active Active
- 2019-01-31 EP EP19913048.5A patent/EP3918063A4/en active Pending
- 2019-01-31 CA CA3124941A patent/CA3124941A1/en active Pending
- 2019-01-31 KR KR1020217025365A patent/KR20210106001A/en not_active Application Discontinuation
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AU2019427773A1 (en) | 2021-07-15 |
KR20210106001A (en) | 2021-08-27 |
DE19913048T1 (en) | 2022-01-27 |
EP3918063A4 (en) | 2022-12-14 |
CN113366104B (en) | 2024-03-26 |
EP3918063A1 (en) | 2021-12-08 |
JP2022521147A (en) | 2022-04-06 |
CN113366104A (en) | 2021-09-07 |
CA3124941A1 (en) | 2020-08-06 |
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